THE 4 th ACADEMIC CONFERENCE ON NATURAL SCIENCE FOR YOUNG SCIENTISTS, MASTER AND PhD. STUDENTS FROM ASEAN COUNTRIES

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2 ISBN THE 4 th ACADEMIC CONFERENCE ON NATURAL SCIENCE FOR YOUNG SCIENTISTS, MASTER AND PhD. STUDENTS FROM ASEAN COUNTRIES December 2015, Bangkok, Thailand PROCEEDINGS Publishing House for Science and Technology

3 PREFACE The 4 th Academic Conference on Natural Science for Young Scientists, Master and PhD students from ASEAN Countries was successfully held at the King Mongkut s University of Technology North Bangkok, Thailand from 15 to 18 December The Conference is significantly devoted to the establishment of the ASEAN Community in 2015 There were about 250 participants in different field of natural sciences in the meeting. The conference is an excellent venue for the young scientists from ASEAN countries to exchange their research results and experiences. More 100 scientific reports were presented in the conference. Furthermore, some senior scientists and professors were invited to give their talks. The Conference created favorable conditions for all participants to establish new cooperative linkages and also strengthen our friendship. The Proceedings of the 4 th Academic Conference on Natural Science for Young Scientists, Master and PhD students from ASEAN Countries has published the papers presented in the conference and selected by the editorial committee with a standard referee procedure. We have to say that the great success of the Conference was due to the active contribution from all the conference committees, the chairmen of sessions and the participants. Especially, we would like to thank Faculty of Applied Science, King Mongkut s University of Technology North Bangkok for all its wonderful cooperation and great contribution. On the occasion, we express special thanks to Co-Sponsors. We have to thank the conference secretariat and technicians for their dedication and hard works./. Co-Chairmen

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5 EDITORIAL BOARD Prof. Nguyen Dai Hung Prof. Meak Kamerane Prof. Wan Azelee Wan Abu Bakar Prof. Arnel Salvador Prof. Tou Teck Yong Prof. Huynh Thanh Dat Prof. Dao Tran Cao Prof. Dang Diem Hong Prof. Dang Dinh Kim Prof. Le Hong Khiem Prof. Tran Dai Lam Prof. Thai Quang Vinh

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7 ORGANIZERS Vietnam Academy of Science and Technology (VAST) King Mongkut s University of Technology North Bangkok (KMUTNB) Suranaree University of Technology (SUT) Vietnam Physical Society (VPS) Vietnam National University, Hanoi (VNU) Vietnam National University, Ho Chi Minh (VNUH) Graduate University of Science and Technology (GUST, VAST) Institute of Physics (IOP), VAST National University of Laos (NUOL) Royal University of Phnom Penh (RUPP) University of Technology of Malaysia (UTM) Multimedia University, Malaysia University of the Philippines, Philippines East Yangon University, Myanmar Yangon University, Myanmar

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9 SPONSORS King Mongkut s University of Technology North Bangkok Vietnam Academy of Science and Technology (VAST) Vietnam National University in Ho Chi Minh Institute of Physics, VAST

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11 CONFERENCE PRESIDENT: Prof. Acad. Nguyen Van Hieu (VAST) CO - CHAIRMEN Prof. Teravuti Boonyasopon Prof. Dr. Nguyen Dai Hung Prof. Dr. Huynh Thanh Dat Prof. Dr. Nguyen Huu Duc Prof. DrSc. Duong Ngoc Hai Prof. Dr. Le Hong Khiem President, KMUTNB, Thailand President, VPS, Vietnam President, VNU Ho Chi Minh City Vice President, VNU Ha Noi Vice President, VAST; Director GUST Director IOP, VAST Prof. Dr. Somchanh Bounphanhmy Dean, National University of Laos Prof. Dr. Meak Kamerane Dean, RUPP, Cambodia Prof. Dr. Wan Azelee Wan Abu Bakar, Univesiti Teknologi Malaysia Prof. Dr. Arnel Salvador Prof. Dr. Tou Teck Yong Prof. Dr. Kyaw Kyaw Khaung, Prof. Dr. Pho Kaung University of the Philippines Multimedia University, Malaysia Rector East Yangon University Rector Yangon University, Myanmar

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13 CONFERENCE SECRETARIAT Prof. Vu Thi Bich Institute of Physics 10 Dao Tan Str. Ba Dinh, Hanoi, Vietnam Phone: (84 4) ; Fax: (84 4) Dr. Anusara Srisrual Faculty of Applied Science, KMUTNB Dr. Le Vu Tuan Hung Vietnam National University in Ho Chi Minh City 227 Nguyen Van Cu Str., Distr. 5, Ho Chi Minh City, Vietnam Dr. Pham Hong Minh Institute of Physics, 10 Dao Tan Str. Ba Dinh, Hanoi, Vietnam Phone: (84 4) ; Fax: (84 4)

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15 CONTENTS OPTICAL SPECTROSCOPY OF Eu 3+ IONS IN ALUMINOSILICATE GLASS... 7 Sengthong Bounyavon, Vu Phi Tuyen, Nguyen Thi Quy Hai, Pham Thi Minh Chau, Phan Van Do, Vu Xuan Quang and Nguyen Trong Thanh EFFECT ON BONDABILITY OF PROLONGED EXPOSURE TIME AFTER PLASMA CLEANING ON IC PACKAGE Pornthep Yaekwong, Kessararat Ugsornrat, Chalermsak Sumithpibul, Neil S. Carunungan, Bancha Arthibenyakul and Surapan Yimman STUDY ON THE FABRICATION AND PROPERTIES OF ALLOYED QUANTUM DOTS AND GRAPHENE QUANTUM DOTS Pham Nam Thang, Le Xuan Hung, Nguyen Hai Yen, Hoang Van Nong, Vu Thi Hong Hanh, Nguyen Ngoc Hai, Phan Ngoc Hong, Pham Thu Nga EXTRACTION OF BIO-OIL RICH IN OMEGA-3-6 FORM ALKYL ESTER FROM HETEROTROPHIC MICROALGA SCHIZOCHYTRIUM MANGROVEI PQ Le Thi Thom, Luu Thi Tam, Nguyen Cam Ha, Hoang Thi Huong Quynh, Pham Van Nhat, Hoang Thi Lan Anh and Dang Diem Hong HYDROCARBON DEGRADATION IN OILY WASTEWATER BY MICROBIAL BIOFILM ATTACHED ON POLYURETHANE FOAM CARRIERS Do Van Tuan, Do Thi To Uyen, Dong Van Quyen, Le Thi Nhi Cong EXTRACTION OF SQUALENE FROM VIETNAM HETEROTROPHIC MARINE MICROALGA Nguyen Cam Ha, Le Thi Thom, Hoang Thi Huong Quynh, Pham Van Nhat, Hoang Thi Lan Anh and Dang Diem Hong REMOVAL OF COPPER (II) FROM AQUEOUS SOLUTION BY ADSORPTION ONTO MnO 2 NANOSTRUCTURE: EQUILIBRIUM AND KINETIC STUDIES Van-Phuc Dinh, Ngoc-Chung Le, Ngoc-Tuan Nguyen EFFECT OF ENGINEERED NANOPARTICLES ON CYANOBACTERIA STRAIN Tran Thi Thu Huong, Duong Thi Thuy, Ha Phuong Thu; Nguyen Trung Kien, Dang Dinh Kim, Dao Trong Hien SEDIMENTATION RATES IN THE U-TAPAO ESTUARY DEMONSTRATED BY 210 Pb-AND 137 Cs-DATING METHODS Santi Raksawong, Miodrag Krmar, Tripob Bhongsuwan ASYMPTOTIC ANALYSIS FOR TRANSMISSION OF GAUSSIAN BEAM THROUGH A PLANE DIELECTRIC INTERFACE Dinh Trong Quang and Trinh Xuan Tho ISSN

16 SELECTIVE HYDROGENATION OF 3-NITROSTYRENE TO 3-VINYLANILINE OVER PT-BASED BIMETALLIC CATALYSTS Sathaporn Tiensermsub and Joongjai Panpranot A NEW SIMPLE ALGEBRAIC PROOF OF THE HOOK-LENGTH FORMULA Nguyen Bich Van TERAHERTZ EMISSION FROM Mn:GaAs Alexander De Los Reyes, Karim Omambac, Jeremy Porquez, Karl Cedric Gonzales, Elizabeth Ann Prieto, Jorge Michael Presto, Kohji Yamamoto, Masahiko Tani, Armando Somintac, Elmer Estacio and Arnel Salvador DETECTING HUMAN EMOTION VIA SPEECH RECOGNITION BY USING ENSEMBLE CLASSIFICATION MODEL Surinee Doungwichain, Sathit Prasomphan DESIGN OF OPTICAL FILTERS IN SLOTTED PHOTONIC CRYSTAL WAVEGUIDES Hoang Thu Trang and Ngo Quang Minh PREPARATION AND CHARACTERIZATION OF Sm 3+ DOPED ZINC BARIUM TELLURITE GLASSES Patarawagee Yasaka and Jakrapong Kaewkhao PHOTOLUMINESCENCE OF LITHIUM GADOLINIUM BORATE GLASSES DOPED WITH CERIUM (III) OXIDE Kitipun Boonin, Warawut Sa-ardsin and Jakrapong Kaewkhao FABRICATION OF POROUS AMORPHOUS SILICON CARBIDE FREE-STANDING MEMBRANES AND MULTILAYERS Cao Tuan Anh, Luong Truc Quynh Ngan, Dao Tran Cao and Nguyen Ngoc Hai A VAPOR SENSOR BASED ON NANO-POROUS SILICON MICROCAVITY FOR DETERMINATION OF FUEL MIXTURES Thuy Van Nguyen, Van Dai Pham, Thi Cham Tran, The Anh Nguyen, Thuy Chi Do, Van Hoi Pham and Huy Bui STUDY ON SYNTHESIS OF Fe-BTC MOF MATERIAL AT LOW TEMPERATURE AND ATMOSPHERIC PRESSURE Tran Dinh Tuan, Nguyen Thi Hoai Phuong, Ngo Hoang Giang, Nguyen Tien Hue, Do Huy Thanh and Ninh Duc Ha TREATMENT FOR SCIATICA AND DEGENERATIVE KNEE: APPLICATION OF LOW POWER SEMICONDUCTOR LASER Trinh Tran Hong Duyen, Tran Minh Thai, Ngo Thi Thien Hoa, Nguyen Thi Hong Hanh, Dinh Thi Thu Hong, Nguyen Thi Huong Linh, Tran Trung Nghia, Nguyen Thanh Tam, Nguyen Quang Trung, Lam Thanh Son, Nguyen Duong Hung 2 ISSN

17 SUPPORT TUBERCULOSIS TREATMENT USING LOW POWER SEMICONDUCTOR LASER Mai Huu Xuan, Tran Minh Thai, Ngo Thi Thien Hoa, Tran Van Be, Huynh Quang Linh APPLICATION OF LOW POWER SEMICONDCTOR LASER TO TREAT DEGENERATIVE LUMBAR SPINE HERNIATED DISC, OSTEOPHYTOSIS Trinh Tran Hong Duyen, Tran Minh Thai, Ngo Thi Thien Hoa, Bui Chi Hung, Dinh Thi Thu Hong, Ngo Thi Tra Huong, Tran Thanh Vinh, Huynh Thanh Hoa, Tran Thi Ngoc Dung, Nguyen Dinh Quang, Nguyen Minh Chau CONDITIONS TO STRETCH PLASMID DNA MOLECULE IN OPTICAL TWEEZERS USING CW LASER GAUSSIAN BEAM Thai Dinh Trung, Mai Van Luu, Chu Van Lanh, Nguyen Van Thinh, Hoang Van Nam, Thai Doan Thanh, Ho Quang Quy APPLICATION OF LOW POWER SEMICONDUCTOR LASER IN MINIMIZING HARMFUL SIDE EFFECTS AFTER CHEMOTHERAPY AND RADIATION THERAPY IN CANCERS Tran Minh Thai, Ngo Thi Thien Hoa, Tran Thien Hau, Dang Nguyen Ngoc An, Le Quoc Dat INITIAL RESULT OF THE TREATMENT OF FATTY LIVER BY LOW POWER SEMICONDUCTOR LASER Tran Minh Thai, Tran Thien Hau, Ngo Thi Thien Hoa, Nguyen Pham Song Toan APPLICATION OF LOW POWER SEMICONDUCTOR LASER TO THE TREATMENT OF ATHEROSCLEROSIS OF INTERNAL CAROTID ARTERY IN TYPE 2 DIABETES Tran Minh Thai, Ngo Thi Thien Hoa, Tran Thien Hau, Ngo Van Trung, Phan Van To Ni CLINICAL TREATMENT RESULT OF THE BENIGN PROSTATIC HYPERTROPHY OF THE OLD MEN BY USING LOW-POWER SEMICONDUCTOR LASER Tran Anh Tu, Tran Minh Thai, Tran Thi Ngoc Dung, Ton Chi Nhan, Ngo Thi Thien Hoa, Nguyen Xuan Quang, Huynh Thanh Hoa and Cao Tan Tieng LASER CHARACTERISTICS OF A DIODE-PUMPED Nd:YAG LASER SYSTEM USED IN INDUSTRIAL APPLICATIONS Giang Manh Khoi, Do Xuan Tien BIOCHARACTERIZATION AND IDENTIFICATION OF LIGNIN DEGRADING FUNGUS CP36 ISOLATED FROM THE FORESTS OF NORTH CENTRAL VIETNAM Nguyen Thi Hong Lien, Nguyen Van Hieu, Pham Thi Bich Hop, Cao Van Son, Dang Van Son, Phan Thi Hong Thao ISSN

18 RESEARCH INTO THERMAL DECOMPOSITION OF A MIXTURE OF RDX AND INSENSITIVE Nguyen Mau Vuong, Ngo Van Giao, Dang Van Duong SYNTHESIS AND BIOLOGICAL ACTIVITY OF NOVEL DERIVATIVES OF MURRAYAFOLINE A Le Duc Anh, Truong Ngoc Hung, Nguyen Thi Nga, Le Mai Huong, Tran Thi Hong Ha, Tran Thi Thu Thuy, Nguyen Manh Cuong, Le Thi Thoa, Tran Minh Cong, Nguyen Trong Dan, Luu Van Chinh EFFECT OF CATALYSt ON THE BURNING RATE OF ENERGY MATERIALS BASEd ON NC-NG-DINA Le Duy Binh, Nguyen Viet Bac, Pham Van Toai and Dam Duc Trung EXPRESSION AND CHARACTERIZATION OF RECOMBINANT L-ASPARAGINASE IN PICHIA PASTORIS Thi Hien Trang Nguyen, Van Van Vu, Huu Quan Nguyen, Dinh Thi Quyen and Thi Tuyen Do OPTIMAZATION OF CULTURE CONDITIONS FOR PRODUCTION OF PROTEASE BY LECANICILLIUM LECANII Nguyen Huu Quan and Vu Van Hanh INVESTIGATION OF STABLE STRAIN OF STREPTOMYCES SP. KB1 FOR STABILITY OF BIOACTIVE SECONDARY METABOLITES PRODUCTION BY CONTINUOUS SUB-CULTURE Kittisak Chawawisit and Monthon Lertcanawanichakul EFFECT OF COMPOSITION ON THE BURNING RATE OF PYROTECHNICS Doan Anh Phan, Ngo Van Giao and Dang Van Duong REDUCING OF CARBON DIOXIDE POLLUTION BY THE BACKWARD DIFFUSION METHOD Vu Ba Dung DEVELOPMENT OF ELECTROCHEMICAL DETECTOR COMBINE WITH DIGITAL MICROFLUDIC SYSTEM Sarunchana Wiboonsak, Chonticha Chunhakit, Paveena Nakbumpen, Kessararat Ugsornrat, Patiya Pasakorn, Thitima Maturos and Adisorn Tuantranont STRUCTURAL AND PHOTOPHYSICAL PROPERTIES OF CURCUMINOID AND METAL- CURCUMIN COMPLEXES DERIVED FROM TURMERIC (CURCUMA LONGA) Nguyen Thanh Binh, Nguyen Dinh Cong, Nguyen Thi Lan Huong, Ngo Thi Nhan, Nguyen Thi Khanh Van, Le Xuan Hung, Vu Thi Bich 4 ISSN

19 FABRICATION OF Fe 3 O 4 MAGNETIC FLUID BY HYDRO THERMAL FOR BIOMEDICAL APPLICATIONS Vuong Thi Kim Oanh, Tran Dai Lam, Do Hung Manh, Le Trong Lu, Phung Thi Thu, Pham Hong Nam and Nguyen Xuan Phuc PREPARATION AND PHOTOCATALYTIC ACTIVITY OF TiO 2 -SiO 2 -Fe 3 O 4 COMPOSITES FOR DECOMPOSITION OF POLY CHLORINATED BIPHENYLS (PCBs) IN WASTE TRANSFORMER OIL Nguyen Quoc Trung, Vu Duc Chinh, Vuong Thi Kim Oanh, Pham Nam Thang and Vu Thi Hong Hanh RESERVOIR OPTIMIZATION METHODS: DYNAMIC PROGRAMMING AND DIFFERENTIAL EVOLUTION Phan Thi Thu Phuong TYPE-2 FUZZY CO-CLUSTERING ALGORITHM FOR COLOR IMAGE SEGMENTATION Van Nha Pham, Duc Thao Nguyen APPLICATION OF GAMMA-GAMMA COINCIDENCE METHODS IN NEUTRON ACTIVATION ANALYSIS Truong Van Minh, Pham Dinh Khang, Nguyen Xuan Hai, Tran Minh Hung, Ho Huu Thang and Nguyen An Son A METHOD TO OPTIMISE THE TOP QUARK MASS IN DI-LEPTON DECAY CHANNEL Phan Thi Hong Ngoc, Kaven Yau Wong and Markus Cristinziani PROPAGATION DYNAMICS OF LASER PULSES IN A THREE-LEVELV-TYPE ATOMIC MEDIUM UNDER ELECTROMAGNETICALLY INDUCED TRANSPARENCY Hoang Minh Dong, Le Van Doai, Pham Van Trong, Mai Van Luu, Dinh Xuan Khoa, Vu Ngoc Sau and Nguyen Huy Bang A STUDY OF CO-PROCESSING WASTES CONTAMINATED OILS IN CEMENT KINLS Hoang Quoc Trong, Pham Gia Dien SCREENING FOR HAEMATOCOCCUS STRAINS CONTAINING ASTAXANTHIN ISOLATED FROM SOME PLACES IN VIETNAM Luu Thi Tam, Le Ha Thu, Le Thi Thom, Nguyen Cam Ha, Hoang Thi Huong Quynh, Pham Van Nhat, Hoang Thi Lan Anh and Dang Diem Hong AUTHORS INDEX ISSN

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21 OPTICAL SPECTROSCOPY OF Eu 3+ IONS IN ALUMINOSILICATE GLASS Sengthong Bounyavong 1,2*, Vu Phi Tuyen 3, Nguyen Thi Quy Hai 2, Pham Thi Minh Chau 2, Phan Van Do 4, Vu Xuan Quang 2 and Nguyen Trong Thanh 5 1 Department of Physics, Faculty of Natural Sciences, National University of Laos, Laos P.D.R 2 Duy tan University, Da Nang City, Vietnam 3 Graduate University of Science and Technology - VAST, 18 HoangQuocViet, Hanoi, Vietnam 4 Thuyloi University, 175 Tay Son, Dong Da, Ha Noi, Vietnam 5 Institute of Materials Science, VAST *: sengthong_bounyavong@yahoo.com Abstract: Eu 3+ -dopedaluminosilicate (AlSiO) glass with the concentrations of 1,0 wt %was prepared by Sol-gel method.optical excitation and emission spectra of Eu 3+ ions have been investigated. The phonon sideband (PSB) associated with the 7 F 0-5 D 2 excitation transition is used to determine the electron phononcoupling constant and the local structure of the local environment around Eu 3+ ions. The luminescence intensity ratio of the 5 D 0-7 F 2 to 5 D 0-7 F 1 transition has been calculated to estimatethe local site symmetry around the Eu 3+ ions. The Judd Ofelt (JO) intensity parameters Ω λ (λ=2,4,6) were calculatedfrom the emission spectra and are used to estimate the transition probability(a), branching ratios (β), the stimulated emission cross-sections (σ λp ) for the excited levels 5 D 0 of the Eu 3+ ions. Keywords: Sol-gel method, aluminosilicate glass, Judd- Ofelt theory. I. INTRODUCTION Rare earth (RE) doped glasses have been attracted the attention of scientists due to their wide applications in many optical devices like lasers, light converters, sensors, highdensity memories and optical amplifiers. Among the RE 3+ ions used to optically activate materials, the Eu 3+ ions are mostly chosen due to Eu 3+ ions emit narrow-band, almost monochromatic light and have long lifetime of the optically active states. Further, the structure and the relative intensities of the optical transitions in Eu 3+ ion strongly depend on the its local environment, so this ion is used as a probe to survey the point group symmetry of the Eu 3+ site and sometimes also information on the coordination polyhedron [1-3]. As for the hosts, alumina is a good network modifier for dispersing RE 3+ ions in silica gel and silicate glass matrices, in which RE 3+ ions were preferably partitioned by alumina, forming Al-O-RE bonds rather than clustering and forming RE-O-RE bonds[4,5]. A. Monteil et al [6] have shown that when Eu 3+ ions doped aluminosilicate glasses, these ions are preferentially located in alunimum-rich domains, while the local structure around ISSN

22 Count Intensity (a.u) The 4 th Academic Conference on Natural Science for Young Scientists, Master & Eu 3+ ions is affected by aluminum through a structuring effect. M. Nogami have reported that the aluminum was effective to gives intense photoluminescence from aluminosilicate glasses doped with Sm 2+ ions [7]. However, the optical properties of Eu 3+ ions in aluminosilicate (AlSi) glass have been studied less than other matrixes. In this paper, Eu 3+ ions are used as probe to study the ligand field around RE 3+ in aluminosilicate (AlSi) glass. In addition, optical properties of AlSi:Eu 3+ glass are analyzed using Judd Ofelt (JO) theory. II. EXPERIMENT Aluminosilicate (90SiO 2 +10Al 2 O 3 ) glass doped with 1,0 wt % of Eu 3+ ions have been prepared by sol-gel method. The glass nature of samples was confirmed by X-ray diffraction (XRD) pattern using a Bruker D8-Advance. Raman spectra were carried out by Micro Raman spectroscopy (XploRA-Hariba). The photoluminescence (PL) and photoluminescence excitation (PLE) were recorded by Fluorolog-3 spectrometer; model FL3-22, Horiba Jobin Yvon. Luminescence lifetime was measured using a Varian Cary Eclipse Fluorescence Spectrophotometer. All the measurements were carried out at room temperature. III. RESULTS AND DISCUSSION Structural analysis 50 AlSi glass 1k Si-O-Si(Al) AlSi glass 40 Si-O 30 SiO 4 -Al Theta-Scale Fig. 1. XRD pattern of AlSi glass Raman shift (cm -1 ) Fig. 2. Raman spectrum of AlSi glass. XRD pattern: The X-ray diffraction pattern of the Eu 3+ doped AlSi glass recorded in the range 10 to 70 o exhibit broad diffusion at lower scattering angles which in turn confirm the amorphous nature of the title glasses and as a representative case XRD pattern of the AlSi glass is shown in Fig. 1. Raman spectroscopy: Figure 2 shows the Raman spectrum of the AlSi glass. It is found that the maximal phonon mode frequency is 1120 cm -1. Among observed bands, the Raman band at 480 cm -1 has the most intense intensity. This band relates vibration of the Si- O-Si(Al) bond. The bands about 970 and 1120 cm -1 are assigned to stretching vibrations of 8 ISSN

23 SiO 4 tetrahedral bound to one and two Al atoms, respectively. Three bands near 600, 706 and 800 cm -1 are due to stretching vibration of the Si-O bond in SiO 4 tetrahedral groups with various number of non-bridging oxygens [8, 9] Photoluminescence excitation spectrum and sideband phonon energy The excitation spectrum of SiAl:Eu 3+ glass was recorded in the spectral region nm by monitoring the emission at 617 nm ( 5 D 0-7 F 2 transition) and shown in Fig. 3. The excitation spectrum consists the sharp bands due to the f-f transitions from 7 F 0 of ions Eu 3+ to the excited levels. The most intense excited band at wavelength of 397 nm corresponds to the 7 F 0 5 L 6 transition, which is often used in fluorescence excitation for Eu 3+. The shoulder appears at wavelength around 508 nm can be related to the phonon sideband (PSB), which is used to understand the vibrational modes around the Eu 3+ [9]. The PSB of Eu 3+ in SiAl glass is associated with the 7 F 0 5 D 1 transition and shown in inset of Fig 3, in which the 7 F 0 5 D 1 excited transition is the pure electronic transition (PET). The PET is set as zero energy shift, the sideband phonon energy in SiAl glass can be calculated to be 805 cm -1. This phonon energy is related to stretching vibration of the Si-O bond in SiO 4 tetrahedral groups [8, 9]. The electron phonon coupling constant (g) have been calculated by [3]: g I I PSB PET ( ) d ( ) d where I PSB is the intensity of the phonon sideband and I PET is the intensity of the pure electric transition. In SiAl:Eu 3+ glass, the g value is found to be This value is much lower than that in lead fluoroborate (LFB) glasses [3] and borotellurite glasses [11]. This behavior shows that the electron phonon coupling in SiAl:Eu 3+ glass is wesker lead fluoroborate and borotellurite glasses Emission spectrum Fig. 4 illustrates the emission spectrum of AlSi:Eu 3+ glass using the 397 nm excitation wavelength of xenon lamp source. The luminescence lines are assigned according to Carnall s paper [10]. The emission spectrum consists 7 observed emission bands at wavelengths of 577, 590, 611, 651, 700, 745 and 802 nm which correspond to the Fig. 3. Excitation spectrum of Eu 3+ in SiAl glass. Fig. 4. Emission spectra of SiAl:Eu 3+ glass. (1) ISSN

24 5 D 0 7 F 0, 6 transitions, respe-ctively. Among emission transitions, the 5 D 0 7 F 2 transition has the most intense intensity whereas the 5 D 0 7 F 5, 7 F 6 transitions are very weak in intensity. The 5 D 0 7 F 2 transition is allowed electric dipole, so the it s intensity strongly depends on asymmetry of ligand and covalency of RE 3+ -ligand bond. The intensity of the 5 D 0 7 F 1 transition is independent with the asymmetry of ligand, because this is allowed magnetic dipole transition [1-3].The fluorescence intensity ratio (R) of 5 D 0 7 F 2 to 5 D 0 7 F 1 transitions of Eu 3+ ions allows one to estimate the deviation from the site symmetries of Eu 3+ ions. For AlSi:Eu 3+ glass, the R values is The luminescence intensity of the 5 D 0 7 F 2 transition of the Eu 3+ ions in the prepared glasses is stronger than that of 5 D 0 7 F 1 transition and further it suggest that Eu 3+ ions take a site with inversion antisymmetry [3].Moreover, the these values are higher than those of lead fluoroborate (LFB) glasses [3] and borotellurite glasses [11].The lower R value is attributed to the higher asymmetry and covalency around the Eu 3+ ions in AlSi glass than those hosts. Fig. 4 shows that the magnetic dipole 5 D 0 7 F 1 transition splits into three components, indicating that the crystallographic site of the Eu 3+ ions in the present glass is as low as orthorhombic, monoclinic or triclinic in a crystalline lattice [2, 3] Judd-Ofelt intensity parameters (Ω λ ) The Judd-Ofelt (JO) theory was shown to be useful to characterize radiative transitions for RE 3+ -doped solids, as well as aqueous solutions, and to estimate the intensities of the transitions for RE 3+ ions [12,13]. This theory defines a set of three intensity parameters Ω λ (λ = 2,4,6), that are sensitive to the environment of the RE ions. Commonly, The JO intensity parameters are usually derived from absorption spectrum. However, owing to the special energy level structure of Eu 3+ ion, these Ω λ could be estimated from the emission spectra. Four main emission peaks 5 D 0 7 F 1,2,3,4 are used to calculate Ω λ. The 5 D 0 7 F 1 is a magnetic dipole (MD) transition and its spontaneous emission probability A md is given by [1-8]: A md n S 3h(2J 1) where h is the Planck constant, is the wave number of the transition in interest, J is the total angular momentum of the excited state, and n is the refractive index. S md is the MD line strength, which is a constant and independent from the host material. The value of A md can be estimated using the reference value of A md published somewhere, and using the relationship A md = (n/n ) 3 A md [1-8], where, A md and n are spontaneous emission probability and refractive index of the reference material. The 5 D 0 7 F 2,4,6 transitions are an electric dipole partially allowed. The spontaneous emission probabilities A ed of electric transition is given using the following expression: md 2 2 n 2 ( ) 2 U J n Aed (3) 3h 2J 1 9 2,4,6 (2) 10 ISSN

25 where J is the wave number of transition 5 D 0 7 F J, e is the electron charge, 2 ( ) U are the squared doubly reduced matrix elements of the unit tensor operator of the rank λ = 2, 4, 6 are calculated from intermediate coupling approximation for a transition J ' J'. These reduced matrix elements did not nearly depend on host matrix as noticed from earlier studies. Thus the parameters could be evaluated simply by the ratio of the intensity of the 5 D 0 7 F J=2,4,6 transitions to the intensity of 5 D 0 7 F 1 transition as follow: I 5 7 J d A( D0 F 5 7 I A( D0 F 1d ,4,6 2 ( ) 2 J n n U 1 ) e ) S md ,4,6 For 5 D 0 7 F 2 transition, U (2) = 0,0033; U (4) = U (6) = 0, 5 D 0 7 F 2 transition, U (2) = 0; U (4) = 0,0023; U (6) = 0 and 5 D 0 7 F 2 transition, U (2) = U (4), U (6) = 0,0003. Using equation (4) and the reduced matrix elements, the JO parameters were calculated. In the AlSi:Eu 3+ glass, the JO parameters are: Ω 2 = cm 2, Ω 4 = cm 2 andω 6 =1, cm 2. The Ω λ parameters are important to study the symmetry of local structure around RE 3+ ions and nature of RE X (X = F, O) bonding. The Ω 4 and Ω 6 are related to the bulk properties such as viscosity and rigidity whereas the Ω 2 is more sensitive to the local environment of the RE 3+ ions and is often related with the asymmetry of the local crystal field. The Ω 2 and Ω 6 parameters in AlSi:Eu 3+ is larger than those of lead fluoroborate (LFB) glasses [3] and borotellurite glasses [11]. The large value of Ω 2 can be attributed to higher asymmetry of the ligand field and covalent in Eu 3+ -ligand bond than other hosts, whereas the larger of Ω 6 parameter shows that the rigidity of the media in which RE ions put into other hosts is lower Radiative properties Table 1. The radiative properties of SiAl:Eu 3+ glass. (4) 5 D 0 7 F 0 7 F 1 7 F 2 7 F 3 7 F 4 7 F 5 7 F 6 A R β cal β mes σ(λ P ) σ(λ P ) Δλ eff σ(λ P ) τ cal The JO parameters have been used to estimate the radiative properties such as the radiative transition rates (A R,s -1 ), branching ratios (β cal, %) and stimulated emission crosssection (σ(λ P ),10-22 cm 2 ) for 5 D 0 7 F J transitions and radiative lifetime (τ R ) of 5 D 0 level of Eu 3+ in AlSi glassby using Eqs in Rer [14]. In addition, the gain band width (σ(λ P ) Δλ eff, ISSN

26 10-28 cm -3 ) and optical gain (σ(λ P ) τ R, cm 2 s -1 ) also calculated for 5 D 0 7 F J transitions. The results are presented in Table 1. The predicted branching ratio (β cal ) of 5 D 0 7 F 2 transition get a maximum value 62.5 % whereas the measured ratio (β mes ) is 60.5 %, thus there is a good agreement between experimental and calculated branching ratios. The fluorescence decay curve of 5 D 0 state of Eu 3+ in AlSi glass is shown in Fig. 4.The measured lifetime is 3.21 ms, where as the calculated lifetime is 3.88 ms. It is observed that the experimental lifetime is smaller when compared with the calculated lifetime. The deviations between measured and calculated lifetime may be owing to the nonradiative relaxation rates of excited Eu 3+ ions. The quantum efficiency of the excited state 5 D 0 is given by the equation: η = τ exp /τ cal. For the AlSi:Eu 3+ glass, η = 82.5 %. Table 1 presents that the branching ratio, stimulated emission crosssection, gain band width and optical gain of 5 D 0 7 F 2 transition are larger than those of other transitions. Further the quantum efficiency of sample is high. These results suggest that the 5 D 0 7 F 2 transition of Eu 3+ ions in AlSi glass is found to be suitable for developing the optical devices such as laser and optical amplifier. Fig. 4. Fluorecence decay profiles of 5 D 0 level of Eu 3+ doped aluminosilicate glass. IV. CONCLUSIONS Aluminosilicate glass doped with 1.0 wt% of Eu 3+ ions have been prepared by solgel method. The XRD indicates that the glass has an amorphous structure. Raman spectrum presents the existence of specific structural groups in silicate glass and the maximal phonon mode frequency is 1120 cm -1.From the excitation spectrum, the PSB was found with the energy phonon about 805 cm -1, which related to stretching vibration of the Si-O bond in SiO 4 tetrahedral groups. The optical properties of Eu 3+ -doped aluminosilicate glass have been investigated. The large value of Rand Ω 2 parameter shows that the coordination structure surrounding the Eu 3+ ions has high asymmetry and Eu 3+ -O bond in AlSi glass has high polarizability. The radiative parameters show that the 5 D 0 7 F 2 transition of Eu 3+ ions in AlSi glass is very useful for optical devices. REFERENCES [1] T.K. Srinivasan, B. Venkatraman, D. Ponraju, A.K. Arora, W.J. Nano Science and Engineering, 2 (2012), [2] K. Binnemans, Coordination Chemistry Reviews 295 (2015) ISSN

27 [3] S. Arunkuma, K.V. Krishnaiah, K. Marimuthu, Physica B 416 (2013) [4] M. J. Lochhead and K. L. Bray, Chem. Mater. 7 (1995) [5] K. Arai, H. Namikawa, K. Kumata et al, J. Appl. Phys. 59 (1986) [6] A. Monteil, S. Chaussedent, G. Alombert-Goget et al, J. Non-Cryst. Solids. 348 (2004) [7] M. Nogami, and Y. Abe, J. Sol-Gel Sci. Technol. 8 (1997) [8] D. Neuville, L. Cormier,D. Masiot, Geochim. Cosmochim. Acta, 68 (2004) [9] D. Zhao, X. Qiao, X. Fan, M. Wang, Physica B 395 (2007) [10] W.T. Carnall, P.R. Flields, K, Rajnak, J. Chem. Phys, Vol 49, No 10 (1963) [11] K. Maheshvaran, P.K. Veeran, K. Marimuthu, Solid State Sciences 17 (2013) [12] B.R. Judd, Physical Review 127 (1962) [13] G.S. Ofelt, The Journal of Chemical Physics 37 (1962) [14] Phan Van Do, Vu Phi Tuyen, Vu Xuan Quang, Nguyen Trong Thanh,Vu Thi Thai Ha, N M. Khaidukov, Yong-Ill Lee, B.T. Huy, J. Alloys Compd, 520 (2012) ISSN

28 EFFECT ON BONDABILITY OF PROLONGED EXPOSURE TIME AFTER PLASMA CLEANING ON IC PACKAGE Pornthep Yaekwong 1*, Kessararat Ugsornrat 1, Chalermsak Sumithpibul 2, Neil S. Carunungan 2, Bancha Arthibenyakul 1 and Surapan Yimman 1 1 Department of Industrial Physics and Medical Instrumentation, King Mongkut s University of Technology North Bangkok, Bangkok, Thailand 2 Department of Engineering, Utac Thai Limited, Bangkok, Thailand *: kessararat_u@hotmail.com Abstract. We studied about an appropriate exposure time of plasma cleaning process on integrated circuit (IC) packaging. In experiment, 5L SOT packages were studied for Au wire bonding. IC packaging process were varied exposure time after plasma cleaning process with 5 mins from 0 to 96 hours with a step of 12 hours to find the appropriate condition for wire bonding process. All packages were tested for contact angle measurement, intermetallic compound (IMC), bondability, and delamination. The results confirmed that plasma cleaning process with 5 minutes to reduce the contamination on die surface and leadframe to enhance bondability performance for all three packages and wires. Moreover, the evaluation showed about not clearly possibility of prolonged exposure time for extending exposure time after plasma cleaning process. Keywords: IC Packaging, Plasma cleaning, Exposure time. I. INTRODUCTION Wire bonding process is one of most critical processes in integrated circuit (IC) packaging and wire bond process is the major interconnecting method between the die and the substrate or leadframe. The requirement of this process need to reliability for high yield product. For failure prevention and cost reduction, the processing parameters of wire bonding must be optimized to make reliable bonds [1]. The bond pad surface effects to wire bond process if a poor bondability quality on pad surface can result in a non-stick-onpad (NSOP) and non-stick-on-lead (NSOL) effect to high lot reject rat. The bond pad surface also effects to the package reliability after stress tests due to its influence on the adhesion between bond pad and wires [2]. The oxidation layer is important factors in bond pad performance. Thickness of oxide layers and some organic contamination effects to result in weak bondability or no electrical conductivity. It has been shown that a thick Al fluoride oxide caused NSOP and oxide layer on lead frame caused NSOL problem. However while staging time after plasma cleaning effect to some problem such as delamination, lifted ball, and poor bondability. To reduce oxidation and organic contamination of bond pad surfaces prior to wire bonding process helps to improve the process ability of wire bonding and reliability of package. Plasma cleaning process to physically and chemically remove surface contamination and 14 ISSN

29 oxide layer on dies and lead frame [3]. Plasma of gases such as argon, hydrogen, oxygen, and mixtures of these are often used removed oxidation and organic contamination from the surface of die and lead frame. The plasma cleaning process operating parameters used for common semiconductor [4, 5]. In this research, argon (Ar) and hydrogrn (H 2 ) gas mixture for plasma cleaning and DC plasma was applied to the Al bond pad surface and pre plated lead frame (PPF) for remove/reduce the oxidation layers on bond pads before to the wire bond process. To compare each staging time result of surfaces analysis without and with plasma cleaning. The effectiveness of plasma cleaning are observed contact angle measurement, ball shear, wire pull and stitch pull test which were analyzed after wire bonding and after stress test. After that, intermetallic compounds (IMC) were also checked and analyzed for the Au wire bond on the Al bond pad. Moreover, the results of delamination were also by scanning electron microscope. II. METHODOLOGY The IC packaging process used for this research as shown in Fig. 1. Aluminum wafers were sawed for 30x30 mils of small package size and attach die with palladium pre plated lead frame (PPF) using epoxy 2200D conductive Ag, wire size is 1.0 mils, and wire Au gold wire used for wire bond process, molding process is available with compound G600. The experiment starts from plasma cleaning plasma cleaning process. The argon (Ar) and hydrogrn (H2) gas mixture for plasma cleaning and DC plasma was applied to the Al bond pad surface and pre plated lead frame (PPF) for remove/reduce the oxidation layers on bond pads with 5 mins before start to wire bonding process. The staging time were varied for comparing each staging time and without staging time effect to surfaces analysis of IC packages. The effectiveness of plasma cleaning checking by contact angle measurement, ball shear, wire pull, and stitch test which experiment both after wire bonding and after stress test. Moreover, intermetallic compounds (IMC) were also checked and ISSN Fig. 1. IC Packaging Process. Fig. 2. Diagram of experimental process flow. 15

30 analyzed for the Au wire bond on the Al bond pad. The diagram of process flow is shown in Fig. 2. III. RESULTS AND DISCUSSION A. The contact angle measurement result This section shows about the contact angle with staging time after plasma cleaning process. From Fig. 3, contact angle of deionized water clearly reduce after plasma cleaning with 5 mins. In each staging time, the contact angle is different and obviously increase from 12 hours to 96 hours of staging time as shown in Fig. 3. Fig. 3. The result of contact angle of deionized water with staging time after plasma cleaning. Fig. 4. The result of ball shear test. B. The result of ball shear test. The result of ball shear test is shown in Fig. 4. Ball shear force slightly increase after plasma cleaning process with 5 mins and not clearly reduce and different from 12 to 96 hours of staging time. C. The result of wire pull test. The results of wire pull test is shown in Fig. 5. Wire pull force slightly increase after plasma cleaning process with 5 mins which is similar to the result of ball shear force. After staging time, wire bull forces are significantly different but is not clearly about tendency of forces from 12 to 96 hours of staging time. Fig. 5. The result of wire pull test. Fig. 6. The result of stitch pull test. 16 ISSN

31 D. The result of stitch pull test. The results of wire pull test is shown in Fig. 6. Stitch pull strength clearly increase after plasma cleaning process with 5 mins. After staging time, stitch force are sligthly different from 12 to 96 hours of staging time. The features of wires after these failure analysis tests after palsma cleaning are shown in Fig. 7. Fig. 7. Feature of Au wire after wire bond process for failure analysis (a) after wire bonding process. Fig. 8. The results of intermetallic compound (IMC) between Au wire and Al pad. E. The result of Intermetallic compound (IMC) The results of intermetallic compound (IMC) between Au wire and Al bond pad is shown in Fig. 8. The results are based on a testing random of 15 units for each staging time. IMC percentage of non plasma cleaning is the worst case. After plasma cleaning, IMC percentage of each staging time at 0 to 36 hours increase and start to drop at 72 hours of staging time as shown in Fig. 7. F. The results of ball shear test after decapsulation process. Fig. 9. The result of ball shear test after decapsulation process. Fig. 10. The result of wire pull test after decapsulation process. ISSN

32 The result of ball shear test after decapsulation process is shown in Fig. 9. Ball shear orce slightly increase after plasma cleaning process with 5 mins and not clearly deferent value from 12 to 96 hours of staging time. G. The results of wire pull test after plasma cleaning process. The results of wire pull test is shown in Fig. 10. Wire pull forces clearly increase after plasma cleaning process with 5 mins. After staging time, wire bull forces are significantly different but is not clearly about tendency of forces from 12 to 96 hours of staging time. H. The result of stitch pull test after decapsulation process. The results of stitch pull test is shown in Fig. 11. Stitch pull forces increase after plasma cleaning process with 5 mins and extremely increase at 12 hours of staging time. After 24 hours of staging time, tendency of forces clearly reduce from 12 to 96 hours of staging time. Fig. 11. The result of stitch pull test after decapsulation process. Fig. 12. Delamination of units at each staging time with C-SAM. I. The results of reliability Table 1. The results of delamination at each staging time. 18 ISSN

33 In order to investigate delamination on package using scanning acoustic microscope AM) with mode C to observe void and delamination between die, epoxy molding mpound (EMC), and lead frame. The experiments separate to two conditions which are er molding process and after reliability test. The results are shown in Fig. 12 and Table 1. IV. CONCLUSION In this research, the effect on bondability of prolonged exposure time after plasma cleaning process on IC package were analyzed. The bondability will be disturbed from the contamination and oxidation effect to increase lot reject rate. All packages were tested for contact angle measurement, bondability, IMC, and delamination. The results confirmed that plasma cleaning process with 5 minutes to reduce the contamination on die surface and leadframe to enhance bondability performance of wires. Moreover, the evaluation showed about not clearly possibility of prolonged exposure time for extending exposure time after plasma cleaning process. V. ACKNOWLEDGMENT The research was supported by UTAC Thai Limited and employees of UTAC Thai Limited for valuable recommendation. REFERENCES [1] W-H. LI and K. Reingruber, Electronics Packaging Technology Conferenc. 2009, pp [2] J. T. Huneke and M. Cowens, IEEE/CPMT Electronic Packaging Technology Conference. 1997, pp [3] S. Yalcin and R. Avci, Applied Surface Science. 214, 2003, pp [4] Y. F. Chong, et al. Microelectron. Reliab. 40, 2000, pp [5] V. L. von Arnim, J. Fessmann, and L. Psotta, Surf. Coat. Tech.. 116, 1999, pp [6] Y. F. Chong et al, Microelectronics Relibility. 40, 2000, pp ISSN

34 STUDY ON THE FABRICATION AND PROPERTIES OF ALLOYED QUANTUM DOTS AND GRAPHENE QUANTUM DOTS Pham Nam Thang 1, Le Xuan Hung 2, Nguyen Hai Yen 1, Hoang Van Nong 1, Vu Thi Hong Hanh 3, Nguyen Ngoc Hai 4, Phan Ngoc Hong 1, Pham Thu Nga 1* 1 Institute of Materials Science, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Vietnam 2 Institute of Research and Development, Duy Tan University, Da Nang City, Vietnam 3 Faculty of Physics, Thai Nguyen University of Education, Thai Nguyen, Vietnam 4 Uong Bi High School, Quang Ninh, Vietnam *: phtnga@ims.vast.ac.vn Abstract. The synthetic methods which have been studied and developed to be suitable for the fabrication of alloy quantum dots (QDs) with uniform size and core/shell structure, and of granphene quantum dots (GQDs), were presented in this report. The study results on the size, shape, crystalline phase and relative optical properties such as absorption, emission in NIR of CdTeSe and CdTeSe/ZnSe alloy QDs are also presented in details, to clarify the best condition to fabricate the best quality alloy QDs. The optical properties of GQDs are also presented in order to clarify the correlation between fabricating conditions and optical properties of GQDs. Keywords: CdTeSe quantum dots, CdTeSe/ZnSe, CdTeSe/ZnTe core/shell alloyed QDs, Raman spectra, Absorption, Emission, Graphene Quantum Dots. I. INTRODUCTION Quantum dots (QDs) with near-infrared (NIR) emission ( nm) are particularly attractive for bio-applications and optoelectronics. So far many kinds of NIRemitting QDs have been reported, such as CdSeTe, InAs, PbSe, CdP and CuInS, etc [1], among which the alloyed CdSeTe QDs have been of immense interest since reported in 2003 [1, 2]. In comparison with other NIR-emitting QDs, alloyed CdSeTe QDs exhibit a very strong nonlinear effect between the composition and the absorption/emission energies, providing them with special optical and electronic properties not available from the parental CdSe and CdTe QDs [2]. Consequently, the emission wavelength of CdTeSe QDs can readily reach NIR region by tuning Te/Se molar ratio. Meanwhile, the parental CdSe QDs can only cover typically nm in emission, and even the CdTe QDs of 7 nm can only reach ~720 nm in emission [3-5]. To date, various methods have been developed for the synthesis of single or core-shell structured CdTeSe QDs [5, 6]. Till now, the CdSeTe QDs are mostly passive with nontoxic ZnS shell to obtain CdSeTe/ZnS core-shell QDs [7]. However, for both CdSe and CdTe, the large lattice mismatches (>10%) relative to ZnS hinder the formation of high-quality QDs. Therefore it is of high importance to develop new aqueous synthetic procedure for NIR-emitting core- 20 ISSN

35 shell CdTeSe QDs with excellent optical properties and low biotoxicity. With the aim to find a new method to fabricate QDs with new core/shell structure for applications in solar cells or biology, we have studied CdTeSe/ZnSe and CdTeSe/ZnTe QDs. In this report, we present the improved method to fabricate the core/shell CdTeSe/ZnSe and CdTeSe/ZnTe, in ODE-OA-OLA medium, with two different molar ratios Cd:Te:Se = 1:1.8:1.8, close to the ratio used in [8] and 10:1:1, which was used in [9, 10]. The results on their crystalline phase, size, shape and optical properties are also presented. In particular, the Raman scattering spectroscopy of these QD samples are also presented to clarify the crystalline phase of these core/shell QDs. Moreover, the graphene quantum dots (GQDs) are also a very new research subject, depending on the change in size of GQDs that we can obtain changing absorption or emission spectra in the visible range. In this report, we also present some initial results on fabricating GQDs and their optical properties. II. EXPERIMENTS 2.1. Synthesis Method We used the following reagents (from Aldrich): cadmium acetate dihydrate (Cd(Ac) 2.2H 2 O, 99.9%), elemental selenium powder (Se, 99.99%), elemental tellurium powder (Te, 99.99%) as Cd, Se and Te sources, respectly, a oleic acid (OA, 90%) and oleylamine (OLA,90%) as surface ligands, 1-octadecene (ODE, 90%)and trioctylphosphine (TOP, 90%) as the reaction medium, zinc acetate (Zn(Ac)2, 99.9%) as Zn source for the shell preparation. For the preparation of GQDs, we use citric acid (CA, C 6 H 8 O 7 ), NaOH and NH 4 OH. All chemicals were used without further purification. CdTeSe cores were prepared following a modified method described in [10, 11]. Core-shell alloy quantum dots were prepared according to a modified successive ion layer absorption and reaction (SILAR) protocol that has been previously published [8]. To carry out the fabrication of CdTeSe QDs with core/shell-structured CdTeSe/ZnSe and CdTeSe/ZnTe, we followed three steps. First is to prepare precursors, then CdTeSe cores, and finally coating the QD cores with ZnSe and ZnTe shells with different thickness counted by n monolayer (ML), with n ranging from 1, 2 to 4 ML (n is the nominal thickness: we calculated the amounts of shell precursors to introduce into the solution in order to have stoichiometric proportions to the concentration of core QDs, depending on the core size estimated from TEM).In this study, we have fabricated 1 mmol CdTeSe quantum dots (QDs) in OLA-ODE medium with two different molar ratios Cd:Te:Se = 1:1.8:1.8and 10:1:1. The fabrication process consists of preparing Cd precursor, Zn precursors, TOP-Se precursor, and TOP-Te precursor, then synthesizing CdTeSe core quantum dots from the solution mixtures of TOP-Se and TOP-Te fabricated above with Cd precursor in N 2 gas. Afterwards, increase the solution temperature, until it reaches 180 o C, 200 o C or 220 o C. ISSN

36 To coat the core quantum dots with ZnSe and ZnTe, we prepared the precursors for the shell. The weight of Zn and Te were calculated for 1 ML, 2 ML and 4 ML of ZnSe and ZnTe. A monolayer thickness is based on the lattice constant a of ZnSe or ZnTe crystals, depending on the shell type. The used molar ratio of Zn:Te was 1:1.We used 1.6 mmol of CdTeSe core QDs for coating at a temperature ~20 o C lower than the core growth temperature. At this temperature, quickly inject the (Zn + Te) precursor mixture solution (respective to a monolayer of Zn ions) into the core solution to grow the shell in 10 min. Do the same when coating ZnSe for CdTeSe QD cores to form CdTeSe/ZnSe. All ternary quantum dots were purified by several rounds of precipitation and centrifugation and were stored at room temperature for later characterization and use. The GQDs were synthesized by pyrolyzing citric acid (CA) referred from publication [12]. Briefly, 2g citric acid was added into a glass bottle and heated to 180 o C, 190 o C and up to 200 o C under nitrogen gas. After the solid citric acid changed to liquid at about 155 o C. The boiling colorless liquid turned to light yellow in about 5 minutes later, and then orange and brown in 30 min which indicated the formation of GQDs. The obtained color liquid was added drop by drop into100 ml NaOH 0.25M under vigorous stirring for the preparation of the GQD solution. Finally, the GQD aqueous solutions were neutralized to ph 7.0 with the same concentration NaOH. The sample series of GQDs were prepared at the different temperatures are obtained Characterization of CdTeSe /ZnSe (Te) core/shell alloyed quantum dots and GQDs The size of the QDs was determined by transmission electron microscopy (TEM) with a JEOL Jem 1010 microscope operating at 100 kv. Powder X-ray diffraction (XRD, Siemens D5005) was used to confirm the wurtzite (w) or zinc-blende (zb) crystalline structure. The XRD patterns were compared with the tabulated values of bulk CdSe (JCPDS card No (zb) and (w)), CdTe: (zb), (w), ZnSe (JCPDS (zb) and (w), ZnTe: (zb), (w) and CdTeSe (w): The ultraviolet-visible (UV Vis) absorption spectra of QDs in toluene were scanned within the wavelength range of nm using a Shimadzu (UV-1800) spectrophotometer. An Acton SpectraPro-2300i spectrometer with He Cd laser emitted at two wavelengths 442 nm and 325 nm also was used to measure photoluminescence (PL) emission spectra. The QD samples were analyzed by Micro Raman spectroscopy (XploRA- Horiba) using 532 nm (90mW) or 785 nm (25 mw) excitation lines. III. RESULTS AND DISCUSSION Figure 1 shows the TEM image of CdTeSe QDs (sample N3-0-15). The TEM image shows that the nanoparticles formed at 180 o C have a bit long shape, the average size determined from the scale of the TEM images is 7-8 nm. Fig 2 is the TEM 22 ISSN

37 image of the nanoparticle QDs (N sample) that are fabricated at a higher temperature: 220 o C. The TEM image shows that the CdTeSe QDs are more round-shaped, the size is also ~ 7-8 nm. Fig. 3 is the powder XRD patterns of CdTeSe QD samples: core CdTeSe (N3-0-15) and two core samples wrapped with ZnTe 1 ML and 2 ML thick. The position of the lines that appear on the XRD pattern of CdTeSe core QD samples is between the characteristic lines of zb CdSe and zb CdTe phase, proving that the CdTeSe QDs crystallize at the alloy phase of CdSe and CdTe. This will be further proven at the Raman spectrum results. When wrapped with a thin layer of ZnTe (1-2 ML), the position of these diffraction lines does not change. This shows that the wrapping with less than 2 ML of ZnTe has yet to change the crystalline phase of CdTeSe QD core. When the core Fig. 1. TEM image of sample CdTeSe QDs, (N3-0-15), prepared at 180 o C. Scale bar: 20 nm. Fig. 2. TEM image of sample CdTeSe QDs, (N4-0-15), prepared at 220 o C. Scale bar: 20 nm. Fig. 3. Powder XRD patterns of CdTeSe ternary QD cores (sample N3-0-15) prepared at 180 o C, and CdTeSe/ZnTe (1 and 2 ML). Bulk diffraction peaks for wurtzite (w) and zinc blend (zb) ZnTe (top), ZnTe (middle) and CdSe (bottom) are shown. Fig. 4. Powder XRD patterns of CdTeSe ternary QD cores (sample N4-0-15) prepared at different temperatures at 180 o C, 200 o C and 220 o C. Bulk diffraction peaks for wurtzite (w) and zinc blend (zb) CdTe (top) and CdSe (bottom) are shown. ISSN

38 growing temperature is increased to 200 o C and 220 o C, the diffraction patterns show that the position of diffraction lines does not change. These QDs still crystallize at the CdTeSe alloy crystal phase (Fig. 4). We have carried out recording the micro-raman spectra of the CdTeSe core QDs to gain additional information and verify the analysis from the XRD patterns. Fig. 5 is the Raman spectra of the QDs fabricated at different temperatures, from 120 o Cto 220 o C. We see that, when the QD growth temperature is low, like at 120 o C and 150 o C, only peaks at wave number cm -1 appear on the spectrum. This wave number is close to the LO CdTe phonon vibration mode of the CdTe [13, 14], which means at this temperature, maybe only CdTe seeds have been formed, the explanation is that thanks to the Te element s strong mobility, it can easily link with Cd to form CdTe [2]. When the temperature is raised to 180 o C, the Se element will become more mobile and it will become easier to form Cd-Se-Te bonds, whose characteristic peak is at wavenumber 192 cm -1, close to CdSe. The second peak is located at 192 cm -1 ; this peak corresponds to the characteristic vibration frequency of CdSe-like LO, as observed in [15, 16]. When the growth time of nanocrystals increases, from 30 min. to 1h, more CdTeSe QDs are formed, it shows that the intensity ratio of the band at 158 cm -1 and 192 cm -1 increases when the nanocrystal growth time increases, from 10 min to 60 min. For the samples fabricated at higher temperatures, the peak at 192 cm -1 was observed with stronger intensity. Hence it is possible to see that for the fabrication of nanocrystals at a higher temperature with the same growth time of 10 min, the nanocrystals crystallize more in the CdTeSe alloyed crystalline phase. The Raman spectra of the CdTeSe samples prepared at above 180 o C are shown in Fig. 5, which reveals longitudinal optical (LO) peaks of both CdTe (near 160 cm -1 ) and CdSe (near 200 cm -1 ) and their overtone modes (2LO: cm -1 ) like those reported in refs. [14, 17]. From the presented results, we came to the conclusion that it is possible to apply the fabrication conditions of 180 o C to 220 o C temperature, better at 220 o C and 10 min. nanocrystal growth time, to the fabrication CdTeSe QDs. Fig. 5. Raman spectra of CdTeSe QDs prepared at different temperatures from 120 o C to 220 o C. Fig. 6. Raman spectra of CdTeSe QDs prepared at 180 o C with two different molar ratios. 24 ISSN

39 The Raman spectra of QDs fabricated with two different molar ratios are presented on Fig. 6. It can be seen that the QDs fabricated with the molar ratio 10:1:1 result in alloyed CdTeSe QDs and on the Raman spectrum appears a band with double peaks. And with the molar ratio 1:1.8:1.8, we only obtain CdTe-rich QDs or CdTe QDs. Therefore, we will only use the ratio 10:1:1 in the next fabrications. Fig. 7 is the Raman spectra of the series of CdTeSe wrapped with ZnTe and ZnSe, when the shell thickness changes from 1 ML to 4 ML. For the cores, we can observe the Raman characteristic double line (two consecutive peaks), as usually seen to appear in the ternary alloy core QD samples studied in this report. Fig. 7. Raman spectra of CdTeSe and with the shell of ZnTe and ZnSe nml (n= 0, 1, 2 and 4) QDs samples series. The frequency position of the first peak lies at cm -1 and that of the second peak lies at cm -1. The position if these two peaks coincide with the relative peaks of the CdTeSe core. The intensity of the peak at cm -1 is stronger compared to that at cm -1 with ZnTe 1ML. When the ZnTe shell thickness increases to 2 ML, on the Raman spectrum only one prevailed peak appears at cm -1, the peak at cm -1 is like a shoulder of the vibration spectra. When the shell thickness increases to 4 ML, only one peak at cm -1 is observed. Their overtone modes (2LO: 340 cm -1 ) can be observed. This line is close with the characteristic line of CdTe vibration rather than of CdSe. It can be seen that when the ZnTe shell thickness increases to higher than 2 ML, the CdTeSe ternary alloy QDs become CdTe-rich QDs. The explanation for this is that Te element has strong chemical activity, thus when a large amount is brought into the reaction flask to create a thick shell, it immediately reacts to the abundant Cd ions from the CdTeSe core fabrication (the Cd molar ratio is 5 times larger than Te and Se), to create a CdTe layer around CdTeSe. The results on the Raman spectra verify the analysis on the crystalline ISSN Fig. 8. Normalized UV vis absorption and PL spectra of CdTeSe, CdTeSe/ZnSe QDs growing at 220 o C. 25

40 phase of CdTeSe QDs with the XRD method. For QDs coated with ZnSe (the three curves above in Fig.7), when the shell thickness increases, we can observe a similar phenomenon, but this time it is the characteristic line of CdSe vibration rather than of CdTe. When the shell thickness exceeds 2 ML, such as 4 ML, we can only observe one vibration line at 200 cm -1 which assigned to longitudinal optical (LO) peak of CdSe (200 cm -1 ). Their overtone modes (2LO: 400 cm -1 ) can be observed. This line coincides with the vibration line of CdSe nanocrystals. Therefore it can be explained that when the ZnSe shell is formed, the Zn and Se precursors have been used. Since the Cd ions are always available in the shell fabricating environment without abundant Te, in this case, the CdSe material layer forms gradually on the CdTeSe core, thus we obtain CdSe-rich QDs. Fig. 8 presents the absorption and PL emission spectra of CdTeSe QDs wrapped with a ZnSe shell. The CdTeSe QDs exhibit dominant band gap emission. Upon prolonging the reaction time, the excitonic absorption peak in the absorption spectra and the emission peak in the PL spectra shift systematically towards longer wavelengths, which demonstrates clearly the growth of nanocrystals.the maximum peak of the excitonic emission band of CdTeSe QDs is located at ~828 nm. When wrapped with ZnSe shell with different thickness, the emission spectrum peak shifts towards longer wavelengths, up to 925 nm with the 4 ML thick shell layer. Fig. 9. Normalized UV vis absorption and PL emission spectra of two samples GQDs growing at 200 o C. It may have involved decay through surface traps created at the shell surface. The reason for this shift is not yet fully understood. When wrapping QDs with ZnTe, the emission spectrum peak position hardly changes (spectrum not provided here). We need to study further to explain this matter. Fig. 9 shows thenormalized UV vis absorption and PL emission spectra of two samples GQDs. The absorption bands have a small absorption peak at 349 nm that can be assigned to π π * transition of the π bond. The emission spectrum is a wide band whose emission peak is located at 481 nm wavelength. This emission band is relative to the recombination transition of electron π* π at the edge of GQDs. This band is also observed by the authors in [18]. IV. CONCLUSION We have fabricated CdTeSe QDs with core/shell structure with the molar ratio Cd: Te: Se = 10:1:1 at the temperature of 220 o C in 10 minutes. The usage of ZnSe and ZnTe allowed protection of the core. These core/shell CdTeSe QDs have a size of ~8 nm that 26 ISSN

41 changes depending on each sample. The research on these QDs with the Raman spectra has shown that it is a strong tool to detect the forming of the ternary alloyed CdTeSe crystalline phase. The CdTeSe/ZnSe QDs can absorb up to nearly 800 nm and emit up to nearly 900 nm. GQDs were prepared and their emission band is ~ 480 nm. Further research is being conducted to optimize the fabrication process and to explain some physical mechanisms that are not yet fully understood in this report. V. ACKNOWLEDGMENTS This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number , the PICS cooperation projects between CNRS and VAST (Project Number 6456 and VAST.HTQT. Phap. 01/15-16). The authors thank the National Key Laboratory for Electronic Materials and Devices IMS and Duy Tan University for the use of facilities. REFERENCES [1] Xie, R., Rutherford, M. & Peng, X. J. Am. Chem. Soc. 131, 2009, pp [2] Bailey, R. E.& Nie, S. M. J. Am. Chem. Soc. 125, 2003, pp [3] Liang, G.-X. et al. Chem. Commun. 46, 2010, pp [4] Taniguchi, S., Green, M., Rizvi, S. B. & Seifalian, J. Mater. Chem. 21, 2011, pp [5] Pons, T. et al. Chem. Mater. 21, 2009, pp [6] Song, L., Duan, J. & Zhan, J. Mater. Lett.64, 2010, pp [7] Lingling Li, Ying Chen, Qian Lu, Jing Ji, Yuanyuan Shen, Mi Xu, Rong Fei, Guohai Yang, Kui Zhang, Jian-Rong Zhang & Jun-Jie Zhu; 3: 1529 DOI: /srep01529 [8] N. H. Yen, W. D. D. Marcillac,C. Lethiec, P. N. Hong, C. Schwob, A. Maître, N. Q. Liem, L. V. Vu, P. Bénalloul, L. Coolen and P. T. Nga, Optical Materials36, 2014, pp [9] Z. Pan, K. Zhao, J. Wang, H. Zhang, Y. Feng, and X. Zhong, ACS Nano 7 (6),2013,pp [10] L. Liao, H. Zhang, X. Zhong, Journal of Luminescence 131, 2011, pp [11] F. Yang, Z. Xu, J. Wang, F. Zan, C. Dong and J. Ren, J. Luminescence 28,2013, p [12] Y. Dong et al. CARBON 50, 2012, pp [13] V. S. Vinogradov, G. Karczewski, I. V.Kucherenko, P.Fernandez, Phy. Sol. Stat., Vol. 50, No. 1, 2008, p.164. [14] S. Li., G. Tan, C. Wisner, N. Leventis, T. Xia, X. Chen., and Z. Peng, J. Elec. Mat., Vol. 42, No. 12, 2013, p ISSN

42 [15] B. T. Spann and X. Xu, Appl. Phys. Lett. 105, 2014, p [16] Z. Chai, W. Wu, D. Kong, Y. Gao, and Q. Chang, J. Non-Cryst. Solids 82, 2013, p [17] D. N. Talwar, Z. C. Feng, J. F. Lee and P. Becla, Physical Review B, 87, 2013, p [18] Lingling Li, Gehui Wu, Guohai Yang, Juan Peng, Jianwei Zhao and Jun-Jie Zhu, Nanoscale, 2014, pp ISSN

43 EXTRACTION OF BIO-OIL RICH IN OMEGA-3-6 FORM ALKYL ESTER FROM HETEROTROPHIC MICROALGA SCHIZOCHYTRIUM MANGROVEI PQ6 Le Thi Thom, Luu Thi Tam, Nguyen Cam Ha, Hoang Thi Huong Quynh, Pham Van Nhat, Hoang Thi Lan Anh and Dang Diem Hong * Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet St., Cau Giay, Vietnam *: ddhong60vn@yahoo.com Abstract. Heterotrophic marine microalga Schizochytrium mangrovei PQ6 was isolated from Phu Quoc Island, Kien Giang province, Vietnam in This strain is potential used for functional food, aquaculture feed, material for biodiesel production and exploitation of natural compounds as squalene. In this study, we present the results of extraction of algal oil rich in omega-3 ( -3) and omega-6 ( -6) polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA; C22:6-3), eicosapentaenoic acid (EPA; C20:5-3) and docosapentaenoic acid (DPA; 22:5-6) in the form of alkyl ester from biomass of PQ6 strain. This microalga was cultured in 30 L bioreactor to supply enough biomass for extraction of algal oil. The cell density, dry cell weight and total lipid content were x 10 6 cells /ml, g/l and 66.3 % of dry cell weight after 96 h of cultivation, respectively. The in situ transesterification producing fatty acids in the form of alkyl ester using catalyst of acid was carried out. A simple procedure involving urea complexation and winterization has been developed in order to separate the total fatty acids into a first fraction enriched with saturated fatty acids and a second fraction enriched with long chain PUFAs. The production of total fatty acids from PQ6 strain resulted in a yield of 44% based on algal dry biomass. The content of -3 and -6 PUFAs reached up to 76.97% of TFA when TFA to urea and methanol ratio was 1:4:20 while DHA content obtained 69% of TFA. The yield of -3 and -6 PUFAs extraction reached up to 10.86% of dry cell weight. Therefore, dry biomass of PQ6 strain has ensured good quality material to produce algal oil rich -3 and -6 PUFAs for functional food. Keywords: Bio-oil, omega-3-6, Schizochytrium mangrovei PQ6, polyunsaturated fatty acid. I. INTRODUCTION Polyunsaturated fatty acids (PUFAs) including docosahexaenoic acid (DHA; C22:6-3), eicosapentaenioc acid (EPA; C20:5-3) and docosapentaenoic acid (DPA; 22:5-6) which are the critical important components of membrane in most eukaryotes and the precursors of many lipid molecules, important nutrients and have many benefits on humans [1]. PUFAs are commercially produced from plants and marine fish. Currently, bio-oil producing from marine fish has some difficulties as decrease in the number of fish species because of overexploitation, unpleasant taste and low stability... [2]. More recently, heterotrophic microalgae are seeing as potential sources for the PUFAs 29 ISSN

44 production because they have some distinct advantages compared with other materials as the stabilization of their lipid composition [2], their high growth rate [3], and their cultivation not dependent on the season so they can produce biomass all year round [4]. In some of the potential microalgae for bio-oil production, heterotrophic microalga Schizochytrium has some favorable characteristics for PUFAs commercial production such as high lipid and DHA contents, fast growth rate [5]. Recently, some published researches have shown the possibility of direct conversion from algal biomass to fatty acid in the form of alkyl esters (methyl ester or ethyl ester) using sulfuric acid as catalyst [6]. Biodiesel production under supercritical methanol and microwave irradiation conditions has high efficiency. Decreased in extractivetransesterification time [7] or in situ transesterification from the wet biomass to fatty acid in the form of alkyl ester [8] was considered as significant improvement for increasing yield of transformation process. The -3 fatty acids can exist in the different forms such as ethyl ester (EE), free fatty acids (FFAs) và triacylglycerols (TAGs). Specific molecular structures of -3 fatty acids can decide their bioavailability [9]. Lawson et al. (1988) [10] were compared the relative absorption of EPA and DHA in the form of TAGs, EE and FFA. EPA and DHA in the form of FFA were well absorbed ( 95%). Absorption of EPA and DHA in the form of TG was 90% and 75% respectively. Absorption of EPA and DHA in the form of EE was 60%. However, fatty acids in the form of EE or methyl ester are more sustainable, stability than that in the form of FFA. In addition, TAGs is storage energy for the body and it has an important role in transformation process. However, when the blood has high TAGs concentration, they can cause some diseases as atherosclerotic and fatty liver disease [11]. To follow up the work of Ha et al. (2014) [12] and Thom et al (2014) [13] for the production of bio-oil in the form of FFA by two-stage method, in this work we use the in situ transesterification process (only one-stage method) from algal biomass using catalysts of acid as described in report of Johnson & Wen (2009) [6] to obtain fatty acids in methyl ester form. Using urea complexation method, saturated fatty acids (SFAs) and mono unsaturated fatty acids (MUFAs) was separated from PUFAs. The technology of in situ transesterification from algal biomass can help reduction of time and made co-products as PUFAs, biodiesel and lower cost of production. II. MATERIALS AND METHODS Algal strain and culture conditions In this work, we used the microalga Schizochytrium mangrovei PQ6 which was isolated from Phu Quoc Island, Kien Giang province, Vietnam (access number SPQKG02) and later deposited at the Department of Algal Biotechnology, Institute of Biotechnology belonging to Vietnam Academy of Science and Technology, Vietnam. Fermentation was carried out using a 30 L bioreactor with working volume of 15 L and a M12 medium that 30 ISSN

45 contained 90 g/l glucose, 10 g/l yeast extract, and 17.5 g/l artificial seawater (ASW). The inoculum size was 2-3% of the total liquid volume of the bioreactor. Temperature was kept at 28 C. The medium ph was controlled within the range of PQ6 biomass was harvested after 96 h of fermentation because the maximum biomass and lipid content were reached then. In PQ6 strain, there was correlation between biomass and cellular lipid content as described in the report of Hong et al. (2011) [14]. Algal biomass was harvested by centrifugation at 4000 rpm for 10 min. The algal paste was washed three times with sterile distilled water, dried to a constant weight in an oven at 80 C and then stored in desiccators. To eliminate batch-to-batch variation, all biomass was grown under the same conditions and thoroughly mixed to ensure a homogeneous biomass stock. Cell growth Cell growth was determined by dry cell weight (DCW) and cell density. Cells (usually from 15 ml culture broth) were collected and washed with sterile distilled water by centrifugation at 4000 rpm for 10 min using Sorvall Legend- RT 1900W centrifuge (Kenddro, Langenfeld, Germany) and then dried at 105 C and measured after constant weight had been attained. Cell density was determined by counting the individual cells with a Burker- Turk chamber (Hirschmann, Laborgerate Hilgenberg, Germany). Oil extraction: Oil extraction was as described in the report of Bligh & Dyer (1959) [15] and some modify as described in the report of Hong et al. (2011) [14]. 100 grams of dried biomass was suspended in 1000 ml of n-hexane and wet milled. The mixture was heated and maintained at o C for 4 h and well mixed. After the reaction, the mixture was allowed to cool to room temperature for 1 h. The de-oiled biomass was separated from the oil-rich n-hexane phase by centrifugation. Oil product was obtained after n-hexane removal step. In situ transesterification from the microalga biomass to get mixture of fatty acids in the form of alkyl ester 100 g DCW of Schizochytrium was methylated with 1000 ml solutions of 10% HCl in methanol and 500 ml dichloromethane. The mixture was heated and maintained at o C for 3 h and then well mixed. After the reaction, the mixture was allowed to cool down to room temperature and filtered to remove biomass residues. Then, mixture was added 200 ml of saturated NaCl solution and 1000 ml n-hexane. The mixture was shaken, separated on the funnel and collected n-hexane phase containing total fatty acid - TFA. Under phase was washed some time by n-hexane. The total n-hexane phase was collected and solvent removal step. Collected product was fatty acid in the form of alkyl ester [6]. Yield of the TFA extraction process was calculated by the following formula: H(%) = m 100 M ISSN

46 where: H (%): Yield of the TFA extraction process; m (g): amount of TFA obtained after transesterifification; M (g): initial amount of biomass. Enriched mixture of -3 and -6 PUFAs from algal oil by urea complexation method 10 g of TFA was added in mix of urea and methanol. The mixture was dissolved on a magnetic stirrer and heated at 60 C for 5 min. Then, the mix was complexed at 4 C for h. Where, the complex was constituted by the combination of SFA and MUFA with urea. The ω-3 and ω-6 PUFAs were not complex with urea. After complexing process, the mix was filtered and removed complexation. Methanol solvent was removed at 70 C and obtained ω-3 and ω-6 PUFAs mixture. Next, ω-3 and ω-6 PUFAs mixture was washed with warm water for 3 time and superfluous urea removed. Then, the mixture was layered over funnel, solvent containing ω-3 and ω-6 PUFAs was collected and the bottom phase consisting of water was removed. Solvent containing fatty acids was filtered with dry Na 2 SO 4 to remove superfluous water. Then, solvent was evaporated in a vacuum rotary evaporator at 70 C and obtained the ω-3 and ω-6 PUFAs mixture. Effect of TFA/urea ratio to the extraction and enrichment yield of the ω-3 and ω-6 PUFAs mixture Ratio of TFA and urea were tested in the range as 1:2.5; 1:4 and 1:5 while the ratio of TFA: methanol was kept in 1:20 and using 10 g of TFA. Yield of the ω-3 and ω-6 PUFAs extraction process was consisted of yield of the PUFAs extraction and urea complexation of SFA, MUFA processes. Yield of the PUFAs extraction process (Hs %) was calculated by the following formula: M1 x 100 Hs (%) = M2 where: Hs (%): Yield of the PUFAs extraction process; M1 (g): amount of PUFAs obtained after complexation; M2 (g): initial amount of TFA Yield of the complexation process of SFA, MUFA with urea (H %) was calculated by the following formula: where: H (%): Yield of the complexation process of SFA, MUFA with urea; m1 (g): amount of SFA and MUFA obtained after complexation; m2 (g): initial amount of TFA. Fatty acids analysis H (%) = m1 x 100 m2 The fatty acid composition was analyzed via gas chromatography (GC). The samples were injected by an autoinjector (injection volume of 0.9 µl) on a gas chromatograph (GC) (Hewlett-Packard 6890, New York, NY, USA) equipped with a flame-ionized 32 ISSN

47 detector and DB23 capillary column (30 m x 0.25 mm x 0.26 m; Agilent Technologies, Santa Clara, CA, USA). The column temperature was increased from 150 o C to 260 o C at a rate of 3.5 o C/min. The fatty acids were identified by comparing the retention times with those of standard fatty acids and quantified by comparing their peak area with that of the internal standard. The experimental data were treated with the software Excel and processing ANOVA statistical at significance level of P value III. RESULTS AND DISCUSSION The cell growth of S. mangrovei PQ6 in 30 L bioreactor Changes in cell density, wet and DCW and total lipid of S. mangrovei PQ6 during cultured in 30 L bioreactor have shown in Table 1. At the initial inoculation with 1.81 x 10 6 cells/ml, the density of PQ6 rapidly increased during the first 24 h and reached x 10 6 cells/ml. The maximum cell density of x 10 6 cells/ml was reached after 96 h. Biomass and lipid content gradually increased with time after inoculation, reaching g/l and % of DCW after 96 h, respectively. Culture time (h) Table 1. Changes of parameters of S. mangrovei PQ6 in 30 L bioreactor. Cell density (x 10 6 cells/ml) Wet weight Dry cell weight (g/l) Total lipid (g/l) (% DCW) 1.81 ± 0.36 Not determined Not determined Not determined ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.43 Obtained results in Table 1 was agree with another reports of Hu et al (2008) [16], Nakahara et al. (1996) [17], Yaguchi et al. (1997) [18], Hien et al. (2013) [19]. Lipid content of microalga can reach maximum of 70% of DCW). Cell morphology of strains PQ6 at different times cultured in 30 L bioreactor has shown in Fig.1. During fermentation time, the cell morphology was not change. ISSN

48 Fig. 1. Images of S. mangrovei PQ6 cell cultured in 30 L bioreactor at various fermentation times under light microscope (x 400 X magnifications). The fatty acid composition of PQ6 strain biomass cultured in 30 L bioreactor The fatty acids composition of PQ6 strain biomass was analysis and shown in Table 2 and Fig. 2. The obtained results shown in Table 2 indicated that total ω-3 and ω-6 PUFAs content was accounted for 56.80% of TFA while DHA content reached up to 46.69% of TFA. Obtained results were in accordance with the publication of other authors in the world in heterotrophic marine microalga Crypthecodium cohnii D31 in which its DHA content was more 60% of TFA [20]. DHA content of some species in Thraustochytrium genus was accounted for 11 to 41% of TFA [21]. Order Table 2. The fatty acid composition of PQ6 strain biomass. Fatty acid composition Scientific name Common name FA Content (% of total fatty acid) 1 14:1-5 Tetradecenoic acid 2.44± :0 Hexadecanoic acid Palmitic 34.19± :1-9 Octadecenoic acid Oleic 4.12± : 2-6 Octadecadienoic acid Linoleic 3.55± :5-6 docosapentaenoic acid DPA 7.56± :6-3 docosahexaenoic acid DHA 45.69±1.24 Total polyunsaturated fatty acid ω-3 and ω ± ISSN

49 Fig. 2. Chromatographic of the fatty acid composition of PQ6 strain biomass cultured in 30 L bioreactor. In situ transesterification from microalgal biomass for PUFAs production in the form of alkyl ester Fatty acid in the alkyl ester form was sustainable. So, fatty acid in this form was extracted from this strain PQ6 by in situ transesterification. Based on obtained results of many repeated experiments, we found that the yield of TFA extraction process was 44% DCW. The mixture of fatty acid obtained after in situ transestirification from algal biomass was included SFA, MUFA and ω-3 and ω-6 PUFAs. Then, SFA and MUFA were separated from the PUFAs mixture by urea complexation method. Optimization of enrichment process of -3 and -6 PUFAs mixture by urea complexation A simple and inexpensive procedure involving oil extraction, in situ transestification, winterization and urea complexation in a sequential way has been developed in order to concentrate docosahexanenoic acid (DHA) from S. mangrovei PQ6 biomass. Based on the properties of SFAs/MUFAs and PUFAs, urea complexation and winterization methods to enrich DHA, EPA, DPA content in the mixture of -3 and -6 FA was carried out. These above mentioned methods is effective and easily in Vietnam Laboratory conditions. The optimum enrichment process of PUFAs as DHA, EPA, DPA from FA mixture was tested using different ratios of TFA/urea/methanol as 1: 2.5: 20; 1: 4: 20 and 1: 5: 20. Result of analyzed FA composition of PUFAs mixture in different TFA/urea ratio is shown in Table 3 and Fig. 3 and Fig. 4. ISSN

50 Table 3. The fatty acid composition of PUFAs mixture extracted using different TFA: urea ratio. Fatty acid composition Scientific name TFA: urea ratio as 1 : 2.5 TFA: urea ratio as 1 : 4 TFA: urea ratio as 1 : 5 C7:0 Heptanoic acid C14:0 Tetradecanoic acid C14: C15: C16:0 Hexadecanoic acid C16: C17:0 Heptadecanoci acid C18:0 Octadecanoic acid C18:1( -9) Octadecenoic acid C18:2-6-t C18:3-6 Octadecadienoic acid C19: C20: eicosenoic C20: C20:3-6 8,11,14-eicosatrienoic acid 0.60 C22: C22: C22:5-3 5,8,11,14, eicosapentaenoic acid - C22:6-3 4,7,11,13,16, docosahexaenoic acid C24: : not detected Fig. 3. Chromatographic of fatty acid composition with TFA: urea ratio 1: ISSN

51 Fig. 4. Chromatographic of fatty acid composition with TFA: urea ratio 1:4. TFA: urea ratio is very important factor in order to purity and recovery PUFAs yield in urea complexation method. Obtained results shown in Fig. 5 have indicated that the yield of complexation and extraction process using different ratios of TFA: urea as 1:2.5; 1:4 and 1:5 were 47% and 53%; 70% and 30%; 74% and 26%, respectively. The enrichment of DHA and -3 and -6 PUFAs when using different in TFA: urea ratio have shown in Fig. 5. In the ratio of 1:2.5, 1:4 and 1:5, PUFAs content reached up to 54.56%; 100% and 100% of TFA, respectively. -3 and -6 PUFAs content reached 46.6%, 76.96% and 100% of TFA, respectively, while DHA content obtained 40.54%; 69% and 85.21% of TFA, respectively. Obtained results have shown that DHA content at ratio of TFA: urea as 1:4 and 1:5 ratios was more higher 1.7 and 2.1 times than that at ratio of TFA: urea as 1:2.5 ratio. Therefore, ratio of TFA: urea as 1:4 and 1:5 were chosen for subsequent experiments. On the other hand, the yield of -3 and -6 PUFAs obtaining at the ratio 1:2.5; 1:4 and 1:5 was reached up to 10.16%; 10.86% and 11.44% of DCW (Fig. 6). Thus, when the ratio of TFA: urea increased from 1:4 to 1:5, urea content was increase in 25% but -3 and -6 PUFAs content was only increased in 0.58% of DCW. Alternatively, at the TFA: urea ratio of 1:5, PUFAs content in solid phase was very high (accounted for 38.64% of TFA) while DHA content was accounted 20.23% of TFA because of excess urea in urea complexation of FA. The excess of urea is not only increase the cost of the final product but also it is waste of the PUFAs (especially DHA). It is the reason for us to select TFA: urea ratio of 1:4 for obtaining the -3 and -6 PUFAs content, especially, for high DHA content; save chemicals and high economic efficiency. ISSN

52 Fig. 5. Yield of the complexation and extraction process of PUFA using difference in TFA: urea ratios. Fig. 6. The fatty acid composition of PUFAs using difference in TFA: urea ratios. IV. CONCLUSION When cultivating S. mangrovei PQ6 in 30 L bioreactor, the maximum cell density, wet cell weight, dry cell weight and total lipid were x 10 6 cells/ml, g/l, g/l and 66.3% of dry cell weight after 96 h of cultivation, respectively. Biomass of PQ6 strain cultured in 30 L bioreactor was ensuring quality for oil extraction. The oil has omega-3 and omega-6 polyunsaturated fatty acid and DHA content which accounted for 56.80% and 45.69% of total fatty acid, respectively. Yield of total fatty acid extraction from the dry biomass of PQ6 strain was 44% of dry cell weight. Total fatty acid to urea and methanol ratio as 1:4:20 for enrichment process was selected. Under this condition, the content of the omega-3 and omega-6 PUFAs was % of total fatty acids while DHA content was accounted to 69% of total fatty acids. Yield of the omega-3 and omega-6 PUFAs extraction from dry biomass of PQ6 strain reached 10.86% of dry cell weight. V. ACKNOWLEDGEMENTS This research is funded by Ministry of Industry and Trade with program of Biotechnology in processing (01/HD-DT /CNSHCB for Assoc./Prof. Dr. Dang Diem Hong, We are grateful for use the facilities of National Key Laboratory, IBT; Department of Algal Biotechnology, Institute of Biotechnology, VAST. 38 ISSN

53 REFERENCES [1] J. G. Metz, P. Roessler, D. Facciotii, C. Levering, F. Dittrich, M. Lassner M, R. Valentine, K. Lardizabal K, F. Domergue, A. Yamada, K. Yazawa, V. Knauf and J. Browse, Science. Vol. 293, 2001, pp [2] L. Sijtsma and M. E. de Swaaf, Appl. Microbiol. Biotechnol. Vol. 64, 2004, pp [3] B. E. Rittmann, Biotechnol. Bioeng. Vol. 100, 2008, pp [4] G. C. Dismukes, D. Carrieri, N. Bennette, G. M. Ananyev and M.C. Posewitz, Curr. Opin. Biotechnol. Vol. 19, 2008, pp [5] O. P. Ward and A. Singh, Process Biochem. Vol. 40, 2005, pp [6] M. B. Johnson and Z. Wen. Ener. Fuels. Vol. 23, 2009, pp [7] P. D. Patil, V. G. Gude, A. Mannarswamy, P. Cooke, N. Nirmalakhandan, P. Lammers, S. Deng. Fuel. Vol. 97, 2012, pp [8] H. Im, H. Lee, M. S. Park, J. W. Yang and J. W. Lee. Bioresour.Technol. Vol. 152, 2014, pp [9] S. Ghasemifard, G. M. Turchini, A. J. Sinclair. Prog. Lipid Res. Vol. 56, 2014, pp [10] L. D. Lawson and B. G. Hughes. Biochem. Biophys. Res. Commun. Vol. 152, 1988, pp [11] S. E. Berry. Nutr. Res. Rev. Vol. 22, 2009, pp [12] N. C. Ha, L. T. Thom, L. T. Tam, N. T. H. Thu, H.T. L. Anh, H. T. M. Hien and D. D. Hong. The 3 rd academic conference on natural science for Master and PhD. students from Asean countries. Phnompenh, Cambodia November, 2014, pp [13] L.T. Thom, L.T. Tâm, N.C. Ha, H.T.L. Anh, N.T.H. Thu, H.T.M. Hien, D.D. Hong, J. Biol. Vol. 36, No. 1, 2014, pp [14] D. D. Hong, H. T. L. Anh and N. T. H. Thu. J. Phycol. Vol. 47, 2011, pp [15] E. G. Bligh and W. J. Dyer. Can. J. Biochem. Physiol. Vol. 37, 1959, pp [16] Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert and A. Darzins. Plant J. Vol. 54, 2008, pp [17] T. Nakahara, T. Yokochi, T. Higashihara, S. Tanaka, T. Yaguchi, D. Honda. J. Am. Oil Chem. Soc. Vol. 73, 1996, pp [18] T. Yaguchi, S. Tanaka, T. Yokochi, T. Nakahara, T. Higashihara. J. Am. Oil Chem. Soc. Vol. 74, 1997, pp [19] H.T.M. Hien, L.T. Tâm, L.T. Thom, N.C. Ha, L.H. Hanh, H.T.L. Anh, N.T.H. Thu, D.D. Hong. J. Biol. Vol. 35, No. 4, 2013, pp [20] T. Okuda, A. Ando, E. Sakuradani. J Am Oil Chem Soc. Vol. 90, 2013, pp [21] A. Gupta, S. Wilkens, J. L. Adcock, M. Puri and C. J. Barrow. J. Ind. Microbiol. Biotechnol. Vol. 40, 2013, pp ISSN

54 HYDROCARBON DEGRADATION IN OILY WASTEWATER BY MICROBIAL BIOFILM ATTACHED ON POLYURETHANE FOAM CARRIERS Do Van Tuan 1*, Do Thi To Uyen 2, Dong Van Quyen 2, Le Thi Nhi Cong 2 1 Son La college, Son La, Viet Nam 2 Institute of Biotechnology, VAST, 18 Hoang Quoc Viet, Ha Noi, Viet Nam *: dotuan.cnsh@gmail.com, lenhicong@ibt.ac.vn Abstract. Microorganisms typically colonize surfaces or interfaces to promote their survival and growth. It is on these faces that communities of microbes have been found to form biofilms comprised of microbial cells and extracellular polymeric substances (EPS) such as proteins and polysaccharides. In biolilms, organisms increase their ability to grow and survive in changing environmental conditions and increase their access to absorbed substrates or nutrients. These microenvironments are sites of comparatively high microbial activity and the many complex interactions that take place within them need to be better understood before rational approaches to soil bioremediation can be developed. Successful application of microbial biofilms in petroleum hydrocarbon degradation and transformation is demonstrated. This study focused on the capacity of hydrocarbon contaminated wastewater removal by biofilm formed by a mixture of bacterial and yeast strains isolated from oil contaminated water in Viet Nam. To promote petroleum hydrocarbon removal, carrier for immobilization of indigenous hydrocarbon-degrading microorganisms was developed using polyurethane foam in 50-liter-modules under laboratory condition. Microbial biomass reached cells g -1 on polyurethane foam carrier. High microbial colonization by spherical and rod shapes were observed on the outer surface of the carrier using electronic microscopy. As the result, biofilm attached on polyurethane foam carrier could degrade 91.2 % of the total amount of diesel oil after 7 days with the initial concentration of mg litre -1. While planktonic type of these hydrocarbon-utilizing organisms could degrade only 72.1 %. This result gave hint to develop new method to treat petroleum oil contaminated water in Vietnam. Keywords: Biofilm, microorganism, oil degradation, waste-water. I. INTRODUCTION Water is one of the most important natural resource on our planet. Today, water pollution is a major global problem which is the leading worldwide cause of deaths and diseases. One of the major reasons makes water be polluted is oily wastewater. Oily wastewater is generated in many industrial processes and the hydrocarbons in oily wastewater such as phenol, polycyclic aromatic hydrocarbons have been linked to many serious risks to human and environmental health. The application of microbial biofilm for the removal of oil and its derivatives from contaminated environment has received much attention (Nhi Cong et al., 2013; Liu et al., 2013). A biofilm can be defined as a complex coherent 40 ISSN

55 structure of cells and cellular products, like extra- cellular polymers (Characklis, 1990). Which either form spontaneously as large, dense granules (Lettinga et al., 1980), or grow attached on a static solid surface (Heijnen, 1984). The microbial biofilm formation on solid support can protect microorganisms from being damaged and maintain continuous cell growth and enhance biodegradation. It was demonstrated that microbial extracellular polysaccharide surface (EPS) build up the biofilm matrix that can serve as protective barrier in which immediate direct contact between the highly toxic organic compound and the cells is minimized (Burdman et al., 2000; Czaczyk, 2007).Several carriers have been tested and used in making microbial biofilmfor wastewater treatment systems. Among them, one can mention: Polyethylene (Levstek & Plazl 2009), composite (Zhao et al., 2006), porous ceramic material (Zellner et al., 1987; Kawase et al., 1989) and polyurethane foam (Huysman et al., 1983; Manohar et al., 2001; Tran & Chowdhury, 1991). Several investigations demonstrated that polyurethane showed many advantages for microbial immobilization (Varesche et al., 1997; Huysman et al., 1983; Manohar et al., 2001).This study focused on assessment of hydrocarbons degradation in oily wastewater by microbial biofilm attached on polyurethane foam carrier. II. EXPERIMENTS Microorganisms strains: The strains used in this study (Table 1) were isolated from oil contaminated water in Viet Nam, such as Quangninh, Haiphong, Thanhhoa, Quangngai, Khanhhoa and Vungtau (Nhi Cong et al., 2014). Assimilation tests and sequencing of the 16S rrna and ribosomal ITS region revealed that these strains were Acinetorbacter, Bacillus, Pseudomonas, Rhodococcus, Serratia, Candida, Debaryomyces, Rhodotorula, Trichosposon, etc... The isolates exhibited an excellent ability to degrade alkanes, anthracene, phenol, pyrene, Table 1. Characteristics of several microorganisms isolated from coastal zones in Vietnam. Hydrocarbon utilization Biofilm formation Colony morphology Acinetorbacter sp. QN Bacillus sp. B ISSN

56 Rhodococcus sp. BN Serratia sp. DX Debaryomyces sp. QNN Rhodotorula sp. QNB : Anthracene; 2: Diesel oil; 3: Naphthalene; 4: Phenol; 5: Pyrene (-: no growth; +: growth; ++: good growth) Biofilm formation test: The experiment was performed using the method described by Shimada et al. (2012). System design: Each 50 litter-module included 3 carrier sheets (54x53x27cm), air system and mixing system placed inside the tank. The carrier sheets were sterilized before using in this module. Study design: Bacterial and yeast strains were cultured in complex medium of MPA and Hanssen under laboratory conditions for microbial biofilm formation on carrier. 50 litter oily waste-water with the initial concentration of mg litter -1 (10 % - volume/volume) was added in module to test the hydrocarbons degradation of microbial biofilm. This experiment Fig litter module. (1, mix system; 2, carrier; 3, carrier box; 4, tank). 42 ISSN

57 was controlled by adding oily wastewater with the same concentration in the tank without carrier (planktonic types). Assessment of microbial biomass on carriers: 1g carrier was collected from module every day. Each carrier sample was vibrated in distilled water several times to wash out cells from carrier. The microbial cell quantity was identified by MPN method (Most probable number). The MPN method was described in detail by Oblinger and Koburger (1975). Analysis of petroleum hydrocarbon degradation by gas chromatography (GC): The GC performance was described in detail by Shimada et al. (2012). These experiments were conducted independently at least three times and calculated standard deviation (SD) values. III. RESULTS AND DISCUSSION Twenty bacterial and two yeast strains which could well form biofilm and degrade hydrocarbon were isolated from a number of oil polluted water and sediment samples taken from coastal zones in Vietnam. Some of them were characterized and presented in Table 1. These isolates were cultured on polyurethane foam carriers to compare their capacity of hydrocarbon degradation with the planktonic types at 50 litter modules. The efficiency of degradation was 82.7 % and 47.2 % of total petroleum oil with the initial concentration of mg litre -1 after 5 days of incubation for microbial biofilm attached on polyurethane foam and planktonic types. After 7 days, the most of petroleum oil was degraded and transformed completely, and the microbial biofilm attached on polyurrethane foam showed the capacity of degradation with 91.2 % of total petroleum oil, while planktonic type could degrade only 72.1 %. Fig litter module after 7 days treatment; (a), Planktonic type; (b), Biofilm attached on polyurethane foam. The microbial biofilm formation was good on polyurethane foam with microbial biomass reached cells g -1. High microbial colonization by spherical and rod shapes were observed on the outer surface of the carrier using electronic microscopy (Fig. 3). ISSN

58 Fig. 3. SEM images of biofilm formed on polyurethane foam material; (a), polyurethanefoam material without organisms; (b), mixed-species biofilm forming on polyurethane material. IV. CONCLUSION Biofilm formed by mixture of bacterial and yeast attached on polyurethane foam carriers could degrade and transform hydrocarbons of petroleum oil better than planktonic type. The result gave hint to develop new method to treat petroleum oil contaminated water in Vietnam. V. ACKNOWLEDGMENTS This research is funded by the Ministry of Scienceand Technology Vietnam under grant number KC04.21/ REFERENCES [1] Burdman, S., Jurkevitch, E., Soria-Diaz, M.E., Serrano, A.M. & Okon, Y. (2000). Extracellular polysaccharide composition of Azospirillum brasilense and its relation with cell aggregation. FEMS Microbiol Lett 189 (2), [2] Characklis, W.G. (1990). Biofilm processes. In: Characklis WG, Marshall KC (Eds), Biofilm. Wiley, New York. [3] Czaczyk, K. M. K. (2007). Biosynthesis of Extracellular Polymeric Substances (EPS) and Its Role in Microbial Biofilm Formation. Polish J Environ Stud., 16, [4] Heijin, J. J. (1984). Biological industrial waste water treatment minimizing biomass production and maximizing biomass concentration. Ph.D. Thesis, Delft University of Technology, Delft. [5] Huysman, P., Meenen, P., van Assche, P. & Vertraete, W. (1983). Factors affecting the colonization of non porous and porous packing materials in model up flow mathane reactor. Biotechnol Lett., 5, [6] Kawase, M., Nomura, T., Najima, T. (1989). An anaerobic fixed bed reactor with a porous ceramic carrier. Water Sci Technol, 21, ISSN

59 [7] Lettinga, G., van Velsen, A.F.M., Homba, S.W., de Zeeuw, W. & Klapwijk, A. (1980). Use of the upflow sludge blanket reactor concept for biological waste water treatment especially for anaerobic treatment. Biotechnol Bioeng 22, [8] Levstek, M. & Plazl, L. (2009). Influence of carrier type on nitrification in the moving bed biofilm process. Water Sci Technol., 59, [9] Liu, G., Ye, Z., Tong, K. & Zhang, Y. (2013). Biotreatment of heavy oil wastewater by combined up flow anaerobic sludge blanket and immobilized biological aerated filter in a pilot scale test. Biochem Eng J., 72, [10] Manohar, S., Kim, C.K. & Karegoudar. Enhanced degradation of naphthalene by immobilization of Pseudomonas sp. Strain NGK1 in polyurethane foam (2001). Appl Microbiol Biotechnol 55, [11] Nhi-Cong, L.T., Cung, C.T.M., Nghiem, N.M. (2013). Aromatic hydrocarbon degradation of a biofilm formed by a mixture of marine bacteria. 5 th International contaminated site remediation conference: Program and proceedings, Clean Up 2013 conference, Melbourne, Australia, September [12] Nhi-Cong, L.T., Cung, T.N.M., Morikawa, M. & Nghiem, N.M. (2014). Transformation of iso-pentylbenzene by a biofilm - forming strain of Candida viswanathii TH1 isolated from oilpolluted sediments collected in coastal zones in Vietnam. J Environ Sci Health, part A 49: [13] Oblinger, J.L. & Koburger, J.A. (1975). Understanding and teachinh the Most Probable Number technique. J Milk Food Technol 38(9), [14] Shimada, K., Itoh, Y., Washio, K. and Morikawa, M. (2012). Efficacy of forming biofilms by naphthalene degrading Pseudomonas stutzeri T102 toward bioremediation technology and its molecular mechanisms. Chemosphere 87, [15] Tran, T.F.& Chowdhury, A.K.M.M. (1991). Petroleum waste biodegradation with porous biomass support system (PBSS) on rotating biological contactor. J Environ Sci (China) 3, [16] Zellner, G., Vogel, P., Kneifel, H. & Winter, J. (1987). Anaerobic digestion of Whey and Perameate with suspended and immobilized complex and defined consortia. Appl Microbiol Biotechnol 27, [17] Zhao, Y., Cao, D., Liu, L. & Wei, J. (2006). Minicipal wastewater treatment by moving bed biofilm reactor with diatomaceous earth as carriers. Water Environ Res 78, ISSN

60 EXTRACTION OF SQUALENE FROM VIETNAM HETEROTROPHIC MARINE MICROALGA Nguyen Cam Ha, Le Thi Thom, Hoang Thi Huong Quynh, Pham Van Nhat, Hoang Thi Lan Anh and Dang Diem Hong* Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam *: Abstract. Squalene is a highly unsaturated aliphatic hydrocarbon (C 30 H 50 ) and belongs to the triterpene group. Squalene is currently known as an important ingredient in skin-care products or antioxidant agents in the industry. Many studies have shown the effect of squalene on essential physiology - biological functions including immunity, antioxidant, and decreases in cholesterol levels, liver detoxification and protection against heart diseases. This work has researched and confirmed the great potential of exploiting squalene from Vietnam heterotrophic marine microalga Schizochytrium mangrovei besides biodiesel, omega-3 fatty acid as DHA and glycerol. S. mangrovei was grown in 30-L bioreactor to supply enough biomass for lipid extraction. The cell density, dry cell weight and total lipid content were x 10 6 cells/ml, g/l and 66.3 % of dry cell weight after 5 days of cultivation, respectively. Squalene content of 35 mg/g of dry cell weight after 5 days of cultivation. Squalene content extracted from the biomass of this strain increased 1.3 times through enrichment process via organic solvents partitioning. In addition, S. mangrovei can grow well in bioreactors and the obtained results have demonstrated the potential of producing squalene from this strain by the fermentation industry in the near future. Keywords: Extraction, Squalene, Heterotrophic marine microalgae, Schizochytrium mangrovei. I. INTRODUCTION Squalene (2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexane) is a highly unsaturated aliphatic hydrocarbon (C 30 H 50 ) and belong to the triterpene group. Squalene is currently known as an important ingredient in skin-care products or antioxidant agents in the industry [1]. Many studies have shown the effect of squalene on essential biological and physiological functions including immunity, antioxidant, decreases cholesterol levels, liver detoxification and protection against heart diseases [2]. Absorbing high amount of squalene plays an important role in reducing incidence of coronary heart disease and cancer in Mediterranean populations (normal intake of mg squalene/person) compared with Southeast Asia populations (only intake of 30 mg/person) [3]. Therefore, a dietary squalene supplementation of 500 mg per day is benefit to human health [4]. In the past few decades, commercial squalene has been produced by different sources. The squalene is mainly contained in deep - sea shark liver oil with high content of 46 ISSN

61 80 % (total amount of oil). Besides, squalene content in olive, wheat germ and rice bran oils are only in the % (total amount of oil) ranges which are not high enough to be considered as a commercial source. The over-exploitation of sharks for taking squalene has decreased in numbers of the marine animals. In addition, depending on soil and plant crop that used as material for exploitation of squalene has prompted scientists to search the potential sources. So microorganisms are considered as an alternative source of squalene. Several studies in the world have mentioned the ability of squalene production from yeasts such as Saccharomyces, Pseudomonas, Candida whose concentration is relatively low. Therefore, they are still considered as not good source for production of squalene on large scale [5, 6]. In recent studies, microalgae have been explored as an alternative source of squalene. The green microalgae Botrycoccus braunii is capable of synthesizing squalene but the very low growth rate and the obligate autotrophic growth characteristic render it unsuitable for commercial production. Of all the microalgae groups, the heterotrophically- grown thraustochytrids are regarded as a promising cell factory for the production of high-value products such as squalene. Many recent studies have demonstrated that Schizochytrium sp. can be used as feedstock for squalene production in commercial scale. According to the report of Lu et al (2003), crude squalene was obtained from microalgae Thraustochytrium ATCC by extraction with organic solvents of n- hexane: methanol (2:1, v/v) and several purification methods. The method yielded 0.2 mg squalene at 96% purity from 150 mg of the crude squalene (0.14 % squalene) with 95% recovery [7]. In previous our researchs, based on colorimetric method, squalene content of seven marine microalgal species were determined. Schizochytrium mangrovei reached the highest squalene content of 122 mg/g but the squalene content of S. mangrovei analyzed by thin - layer chromatography (TLC) and high - performance liquid chromatography (HPLC) methods reached only mg/g of dry biomass published by Mai et al (2013) [8]. The different calculation results of squalene in two different methods may be due to the loss of squalene after the extraction and purification steps. On the other hand, in the colorimetric method, calculation result of squalene included in not only squalene but also other sterol. According to Hien et al (2014) published that the squalene content of S. mangrovei PQ6 biomass reached to ± 0.02 mg/g of dry cell weight (DCW) in 30 L bioreactor after 96 h of fermentation [9]. In this paper, purification and enrichment of squalene from marine microalga S. mangrovei PQ6 cultured in 30 L bioreactor was developed in order to improve productivity and quality of extracted squalene. II. MATERIALS AND METHODS Materials ISSN

62 We used heterotrophic marine microalgae S. mangrovei PQ6 which was isolated from Phu Quoc Island, Kien Giang province in , belong to the culture collection at the Department of Algal Biotechnology, Institute of Biotechnology, Vietnam Academy of Science and Technology. Methods Culture of Schizochytrium mangrovei PQ6 S. mangrovei PQ6 cultured in a 30 L bioreactor (working volume of 15 L) with M12 medium containing 90 g/l glucose, 10 g/l yeast extract, and 17.5 g/l artificial seawater. The culture temperature was kept at 25 o C - 28 C, the stirring speed at maximum 450 rpm, the aeration rate of 0.5 vol air/vol medium/min after filtering through a 0.2 µm filter. The algal biomass was washed three times with sterile distilled water, dried to a constant weight in an oven at 80 C and then stored in desiccators as material for extraction of squalene. Growth of S. mangrovei PQ6 Cell growth was determined by DCW and cell density. Cell density was determined by counting the individual cells with a Burker- Turk chamber (Hirschmann, Laborgerate Hilgenberg, Germany). Oil extraction The total lipid content was determinate by Bligh & Dyer method (1959) with some modifications [10]: 100 grams of dried algal biomass was added distilled water, glass sand and milled in blender machine. Then n-hexane was added into the biomass at the ratio of 10:1. The reaction mixture was stirred continuous at o C for four hours. After the reaction, the mixture was allowed to cool to room temperature. Centrifuge the mixture for 10 minutes at 4000 rpm was carried out. Carry out collection of lipid-containing upper layer which was removed solvent by rotary evaporators. Total lipids obtained is stored in a thick brown glass bottle and kept at temperatures 4-5 C. Extraction of the unsaponifiable lipid The unsaponifiable lipid was extracted by method of Lu et al (2004) [11]. The extraction process as follow: 1 gram of dried biomass was saponified by 40 ml of 10% (w/v) KOH-75% (v/v) ethanol solution at 50 C for 30 minutes. After the reaction, the mixture was allowed to cool to room temperature and added 10 ml n-hexane. Vortex by magnetic mixer for 10 minutes and keeps the mixture naturally for 10 minutes. After the mixture, it was divided into two classes: carry out collection of the upper layer containing unsaponifiable lipid, lower layer containing saponifiable lipid. This step was repeated 5 or 6 times to obtain thoroughly unsaponifiable lipid. Solvent was removed by rotary evaporators for collection of unsaponifiable lipid. Confirmation of squalene by TLC method Unsaponifiables were checked by silica gel TLC using n-hexane/diethyleter/acetic 48 ISSN

63 acid (70: 30: 4 v/v/v) as the solvent. After development, squalene on the TLC plate was visualized by spraying 20% sulfuric acid and heating at 100 C for 5 minutes. Confirmation of squalene by HPLC Analysis of squalene in the sample was performed using the HPLC and a Thermo Hypersild Gold C18 column (150 x 2.1 mm, 3 µm). The mobile phase composed of acetonitrile: distilled water (9:1 v/v) was eluted at a flow rate of 250 µl /min. Squalene identification is performed using a PDA detector with scanning of nm, UV wavelength at 198 nm UV and compression the retention time of the sample to standard. Squalene content in the sample is determined based on comparison of the peak area with that of the standard. Extraction and enrichment of squalene from dry biomass * Step 1: The reaction of methyl ester: 66 g total lipid was mixed with 43 ml methanol and 0.66 g NaOH at C for 3 hours. After the reaction, the mixture was allowed to cool down to room temperature. The mixture has classification. Add 110 ml of distilled water into the mixture to remove glycerol (distilled water: sample = 1:1). The mixture was divided into two layers. Collect the entire sample that as material for next step. * Step 2: 70 g obtained from step 1 was dissolved in a mixture of 116 ml KOH 10% and 465 ml ethanol at C for 3 hours. After the reaction, add 233 ml ethanol, 466 ml hot distilled water and 700 ml n-hexane, mixing. The mixture was divided into two layers, the upper layer containing unsaponifiable lipid, and lower layer containing saponifiable lipid. The lower layer was washed 4 times in a mixture of n-hexane: distilled water (9:1, v/v). The n-hexane layer was collected and removed solvent to take unsaponifiable lipid. Repeat step 1 and step 2 until we obtained enough sample amount to start step 3. * Step 3: Add 35 ml ethanol, 35 ml n-hexane, 3.5 ml distilled water into 2.8 g obtained from step 2, mixing. The mixture was divided into two layers: the upper layer was collected, the lower layer was washed many time using n-hexane. The upper layer was removed solvent. * Step 4: Add 32 ml n- hexane, 9.6 ml methanol, 3.6 ml distilled water into 2.5 g obtained from step 3, mixing. Heat slowly the mixture to 70 C. The mixture after heating was allowed to cool down to room temperature. The mixture was divided into two layers, crystalline solids in the upper n-hexane phase were filtered through filter paper and the lower layer was washed, using 100 ml n-hexane to take entire sample. Remove solvent for collecting the product. * Step 5: Add 38.5 ml n-hexane, 1.1 ml methanol, 1.1 ml distilled water into 2 g obtained from step 4, mixing. Heat slowly the mixture to 70 C. The mixture after heating was allowed to cool down to room temperature. The mixture was divided into two layers; the upper n-hexane phase was filtered through filter paper. The filter paper and the lower 49 ISSN

64 layer were washed, using 60 ml n-hexane to take entire sample. Remove solvent for collecting the product. * Step 6: Add 11.6 ml n-hexane, 0.23 ml methanol, 0.46 ml distilled water into 0.8 g obtained from step 5, mixing. Heat slowly the mixture to 70 C. The mixture after heating was allowed to cool down to room temperature. The mixture was divided into two layers; the upper n-hexane phase was filtered through filter paper. The filter paper and the lower layer was washed using 40 ml n-hexane to take entire sample. Remove solvent for collecting the product. * Step 7: Add 8.7 ml n-hexane, 0.34 ml methanol, 0.34 ml distilled water into 0.62 g obtained from step 6, mixing. Heat slowly the mixture to 70 C. The mixture after heating was allowed to cool down to room temperature. The mixture was divided into two layers; the upper n-hexane phase was filtered through filter paper. The filter paper and the lower layer was washed using 40 ml n-hexane to take entire sample. Remove solvent for collecting the product. * Step 8: Add 32 ml n-hexane and 13 ml methanol into g obtained from step 5, mixing. The mixture was divided into two layers: the upper n-hexane layer was collected, the lower layer was washed many time using n-hexane to take entire sample. Remove solvent for collecting the product. * Step 9: Add 20 ml n-hexane and 12 ml methanol into 0.31 g obtained from step 7, mixing. The mixture was divided into two layers, the upper n-hexane layer was collected, the lower layer was washed many times using n-hexane to take entire sample. Remove solvent for collecting the product. * Step 10: Add 13.7 ml n-hexane and 8 ml methanol into 0.4 g obtained from step 8, mixing. Put into cold area of 12 C for minutes. The mixture was divided into two layers: the upper n-hexane layer was collected, the lower layer was washed many time using n-hexane to take entire sample. Remove solvent for collecting the product. * Step 11: Add 8.3 ml n-hexane and 5 ml methanol into 0.2 g obtained from step 9, mixing. Put into cold area of 12 C for minutes. The mixture was divided into two layers, the upper n-hexane layer was collected, and the lower layer was washed much time using n-hexane to take entire sample. Remove solvent for collecting the product. III. RESULTS AND DISSCUSION Growth and squalene content of S. mangrovei PQ6 in bioreactor of 30 L As we have reported earlier [12] about effects of glucose concentrations on squalene production of S. mangrovei PQ6 in flasks, squalene content was the highest in medium with 3% glucose after 5 days of cultivation. However we choose concentration of 9% glucose in cultivation of microalgae using 30 L bioreactor for harvesting large amounts of biomass as feedstock for extraction of squalene. The result showed in Table 1 and Fig ISSN

65 Table 1. Changes of parameters of S. mangrovei PQ6 in 30 L bioreactor. Culture time (day) Cell density ( 10 6 cells/ml) ± ± ± ± ± 1.45 Fresh cell weight (g/l) ± ± ± ± ± Dry cell weight (g/l) ± ± ± ± ± 0.11 Total lipid (% dry cell weight) ± ± ± ± ± 1.31 Unsapofiable lipid (% dry cell weight) 3.21 ± ± ± ± ± 0.41 Squalene (mg/g dry cell weight) ± ± ± ± ± 0.02 Cholesterol (mg/g dry cell weight) 1.36 ± ± ± ± ± 0.35 Isolated squalene was determined by TLC and then the sample was analyzed content by HPLC. Data in Table 1 showed that the highest squalene content was obtained by extraction using Lu et al (2004) method after 1 day of cultivation (43 mg/g DCW). However, cell density reached low amount at the 1 st day. Thus, harvesting biomass after 5 days of cultivation was chosen. By this time, cell density of ± cells/ml and lipid content of ± 1.31 % of DCW was highest. Compared with our previously published paper (Hien et al 2014) [8], the squalene content of S. mangrovei PQ6 in 30 L bioreactor in this paper was slight higher than that in Hien et al (2014) (35.07±0.02 mg/g of DCW compared with ± 0.02 mg/g of DCW, respectively). Total lipid (% DCW) in this paper was higher than that in paper of Hoang et al (2014) published (66.37±1.31 and ±0.08 of DCW after 5 days and 96 h of fermentation, respectively). The different in squalene and total lipid content in this paper compared with Hien et al (2014) published was due to different fermentation conditions as temperature and stirring speed (at 28 o C and at from 25 o C to 28 o C; increasing the stirring speed from 250 rpm to maximum of 450 rpm and increasing and keeping the stirring speed at maximum of 450 rpm only, respectively). According to a recent report of Nakazawa et al (2012), through TLC method, squalene content of Aurantiochytrium limacinum 9F-4a, 4w-1b, 18w-13a and Schizochytrium limacinum SR21 reached 0.6; 0.5; and 0.2 mg/g DCW, respectively [13]. Squalene content of 18w-13a strain was higher than that of our PQ6 strain with mg/g of DCW. Looking at the chromatogram (Fig 1.), squalene isolated also find plenty of other sterol and therefore we should seek a method for purification and enrichment of squalene content that more efficiently from biomass S. mangrovei PQ6. ISSN

66 A) B) Fig. 1. Typical chromatography of squalene standard solution (A) and squalene isolated from S. mangrovei PQ6 biomass after 5 days of cultivation in 30 L bioreactor (B). Extraction and enrichment of squalene by solvent We extracted and enriched squalene from dry biomass PQ6 after cultivation in 30L bioreactor. Algae biomass was harvested after 5 days of fermentation as feedstock input for total lipid extraction. Squalene is a composition belongs to unsapofiable lipid. The product amount obtained after 11 steps as extraction process (Fig. 2). Here we mainly used n- hexane solvent to extract the unsaponifiable lipids from total lipid. Compared with method 52 ISSN

67 of Lu et al. (2004) [11], in this method, squalene was purificated and enriched though 11 steps. After every step, we isolated and determined squalene content by TLC and HPLC (Table 2, Figure 2 and 3). Table 2. Squalene content in 11 steps of extraction continue. Order Steps Squalene content (% sample) Squalene content (mg/g of DCW) 0 Step 1 Not determined ± Step ± ± Step ± ± Step ± ± Step ± ± Step ± ± Step ± ± Step ± ± Step ± ± Step 10 from step ± ± Step 11 from step ± ± 0.10 Fig. 2. Extraction and enrichment of squalene by solvent in 11 step process. ISSN

68 Fig. 3. Squalene content was analyzed by TLC. C: squalene standard; 1-11: squalene obtained from step 1 to step 11 respectively A B Fig. 4. Typical chromatogram of squalene standard solution (A) and squalene isolated by TLC from 8 th step extractions (B). 54 ISSN

69 Chromatogram in Fig. 3 showed squalene extracted in each steps have similar to standard. In addition, squalene content in step 8 reached highest content of ± 0.15 mg/g of DCW. Squalene was higher content and more purificated comparing with method of Lu et al (2004) [11]. Squalene contents of step 9, 10 and 11 have no significant difference with its content of step 8 thus we suggested that using solvent method should stop at step 8 for concentration and purification achieved highest. Squalene content extracted from the biomass of PQ6 strain increased 1.3 times through enrichment process via organic solvents partitioning (compared content of squalene at step of 8 with first step). Figure 4 showed the chromatogram of squalene standard and squalene extracted in step 8 as an example for squalene analysis by TLC and HPLC in samples obtained from different extraction and purification steps. IV. CONCLUSION Heterotrophic marine microalga Schizochytrium mangrovei PQ6 cultured in 30 L bioreactor obtained squalene content of ± 0.02 mg/g DCW after 5 days of cultivation. For 11 step of squalene extraction by solvent from biomass PQ6, we had chosen stopping at step 8 with squalene content of ± 0.15 mg/g DCW. Squalene content extracted from the biomass of PQ6 strain increased 1.3 times through enrichment process via organic solvents partitioning. Therefore we also have initially enriched and purified squalene from algae biomass cultured in 30 L bioreactor to obtain products which have high content and qualification of squalene as a supplement material for functional foods. V. ACKNOWLEDGEMENTS This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number for A/Prof. Dr. Dang Diem Hong. We are grateful for the use of the facilities of National Key Laboratory, Institute of Biotechnology, VAST. REFERENCES [1] K. Wolosik, M. Knas, A. Zalewska, M. Niczyporuk and A. W. Przystupa. J. Cosmet. Sci. Vol. 64 (1), 2013, pp [2] H.A. Martinez-Correa, D.C.A. Gomes, S.L. Kanehisa and F.A. Cabral. J. Food En. Vol. 96, 2010, pp [3] Z.R. Huang, Y.K. Lin and J.Y. Fang. Molecules. Vol. 14, 2009, pp [4] L.R. Alvaro and D.S. Eduardo. Funct Foods Health Dis. Vol. 3, 2013, pp [5] C. Formaron-Bonnefond, V. Demaretz, E. Rosenfeld and J.M. Salmon. J. Biosci. Bioeng. Vol. 93, 2002, pp [6] M. Garaiová, V. Zambojová, Z. Šimová, P. Griač and I. Hapala. FEMS Yeast Res. Vol. 14, 2014, pp ISSN

70 [7] H.T. Lu, Y. Jiang and F. Chen. J. Chromatogr. A. Vol. 994, 2003, pp [8] D.T.N. Mai, N.C. Ha, L.T. Thom, DD Hong. J. Biol. (in Vietnamese) Vol, 35, 2013, pp [9] H.M.Hien, N.C.Ha, L.T. Thom, L.T. Tam, H.T.L. Anh, N.T.H. Thu, D.D. Hong. J. Biosci. Bioeng., Vol 118, 2014, pp [10] E.G. Bligh and W.J. Dyer. J. Biochem. Physiol. Vol. 37, 1959, pp [11] HT. Lu, Y. Jiang, F. Chen. Chromatographia. Vol. 59, 2004, pp [12] L.T. Thom, N.C. Ha, H.T.M. Hien, H.T.L. Anh, N.T.H. Thu and D.D. Hong. The 3 rd academic conference on natural science for master and PhD. students from Asean countries. Phnompenh, Cambodia November 2013, 2013, pp [13] A. Nakazawa, H. Matsuura, R. Kose, S. Kato, D. Honda, I. Inouye, K. Kaya and M.M. Watanabe. Biores. Technol. Vol. 109, 2012, pp ISSN

71 REMOVAL OF COPPER (II) FROM AQUEOUS SOLUTION BY ADSORPTION ONTO MnO 2 NANOSTRUCTURE: EQUILIBRIUM AND KINETIC STUDIES Van-Phuc Dinh 1*, Ngoc-Chung Le 2, Ngoc-Tuan Nguyen 3 1 Dong Nai University, 04 Le Quy Don, Tan Hiep Ward, Bien Hoa, Dong Nai Province, Vietnam 2 Dalat University, 01 Phu Dong Thien Vuong, Dalat city, Lam Dong Province, Vietnam 3 Vietnam Atomic Energy Institute, Viet Nam *: dinhvanphuc82@gmail.com Abstract. Cu(II) ion was removed from aqueous solution in optimal condition as ph, adsorption time and initial concentration by using MnO 2 nanostructure as an adsorbent. The experimental results were analyzed using five non-linear isotherm models: Langmuir, Freundlich, Sips, Tempkin and Dubinin Radushkevich. The adsorption capacity (qm) calculated from the Langmuir isotherm model for Cu(II) is founded as mg/g (RMSE = , R 2 = , 2 = ), which is higher than some other manganese oxide materials. The heat of adsorption process and the mean free energy were estimated from Temkin and Dubinin Radushkevich isotherm models to be and KJ/mol which vividly proved that the adsorption followed a physical process. Although kinetics were investigated using the pseudo first order model and the pseudo second order model, the pseudo-second-order model was found to explain the adsorption kinetics most effectively. Keywords: MnO 2 nanostructrure, adsorption, isotherm, kinetic, copper. I. INTRODUCTION Although copper is essential to human life and health in trace amount, it is potentially toxic, especially at high concentrations. Copper and its compounds are ubiquitous in the environment and are thus found frequently in surface waters. Potential sources of copper bearing waste include plating baths, fertilizer industry, paints and pigments, municipal and storm water run-offs. Intake of excessively large doses of copper by man leads to severe mucosal irritation and corrosion, widespread capillary damage, hepatic and renal damage, and central nervous system irritation followed by depression. Severe gastrointestinal irritation and possible necrotic changes in the liver and kidney could occur. The maximum recommended concentration for drinking water that is regulated in Environmental Quality Act 1974 is 0.2 mg/l [1 8]. Manganese oxides with many types of crystalline structures, such as -, -, - and so on, have been extensively studied due to their structural varieties and excellent chemical characteristic. Therefore, they were applied for different areas, such as batteries, molecular sieves, catalysts, and adsorbents [9-10]. However, the use of MnO 2 nanoparticles to remove copper (II) from aqueous solution has not been widely studied. ISSN

72 In this study, Manganese dioxide was used as a adsorbent to remove Cu(II) ions from aqueous solutions. The influence of various experimental parameters on Cu 2+ adsorption and the optimum adsorption conditions were studied. This paper also describes equilibrium and kinetics the adsorption of copper (II) onto MnO 2 nano material. II. EXPERIMENTS 2.1. Chemicals and instruments Chemicals: Potassium permanganate (KMnO 4 ), ethyl alcohol (C 2 H 5 OH), HNO 3 and NaOH (Merck); Cu(II) ions were used as adsorbate mg/l standard stock solution of each metal ions were prepared by dissolving Cu(NO 3 ) 2 respectively in distilled water. Instruments: Atomic Absorption Spectrophotometer (Spectrometer Atomic Absorption AA 7000 made in Japan by Shimadzu.). The ph measurements were done with a ph-meter (MARTINI Instruments Mi-150 Romania); the ph-meter was standardized using HANNA instruments buffer solutions with ph values of 4.01±0.01, 7.01±0.01, and 10.01±0.01. Temperature-controlled shaker (Model IKA R5) was used for equilibrium studies Adsorption study Place 0.1 g -MnO 2 nanostructrure to 50 ml heavy metal ion solution in a 100 ml conical flask. Effect of ph (2 6), contact time ( minutes) and initial Cu 2+ ion concentration (Co) ( mg/l) were examined. The obtained mixture was centrifugal at 5500 rpm within 10 minutes, then was purified by PTFE Syring Filters with 0.22 µm of pore size to get the filtrate. Atomic Absorption Spectrophotometer (Spectrometer Atomic Absorption AA 7000) was used to analyze the concentrations of the different metal ions in the filtrate before and after adsorption process. Adsorption capacity was calculated by using the mass balance equation for the adsorbent [1-14]: Co Ce. V q m Here, q is the adsorption capacity (mg/g) at equilibrium, C o and C e are the initial concentration and the equilibrium concentration (mg/l), respectively. V is the volume (L) of solution and m is the mass (g) of adsorbent used. III. RESULTS AND DISCUSSION 3.1. Characterization of the MnO 2 nanostructure MnO 2 nanostructure was synthesized via the reduction-oxidation reaction between KMnO 4 and C 2 H 5 OH at room temperature at Institute for Enviromental Studies, Dalat University, Vietnam [10]. The morphology surface of MnO 2 was observed by SEM and 58 ISSN

73 TEM pictures (Fig. 1a and 1b). From these results, the product of - MnO 2 consisted of a large amount of uniform nanospheres, with size of about 10 nm which created a porous surface. This also indicate that its may offer more adsorption sites for adsorbate. The TEM image further demonstrate that the obtained product has a uniform sphere morphology. The TEM image also provides the size of -MnO 2 nanospheres from 10 to 18 nm. Fig. 1. SEM image (a) and TEM image (b) of - MnO 2. The specific surface area (m 2 /g) and pore volume distribution were determined by BET and BJH-analysis of N 2 adsorption desorption isotherms measured on the -MnO 2 samples (Table 1). In this study, -MnO 2 was corresponded to mesopores with average pore diameter higher than 20 A 0 and smaller than 500 Å. Moreover, -MnO 2 has high BET surface area (average 65 m 2 /g) that facilitates the adsorption of Cu 2+. Table 1: B.E.T and B.J.H analysis results. MnO 2 BJH Adsorption average pore Å BJH Desorption average pore Å B.E.T surface m 2.g Affecting Factors In Fig. 2a, it was observed that with increase in the ph (2-6) of the aqueous solution, the adsorption percentage of Copper (II) ion increased up to the ph = 4. At ph > 6.0, the Cu(II) gets precipitated due to hydroxide anions forming a copper hydroxide precipitate. For this reason, the maximum ph value was selected to be 4.0. The increase in adsorption percentage of the Cu 2+ ions may be explained by the fact that at higher ph the adsorbent surface is deprotonated and negatively charge; hence attraction between the positively metal cations occurred [10]. Fig. 2. Effect of ph (a); contact time (b) on adsorption Cu (II) onto - MnO 2. ISSN

74 The effect of contact time was studied at a room temperature of 24 o C, at intervals of 240 min with different concentration (50 ppm, 100 ppm and 200 ppm). From the obtained result (Fig. 2b), it is evident that the adsorption of Cu 2+ ions increased as contact time increases and the adsorption of Cu 2+ ion approached equilibrium within 150 min Adsorption isotherm study Adsorption isotherms are mathematical models that describe the distribution of the adsorbate specie among liquid and solid phases, based on a set of assumptions that related to the heterogeneity/homogeneity of the solid surface, the type of coverage, and the possibility of interaction between the adsorbate specie. In this study, equilibrium data were analyzed using the Freundlich, Langmuir, Sips, Tempkin and Dubinin Redushkevich isotherms expression (Table 2) and the plot of nonlinear form of these isotherms were shown in Fig. 3. From these results (Table 2) can conclude: The sorption capacity (q m ) caculated from non-linear Langmuir isotherm, which is a measure of the maximum sorption capacity corresponding to complete monolayer coverage, is mg/g. The low Freundlich isotherm parameter 1/n value (0.0598) smaller than 1 represents of favorable sorption and confirms the heterogeneity of the adsorbent. Evaluating the correlation coefficients showed that the Sips isotherm described the data appropriable than Langmuir and Freundlich isotherms. This indicates the adsorption Cu 2+ onto MnO 2 surface followed the both Langmuir and Freundlich isotherms. Table 2. Lists of adsorption isotherms models and Equilibrium Isotherm Parameters. Isotherm Nonlinear form Linear form Equilibrium Isotherm Parameters K L Langmuir q q m.k L.Ce Ce C m (mg/g) e q= e = + 1+K L.C q e e qm q m.k RMSE L R Freundlich q = K.C log q e = logk F + logce 1/n e F e βs K S.C e q= 1+α.C e s β e S 60 ISSN n K s ln C ln ln a qe s e s 1/n K F RMSE R K s s s RMSE R

75 Tempkin q Ln K C Dubinin - Radushkevich q RT RT RT q ln K ln C b b b e T e T. q e e m. 2 e T e T T 2 lnq lnq. e m K T (L/mg) 1, b T (kj/mol) RMSE R q m (mol/g) E (kj/mol) RMSE R where: q e : the adsorption capacity at equilibrium (mg/g); q m : the maximum adsorption capacity (mg/g); C e : the equilibrium concentration (mg/l); K L : Langmuir constant; K F : Freundlich constant; n: adsorption intensity; K S : Sips constant; s : Sips isotherm model constant (L/mg); s : Sips isotherm model exponent; K T : Tempkin isotherm equilibrium binding constant; (L/g); b T : Tempkin isotherm constant; : Dubinin Radushkevich isotherm constant (mol 2 /kj 2 ); :Dubinin Radushkevich isotherm constant; E: mean free energy (kj/mol); R 2 : correlation coefficient; RMSE: Root mean square error; : Nonlinear chi-square test. The heat of sorption process and the mean free energy were estimated from Temkin ans D R isotherm models to be less than 8 KJ/mol. Its maybe indicate that the sorption process of Cu(II) onto MnO 2 is essentially physical Kinetic studies Fig. 3. Pseudo-first-order kinetic plots (a), Pseudo-second-order kinetic plots (b) for the adsorption of Cu (II) ion onto - MnO 2. The kinetic of adsorption describes the rate of Cu (II) ions uptake on - MnO 2 and this rate controls the equilibrium time. The kinetics of adsorbate uptake is required for selecting optimum operating conditions for the full-scale batch process. The pseudo-firstorder model and pseudo-second-order model were given in Table 3. Kinetic constants can be obtained from the slope of plots as shown in Fig.4 and Table 3. From these results, the theoretical q e values calculated from pseudo-second order kinetic model were found to be very close to the experimental values of equilibrium sorption, q e (exp), with correlation ISSN

76 coefficients of determination higher than 0.99 whereas the q e values caculated from pseudo-first order kinetic model were not in accordance with the experimental values with the lowest correlation coefficient of determination. Fig. 3. The plots of Non- Linear Isotherms for the adsorption of Cu 2+ onto MnO 2. Table 3. Kinetic models and Kinetic parameters. Kinetic models When q = 0 to q = qt at t = 0 to t = t Kinetic parameters (at Co = 165 ppm) Pseudo-firstorder model kt log - log - 2,303 dq k1 qe q dt q 1. e qt qe q e(exp) (mg/g) K 1 (min -1 ) R q e (cal) (mg/g) ISSN

77 Pseudosecond-order model dq 2 k q q 2 e dt q k2qe qe t t K 2 (g.mg -1.min -1 ) h (mg/g.min) 6.40 R q e (cal) (mg/g) IV. CONCLUSION The experimental results were analyzed using five adsorption isotherm models: Langmuir, Freundlich, Sips, Tempkin and Dubinin - Radushkevich. By using the Langmuir isotherm, the maximum adsorption capacity for Cu(II) is found as mg/g. Comparision of three correlation coefficients of Langmuir, Freundlich and Sips models show that the adsorption process follows the both Langmuir and Freundlich models. The heat of sorption process and the mean free energy caculated from Temkin and Dubinin Redushkevich isotherm models less than 8kJ/mol can be estimated that the adsorption experiment followed a physical process. Kinetic studies showed that a pseudo second order model was more suitable than the pseudo first order model. REFERENCES [1] P. King, P. Srinivas, Y. Prasanna Kumar and V.S.R.K. Prasad, Sorption of copper (II) ion from aqueous solution by Tectona grandis l.f. (teak leaves powder). Journal of Hazardous Materials, 136 (2006) [2] B. Yu, Y. Zhang, A. Shukla, S. S. Shukla and K. L. Dorris, The removal of heavy metal from aqueous solutions by sawdust adsorption-removal of copper. Journal of Hazardous Materials, B80 (2000) [3] B. Acemioglu and M. H. Alma, Sorption of copper (II) ions by pine sawdust, Holz als Rohund Werkstoff, 62 (2004) [4] Anees Ahmad, Mohd. Rafatullah, Othman Sulaiman, Mahamad Hakimi Ibrahim, Yap Yee Chii, Bazlul Mobin Siddique, Removal of Cu(II) and Pb(II) ions from aqueous solutions by adsorption on sawdust of Meranti wood, Desalination 247 (2009) [5] M. Rafatullah, O. Sulaiman, R. Hashim, A. Ahmad, Adsorption of copper (II), chromium (III), nickel (II) and lead (II) ions from aqueous solutions by meranti sawdust, Journal of Hazardous Materials 170 (2009) [6] Necla Caliskan, Ali Riza Kul, Salih Alkan, Eda Gokirmak Sogut, I hsan Alacabey, Adsorption of Zinc(II) on diatomite and manganese-oxide-modified diatomite: A kinetic and equilibrium study, Journal of Hazardous Materials (2011). [7] Mirjana Minceva, Liljana Markovska, Vera Meshko - Removal of Zn 2+, Cd 2+ and Pb 2+ from binary aqueous solution by Natural Zeolite and Granulated activated carbon, Macedonian Journal of Chemistry and Chemical Engineering, 26, No. 2(2007), pp [8] Singh S. P., L. Ma Q. and Harris W. G., Heavy Metal Interactions with Phosphatic Clay: Sorption and Desorption Behavior, J. Environ. Qual. 30 (2001), ISSN

78 [9] Jingfa Li, Baojuan Xi, Yongchun Zhu, Qianwen Li, Yan Yan, Yitai Qian A precursor route to synthesize mesoporous -MnO 2 microcrystals and their applications in lithium battery and water treatment, Journal of Alloys and Compounds, 509 (2011), [10] Ngoc Chung Le and Dinh Van Phuc, Sorption of Lead (II), Cobalt (II) and Copper (II) ions from aqueous solutions by nanostructured gamma MnO 2, Advances in Natural Sciences: Nanoscience and Nanotechnology, issue 2, 6 (2015), (8pp), ISSN: , doi: / /6/2/ [11] Foo K.Y., Hameed B. H., Insights into the modeling of adsorption isotherm systems, Chemical Engineering Journal, 156 (2010), [12] Ho Y.S., McKay G., Pseudo-Second order model for sorption processes, Process Biochemistry. 34 (1999) [13] Igwe J.C., Abia A.A. - Sonde C. U., Pseudo-kinetics and Intraparticle diffusion models for sorption of Zn (II), Cd (II) and Pb (II) ions onto maize cob ABSU, Journal of Environment, Science and Technology. 1 (2011) [14] Yuh-Shan Ho, Augustine E. Ofomaja, Kinetic studies of copper ion adsorption on palm kernel fibre, Journal of Hazardous Materials. B137 (2006) ISSN

79 EFFECT OF ENGINEERED NANOPARTICLES ON CYANOBACTERIA STRAIN Tran Thi Thu Huong 1,2, Duong Thi Thuy 2*, Ha Phuong Thu 3 ; Nguyen Trung Kien 2, Dang Dinh Kim 2, Dao Trong Hien 2 1 Faculty of Environment, Hanoi University of Mining and Geology, Ha Noi, Viet Nam. 2 Institute of Environmental Technology, VAST, Ha Noi, Viet Nam. 3 Institute of Materials Science, VAST, Ha Noi, Viet Nam. *: duongthuy0712@yahoo.com Abstract: The aim of the present study was to investigate the effect of three engineered nanoparticles (silver, copper and titan oxide) on Cyanobacteria strain (Microcystis aeruginosa). The silver, copper nanoparticles were produced by reduction methods and titan oxide was synthesized by using sol-gel method. The silver and copper nanomaterials showed growth inhibition against M. aeruginosa at three concentrations 3, 5 and 10 ppm after 6 days. Nano titan oxide had no toxic effect on M. aeruginosa Keywords: Cyanobacteria, engineered nanoparticles, inhibition, Microcystis aeruginosa. I. INTRODUCTION Cyanobacteria are a group of microorganisms with a broad distribution in the marine, freshwater, terrestrial, and lithoidal environment. The increasing intensity of the mass occurrence of cyanobacteria in eutrophic to hypertrophic waterbodies can cause a variety of water quality problems. Cyanobacteria blooms cause a variety of water quality problems including unpleasant odors, dissolved oxygen depletion, increased ph Cyanobacteria can produce several potent toxins and the presence of these compounds in drinking and bathing water supplies poses a serious hazard to human health. Various methods have been developed and applied to dealing with cyanobacteria bloom problems including reduction of external and internal phosphorus, UV radiation, biological control and chemical, physical and mechanical methods Nanotechnology has recognized as an effective method for water treatment. Nano-materials including natural and engineered materials are applied in many different industries and fields such as medical devices, cosmetics, electronics, chemical catalysis, environment treatment [9]... They have more advantage characteristics than other materials because their specific size is smaller than 100 nm, larger surface to volume ratio, crystalline structure, high reactivity potential, creating the effect of resonance Plasmon surface [8, 17]... and especially, for treatment of sewage contaminated such as usingnanoiron to remove phosphorus in water, destroying algae cells to prevent the development of ISSN

80 cyanobacteria [1] or taking advantage of the presence of dissolved organic matter in water to increase the toxicity of copper nano to kill algae [23]. The aim of current study was to assess the effect of different concentrations of the three engineered nanoparticles (silver, copper and titan oxide) on the growth and development of M. aeruginosastrains. II. EXPERIMENTS 2.1. Materials and methods Algal cultures M. aeruginosa was obtained from Department of Environmental Hydrobiology collection (IET, VAST). It was isolated from eutrophication water in Ke Go Lake, Ha Tinh province, VietNam and cultured in CB medium at 25 ± 2 o C under fluorescent light (1000 lux, 14 h light/8 h dark). The CB medium was composed of 0.15 g/l Ca(NO 3 ) 2. 4H 2 O, 0.1 g/l KNO 3, 0.04 g/l MgSO 4, 0.05 g/l β-sodium glycerophotphate and mg/l Bicine. It was controlled ph = 7.3 before autoclaving by adding NaOH 0,1 M or HCl 0.1 M solutions. All experiments were carried out in this medium Nano-Cu treating method Copper nanomaterial powder was synthesized by reduction method with reducing agent NaBH 4 (Cica-Japan) and precursor CuSO 4 (Merck, Germany). Take 300 ml of distilled water into the beaker (500 ml) containing 7.5 g CuSO 4 power;stir the mixture by IKA RW 20 digital stirrer with stirring speed of 1500 r/min for 15 minutes. Then, gradually pour NaBH 4 (in ratio CuSO 4 :NaBH 4 = 2:1) into the mixture, stir until the solution turns black color. Wash the solution by alcohol 96 o C for 5 times to remove residual ions. Nano copper powder was obtained by drying in 70 C vacuum oven for 24 hours then stored in the dark [19, 24] Nano-Ag treating method In this study, the reduction method has been used to fabricate silver nanomaterial. Chemicals used include natri borohydrit (Cica - Japan) as reducing agent, Chitosan as Stabilizer (Sigma) and silver nitrate (Merck) as precursor agent mmolagno 3 and 15 mmol NaBH 4 are prepared with deion water and chitosan 500 ppm solution is prepared by 10% acetic acid. Stir the silver nitrate solution with chitosan (ph 5-6) and add NaBH 4 solution by drops until the solution turns yellow color, we obtain silver nanoparticle solution Nano-titan treating method Nano-titan material using in this study was received from the Department of Environmental Physico-Chemistry-IET, VAST and manufactured by sol - gel method [21] Determined Characteristicsof synthesized nanoparticles 66 ISSN

81 The morphological features of Ag, Cu and TiO 2 nanoparticles were further characterized by SEM, TEM, and XRD system. Similarly, the preliminary characteristics of nanoparticles were determined by measuring the UV-VIS spectra at nm wavelengths The effect of nanoparticles on growth of cyanobacteria Mycrocystis aeruginosa KG M. aeruginosa KG was cultured in CB medium [20]. Growth of cyanobacteria strain M. aeruginosa KG is assessed by optical density (OD) at 680 nm wavelength using a UV- VIS spectrophotometer (Simadzu) [2] and the efficiency of growth inhibition is calculated by the following formula [17]: Cyanobacteria inhibition efficiency (%) = [(control - treatment)/control] x 100. Briefly, varied concentrations of copper and silver nanoparticles [3, 5 and 10 ppm] were added into flask containing 97 ml CB medium and 3ml biomass of M.aeruginosa. Concentrations of titan added were [50, 100 and 150 ppm]. The growth dynamics of cyanobacteria strain M. aeruginosa KG is monitored at T0, T2 and T6 of experiment period Statistical analysis All experiments were done in triplicate and the data were analyzed and drawn by the software GraphPad Prism 6(one-way ANOVA). Statistical significance was accepted at a level of p <0.05. III. RESULTS AND DISCUSSION 3.1. Characteristics of synthesized nanoparticles Characteristics of copper and silver nanoparticles Both silver and copper nanoparticles using in this study are synthesized by chemical reduction method. The results of SEM and TEM images are shown in Fig. 1. The synthesized copper nanoparticles show to be spherical, non homogeneous in distribution and have an average particle size of nm (Fig. 1a, b). Similarly, the TEM image has showed Ag-nanoparticles have also spherical shape and size smaller than 10 nm (Fig. 1c). ISSN Fig. 1. The SEM, TEM images of copper and silver nanoparticles. 67

82 Silver nanoparticles are distributed more homogeneously than Cu-nano and no cluster. The UV-VIS and XRD results are shown in Fig. 2. The UV - VIS measurement results have showed the silver nano solution is absorbed in nm wavelengths. The ratio BH 4 - /Ag + is investigated in 0.2 and 1.0 range. From 0.2 to 0.6, the maximum absorption peak is quite acute, wide range of the absorption peak is relatively narrow. However, with the concentration ratio NaBH 4 /Ag + of 1.0, the maximum absorption peak magnitude is low and relatively obtuse; it proves that size of Ag nanoparticles is distributed in a wide range (Fig. 2a). The XRD results of copper nanomaterial have shown the characteristic peaks in the diagram are intense, sharp and wide range of the absorption peak is narrow, indicating high level of crystallization of the materials (Fig. 2b). Fig. 2. a) The UV - VIS with concentration ratio BH 4 - /Ag + : 0,2 (1); 0,4 (2); 0,6 (3); 1 (4); b) The XRD image of copper nanomaterial with concentration ratio BH 4 - /Cu 2+ M 1 -M 5 : 1:1; 1,5:1;2:1;3:1; 4: Characteristics of titan dioxide nanoparticles TiO 2 nanomaterial used in this study was synthesized by sol-gel method at room temperature and using tetra-nbutyl orthotitanate Ti(OBu) 4 (99% Merck, Germany) as precursor. SEM and TEM images are shown in Fig. 3. The nanoparticles show to bespherical, homogeneous and have an average size of nm. Fig. 3. SEM (a) and TEM (b) images of TiO 2 nanomaterial. The XRD results of nanomaterial are shown in Fig. 4, the maximum spectral peaks of anatase phase and rutile phase are at 2θ = 25.3 and 2θ = 27.4, respectively. The average crystal size of TiO 2 nanoparticle calculated from XRD data with Scherrer equation is nm. The percentage of anatase, A (%), was determined by the IR peak intensity of 68 ISSN

83 T h e e f f e c t o f g r o w t h i n h i b i t i o n ( A b s n m ) T h e e f f e c t o f g r o w t h i n h i b i t i o n ( A b s n m ) The 4 th Academic Conference on Natural Science for Young Scientists, Master & rutile at 2θ = 27.4 and IA of anatase at 2θ = The calculation rate for anatase and rutile phases are 90.68% and 9.32%, respectively. Fig. 4. The XRD images of TiO 2 nanomaterial synthesized by sol-gel method. R: rutile, A: anatase The effect of silver, copper and TiO 2 nanomaterials on the growth of cyanobacteria strain Microcystis aeruginosa KG. The initial investigated results are shown in Fig. 5. Both silver and copper metal nanomaterial sinfluence the growth and development of M. aeruginosa KG at all three concentrations 3, 5 and 10 ppm after 6 days. OD valuesof silvernano at initial time (T0) and at the end of the experiments (T6) were significantly different, OD (T0) was 0.04±0.005 and OD (T6) 0.02±0.008, respectively. For coppernano, OD value changed less, OD (T0) was 0,06±0,02, after 6 days OD (T6) was fell to 0,03±0,006. C u r e d u c t i o n A g r e d u c t i o n p p m 5 p p m a) b) p p m C o n t r o l 1 0 p p m 5 p p m 3 p p m C o n t r o l D 0 D 2 D 6 D 0 D 2 D 6 T i m e ( d a y s ) T i m e ( d a y s ) Fig. 5. The effeciency of nanomaterials on growth and development of cyanobacteria strain M. aeruginosa KG at different concentrations (3, 5 and 10 ppm): a) nano Cu; b) nano Ag. The growth inhibition efficiency of Ag and Cu nanomaterial concentrations on growth of M. aeruginosa KG after 48 h and 6 days are shown in Fig. 6. For the silver nanomaterial, the inhibition > 70% was observed in both 3 experiment concentrations after 6 days, at the time of 48 h this result only reached to be 50%. Meanwhile, the inhibition > 55% of copper nanomaterial was only observed after 6 days at all three concentrations. ISSN

84 T h e e f f e c t o f g r o w t h i n h i b i t i o n ( A b s n m ) The 4 th Academic Conference on Natural Science for Young Scientists, Master & The results in Fig. 7 shows that the concentration of the solution of nano titanium added to the culture medium not only do not affect the growth and development of the algae population but also because of photochemical catalytic properties of the material, spur algae grow better. This is evident at all three concentrations, measured OD increased linearly with control sample, the lowest OD value was 0.08 (50 ppm) and in the highest was 0.17 (150 ppm); the OD of control samples were 0.05 and 0,11, respectively. The average OD value at the initial time (T0) was 0.08 ± 0.02 and after six days of exposure OD (T6) increased to 0.13 ± These results provedat all concentrations, titanium nanomaterial cannot be toxic against M. aeruginosa KG algae. T i O p p m p p m p p m C o n t r o l D 0 D 2 D 6 T i m e ( d a y s ) Fig. 6. The growth inhibition efficiencyof concentrations of silver and copper solution (3; 5, 10 ppm) after 48 h and 6 days on M. aeruginosa KG. Fig. 7. The efficiency of titan nanomaterial on growth and development of cyanobacteria strain M. aeruginosa KG at different concentrations (50, 100, 150 ppm). Different nanoparticles exhibited differently the growth against M.aeruginosa. Two silver and copper metal nanomaterials influenced cyanobacteria strain M.aeruginosabut TiO 2 nanoparticle failed to inhibit M.aeruginosa at all tested concentrations. Some reports published recently have also the same results [12, 13, 16, 19, 25]. Semiconducting properties of titanium materials play a major role in the process of removing organic contaminants, rapid recombination of photochemical electron pairs and no selectivity of the system are the main reasons for application restrictions of photochemical catalytic process [18]. According to Konrad et al (2012), at concentrations 50, 100, 200 and 300 ppm, titan-nanomaterial doesnot affect the growth of M. aeruginosa. On the contrary, TiO 2 was found to be aggregated, forming a change of the cyanobacterial cell, reducing their biomass [12]. This is explained that the titanium nanoparticles in liquid media has surrounded the cell surface, reducing surface activity, preventing photosynthesis and metabolism between cells and their surroundings, resulting in inhibition of the growth of algae [13, 19]. Titan nanomaterial was significantly less sensitive to M. aeruginosa than 70 ISSN

85 the four algal species tested [5]. Therefore, the mechanism of action of titanium nanomaterials to prokaryotes still needs to be investigated more. Cu and Ag nanomaterials influence on growth of M. aeruginosa at all three concentrations. However, the efficiency of growth inhibition of copper nano is lower than Ag nano. According to Renu Sankar et al, (2014) copper dioxide nanoparticle inhibited the growth of M.aeruginosa at all three concentrations 12,5; 25 and 50 ppm, the efficiency reached to 31,8% (at 12,5 ppm) and 89,7% (50 ppm). In addition, nano oxides (TiO 2 and CuO) were less toxic than their formulations (Ti and Cu) through measuring EC50 index [22]. On the other hand, the presence of dissolved organic matter also increases toxicity of CuO against M. aeruginosa [25]. Nano silver is very sensitive to bacteria; at very low concentrations they also have the ability to inhibit the growth of cyanobacteria. At a concentration of 1 mg/l, silver nanomaterial inhibited thegrowth of M. aeruginosa population to 87% compared with control sample [15]. Besides, the nano-ag + synthesized by in vivo method is capable of inhibiting cancer promoters when it decreases cell count and total chlorophyll content of M. aeruginosa [14]. Until now, the mechanism of action of nanomaterials for prokaryotic cells has remained controversial. These outstanding features of nanomaterials are increasingly being studied but the interaction of nanomaterials with aquatic organisms like algae, phytoplankton remains limited. IV. CONCLUSION The initial results have showed the silver and copper nanomaterials synthesized by reduction method affect the growth and development of M. aeruginosa KG population, the efficiency of growth inhibition of copper nano is lower than that of silver nano. Titanium nanomaterialsynthesized by sol - gel methodhas no effect on the growth of this cyanobacteria strain. However, this is only a range of preliminary concentrations investigated; the study should be carried out at the different concentrations to find the most appropriate concentration range which has the ability to influence and the greatest inhibition against M. aeruginosa KG cyanobacteria population. V. ACKNOWLEDGMENT The present study was supported by the VAST0701/15-16 project and IFS W projects. The authors are grateful for the financial support from VAST. ISSN REFERENCES [1] Blahoslav Marsalek, et al, Multimodal action and selective toxicity of zerovalent iron nanoparticles against cyanobacteria, Environmental Science and Technology, (2012), (46), ( ). [2] Chaturvei V et al, Fabrication of silver nanoparticles from leaf extract of Butea monosperma (Flame of Forest) and their inhibitory effect on bloom-forming cyanobacteria, Bioresources and Bioprocessing 2:18, (2015). 71

86 [3] Chorus I., et al, In Monitoring and Management, World Health Organization, (1999), 400 pp. [4] Codd et al, Harmful cyanobacteria: from mass mortalities to management measures, Springer, Dordrecht, (2005), 1-23 [5] Chu Thi Thanh Binh et al, Comparing acute effects of nano-tio 2 pigment on cosmopolitan freshwater phototrophic microbesussing high throughput screening, Plus one 10(4) (2015). [6] Drabkova M., et al, Photodynamic therapy against cyanobacteria, Environ Toxicol, 22, (2007). [7] Han FXet al, Accumulation, distribution, and toxicity of copper in sediments of catfish ponds receiving periodic copper sulfate applications, J. Environ Qual. 30, (2001), [8] Jayatissa L.P. et al, Occurrence of toxigenic cyanobacterial blooms in fresh waters of Sri Lanka. Systematic and Applied Microbiology 29, (2006), [9] Jiangxin Wang, Toxicity assessment of manufactured Nano-materials using the unicellular green alga Chlamydomonas reinhartii, Chemosphere 73, (2008), [10] Johnk, K.D, et al, Summer heat waves promote blooms of harmful cyanobacteria. Glob, Change Biol 14, (2008), [11] Klaine S.J. et al, Nano materials in the environment: behavior, fate, bioavailability and effects. Environmental Toxicology and Chemistry 27 (9), (2008), ( ). [12] Konrad J. Kulacki, Effects of Nano-Ti titanium Dioxide on Freshwater Algal Population Dynamics, Plus one vol. 7, (2012). [13] Lanzhou Chen et al, Toxicological effects of nanometer titanium dioxide (nano-tio 2 ) on Chlamydomon as reinhardtii, Ecotoxicology and Environmental Safety 84, (2012), [14] Mostafa Mohamed El-Sheekh, Application of Biosynthesized Silver Nanoparticles Against a Cancer Promoter Cyanobacterium Microcystis aeruginosa, APJCP, Vol. 15, (2014). [15] Myung-Hwan Park et al, Selective inhibitory potential of silver nanoparticles on the harmful cyanobacterium Microcystis aeruginosa, Biotechnol Lett. (2009). [16] O.V. Makarova, T. Rajh et al, Surface modification of TiO 2 nanoparticles for photochemical reduction of nitrobenzene, Environ. Sci. Technol 34, (2000). [17] Park MH et al, Growth inhibition of bloom-forming cyanobacterium M.aeruginosa by rice straw extract. Lett. Appl Microbiol 43, (2006), [18] Roduner E, Size matters: why nanomaterials are different. Chem. Soc. Review 35, (2006). [19] Selvarani M et al, Evaluation of antibacterial efficacy of chemically synthesized copper and zerovalent iron nanoparticles. Asian J Pharm Clin Res 6 (3), (2013), (223-22). [20] Shirai M., et al, Development of a Solid Medium for Growth and Isolation of Axenic Microcystis Strains, Applied an environmental Microbiology, (1989), ( ). [21] Thanh Son Le, et al, Air purification equipment combining a filter coated by silver nanoparticles with a nano-tio 2 photocatalyst for use in hospitals. Nanotechnol. 6, 8pp, (2015). 72 ISSN

87 [22] Villem Aruoja et al, Toxicity of nanoparticles of CuO, ZnO and TiO 2 to microalgae Pseudokirchneriella subcapitata, Science of the total environment 407, (2009), [23] Yan R., et al, The decoction of radix Astragali inhibits the growth of Microcystis aeruginosa. Ecotoxicology Environmental Safety 74, (2011), [24] Zhang Q., Yang Z et al, Preparation of copper nanoparticles by chemical reduction method using potassium borohydride. Trans. Nonferrous Met. Soc. China (2010), ( ). [25] Zhenyu Wang et al, Toxicity and internalization of CuO nanoparticles to prokaryotic alga Microcystis aeruginosa as affected by dissolved organic matter, Environ. Sci. Technol 45, (2011), ( ). ISSN

88 SEDIMENTATION RATES IN THE U-TAPAO ESTUARY DEMONSTRATED BY 210 Pb-AND 137 Cs-DATING METHODS Santi Raksawong 1, Miodrag Krmar 2, Tripob Bhongsuwan 1* 1 Department of Physics, Faculty of Science, Prince of Songkla University, Thailand 2 Department of Physics, Faculty of Science, University of Novi Sad, Serbia *: tripop.b@psu.ac.th Abstract. The study of sediment dynamics will provide an understanding of the dynamics of pollutants, and aid in developing sustainable effective management. The 210 Pb and 137 Cs dating techniques have been widely used to reconstruct the sedimentation histories in the lake sediment. In this contribution, we present the preliminary analyses and results of sedimentation rates. One site from U-Tapao estuary located in the Outer Songkhla lagoon, southern Thailand was selected and the vertical profiles of excess 210 Pb and 137 Cs were used for analyses. The average accumulation rates determined by 137 Cs method are 5.7 and 6.5 kg m -2 year -1 from 1963 to 1986 and 1986 to sampling time, respectively. Moreover, the sediment accumulation rates between 8.8 and 3.3 kg m -2 year -1 during sampling time to 1954 were obtained by the CIC and CRS models. Two-marker events based on 137 Cs activity were used to validate age of sediment layers obtained by 210 Pb method. Keywords. Songkhla lagoon, 210 Pb-dating, 137 Cs- dating, Sedimentation rates. I. INTRODUCTION Nowadays, the Songkhla lagoon is facing the pollution problems as evidenced by several factors: overall low water quality, contamination by toxic pollutants/nutrients, and rapid sedimentation in some areas [1-9]. Especially, the unsanitary drainage from Hat Yai urban area, and the wastewater from large factories along the U-Tapao canal and its watershed drain into the U-Tapao estuary [6, 8-10, 13]. Therefore, studying the sedimentation dynamics in this area will provide an understanding of the dynamics of pollutants to develop effectively sustainable management. Over the past four decades in the sediment chronology, the 210 Pb and 137 Cs dating techniques have been widely used to reconstruct the sedimentation histories from a year up to 150 years [14-17]. 210 Pb is naturally occurring radionuclide of the 238 U series. Its closed parent is 222 Rn, a noble gas having short half-life, 3.8 days. This gas escapes into the atmosphere from surface soil layers and provides 210 Pb with mobility in atmosphere. The 210 Pb is introduced into the estuarine environment through atmospheric precipitation, terrestrial runoff and in situ production from 226 Ra in the water column and soil or sediment. 210 Pb falling directly into the lake is removed quickly to sediments by adsorption process and deposited on the bottom of the lake together with the sediment [18]. Two simple models known as the CIC and CRS models are usually applied. For constant sedimentation rates, the CIC model is the simplest method based on the supply of 210 Pb to the sediments which is constant initial 74 ISSN

89 concentration [18, 19]. Moreover, the CRS model is based on the assumption of the constant rate of supply unsupported 210 Pb or 210 Pb ex to the sediment and insignificant mobility of 210 Pb in the sediment column [18]. Sometime, the chronological results provided by the 210 Pb-dating models could probably provide less reliable information on sediment accumulation rates. When the researchers need a high level of confidence in chronology, the 210 Pb-dating models must always be validated by the 137 Cs profile for identifying the peaks corresponding to sediment deposition in 1963 and 1986, or other records of known contaminant inputs [15, 18, 20]. In this paper, one selected core was taken from the U-Tapao estuary located in the Outer Songkhla lagoon. Results of sedimentation rates are estimated by the vertical profiles of excess 210 Pb and 137 Cs. II. METHODOLOGY Study area The Outer Songkhla lagoon is located in southern Thailand between latitudes 7 o 05 and 7 o 50 N and longitudes 100 o 05 and 100 o 37 E, a part of the east coast of the Malay Peninsula. The Outer Songkhla Lagoon is a shallow coastal lagoon with an average depth around 2.0 m in rainy season and 1.5 m in summer season. Fig. 1. Study area in the Outer Songkhla lagoon, black star ( ) is the location of the collected sediment core. At the lower east side near the Songkhla harbor and the Songkhla town, it has a deep channel (5 12 m depth) connecting the lagoon with the sea of the Gulf of Thailand and allowing the tides to propagate into the lagoon as shown in Fig. 1. Therefore the aquatic environment in this lagoon is a combination of seawater and freshwater, and the hydrodynamic complexity of this system is mainly controlled by tide, runoff, wind and wave. 75 ISSN

90 Sample preparation and measurements The sediment core was collected using a hand-operated corer equipped with the PVC tube (8.5 cm internal diameter, 150 cm long). In the laboratory, the sediment core was sliced horizontally at 1.0 cm intervals through its depth using the handheld extruder. All the specimens were dried at 105 C for 24 hours by electric oven, and grinded by ceramic mortar, and sieved to < 2 mm fraction, homogenized, weighed and put into a polyethylene containers of a similar geometry with the calibration source. The selected sediment samples at depth layers (2, 4, 6, 8, 10, 14, 18, 24, and 30 cm) were sent to the nuclear laboratory of the Faculty of Sciences, University of Novi Sad, Serbia, to measure the 210 Pb activity usinga low background 100% relative efficiency gamma spectrometer at 46.5 kev gamma line. Other sediment samples were measured in the Nuclear Laboratory at the Prince of Songkla University, Thailand, using a 80% relative efficiency gamma spectrometer coupled with a DSA 1000 multichannel analyzer. Due to a very low activity of 137 Cs in sediments, its gamma ray energy kev was measured by counting-time periods of minimum 40,000s to maximum 100,000s to provide the lowest reasonable analytical error. The IAEA TEL WWOPT soil sample-04 with known radionuclide activities was used to calculate the relative efficiency of 137 Cs at gamma energy line kev. III. RESULTS AND DISCUSSIONS Fig. 2. a) 137 Cs depth profile, b) 210 Pb activity profiles from the Outer Songkhla lagoon. The curve corresponds to those used by the CIC model to estimate sedimentation rate. This paper shows the results of sediment samples from a single core coded SK02 selected for interpretation of two different sedimentation processes. It can be seen that the 137 Cs time-markers correspond to the fallout from the Chernobyl nuclear accident in 1986 and the highest atmospheric thermonuclear weapon testing in Both chronologic markers can be identified as shown in Fig ISSN

91 The 137 Cs activity in each sediment layers varied between the level of detection limit and 2.1 Bqkg -1. The peak values of 137 Cs activity at the mass depths of 18.1 and 31.2 gcm -2 correspond to the sedimentation years of A.D.1986 and A.D.1963, respectively. The average accumulation rates calculated in the period from A.D.1963 to 1986 and A.D.1986 till the moment of sampling from those markers are 5.6 kgm -2 y -1 (linear sedimentation rate 0.52 cmy -1 ), and 6.7 kgm -2 y -1 (linear sedimentation rate 0.78 cmy -1 ), respectively. The previous sedimentation rate of 0.57 cmy -1 near the Pak Ro channel, reported by Chittrakarn et al [2] was the result of the 137 Cs method. The 210 Pb dating method can be used to estimate the sedimentation rates in this site. The depth distribution of 210 Pb ex activity shows some regular decreasing trend. It was supposed that the simplest CIC model can be applied in the estimation of sediment mass accumulation at this location. The mean sedimentation rate is 9.62 kgm -2 y -1, calculated from the slope of the graph between the logarithmic scale of the 210 Pb ex activity and mass depth. Moreover, the mass accumulation rates obtained by the CRS model are between 8.3 and 3.3 kgm -2 y -1. The CRS model provides the best result that the marker-peak values of 137 Cs are validated as shown in Fig 3. Fig. 3. a) results obtained by estimating from CIC and CRS models and validated by 137 Cs markers,b) comparison between mass accumulation rates obtained from CRS model and 137 Cs method. The mass accumulation rate that provides from dating by CRS model in recent year is high value about 8.0 kg m -2 y -1 and it gradually decreases. So it indicates that in the past, before 1960s the Songkhla lagoon experienced a low sedimentation rate. IV. CONCLUSIONS The mass accumulation rates of the sediment at aselected location inthe Outer Songkla lagoon were determined based on both vertical profiles of 137 Cs and 210 Pb in sediment core. Use of 210 Pb-dating models was validated by using fallout of artificial radionuclide 137 Cs, that showed a very good correlation between them. Also CRS model indicates that the sedimentation rates show a constant increasing trend with time. ISSN

92 V. ACKNOWLEDGEMENTS This research was financially supported by the Prince of Songkla University (project code: SCI550111S), the National Research Council of Thailand (project code: SCI560125S), the Physics Department, Geophysics Research Center, Faculty of Science and the graduate fellowship from the Graduate School, Prince of Songkla University. In addition, the first author would like to thank Prince of Songkla University, Graduate Studies Grant and the student exchange program between Prince of Songkla University and University of Novi Sad. REFERENCES [1] T. Bhongsuwan and D. Bhongsuwan, Songklanakarin J. Sci. Technol., Vol. 24, 2002, pp [2] T. Chittrakarn, T. Bhongsuwan, P. Nunnin, and T. Thong-jerm, The determination of Sedimentation rate in Songkhla Lake Using Isotopic Technique, [3] S. Gyawali, K. Techato, S. Monprapussorn, and C. Yuangyai, Procedia - Soc. Behav. Sci., Vol. 91, 2013, pp [4] K. Kitbamroong, P. Sompongchaiyaku, and G. Padmanabhan, J. Appl. Sci., Vol. 9, 2009, pp [5] R. Ladachart, C. Suthirat, K. Hisada, and P. Charusiri, J. Appl. Sci., Vol. 11, 2011, pp [6] K. Pornpinatepong, S. Kiripat, S. Treewanchai, S. Chongwilaikasaem, C. Pornsawang, P. Chantarasap, C. Chandee, and P. Jantrakul, Pollution conrol and sustainable fisheries management in Songkhla Lake, Thailand, Songkhla, [7] S. Pradit, M. S. Pattarathomrong, and S. Panutrakul, Procedia - Soc. Behav. Sci., Vol. 91, 2013, pp [8] W. Sirinawin, D. R. Turner, S. Westerlund, and P. Kanatharana, Mar. Chem., Vol. 62, 1998, pp [9] W. Sirinawin and P. Sompongchaiyakul, Mar. Chem., Vol. 94, 2005, pp [10] S. Angsupanich and R. Kuwabara, Lakes Reserv. Res. Manag., Vol. 4, 1999, pp [11] P. Chevakidagarn, Songklanakarin J. Sci. Technol., Vol. 28, 2006, pp [12] S. Maneepong, SongklanakarinJ. Sci. Technol., Vol. 18, 1996, pp [13] S. Maneepong and S. Angsupanich, Songklanakarin J. Sci. Technol., Vol. 21, 1999, pp [14] Y. S. Ahn, F. Nakamura, and K. W. Chun, Geomorphology, Vol. 114, 2010, pp [15] M. Baskaran, J. Nix, C. Kuyper, and N. Karunakara, J. Environ. Radioact.,Vol. 138, 2014, pp ISSN

93 [16] J. O Reilly, L. L. Vintró, P. I. Mitchell, I. Donohue, M. Leira, W. Hobbs, and K. Irvine, J. Environ. Radioact., Vol. 102, 2011, pp [17] S. P. Rai, V. Kumar, and B. Kumar, Hydrol. Sci. J., Vol. 52, 2007, pp [18] P. G. Appleby, Chronostratigraphic techniques inrecent sediments, in Tracking Environmental Change Using Lake Sediments. Vollume 1: Basin Analysis, Coring, and Chronological Techniques, vol. 1, W. M. Last and J. P. Smol, Eds. Dordrecht, The Netherlands: Kluwer Academic Publishers, 2001, pp [19] S. C. Yao, S. J. Li, and H. C. Zhang, J. Radioanal. Nucl. Chem., Vol. 278, 2008, pp [20] J. M. Abril, J. Paleolimnol., Vol. 30, 2003, pp ISSN

94 ASYMPTOTIC ANALYSIS FOR TRANSMISSION OF GAUSSIAN BEAM THROUGH A PLANE DIELECTRIC INTERFACE Dinh Trong Quang* and Trinh Xuan Tho Academy of Military Science and Technology, 17 Hoang Sam, Hanoi *: dtquang.vn@gmail.com Abstract. We have analyzed the phenomenon of the transmission and scattering of the Gaussian beam that is incident on a plane dielectric interface from a denser medium to a rarer one. The asymptotic solutions for the transmitted wave and scattered wave have been derived by using the saddle point technique. We have confirmed the validity of the asymptotic solutions by compared with the reference solution calculated numerically from the integral representation. We have shown the very interesting phenomenon that, like the reflection and scattering of the Gaussian beam, the beam shift can be observed in this case. Keywords: transmitted Gaussian beam, asymptotic analysis, plane dielectric interface. I. INTRODUCTION It have been studied by many researcher when the cylindrical, spherical, and beam waves incident a plane dielectric interface from a denser medium to a rarer one [1-11]. It is known that when the Gaussian beam is incident from a denser medium to a rarer one and is reflected by single and multiple media interfaces does not exactly follow the path of the geometrical optics [1-7, 9-11] and exhibits the nonspecular effect including lateral shift, and angular shift [10, 11]. Recently, we have shown that when the cylindrical wave or the spherical wave is incident on a plane dielectric interface from a denser medium, the observation point placed in the rarer medium may receive only the transmitted geometrical ray in the short distance from the source and both the transmitted geometrical ray and the evanescent wave in the long distance. We have derived the uniform asymptotic solution to connect the one solution in the near region to the other solution in the far region smoothly through the transition region by using the parabolic cylinder function on the recent researches. We have also shown the very interesting phenomenon that the lateral wave type transmitted wave observed in the far and shallow region. In this paper, extending the results of previous papers, we will derive the asymptotic solution for the transmitted Gaussian beam observed in the rarer medium by using the parabolic cylinder function. The validity and the applicable range of the asymptotic solution are confirmed by comparing with the reference solution calculated numerically from the integral representation of the transmitted Gaussian beam. We show that like the reflected Gaussian beam, the beam shift of the transmitted Gaussian beam is also appeared in the rarer medium. 80 ISSN

95 II. FORMULATION AND INTEGRAL REPRESENTATION FOR GAUSSIAN BEAM Fig. 1 shows the schematic figure for the transmission and scattering of the Gaussian beam through a plane dielectric interface z = 0, the Cartesian coordinate system (x, y, z), the incident beam coordinate system (x i, z i ), and the transmitted beam coordinate system (x t, z t ). We have divided the medium 2 into 3 regions: near region, far region and transition region along the z-direction. We assume that the medium 1 (ε 1, μ 0 ) is denser than the medium 2 (ε 2, μ 0 ). medium 1 medium 2 geometrically transmitted beam incident Gaussian beam beam shift near region critical angle transition region beam shift evanescent beam wave far region Fig. 1. Schematic figure for transmission and scattering of Gaussian beam through a plane dielectric interface and coordinate systems(x, y, z), (x i, z i ), and (x t, z t ). We also assume that the incident Gaussian beam polarized in y-direction along the aperture plane x i = 0 in the incident coordinate system (x i, z i ) is given by [1, 2, 4, 7, 9, 11]: U i (x i, z i = 0) 1 πw exp x i W 2 (1) where 2W denotes the beam width of the Gaussian beam. By applying Fourier integral representation and transforming from the incident beam coordinate system (x i, z i ) to the Cartesian coordinate system (x, y, z), one may obtain the transmitted Gaussian beam observed at the observation point P 1 (x, z) (or P 2 (x, z)) (Fig. 1) in the rarer medium 2 as follows [4, 9, 11]: U t (x, y) = k 1 2π T θ B(θ, θ b ) e ikq (θ) dθ (2) P θ where the beam function B(θ, θ b ), the phase function q(θ), and the transmission coefficient T θ are defined as follows: B θ, θ b = exp k 1Wsin (θ θ b ) 2 2 cos (θ θb ) (3) q θ = Rcos θ θ s + z n 2 sin 2 θ (4) T θ = 2 cos θ cos θ+ n 2 sin 2 θ (5) Note that propagation distance R, and the beam incident angles θ 0, θ b are defined geometrically in Fig. 1 and that n = ε 2 /ε 1 < 1 denotes the refractive index. The 81 ISSN

96 original integration path P θ in (2), branch point δ(= sin 1 n), and the branch cut are shown in the complex θ-plane in Fig. 2(a) and Fig. 2(b). The branch point δ corresponds to the critical angle of total reflection (Fig. 1). Im [ ] valley complex -plane branch cut Im [ ] valley complex valley -plane original path O Re [ ] original path O Re [ ] valley original path valley (a) Fig. 2. Saddle points θ s and θ e and steepest descent paths SDP θs and SDP θe passing through saddle points θ s and θ e in the complex θ-plane. (a): the observation point is located in the near region and (b): the observation point is located in the far region. (b) III. ASYMPTOTIC SOLUTIONS FOR TRANSMITTED GAUSSIAN BEAM 3.1. Asymptotic Solution in the Near Region When the observation point is located at P 1 in the near region on the left of the curve AG (Fig. 1), the saddle point equation d(q(θ) )/d(θ) = 0 or: Rsin θ θ s + z sin θ cos θ n 2 sin 2 θ = 0 (6) possesses one saddle point θ s that contributes to the integral (2). In Fig. 2(a), we have shown the saddle point θ s, and the steepest descent path SDP θs passing through the saddle point θ s in the complex θ - plane. By applying the isolated saddle point technique [12], one may obtain the asymptotic solution for the transmitted Gaussian beam U t as follows [9, 11]: U t U go, U go ~T θ s B(θ s, θ b ) k 1 2π q (θ s ) eik 1l 1 +ik 2 l 2 iπ/4 (7) q θ s = cos θ s sec 2 θ s + z n2 cos θ s n 2 sin 2 θ 2 (8) The asymptotic solution for the geometrically transmitted beam U go represents clearly the transmitted geometrical ray O i C P 1 observed at P 1 where the propagation distances l 1 and l 2 are defined geometrically in Fig Asymptotic Solution in the Far Region When the observation point is located at P 2 in the far region on the right of the curve AH (Fig. 1), the saddle point equation (6) produces two saddle points θ s and θ e contribute to the integral. The steepest descent paths SDP θs and SDP θe passing through the saddle 82 ISSN

97 points θ s and θ e are shown in Fig. 2(b). By deforming the original integration path P θ into the steepest descent paths SDP θs and SDP θe, one may express the transmitted Gaussian beam as follows [9, 11]: U t U go + U eva (9) where the integral representations of geometrically transmitted beam U go and evanescent beam wave U eva are given by: U go = k 1 2π B(θ s, θ b ) SDP θ s T θ e ikq (θ) dθ (10) U eva = k 1 2π B(θ e, θ b ) SDP θ e T θ e ikq (θ) dθ (11) Since the saddle point θ s is located near the branch point δ and far from the beam incident angle θ b (Fig. 2(b) and Fig. 1), the beam function B(θ s, θ b ) in (10) and therefore the geometrically transmitted beam U go become sufficiently small. Thus, by applying the saddle point technique for the integral in (11) and approximating the saddle point θ e around θ 0, i. e. θ e θ 0, one may obtain the transmitted Gaussian beam U t expressed by using only the evanescent beam U eva : O i D P 2 as follows: U t = U eva, U eva ~ k 1 2πR B(θ 0, θ b )e ik 1R iπ/4 T θ 0 e z sin 2 θ 0 n 2 (12) The aympotic solution for evanescent beam U eva shows that when the observation point move from the shallow region to the deep region, the magnitude of evanescent beam will decrease exponentially with depth z Asymptotic Solution in the Transition Region Observation point located in the transition region and in the near region When the observation point located in the transition region between the dotted curves AH and AG (x < P 4 ), the saddle point equation (6) obtain two saddle points θ s and θ e but only one saddle point θ s contributes to the integral (Fig.3(a)). By deforming the integral P θ into the steepest descent path SDP θs, the transmitted Gaussian beam can be approximated as follow: U t ~U go + U tran, U tran = U U go (13) where the integral U is along the steepest descent path SDP θs and can be analyzed asymptotically by using the approximation of beam function around θ θ s : U~ k 1 2π B(θ s, θ b ) SDP θ s T θ e ikq (θ) dθ (14) In this case, the saddle point θ s is located near the branch point θ s and the integral U can be approximated as follow: ISSN

98 beam magnitude [V/m] beam magnitude [V/m] The 4 th Academic Conference on Natural Science for Young Scientists, Master & U~U 1 + U 2 + U 3 (15) U 1 = k 1 T θ 2π s e ik 1l 1 +ik 2 l 2 iπ 4 α j j =0 I(j) (16) j! k 1 Rcos θ s θ 0 U 2 + U 3 = k 1cosθ π 1 n 2 eik 1l 1 +ik 2 l 2 iπ 4 sin (δ θ s )cos (δ θ s )α j j =0 I(j + 1) (17) e iπ /8 j! k 1 Rcos θ s θ 0 3/4 where the function I(j) can be calculated by applying the parabolic cylinder function D as follow: I j = i m j 2 jm=0 2π C m β j m e γ 2 2 γ β 2 2 D m/2 i γ β (18) The parameters use in above equations are defined as follow: α = ik 1 ze iπ /8 sin δ +θ s cos δ θ s (19) k 1 Rcos θ s θ 1/4 0 β = e iπ /4 tan δ θ s k 1 Rcos θ s θ 0 (20) iπ /4 γ = e k 1 Rsin θ 0 θ s k 1 Rcos θ s θ 0 (21) Observation point located in the transition region and in the far region When the observation point is located near the transition region in the far and shallow regions, the saddle point equation (6) possesses two saddle points θ s and θ e that located near the branch point δ (Fig. 2(b)). In this case, the asymptic solution for the transmitted Gaussian beam can be derived by using similar method used in Sec IV. CALCULATION RESULTS AND DISCUSSION : reference beam shift 0.2 : reference beam shift a θ b = 30 b θ b = 60 Fig. 3. Comparisons of the asymptotic solutions for the transmitted Gaussian beam with the reference solutions. 84 ISSN

99 beam magnitude The 4 th Academic Conference on Natural Science for Young Scientists, Master & Fig. 3(a) shows the results of the numerical calculation for the transmitted Gaussian beam when the observation point is located in the near region in the case that the beam angle θ b = 30 is smaller than the critical angle δ = The numerical parameters are W = 1.5λ, f = 3GHz, L 1B = 70λ, L 2B = 30λ, ε 1 = 2.3ε 0, and ε 1 = ε 0. The beam magnitude is calculated as the function ofx t, the beam coordinate x t [λ] (Fig. 1). It is clarified that geometrically transmitted beam U go ( ) obtained from (7) agrees excellently with the reference solution ( ) calculated numerically from (2). Fig. 3(b) shows the calculation results when the beam incident angle θ b = 60 is larger than the critical angle. The numerical parameters are W = 1.5λ, = 200λ, and z t = 0.2λ. The beam magnitude has been calculated along the beam coordinate x t (Fig. 1). It is shown that the evanescent beam wave U eva ( ) in (12) agrees excellently with the reference solution ( ). It is shown that, when the observation point is located in the far region, only the evanescent beam can provide the excellent solution. Fig. 4, we have shown the calculation of the transmitted Gaussian beam when the beam incident angle θ b = 42 located near the critical angle δ = The numerical parameters are W = 1.5λ, f = 3GHz, = 200λ, z = 0.1λ, ε 1 = 2.3ε 0, and ε 1 = ε 0. We have calculated the transmitted Gaussian beam U t by using the asymptotic solution (13) in the transition region. It is clarified that, the asymptotic solution ( ) agrees well with the reference solution ( ) calculated numerically from (2) in whole region. It is also shown that transition beam ( ) calculated in (13) by exciting the asymptotic solution for integral play important role to connect the solution in the near region and the one in the far region. It is very interesting to observe the phenomenon that beam shift is occurred when the Gaussian beam is incident through the plane dielectric interface in whole observation regions: in the near region, in far region an in transition region : asymptotic : reference : transition region Fig. 4. Comparisons of the asymptotic solutions for the transmitted Gaussian beam with the reference solutions when the observation point located in the transition region. V. CONCLUSION We have derived the asymptotic solution for the transmitted Gaussian beam observed in the rarer medium when the Gaussian beam is incident on a plane dielectric interface from the denser medium by applying the saddle point technique and using the parabolic cylinder function. By comparing with the reference solution calculated numerically, we 85 ISSN

100 have confirmed the validity of the asymptotic solution proposed in this paper. It is shown that, transition beam that we have defined in this paper plays a very important role to connect the solution in the near region and the other in the far region. We have shown that beam shift of the transmitted Gaussian beam can be observed in the whole observation region. REFERENCE [1] H. L. Bertoni, L. B. Felsen, and J. W. Ra, "Evanescent fields produced by totally reflected beams," IEEE Trans. on Antennas and Propag., pp , Sept [2] B. R. Horowitz and T. Tamir, "Unified theory of total reflection phenomena at a dielectric interface," Appl. Phys., vol. 1, pp , [3] H. L. Bertoni, L. B. Felsen, and J. W. Ra, "Evanescent fields produced by totally reflected beams," IEEE Trans. on Antennas and Propag., pp , Sept [4] B. C. S. Kozaki and H. Sakurai, "Characteristics of a Gaussian beam at a dielectric interface," J. Opt Soc. Am., vol. 68, no. 4, pp , April [5] T. Tamir, "Nonspecular phenomena in beam fields reflected by multilayered media," J. Opt Soc. Am., vol. 3, no. 4, pp , Apr [6] H. Shirai, "Transient scattering responses from a plane interface between dielectric half spaces," IEICE, C-I, vol. J78-C-I, no. 3, pp , March, [7] H. Yamada, T. Kawano, K. Goto, and T. Ishihara, "A uniform asymptotic analysis of a Gaussian beam scattered by a dielectric interface," IEEJ Technical Report, IEE Japan, EMT-07-40, pp , May [8] J. R. Thomas and A. Ishimaru, "Wave packet on negative index media," IEEE Trans. on Antennas and Propag., vol. 53, no. 5, pp , May [9] D. T. Quang, K. Goto, T. Kawano, and T. Ishihara, "A uniform asymptotic solution for reflection and beam shift of a Gaussian beam at a plane dielectric interface," IEICE Trans. Electron., vol. E95-C, no. 1, pp , Jan [10] H. Yamada, T. Kawano, K. Goto, and T. Ishihara, "A uniform asymptotic analysis of a Gaussian beam scattered by a dielectric interface," IEEJ Technical Report, IEE Japan, EMT-07-40, pp , May [11] D. T. Quang, K. Goto, and T. Ishihara, "Asymptotic solutions for transmitted Gaussian beam through a plane dielectric interface," Proc. of International Symposium on Antennas and Propagation 2012, pp , Nagoya, Japan, Oct [12] L. B. Felsen and N. Marcuvitz, eds., Radiation and Scattering of Waves, chap.4, IEEE Press (Classic Reissue), New Jersey, USA, ISSN

101 SELECTIVE HYDROGENATION OF 3-NITROSTYRENE TO 3-VINYLANILINE OVER PT-BASED BIMETALLIC CATALYSTS Sathaporn Tiensermsub 1,* and Joongjai Panpranot Center of Excellence on Catalysis and Catalytic Reaction, Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand *: Abstract. In this research, a series of Pt catalysts supported on P-25 titania and Pt-X/titania in which X were Ni, Co, Zn, Ga, Cr, and Fe were prepared by the incipient wetness impregnation method (co-impregnation). The X-ray diffraction results showed only anatase and rutile phase TiO 2. The peaks corresponding to metal or metal oxide species were not detected. There were no differences in particle morphology as confirmed by the scanning electron microscopy results. However, the amount of active Pt surface determined from CO chemisorption for all the bimetallic catalysts decreased from the monometallic Pt/TiO 2 (85% dispersion) to 66-75% on the Pt-X/TiO 2. The catalytic performances were tested in the 3- nitrostyrene hydrogenation at 50 ºC and 20 bar of H 2 gas. The catalysts that exhibited an improved performance for the selective hydrogenation nitrostyrene to vinylaniline were Pt- Co/TiO 2 and Pt-Ga/TiO 2 in which higher yield of vinylaniline was obtained as compared to the monometallic Pt/TiO 2. The addition of a second metal may modify Pt surface (formation of edge/corner/terrace sites). The edge/corner Pt sites has been shown to promote the selectivity to 3-vinylaniline. Keywords: 3-Nitrostyrene hydrogenation, Incipient wetness impregnation, Platinum catalyst, bimetallic. I. INTRODUCTION The liquid phase chemoselective hydrogenation of nitroanilines is essentially utilized in industrial production of valuable intermediates for agrochemicals, pharmaceuticals, pigments, polymers, dyes, and other fine chemicals [1-3]. The liquid phase selective hydrogenation of nitrostyrene has been carried out using Pt, Au and Pd catalysts supported on TiO 2 and high conversion (nearly 100%) with supreme selectivity of vinylaniline reached up to 73-98% [1, 4-6]. In an earlier study by Serna, P. et al [7], TiO 2 supported bimetallic gold - platinum which was synthesized by a deposition - precipitation technique and increasing Pt content in Au-Pt/TiO 2 enhanced the TOF. However, when Pt content reached over 100 ppm, the chemoselectivity to reduce the nitro group decreased. It is known that the catalytic behavior of metal that strongly interacts with a support is essentially different from that of the same metal supported on inert carriers such as SiO 2, Al 2 O 3 or C. Due to the strong metal-support interactions (SMSI) effect, TiO 2 can enhance the selectivity and reaction rate when compared to the other supports in the liquid phase hydrogenation of 3- nitrostyrene [2]. ISSN

102 Pt-based bimetallic catalysts were synthesized using a wide range of synthesis techniques such as incipient wetness impregnation, slurry synthesis method, electroless deposition, and dendrimer synthesis. Selective hydrogenation reactions over Pt-based bimetallic catalysts were carried out using various bimetallic catalysts including Pt-Ni/γ- Al 2 O 3, Pt-Co/γ- Al 2 O 3, Pt-Pd/γ- Al 2 O 3, Pt-Au/ SiO 2, Pt-Cu/ SiO 2, Pt-Fe/ SiO 2, Pt-Zn/ SiO 2, Pt-Ga/CNF, and Pt-Ru/CNT [8]. Selective hydrogenation of a nitro group (NO 2 ) and vinyl group (C=C) is an addition of H 2 atoms to a C=C either or N=O double bond for generate nitro group product. The ordinary source of two hydrogen atoms is molecular hydrogen (H 2 ). Although, a careful hydrogenation proceeds is very rapidly hydrogenated to a nitro group product. Then the overall hydrogenation reaction is exothermic, the high activation energy also prevents it from taking place under normal conditions. Scheme 1 exhibited the reaction path way of 3- nitrostyrene that can be transformd into 3 main products as a vinilaniline or aminostyrene (VA or AS), ethylnitrobenzene (ENB), and final product ethylaniline (EA) [9]. Scheme 1. The hydrogenation pathways of 3-nitrostyrene focused on product of reaction mixture. Although several Pt-based bimetallic catalysts were investigated in the selective hydrogenation, they were not systematically compared because different preparation methods and supports were used. In the present study, a series of Pt-X/TiO2 were prepared by co-impregnation method and studied in the selective hydrogenation of 3-nitrostyrene. The catalysts were also characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and CO pulse chemisorption. II. EXPERIMENTS Catalyst preparation by incipient wetness impregnation The Pt precursor (H 2 PtCl 6.6H 2 O) was dissolved with deionized water to obtain 0.5 wt% Pt loading. Then, the solution was added to the TiO 2 P25 support drop wise and grinded until homogeneous solid was obtained. After that, the catalysts were dried overnight at 110ºC and calcined in air at 450ºC for 2 h. The series of bimetallic catalysts was synthesized by co-impregnation with 0.5 wt% Pt and 0.5 wt% of the second metal. 88 ISSN

103 The catalysts were reduced under H2 flow for 2 h at 200 C before the catalytic reaction tests Catalytic performance tests A magnetically stirred 20 cm 3 of Teflon lined stainless steel autoclave reactor was employed with 20 mg of reduced catalyst, 10 cm 3 of ethanol, and 0.4 cm 3 of 3-nitrostyrene. The reaction was performed isothermally at 50 C in water bath and 20 bar of H 2. Afterwards the reactor was purged with H 2 to vent air from the reaction system for 3-4 times. The reaction mixture was stirred continuously with a magnetic stirrer. The reaction was carried out for 1 hour, then the reactor was carefully depressurized and cooled to ambient temperature. The product was centrifuged for 5 min before analyzed by a gas chromatograph. III. RESULTS AND DISCUSSION 3.1. Catalysts characterization X-ray diffraction (XRD) The series of Pt/TiO 2 and Pt-X/TiO 2 catalysts with different second metals displayed the XRD patterns as shown in Fig. 1. The XRD diffraction peaks showed the crystalline phases of anatase-tio 2 and rutile-tio 2. The XRD diffraction peaks of anatase-tio 2 were detected at 2θ degrees of 25.2º, 37.7º, 48.0º, 53.7º, 54.9º, 62.5º, 68.5º, 69.8º,and 74.7º [10] which corresponded to the (101), (004), (200), (105), (211), (204), (116), and (215) planes, respectively. The characteristic diffraction peaks of the rutile-tio2 phases appeared at three main diffraction peaks i.e. 2θ degrees of 27.3º, 35.8º, and 40.8º which corresponded to the (110), (101), and (111) planes, respectively. Fig. 1. The XRD patterns of Pt/TiO2 and Pt-X/TiO 2 catalysts with different second metals. ISSN

104 The XRD patterns corresponded to the metal/metal oxide species such as platinum oxides, nickel oxides, cobalt oxides, zinc oxides, gallium oxides, chromium oxides or iron oxides were not detected. This suggests that the crystallite size of these metal/metal oxides platinum was smaller than the detection limit of the XRD or maybe due to the insufficient amount present Scanning electron microscopy (SEM) The SEM images of the Pt/TiO 2 and Pt-X/TiO 2 catalysts are shown in Fig. 2. All the catalysts showed non-uniform particle size and shape. There were no significant differences in the catalyst morphology when a second metal was added by co-impregnation technique. Fig. 2. The SEM images of a) Pt/TiO 2, b) Pt-Co/TiO 2, c) Pt-Ga/TiO 2, d) Pt-Fe/TiO 2, e) Pt-Zn/TiO 2, and f) Pt-Cr/TiO Pulse carbon monoxide chemisorption CO chemisorption was used to compare the amount of Pt surface active sites on the catalysts and the results are shown in Table 3. The CO chemisorption at room temperature was analyzed by measuring the CO responses upon passing CO gas through the catalyst. 90 ISSN

105 The response curves area corresponding to the empty and loaded reactors is equivalent to the uptake of CO molecules on the catalyst, assuming that one CO molecule chemisorbs on one Pt site [11]. The number of surface Pt atoms on the Pt bimetallic catalysts derived from CO uptake were ranged between x 1018 molecules CO/g cat. Whereas the monometallic Pt/TiO 2 exhibited higher CO chemisorption at 13.2 x1018 molecules/g cat. It is suggested that part of the Pt surface was covered by the second metal and/or alloy formation occurred, resulting in lower Pt surface. In addition, the second metal should be formed with Pt, not isolated. This is confirmed by the decrease in the amount of CO chemisorption. If Ni or Co is present as individual particles, the amount ofco chemisorption would increase. The calculated %Pt dispersion decreased from the monometallic Pt/TiO 2 (85% dispersion) to 66-75% dispersion on the bimetallic catalysts. Table 1. CO chemisorption of the series of platinum based bimetallic catalyst Catalytic performance Table 2. The selective hydrogenation of 3-nitrostyrene into 3-vinylaniline using series of Pt based bimetallic catalyst. Reaction conditions: catalyst, 20 mg; solvent, 10 ml (ethanol); atmosphere, 2 MPa; 3- nitrostyrene, 0.4 ml; reaction time, 1 h; (3-vinylaniline 1, 3-ethylnitrobenzene 2, 3- ethylaniline 3 ) ISSN

106 The catalyst performances in 3-nitrostyrene hydrogenation in terms of conversion of 3- nitrostyrene and product selectivity are shown in Table 2. The conversion of 3- nitrostyrene at reaction time 1 hr. of the Pt-based catalysts were ranged between % in the order: Pt-Co/TiO 2 > Pt-Ga/TiO 2 > Pt/TiO 2 > Pt-Zn/TiO 2 >Pt-Fe/TiO 2 >Pt- Ni/TiO >Pt-Cr/TiO 2. Surprisingly, two of the bimetallic catalysts, Pt-Co/TiO 2 and Pt- Ga/TiO 2 with lower Pt active sites (determined by CO chemisorption results), showed higher hydrogenation activity in the 3-nitrostyrene hydrogenation than the Pt/TiO 2. These catalysts also exhibited comparable selectivity towards vinylaniline at around 60-65%. As a consequence, higher yield of 3-vinylaniline can be obtained under the similar reaction conditions. According to the literature [1], the edges/corner Pt surface sites promoted the selective hydrogenation of nitrostyrene to vinylaniline whereas the terrace Pt sites facilitated the formation of ethylnitrobenzene. The addition of these second metals may modify the formation of Pt active sites and/or result in an alloy formation so that their catalytic properties could be improved. Furthermore, higher vinylaniline selectivity was observed on the Pt-Fe/TiO2, but at relatively low conversion of nitrostyrene. IV. CONCLUSIONS A series of Pt-X/TiO 2 bimetallic catalysts (X= Ni, Co, Zn, Ga, Cr, and Fe) were investigated in the 3-nitrostyrene hydrogenation. The Pt-Co/TiO 2 and Pt-Ga/TiO 2 were found to be the most efficient catalysts, giving the highest yield of vinylaniine under the reaction conditions used. The catalyst performances may be related to the formation of specific Pt surface sites such as edges/corner sites and/or alloy formation. V. ACKNOWLEDGMENTS Financial supports from the Thailand Research Fund (TRF) and the Grant for International Research Integration: Chula Research Scholar, Ratchadaphiseksomphot Endowment Fund are gratefully acknowledged. REFFERENCES [1] S.-i. Fujita, H. Yoshida, K. Asai, X. Meng, M. Arai, J. Supercrit. Fluid 60 (2011) [2] P. Serna, M. Boronat, A. Corma, Top Catal. (2011) 54: [3] S. Furukawa, Y. Yoshida, and T. Komatsu, ACS Catal. 2014, 4, [4] M. Boronat, P. Concepcio n, A. Corma, S. Gonza lez, F. Illas, and P. Serna, J. AM. CHEM. SOC. 2007, 129, [5] M. Makosch, W.-I. Lin, V. Bumbálek, J. Sá, J.W. Medlin, K. Hungerbühler, J.A. van Bokhoven, ACS Catal. 2 (2012) ISSN

107 [6] S. Pisduangdaw, O. Mekasuwandumrong, H. Yoshida, S.-i. Fujita, M. Arai, J. Panpranot, Appl. Catal A: Gen. 490 (2015) [7] P. Serna, P. Concepción, A. Corma, J. Catal. 265 (2009) [8] W. Yu, M. D. Porosoff, and J. G. Chen, Chem. Rev. 2012, 112, [9] M. J. Beier, J.-M. Andanson, and A. Baiker, ACS Catal. 2012, 2, [10] W. Zhao,* M. Zhang, Z.Ai, Y.Yang, H. Xi, Q. Shi, X. Xu, and H. Shi, J. Phys. Chem. C 2014, 118, [11] Zhaolong, Z. and Xenophon, E.V., Applied Catalysis A: General, 138, 1996, ISSN

108 A NEW SIMPLE ALGEBRAIC PROOF OF THE HOOK-LENGTH FORMULA Nguyen Bich Van Institute of Mathematics, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Abstract. In this work a new simple algebraic proof of the hook- length formula is presented. This proof is based on elementary properties of skew-symmetric and linear polynomials. No knowledge of representation theory is required in order to understand the proof. Keywords: Standard Young tableaux, Specht module, Hook-length formula, skewsymmetric. I. INTRODUCTION The hook-length formula is discovered by Frame, Robinson and Thrall in 1954 to compute the number of standard Young tableaux of a given shape [4]: Theorem 1.1. Let be a Young diagram with n boxes. The number of standard Young tableaux of shape is equal to: n! f (1) h i, j 94 ISSN i, j where i, j respectively vary rows and columns of, h i, j is the hook-length of the box in the i-th row and the j-th column. In the original proof [1] hook lengths appear in the course of rearranging terms in another formula for f due independently to Frobenius [2] and Young [6]: n! V ( k, k 1 1,..., 1 k 1) f!( 1)!...( k 1) k k 1 1 where,..., 1 k are lengths of the rows, V is the Vandermonde determinant. The original proofs of (2) use complicated algebraic methods (group characters, schur polynomials). The search for a short, intuitive explanation be tting such a simple result gave rise to many alternate proofs for the hook-length formula, for example a probabilistic proof by Greene et al [4], a direct bijective proof by Novelli et al [5]. These proofs are amazing, but some of the details are complicated and for nonspecialists they are not easy to understand. In this work we present a new simpler algebraic proof of the hook-length for-mula, using induction on the numbers of columns of Young diagrams and well-known properties of skew-symmetric and linear polynomials. For convenience of the readers first of all we recall some basic de nitions. (2)

109 II. BASIC DEFINITIONS Definition 2.1. A partition of n N is a sequence of positive integers ( 1,..., k ) such that k 0 and 1... k n Definition 2.2. A Young diagram is a finite collection of boxes arranged in leftjustif-i ed rows with the row sizes weakly decreasing. The Young diagram associated to the partition ( 1,..., k ) of n is the one that has k rows and i boxes on the i-th rows. Example 2.1. For n = 4 we have the following partitions and their Young diagrams: Definition 2.3. Let be a partition of n. A Young tableau T of shape is obtained by lling in the boxes of a Young diagram with numbers 1,,n, where each number occurs exactly once. In this case, we say that T is a -tableau. Definition 2.4. A standard Young tableau is a Young tableau whose the entries increase accross each row and each column. Example 2.2. For = (2; 1) we have two standard Young tableaux: Definition 2.5. Let be a Young diagram. For a box u in the diagram, we define the hook of u to be the set of all boxes directly to the right of u or directly below u, including u itself. The number of boxes in the hook of u is called the hook-length of u and denoted by h(u). If u is located in the i-th row and the j-th column of, we also denote the hooklength of u by h i;j. ISSN

110 Example 2.3. The hook of u contains u itself and all boxes filled with bullets, h 2,2 = 6. Remark 2.1. The hook-length formula (1) is used to compute the number standard Young tableaux of a given shape. This number is also well-known in represen-tation theory as the dimension of the Specht module S (the irreducible represen-tation of the symmetric group S n associated to the partition). The readers can see the construction of Specht modules in [6]. III. A NEW SIMPLE ALGEBRAIC PROOF OF THE HOOK-LENGTH FORMULA For partition ( 1,..., k ) we denote l i i k i. It is easy to see l 1 l 2... lk. l1!... lk! Lemma 3.1. hi, j ( l l ) i, j p q 1 p q k (3) Proof: Let us at first remark that the right hand side of (3) will not change if we continue by adding zero parts. In fact, if we consider as a partition with k + 1 parts, then we have k 1 = lk 1 = 0, the right hand side of (3) will be equal to l1!... lk! lk 1! ( l1 1)!...( lk 1)! ( l l ) l ( l l ) p q p p q 1 p q k 1 p k 1 p q k (4) i.e. is equal to the right hand side of (3) by considering as a partition with k parts. We will prove (3) by induction on the numbers of columns of. For the first column we have: h1,1 1 k 1 l1, h2,1 2 k 1 1 l2,, hk,1 k k k lk. If has more than two columns, we have: k hi,1 l1... lk. (5) i 1 96 ISSN

111 ( i, j) k i 1 h h i, j i,1 ( i, j) ' h' i, j (6) where ' is the Young diagram obtained from by deleting the first column, h ' i, j is the hook length of cell (i,j) in '. By induction assumption we get: h' ( l 1)!...( l 1)! ( l 1)!...( l 1)! (( 1) ( 1)) ( ) 1 k 1 k i, j ( i, j) ' lp lq lp lq 1 p q k 1 p q k (7) h l!... l! ( ) 1 k i, j ( i, j) lp lq 1 p q k (8) We denote the right hand side of (1) by F( ). Since (1) f F((1)) 1 prove that F( ) satisfies the same recursive relation of f, i.e. we need to prove:, we need to (9) where λ λ means that is a Young diagram obtained from by deleting a box. If we ( n 1)! devide two sides of (9) by, we will get: l!... l! 1 k k nv ( l,..., l ) lv ( l,..., l 1,..., l ) (10) 1 k i 1 i k i 1 where V ( l1,..., l k ) ( lp lq). 1 p q k Remark that (10) is obtained from the following identity by letting x l, x 1: ( x... x C x) V ( x,..., x ) 2 1 k k 1 k k i 1 xv ( x,..., x x,..., x ) i 1 i k In order to prove (11) let us at first remark that the right hand side of (11) (denoted by g( x, x1,..., x k )) is a skew-symmetric polynomial in variables x,..., 1 x k. In fact, g( x, x,..., x,..., x,..., x ) xv( x,..., x x,..., x,..., x ) 1 i j k i 1 i j k x V( x,..., x,..., x x,..., x ) j 1 i j k i i (11) ISSN

112 xuv ( x1,..., xi,..., xu x,..., x j,..., xk ) (12) u i, j g( x, x,..., x,..., x,..., x ) x V( x,..., x x,..., x,..., x ) 1 j i k j 1 j i k xv( x,..., x,..., x x,..., x ) i 1 j i k xuv ( x1,..., x j,..., xu x,..., xi,..., xk ) (13) u i, j Since V (x 1,,x k ) is a Vandermonde determinant, it is skew-symmetric: V( x,..., x,..., x,..., x ) V( x,..., x,..., x,..., x ) (14) 1 i j k 1 j i k From equalities (12), (13) and (14) we deduce that g is a skew-symmetric polynomial in variables ( x1,..., x k ), hence g V ( x1,..., x k ). On the other hand, g is linear g with respect to each of variables x, x 1,, x k, and from degrees we see that is V ( x,..., x ) a linear polynomial. Hence it is sufficient to check (11) when x = 0 and when x is equal to some nonzero value and x 1,, x k get some values s.t. V( x1,..., xk ) 0. Suppose that g aox a1x1... ak xk (15) V ( x,..., x ) 1 k Let x = 0, we get g(0, x 1,, x k ) = xv i ( x1,..., xk) a1... a k 1. g... x k x 1 i 1 V ( x1,..., xk ) Let x i = k-i; i = 1,, k and x = -1, we get the left hand side of (11) is equal to 0, g( 1, k 1, k 2,...,0) 0 since V( k 1, k 2,..., k i 1, k ( i 1),...,0) 0 (a Vandermonde determinant with two equal columns). 1 k k IV. CONCLUSION In this paper the author has proposed a new algebraic proof of the hook-length formula, which is based on the induction on the numbers of Young diagrams and wellknown basic properties of skew-symmetric and linear polynomials. The advantage of the proof in comparison with other old proofs (probabilistic, directive proof) is that it is shorter, simpler and easier to understand even for nonspecialists. No knowledge of representation theory or probability is required during the discussion. 98 ISSN

113 REFERENCES [1] J.S. Frame, G. De B. Robinson, and R.M. Thrall, The hook graphs of the symmetric group, Canad. J. Math. 6, 1954, pp [2] G. Frobenius, Uber die charaktere der symmetrischer gruppe, Preuss, Akad. Wiss. Sitz., 1900, pp [3] W. Fulton, Young Tableaux with applications to representation theory and geometry, Cambridge University Press, [4] C. Greene, A. Nijenhuis, and H.S. Wilf, A probabilistic proof of a formula for the number of Young tableaux of a given shape, Advances in Math. 31, 1979, pp [5] J.C. Novelli. I. Pak, and A.V. Stoyanovskii, A direct bijective proof of the hook-length formula, Disc. Math. And Theoretical Computer Science 1, 1997, pp [6] A. Young, Quantitative substitutional analysis II, Proc.London Math. Soc., Ser.1, 35, 1902, pp ISSN

114 TERAHERTZ EMISSION FROM Mn:GaAs Alexander De Los Reyes 1*, Karim Omambac 1, Jeremy Porquez 1, Karl Cedric Gonzales 1, Elizabeth Ann Prieto 1, Jorge Michael Presto 1, Kohji Yamamoto 2, Masahiko Tani 2, Armando Somintac 1, Elmer Estacio 1 and Arnel Salvador 1 1 National Institute of Physics, University of the PhilippinesDiliman, Quezon City 1101, Philippines 2 Research Center for Development of Far-Infrared Region, University of Fukui, Fukui , Japan *: adelosreyes@nip.upd.edu. Abstract. We investigate the effects of manganese (Mn) doping on the terahertz (THz) emission of GaAs films gown via Molecular Beam Epitaxy (MBE). A pump-probe type THz Time-Domain Spectroscopy (THz-TDS) set-up was used to measure the THz-emission from the Mn:GaAs films. Magnetic-field polarity dependence of the THz waveform suggested that the dominant THz-emission is via drift mechanism. Furthemrore, THz emission from low content Mn:GaAs films was found to be comparably higher than the high content Mn:GaAs films. Keywords: Optical properties of semiconductors, infrared and Raman spectra of semiconductors, ultrafast spectroscopy. I. INTRODUCTION Terahertz (THz) refers to frequencies of the electromagnetic radiation between the range THz [1]. Its applications include the high-resolutionimaging of biomedical tissues [2], personnel and security screening [3], among others. A common source of THz radiation are semiconductors illuminated with ultrashort femtosecond laser pulses [4]. The laser excitation of the material create electron-hole (e-h) pairs and the dynamics of these photo-generated e-h pairs determine the THz radiation mechanism - either via surface electricfield-related carrier drift or via Photo-DemberEffect [5]. In carrier drift, photogenerated e-h pairs are accelerated by the built-in surface electric field E s [5]. For p-type semiconductors, E s is directed from the air-semiconductor interface into the bulk of the materialand to the opposite direction for n-type semiconductors [6]. Thus, the direction of carrier drift would depend on the direction of E s and type of doping. This dependence is manifested as a change in the polarity of the Time-Domain Spectrum (TDS) waveform in p-type and n-type semiconductors [5]. Meanwhile, in the photo-dembermechanism, photogenerated carriers are accelerated both towards the bulk of the semiconductor due to the presence of a large carrier concentration gradient, thus producing a current surge as described by the diffusion equations [5]: J i ed i dδn i dz 100 ISSN

115 Where : i = e, h. In both cases, the THz radiation is proportional to the time-derivative of the surgecurrent [5]: E THz J t Mn:GaAs are candidates for good magnetic materials with possible applications in spintronics and other related applications [6]. In this work, our aim is to study the effects of Mn doping on the THz emission from GaAs films grown on semi-insulating GaAs (SI- GaAs) substrates. II. EXPERIMENTS The samples used in this work were grown using a Riber 32P MBE Facility available at the National Institute of Physics. High and low Mn:GaAs films were grown on semiinsulating GaAs. A 0.8 μm GaAs buffer was initially at substrate temperature of T s -580 ⁰C. The growth was interrupted by ramping down T s to 330 ⁰C while the As beam shutter was opened and the Ga beam shutter was closed. At T s =330 ⁰C, a 0.2 μm low-temperature GaAs (LT-GaAs) was grown followed by a 200 nm Mn-doped LT-GaAs film. High and low Mn:GaAs films were achieved by setting the Mnsurce cell temperature to 650 ⁰C and 575 ⁰C, respectively. A pump-probe type THz-TDS was used to measure the THz emission from the samples. A mode-lockd Ti:Sapphire with central wavelength of 800 nm, reperirion rate of 80 MHz giving 100 fs of optical pulses was used to excite the samples. The radiated THz emission were collected using off-axis paraboloids and redirected into the optical gap of BATOP GMBH photoconductive antenna. An external magnetic field was applied using a 650 mt permanent magnet and the B-field dependence of the THz emission was investigated. III. RESULTS AND DISCUSSION Figures 1 shows the TDS waveforms for the SI-GaAs (dashed green lines), low Mn:GaAs (black dotted lines) and high Mn:GaAs (dashed blue lines). Inset in the figure are the Reflection High Energy Electron Diffraction (RHEED) patterns that confirms the presence of Mn during the Mn:GaAs deposition. Fig. 1. TDS-waveform and FFT specta of p-type InAs. ISSN

116 The THz emission from the lownmn:gaas is greater than the THz emission from the SI-GaAs by a factor of 3. The increase in the THz emission from the low Mn-GaAs sample is attributed to a drift-related current surge from the surface dpeletion field. Mn-doping introduces acceptor impurities in Mn:GaAs films grown at low T s [7, 8]. The charge compensation of the Mn acceptor impurities pin the Fermi-level at the surface. This results to higher surface depletion field for low Mn:GaAs than SI-GaAs. Further increase in Mn acceptor impurities however, reduces the crystallinity of the Mn:GaAs. This decreases carrier mobility hence the decrease in the THz emission for the high Mn:GaAs. To further investigate the THz radiation mechanism in our samples, we measured the TDS signal of our samples under an externally applied magnetic field of 650 mt. Figure 2 shows the magnetic field dependence of the TDS waveforms. Fig. 2. Magneitc field dependence of the TDS waveform for SI-GaAs (upper left), low Mn:GaAs (upper middle) and high Mn:GaAs (upper right). The B+ and Bi components of the SI-GaAs (lower left), low Mn:GaAs (lower middle) and high Mn:GaAs (lower right). The B+ (B-) component was obtained by subtracting the TDS waveform of the no field case with the TDS waveform of the upward (downward) magnetic field case (Figure 2 lower figures). Figure 1b shows the B-field polarity dependence of the THz signal for the low Mn:GaAs. There was an observed 180 -flipping of the THz signalupon reversal of the magnetic field orientation affirming the dominant carrier drift THz radiation mechanism [9]. IV. CONCLUSION In summary, we have investigated the effects of Mn-doping in the THz emission from GaAs films grown on SI-GaAs. The THz emission is dominantly drft-related. The THz emission from the Mn-doped films are greater than in SI-GaAs. Furthermore, the THz emission from low Mn:GaAs is higher than the THz emission from high Mn:GaAs. This is attributed to reduction in crystallinity and decreased carrier mobility in the high Mn:GaAs. 102 ISSN

117 Magnetic field polarity dependence of the TDS waveform confirmed carrier drift as the dominant THz radiation mechanism. V. ACKNOWLEDGMENTS This work was supported in part by grants from the following institutions: Office of the Vice-Chancellor for Research and Development (OVCRD), Philippine Council for Industry, Energy and Emerging Technology Research and Development (PCIEERD) and Department of Science and Technology Grants-In-Aid Program (DOST-GIA). REFERENCES [1] Zomega, The Terahertz Wave Ebook, Zomega THz Coorparation (2012). [2] YuefangHua and Hongjian Zhang, IEEE Transactions on Microwave Theory and Techniques, Vol. 58 (7), [3] Salih Ergun and Selcuk, Sonmez, Journal of Military and Information Science, Vol. 3 (1), [4] P. K. Schroder, Wiley, New Jersey, [5] Kiyomi Sakai, Springer, Berlin, [7] J. Sadowski, et al., J. Vac. Sci. Technol. B 18 (3), p.1697, [8] R. Šustaviciutea, et al., Acta. Phys. Pol. A 112, p.311, [9] M. Migita, et al., Appl. Phys. Lett. 79(21), p.3437, ISSN

118 DETECTING HUMAN EMOTION VIA SPEECH RECOGNITION BY USING ENSEMBLE CLASSIFICATION MODEL Surinee Doungwichain 1, Sathit Prasomphan 1 1) Department of Computer and Information Science, King Mongkut's University of Technology North Bangkok, 1518 Pracharat 1 Road, Wongsawang, Bangsue, Bangkok surinee31@hotmail.com, sathit_p26@yahoo.com Abstract. Speech Emotion Recognition is one of the most challenging researches in the field of Human-Computer Interaction (HCI). The accuracy of detecting emotion depends on several factors for example, type of emotion and number of emotion which is classified, quality of speech. In this research, we introduce the process of detecting 4 different emotion type (anger, happy, natural, sad) from Thai speech which is recorded from Thai drama show. This dataset is most similar with daily life speech. The proposed algorithms used the combination of Support Vector Machine, Neural Network and K-nearest neighbor for emotion classification by using the ensemble classification method with majority weight voting. The experimental results show that emotion classification by using the ensemble classification method by using the majority weight voting can efficiency give the better accuracy results than the single model. The proposed method has better results when using with fundamental frequency (F0) and MFCC of speech which give the accuracy results at 70.69%. Keywords: Speech Emotion recognition, Feature extraction, Ensemble Classification, Weight Majority Vote, K-nearest neighbor, Neural Network, Support Vector Machines. I. INTRODUCTION Speech Emotion Recognition (SER) is a challenging research area in the field of Human-Computer Interaction (HCI). The purpose of SER is to recognize emotions, such as anger, disgust, fear, happiness, sadness, etc. from tonal variations in human speech [1, 2]. Several algorithms were introduced to make computer to be able to understand and to be able to classify the several type of emotion in human speech. Some benefit of knowing this emotion from speech is to use with the application which requires a man-machine interaction such as computer tutorial, automatic translation, mobile interaction, health care, children education, etc. Emotion is an importance mental and physiological state. In natural, baby learns to recognize emotional information before understanding semantic information in his/her mother s utterance [3]. Reliable emotion detection in usability tests will help to prevent negative emotion [4]. Detecting emotion can help particularly for user opinion mining or stress prevention [3, 4]. Computer may not be able to exactly understand the natural of these emotions unless we employ the speech processing. Many researchers have used the statistics of the difference attributes of speech for being a representation of each sound such as pitch, formant, amplitude or power of the speech. These features can be 104 ISSN

119 classified to one of these three categories: prosodic features such as pitch (F0), intensity and duration, voice quality and spectral features such as Mel-Frequency Cepstral Coefficients (MFCC) or Linear Prediction Cepstral Coefficients (LPCC). In case of classification model, researchers offer several model such as Support Vector Machines (SVM) [3] [8], Gaussian Mixture Model (GMM) [2], Hidden Markov Modeling (HMM) [7], K-nearest neighbor (k-nn) [10], and Neural Network (NN) [12]. Although the above techniques provided the better classification accuracy, but these techniques are single model that resulted in a used data set in the study must include the parameter configuration step. Moreover, each parameter will be fixed which cause to bias and poor performance. Another way to reduce bias is to use common decision (ensemble), which can create diversity and minimize the errors caused by the variance [9]. Researchers have attempted to make together decision applied to enhance the emotion classification. T.Anagnostopoulos et al [11] presented a research on ensemble majority voting classifier for speech emotion by using a decision from the base classifier. KNN, C4.5 and SVM with polynomial kernel which using majority voting in the decision were used to find a suitable model to classify the HUMAINE database. These framework provided accuracy by 96%. D.Morrison [14] presented a technique for searching feature that combined ensemble model by using the base classifier SVM with RBF kernel, random forest, KNN, K * and multilayer perceptron. The algorithm provided accuracy by 79.43% and 73.29% for NATURAL and ESMBS database in ordering. The results showed that if a dataset has different types of information and emotion, the feature selection methods and ensemble model will different. P.Vasuki [17] focused on searching frame work to reject noisy and weak input file by using the weight factor ensemble model with SVM classifier to detect outliers. If input is unusual, it will be rejected from the training dataset. This framework showed the accuracy by 74.70%. From all of these research shows that the ensemble model can increase the performance of emotion classification from speech. It also emphasizes that the effectiveness of methods for separating emotion depends on several factors such as the properties of the selected feature in the experiments, number of emotion, the quality of the audio data is also affected as well. Therefore, we have selected a set of features that are critical to the dataset and methodology to optimize performance of classification by using ensemble. The paper is organized as follows. Following this, section II provides the details in SERS. Section III discussed the experimental setup. Results and performance comparison are given in section IV. Section V gives conclusions and discussions. II. SPEECH EMOTION RECOGNITION SYSTEM The speech emotion recognition system is described in Figure 1. In this section, the pre-processing of speech signal which is the pre-emphasis, frame blocking and Hamming windowing was described. The following speech feature: energy, zero-crossing rate (ZCR), ISSN

120 pitches, MFCC was used. The feature normalization is calculated for every window of a specified number of frames by statistical method. Classifier is modeled to classify emotions. Finally, the ensemble model is applied to integrate the result of classifier with weighted majority vote. Details of each process can be described as followed: Fig. 1. Speech Emotion Recognition System. A. Signal pre-processing: The basic operations used in the speech pre-processing include the following: pre-emphasis, frame blocking and Hamming windowing. 1. Pre-emphasis: The speech signal s(n) is sent to a high-pass filter show in Eq. (1). where is the output signal and the value of a is usually between 0.9 and 1.0. The z- transform of the filter is given in Eq. (2). The goal of pre-emphasis is to compensate the high-frequency part that was suppressed during the sound production mechanism of humans. Moreover, it can amplify the importance of high-frequency formants. 2. Frame blocking: The speech signal is divided into a sequence of frames where each frame can be analyzed independently and represented by a single feature vector. Frame shift is the time difference between the start points of successive frames, and the frame length is the time duration of each frame. The frame block is of length 10 msec to 40 msec from the filtered signal at every interval of 1/2 or 1/3 of frame length. 3. Hamming windowing: In order to keep the continuity of the first and the last points in the frame. If the signal in a frame is denoted by then the signal after Hamming windowing is where the Hamming window is defined in Eq. (3). (1) (2) (3) 106 ISSN

121 B. Feature extraction: The speech feature extraction which is also called speech coding is a very important and is basically part in the automatic speech processing systems. Features of the speech are generally obtained from the digital speech. Various methods are utilized that aim to extract speech features which are useful. In this research, feature which is extracted is energy, ZCR, pitch, and MFCC. C. Feature normalization: The speech segments have different lengths. In order to obtain isometric speech segments and reduce redundancy of data, the statistical method [3] was adopted to normalize the states. For each coefficient, mean, variances, median, maximum and minimum across all frames are calculated. D. Classifiers: Classifier is another component of a speech emotion recognition system. In this research, we used three classification methods: SVM, Neural Network and k-nearest neighbor. E. Ensemble classification method: Ensemble classifier is the model which combines several classifiers technique for solving the same problem by using the results from all of classifiers for decision in the final step. Ensemble model composed of several model for example, vote ensemble which uses the same training data with several classifier, bootstrap aggregating (Bagging) which uses the random training data and constructs the single ensemble model, and random forest which similar to bagging technique but instead of using random data, it randomly selects attribute from dataset and uses several decision tree for becoming classifier in the ensemble model. In this research, the vote ensemble with base classifiers which has low computational complexity and difference theoretical background was selected. The proposed model aims to reduce bias and redundancy [11] by using the combined model with weighted majority vote. If the classifiers in the ensemble are not giving the identical classification result, then it is reasonable to attempt to give the more competent classifiers more power in making the final decision. We called this step is weighted majority vote. The formula for weighted majority vote is shown in Eq. (4). The T classifiers are class-conditionally independent with accuracies p 1,,p T. The optimal weights for the weighted majority voting rule can be shown to be w t α p t 1 p t. (4) III. EXPERIMENTAL SETUP The Thai Emotional speech corpus [6] is used to classify emotion states. This corpus construction has been funded by National Electronics and Computer Technology Center (NECTEC). All emotional speech, collected from conversations by professional actors and actresses in a Thai drama show that contains many background music and noise within the speech. There are two groups of emotion were used to annotate in this corpus. The first ISSN

122 group consists of four basic emotions: neutral, happy, sad, and angry. The second group consists of twelve labels: happiness, satisfaction, fear, surprise, anger, jealousy, rage, doubt, hate, excitement, sadness, and fun. For this research, we firstly focused on detecting emotions from the first group. It is possible to recognize four real emotions of human. We used only 352 utterances from 2908 utterances in the corpus were utilized in this work. The details of each emotion are shown in Table 1. Table 1. Number of emotions in Thai emotion speech corpus Emotion Male Female Male + Female Anger Happy Neutral Sad Total In Thai emotion speech corpus, we have randomly selected 352 speech for study which with and without noise, background music or one of them for the diversity of speech in the experiment. We use the cross-validation with holdout 1/3 to split the data into two sets for training and testing, 236 training speech and 116 testing speech. In case of signal pre-process, the pre-emphasis was set the coefficient with In the framing process, frame has been segmented with size of 480 sample or approximately 30 milliseconds, and the distance between the frame (frame overlap) is 240 sample or about 15 milliseconds. And then using Hamming window to emphasize the importance of signal in the middle frame signal. The speech feature used in this research is energy, ZCR, F0 and MFCC. After feature extraction has been process, the feature was normalized by using statistical methods. The importance feature was combined to see if it most affects to emotional classification. We used MFCC to combine with prosodic feature (energy, ZCR, F0) due to the MFCC features give the highest accuracy compared to prosodic feature as shown in Table 2. Emotion classification has been created by using the ensemble model from the same set of data. When each classifier give the predicted class, these results will be weight for each classifier which is [7, 6, 2] for SVM (RBF 7), KNN, and NN. The weight values was set depends on the prediction accuracy. After that, vote answer by the sum of predicted class and predicted weight in each classifier. The performance of proposed model was based on evaluation of data classification performance by using Eq. (5), where, TP is true positive, TN is true negative, FP is false positive, FN is false negative. (5) 108 ISSN

123 IV. EXPRIMENTAL RESULT In this research, 5 model was tested for the speech classification accuracy which is 3 single model: SVM-RBF kernel at sigma 7 (RBF7), KNN, and NN, and 2 ensemble models: bagging which uses base classifier by using decision tree and weighted majority vote with 3 base classifier model: SVM (RBF 7), KNN and NN. In addition, the feature was also compared its classification accuracy. From Table. 2, it shows that model which can give the best classification accuracy for speech emotion classification is ensemble weighted majority vote by using F0 and MFCC. No Table 2. Classification accuracy in different feature and different model. Feature No. Feature SVM (RBF 7) Accuracy (%) KNN NN Bagging(tree) Weighted Majority Vote 1 F Energy ZCR MFC MFCC + F MFCC + Energy MFCC + ZCR MFCC + F Energy MFCC + F0 + ZCR MFCC + Energy ZCR MFCC + F Energy + ZCR Table 3. Confusion matrix for the feature set MFCC + F0 of ensemble weighted majority vote. Emotion Recognized Emotions (%) Anger Happy Neutral Sad Anger Happy Neutral Sad The confusion matrix in Table 3 showed that ensemble weighted majority vote model with F0 and MFCC give the best accuracy with 70.69%. ISSN

124 V. CONCLUSION This research presents a novel algorithm for detecting human emotion via speech recognition by using ensemble classification model. The proposed algorithm aims to detect the emotional by using information with the combination of SVM classifier, Neural Network classifier and K-nearest neighbor with the weighted majority voting ensemble method with combine speech feature Fundamental Frequency (F0) and Mel Frequency Cepstral Coefficient (MFCC) for Thai emotional speech corpus. The experimental results show that the proposed framework can efficiently find the correct speech emotion compared to by using the comparing method. For the future work, the process for noise removal and background music should be considered. In addition, the feature selection and model selection for improve the classification accuracy should be focused. REFERENCES [1] M.M.H.E.Ayadi, M.S.Kamel and F.Karray, "Survey on speech emotion recognition: Features, classification schemes, and databases", Pattern Recognition, pp , 2011 [2] S.Xu,Y. Liu and X.Liu, "Speaker Recognition and Speech Emotion Recognition Based on GMM", 3rd International Conference on Electric and Electronics, [3] T.Seehapoch, S.Wongthanavasu,"Speech emotion recognition using Support Vector Machines", The 5th International Conference on Knowledge and Smart Technology (KST), pp , [4] C.Stickel, M.Ebner, S.Steinbach-Nordmann, G.Searle, and A.Holzinger, "Emotion Detection: Application of the Valence Arousal Space for Rapid Biological Usability Testing to Enhance Universal Access," in Universal Access in Human-Computer Interaction. Addressing Diversity, Lecture Notes in Computer Science, LNCS 5614, C. Stephanidis, Ed., ed Berlin, Heidelberg: Springer, pp , [5] F. Burkhardt, A. Paeschke, M. Rolfes, W. Sendlmeier, B. Weiss, A database of German emotional speech, Proc. Interspeech, [6] S.Kasuriya, T.Teeramunkong and C.Wutiwiwatchai, Developing a Thai emotional speech corpus, International Conference on Asian Spoken Language Research and Evaluation, [7] S.Kasuriya, T.Banchaditt, N.Somboon, T.Teeramunkong and C.Wutiwiwatchai, Detecting Emotional Speech in Thai Drama, 2 nd ICT International Student Project Conference (ICT- ISPC), [8] P.Shen and Z.Changjun, Automatic Speech Emotion Recognition Using Support Vector Machine, International Conference on Electronic & Mechanical Engineering and Information Technology, pp , [9] D.Thamsiri and P.Meesad, Ensemble Data Classification Based on Decision Tree, Artificial Neuron Network and Support Vector Machine Optimized by Genetic Algorithm, The Journal of King s Mongkut s University of Technology North Bangkok, Vol. 21, No. 2,pp , ISSN

125 [10] S.A.Rieger Jr., R.Muraleedharan and R.P.Ramachandran, Speech Based Emotion Recognition Using Spectral Feature Extraction and an Ensemble of knn Classifiers, 9th International Symposium on Chinese Spoken Language Processing (ISCSLP), pp , [11] T.Anagnostopoulos, C.Skourlas, "Ensemble majority voting classifier for speech emotion recognition and prediction", Journal of Systems and Information Technology, Vol. 16 Iss: 3, pp , 2014 [12] J.Nicholson, K. Takahashi and R.Nakatsu, Emotion recognition in speech using neural networks, 6th International Conference on Neural Information Processing, Vol. 2, pp , [13] X.Mu, J.Lu, P.Watta and M.H.Hassoun, Weighted Voting-based Ensemble Classifiers with Application to Human Face Recognition and Voice Recognition, Proceedings of International Joint Conference on Neural Networks, Atlanta, Georgia, USA, June 14-19, pp , [14] D.Morrison, R.Wang and L.C.D.Silva, Ensemble methods for spoken emotion recognition in call-centres. [15] D.Aha, D. Kibler, Instance-based learning algorithms, Machine Learning 6, pp , [16] A. J. C. Sharkey, Combining Artificial Neural Nets. Ensemble and Modular Multi-Net Systems, Springer-Verlag, London, [17] P. Vasuki, Speech Emotion Recognition Using Adaptive Ensemble of Class Specific Classifiers, Research Journal of Applied Sciences, Engineering and Technology 9(12): pp , ISSN

126 DESIGN OF OPTICAL FILTERS IN SLOTTED PHOTONIC CRYSTAL WAVEGUIDES Hoang Thu Trang * and Ngo Quang Minh Institute of Materials Science, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam *: tranght@ims.vast.ac.vn Abstract. In this work, we present the theory and numerical calculations of the optical filters using slot two dimensional photonic crystal waveguides. Slotted waveguide is designed for enhanced guiding and confining of light in the narrow area with low refractive index, so that the figure of merit (Q/V) of the resonator is significant enhanced. In addition, the coupling coefficients through the slot waveguides were analyzed by using the coupled-mode theory in time (CMT). We perform two-dimensional (2-D) finite-difference time-domain simulation to confirm the CMT analysis. The results showed the good agreement between the theory and the simulation. Keywords: Photonic crystals, optical filters. I. INTRODUCTION Photonic crystals (PCs) based optical devices have attracted great interest due to their compactness and suitability for photonic intergrated circuits [1]. In PCs, microcavities or resonators with high Q factors can be quite easily fromed by introducing appropariate defects. Higher order filters can be created by coupling multiple resonators through a slot photonic crystal waveguide [2, 3]. Slotted waveguide is confined in a narrow slot region with low refractive index into a high refractive index medium, as a result of merit (Q/V) of the resonator is significant enhanced [4, 5]. In this paper, we propose design of higher order optical resonant filters that uses coupled point defect resonators embedded in the slotted 2D photonic crystal waveguides. A higher order filter has been designed. The coupling between the resonators is analyzed theoretically, based on the coupled-mode theory in time (CMT). We perform the finitedifference time-domain (FDTD) simulation to confirm the CMT analysis. It is shown that the good agreement between the theory and the simulation. Section II of this paper describes coupled-mode theory anlysis of transmission in higher order filter. Section III describes the design of a higher order filter and transmission spectra based on FDTD method. Section IV presents some conclustions. II. DESIGN AND NUMERICAL CALCULATIONS Fig. 1a shows schematic diagram of the higher order coupled resonator filter in a PCs through a slot waveguide. If each resonator has a large enough Q factor, which is an 112 ISSN

127 appropriate assumption for the resonators in the PCs, the coupling between the resonators can be treated as if the resonators interact through a slot waveguide. Fig. 1. (a) Sketch of the higher orders coupled resonator filter. b) Transmission spectra of this structure by using the CMT method. In order to analyze the coupling of two resonators with an arbitrary phase shift was modeled using the coupled-mode theory in time. In this case, the resonator mode has a resonant frequency ω 0 and 1/τ 1 and 1/τ 2 are the decay rates into the slot waveguide from each resonator. Where a 1 and a 2 are the complex mode amplitude of the resonator; s +1 and s -2 are the amplitudes of the incoming and transmitted waves; a 2 and s 2 refer to the energy stored in the resonator and the power of the wave, respectively [6-8]: da dt 1 da dt j( ) a a s j( ) a a s here δ and µ represent the coupling-induced resonance frequency shift and effective coupling coefficient, respectively, which are given by δ = cotϕ /τ and µ = -jcscϕ/τ. Using CMT method, the transmission can be given by: T( ) s s j 0 1 j Using Eq. (2) gives us the transmission spectrum shown in Fig. 1(b). Fig. 1(b) shows the calculated transmission spectra for higher order coupled resonator filter with ϕ = π/2. The choice of ϕ = π/2 (δ = 0) is for the higher order coupled resonator filter among transmission spectra for one and two resonators by keeping the center frequency the same. 2 (1) (2) ISSN

128 In order to confirm the coupled-mode theory analysis, the higher order coupled resonator filter is depicted in Fig. 2(a), and is investigated numerically using FDTD method. In this structure, the refractive index of the 2D PCs is n = , the holes radius r is 0.3a (where a is the lattice constant of the triangular lattice a = 380 nm). We focus on a cavity with a length equal to four lattice constants L = 4a, the resonant state of which is found between the cutoff of the 120 nm and 50 nm wide slot waveguides. The slot waveguide is filled with DDMEBT (n fill = 1.8). The DDMEBT material, known as one of the best organic materials, has a high optical quality. After calculations, this structure has been found that a Q factor of Fig. 2. (a) Structure of the higer order coupled resonator filter in a PCs. (b) Transmission spectra of this structure by using the FDTD method. Fig. 2(b) shows the FDTD calculated transmission spectra for two resonators. The resonator and barrier length were chosen to satisfy ϕ = π/2. The transmission spectra are approximate to a Lorentzian shape, with the resonant wavelength λ = nm. At the resonant wavelength, the transmission peak to 100%. E y profile of the resonant mode calculated by FDTD is presented in Fig. 3. Fig. 3. E y profile of the resonant mode calculated by 2D FDTD. 114 ISSN

129 III. CONCLUSION In conclusion, we have proposed a higher order coupled resonator filter, whose performances are based on the coupling between the resonators through a slot photonic crystal waveguide. Slotted waveguide is designed for enhanced guiding and confining of light in the narrow area with low refractive index, so that the figure give Q factor of The designed higher order filter has a center wavelength of nm. The coupling between the resonators is analyzed theoretically, based on the coupled-mode theory in time (CMT). We perform the finite-difference time-domain (FDTD) simulation to confirm the CMT analysis. It is shown that the good agreement between the theory and the simulation. IV. ACKNOWLEDGMENTS This research is funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number REFERENCES [1] J. D. Joannopoulos, S. G. Johnson, J. N. Winn, and R. D. Meade, Photonic Crystals: Molding the Flow of Light, second edition (Princeton Univ. Press, 2008). [2] S. Fan, P. R. Villeneuve, and J. D. Joannopoulos, J. Opt. Soc. Am B. Vol. 12, 1995, pp [3] J. C. Chen, H. A. Haus, S. Fan, P. R. Villeneuve, and J. D. Joannopoulos, J. Lightw. Technol. Vol. 14, 1996, pp [4] R. Costa, A. Melloni, and M. Martinelli, IEEE Photo. Technol. Lett, Vol. 15, 2003, pp [5] A. Yariv, Y. Xu, R. K. Lee, and A. Scherer, Opt. Lett. Vol. 24, 1999, pp [6] D. S. Park, S. Kim, I. Park and H. Lim, J. Lightw. Technol. Vol. 23, 2005, pp [7] B. Suthar, A. Bhargava, Journal of Ovonic Research. Vol. 6, 2010, pp [8] Q. M. Ngo, S. Kim, S. H. Song, and R. Magnusson, Opt. Express. Vol. 17, 2009, pp [9] Q. M. Ngo, T. T. Hoang, V. L. Nguyen, D. L. Vu, and V. H. Pham, J. Opt. Vol. 15, 2013, pp [10] Q. M. Ngo, S. Kim, J. Lee, and H. Lim, J. Lightwave Technol. Vol. 30, 2012, pp ISSN

130 PREPARATION AND CHARACTERIZATION OF Sm 3+ DOPED ZINC BARIUM TELLURITE GLASSES Patarawagee Yasaka 1,2* and Jakrapong Kaewkhao 1,2 1 Center of Excellence in Glass Technology and Materials Science (CEGM), Nakhon Pathom Rajabhat University, Nakhon Pathom 73000, Thailand 2 Science Program, Faculty of Science and Technology, Nakhon Pathom Rajabhat University, 73000, Thailand *: pyasaka@hotmail.com Abstract. Sm 3+ -doped zinc barium tellurite glasses (ZBT: ZnO BaO TeO 2 ) were prepared by melt quenching technique. The physical and optical studies are characterized through density, molar volume, absorption and photoluminescence measurement, respectively. The results showed that the density of the glasses increased whereas the molar volume decreased with increasing of Sm 2 O 3 concentrations. The optical absorption spectra of glasses were measured in the wavelength range of 350 2,000 nm. The intensity of all absorption bands increased with increasing of Sm 2 O 3 contents. In addition, the photoluminescence properties of Sm 3+ doped ZnO BaO TeO 2 glass system were carried out using excitation wavelengths of 404 nm. four luminescence bands were observed at 563 nm ( 4 G 5/2 6 H 5/2 ), 600 nm ( 4 G 5/2 6 H 7/2 ), 646 nm ( 4 G 5/2 6 H 9/2 ) and 707 nm ( 4 G 5/2 6 H 11/2 ), intense red orange emission was found at 600 nm. Keywords: Tellurite glasses, Sm 3+ -doped glasses, zinc barium glasses. I. INTRODUCTION Among motivated studies of glassy materials, tellurite based glasses drew much interest because of their unique properties such as high dielectric constant and excellent transmission in the visible as well as IR wavelength regions, good mechanical strength and chemical durability [1-4]. These glasses also possess higher refractive index, which is approximately in the range of [5-8], and their low melting temperature (about 800 C) contributes to the high possibility of stable glass forming using a conventional melt quenching method [4]. Pure tellurium oxide cannot form glass by itself; it needs another element known as a glass modifier such as alkali metal, alkaline earth metal oxide and transition metal oxide (TMO) to improve the network connectivity to produce a stable tellurite glass [9, 10] with increasing non-bridging oxygen [9]. In fact, it is believed that the properties of oxide glasses strongly depend on the nature and the concentration of the constituent oxides [11]. Optical spectroscopy is an important tool to study the nature of glasses for the past many years. Investigation of the absorption and luminescence properties of rare earth (RE) doped luminescent materials have found diverse applications in the fields of lasers, telecommunications, optical fiber cables, solar concentrators, optical detectors and the production of wide variety of optical components (as windows, prisms, beam splitters, etc.,). In order to identify new optical devices for specific applications with 116 ISSN

131 enhanced performance, active work is being carried out by selecting appropriate new hosts doped with RE ions [12]. The Sm 3+ ion is one of the most interesting RE ions for analyzing the luminescence properties due to its use in high density optical storage, under sea communication, color displays and visible solid state lasers [13]. Moreover, it has 4f 5 electronic configuration which exhibits orange-red fluorescence in the visible region with strong fluorescence intensity, large emission cross-section and high quantum efficiency [14]. Further, the Sm 3+ ion is well suited to analyze the energy transfer processes, since its emitting 4 G 5/2 level has comparatively high quantum efficiency and shows different quenching channels [15]. The luminescence intensity of Sm 3+ ion depends on the concentration of RE ions as well as surrounding environment. The present work reports the physical, optical and luminescence properties of Sm 3+ ion in zinc barium tellurite glasses. On the basis of the density, molar volume, absorption spectra, luminescence and analysis has been applied to investigate phenomena. II. EXPERIMENTS The preparation of glass samples (ZnO BaO TeO 2 ) was doped with Sm 2 O 3 base on formular (80-x)TeO 2 15ZnO 5BaO xsm 2 O 3. Percentage molecular weight of rare earth varies between x = mol% (see Table 1). All component powders of chemical weight 15 g of the batch materials were thoroughly mixed and crushed in an agate mortar and the homogeneous mixture was transferred into an alumina crucible. Glass samples were melted in alumina crucibles in an electrical furnace for 30 minutes, at 850 C by using melt quenching technique. These melting components were quenched between graphite plates. Thus these glasses obtained were all annealed at 350 C for 3 hours before cooled down to room temperature to remove thermal strains in the glasses. Finally, glass samples were cut and then finely polished to a dimension of 1.0 cm 1.5 cm 0.3 cm. Table 1. Fractional doping of Sm 2 O 3 for zinc barium tellurite glasses. Samples no. Sm 2 O 3 Glass composition (mol%) TeO 2 15ZnO 5BaO TeO 2 15ZnO 5BaO 0.50Sm 2 O TeO 2 15ZnO 5BaO 1.00Sm 2 O TeO 2 15ZnO 5BaO 1.50Sm 2 O TeO 2 15ZnO 5BaO 2.00Sm 2 O 3 The densities (ρ) were measured by Archimedes method using xylene as an immersion liquid. The optical absorption spectra of the glasses sample were recorded in the UV VIS and NIR regions in the range of 350 2,000 nm using a UV 3600 Shimadzu UV VIS NIR spectrophotometer. The luminescence spectra measured were carried out using Cary Eclipse Fluorescence Spectrophotometer with 404 nm excitation of xenon flash lamp. ISSN

132 III. RESULTS AND DISCUSSION The densities and molar volumes of the investigated ZBT glasses are shown in Fig. 1. The density was increased with increase of Sm 2 O 3 concentration. The molar volume depends on the density of glasses and as expected in the present case, it follows a trend opposite to density. The molar volumes of glasses were decreased with increasing of Sm 2 O 3 concentration, reflecting that the glass structure was compactness with higher concentration of Sm 2 O 3, which leads to decrease average atomic separation. The values of density ( ) are in the range of 5.11 to 5.32 g/cm 3 while their molar volumes (V M ) are in the range of to cm 3 /mol. Fig. 1. Densities and molar volumes of Sm 3+ : ZBT glass system. Fig. 2. Absorption spectra of Sm 3+ : ZBT glass system. The optical spectra at room temperature were measured in the wavelength range of 350 2,000 nm using UV VIS NIR spectrophotometer (UV 3600, Shimadzu). The absorption spectra of Sm 3+ doped zinc barium tellurite glasses in the UV VIS NIR are shown in Fig. 2. The spectra obtained for all Sm 3+ doped zinc barium tellurite glasses are similar in nature except for the band intensities. It is clearly observed that the absorption intensity of the absorption band increases with the increase of Sm 2 O 3 concentration. The 118 ISSN

133 absorption bands of Sm 3+ represent the transitions from the ground state 6 H 5/2 to various excited states. The transitions are observed as follows: 6 P 3/2 (402 nm) and 4 F 5/2 + 4 I 13/2 (470 nm) for the UV-VIS range, and 6 F 11/2 (946 nm), 6 F 9/2 (1079 nm), 6 F 7/2 (1226 nm), 6 F 5/2 (1370 nm), 6 F 3/2 (1472 nm), 6 H 15/2 (1524 nm), and 6 F 1/2 (1586 nm) for NIR range indicating the Sm 3+ in glass matrices. Fig. 3. Emission spectra (λ ex = 404 nm) of ZBT glasses doped with Sm 3+ ion. Fig. 3 shows the luminescence spectra of Sm 3+ doped zinc barium tellurite glasses using 404 nm wavelengths using the xenon flash lamp as an excitation source with wavelength consist of four emission bands in yellow, orange, red and NIR regions at 563, 600, 646 and 707 nm corresponding to the transitions 4 G 7/2 6 H 5/2, 4 G 7/2 6 H 7/2, 4 G 7/2 6 H 9/2 and 4 G 7/2 6 H 11/2, respectively with slight difference in intensity. Fig. 4 shows the partial energy level diagram showing the energy level scheme involved in the emission mechanism and cross relaxation channels involved in Sm 3+ ion in the ZBT glasses. Fig. 4. Partial energy level diagram showing the possible emission transitions of Sm 3+ ion in ZBT glasses system. ISSN

134 IV. CONCLUSION The Sm 3+ -doped zinc barium tellurite glasses were prepared at various doping concentration of Sm 2 O 3 and characterized for their physical, optical and luminescence properties. The density of glass samples were increase with the increase of Sm 2 O 3 concentration. The behavior of molar volume mainly depends upon the density of glasses, as expected in the present case, it follows a trend opposite to density. The absorption bands corresponding to the transitions from the ground state 6 H 5/2 to the excited states: 4 K 11/2 (344 nm), 4 H 7/2 (359 nm), 6 P 7/2 (373 nm), 4 F 7/2 (404 nm), 6 P 5/2 + 4 P 5/2 (419 nm), 4 G 9/2 (440 nm), and 4 I 11/2 (482 nm) of Sm 3+ ion. The emission spectra of Sm 3+ were stored by monitoring an intense emission peak at 600 nm (red orange) under excitation 404 nm and increases with the increase of Sm 2 O 3 concentration. V. ACKNOWLEDGMENTS P. Yasaka and J. Kaewkhao would like to thank National Research Council of Thailand (NRCT) for partially support of this research. REFERENCES [1] V.A.G. Rivera, S.P.A. Osorio, D. Manzani, Y. Messaddeq, L.A.O. Nunes, E. Marega, Opt. Mater. 33 (2011) 888. [2] R.A.H. El-Mallawany, Tellurite Glasses Handbook: Physical Properties and Data, CRC Press, New York, [3] A. Jha, B. Richards, G. Jose, T. T-Fernandez, P. Joshi, X. Jiang, J. Lousteau Prog., Mater. Sci. 57 (2012) [4] P. Babu, H.J. Seo, C.R. Kesavulu, K.H. Jang, C.K. Jayasankar, J. Lumin. 129 (2009) 444. [5] P.G. Pavani, S. Suresh, V.C. Mouli, Opt. Mater. 34 (2011) 215. [6] S. Sakida, T. Nanba, Y. Miura, Mater. Lett. 60 (2006) [7] P.G. Pavani, K. Sadhana, V.C. Mouli, Physica B 406 (2011) [8] E. Yousef, M. Hotzel, C. Russel, J. Non-Cryst. Solids 342 (2004) 82. [9] V. Rajendran, N. Palanivelu, B.K. Chaudhuri, K. Goswami, J. Non-Cryst. Solids, 320 (2003) 195. [10] J.C.S. Moraes, J.A. Nardi, S.M. Sidel, B.G. Mantovani, K. Yukimitu, V.C.S. Reynoso, L.F. Malmonge, N. Ghofraniha, G. Ruocco, L.H.C. Andrade, S.M. Lima, J. NonCryst. Solids 356 (2010) [11] B.V.R. Chowdari, P.P. Kumari, Solid State Ionics (1998) ISSN

135 [12] G. Venkataiah, C.K. Jayasankar, K. Venkata Krishnaiah, P. Dharmaiah, N. Vijaya, Opt. Mat., 40, 2015, [13] K.K. Mahato, D.K. Rai, S.B. Rai, Solid State Commun. 108 (1998) [14] H. Lin, E.Y.B. Pun, L.H. Hung, Appl. Phys. Lett. 80 (15) (2002) [15] C.K. Jayasankar, K. Upendra Kumar, V. Venkatramu, P. Babu, Th. Troster, W., Sievers, G. Wortmann, J. Lumin. 128 (2008) ISSN

136 PHOTOLUMINESCENCE OF LITHIUM GADOLINIUM BORATE GLASSES DOPED WITH CERIUM (III) OXIDE Kitipun Boonin 1,2*, Warawut Sa-ardsin 1,2 and Jakrapong Kaewkhao 1,2 1 Center of Excellence in Glass Technology and Materials Science (CEGM), Nakhon Pathom Rajabhat University, Nakhon Pathom 73000, Thailand 2 Science Program, Faculty of Science and Technology, Nakhon Pathom Rajabhat University, 73000, Thailand *: kboonin@hotmail.com Abstract. The Li 2 O-Gd 2 O 3 -B 2 O 3 glasses doped with Cerium (III) oxide were prepared using melt quenching technique. Some properties i.e. density, molar volume, absorption and photoluminescence spectra were investigated. The absorption spectra of glass were measured in the wavelength range 200-2,500 nm. The result show that the absorption edge shifted to longer wavelength. Using a 270 nm light for excitation into the Ce 3+ 5d level. All the spectra exhibited with one emission bands corresponding to the 5d 2 F 7/2 (328 nm). All bands are increase with increasing of Ce 3+ content. Keywords: Lithium gadolinium, Luminescence spectra, LGBO, Glass, CeF 3. I. INTRODUCTION Glass is popular materials which have been applied in several fields for a long time. It has many advantages compared with crystals. Many properties of Lithium borate glasses such as isotropic properties and adaptable compositions made them the alternative choice. Borate glasses have higher bond strength, high transparency, low melting point, high thermal stability and good rare earth ions solubility which are better than other types of glasses [1, 2]. RE ions are being used as dopants to improve absorption bands and modify glass structure affecting energy level transition so that they are suitable for applications like lasers, sensors, light converters, hole-burning high density memories, optical fibers and amplifiers [3-5]. The optical and spectroscopic properties of Ce 3+ -doped crystal have been studied to make scintillators for high-energy physics and medical imaging application. In a research, B 2 O 3 SiO 2 Gd 2 O 3 BaO glass was alternatively prepared and investigated. This made the attraction to find the way to prepare higher concentration cerium-doped oxide glass to make better scintillation material [6]. In this study, the Ce 3+ doped Lithium-gadoliniumborate glasses of the composition: [60Li 2 O:10Gd 2 O 3 :(30-x)B 2 O 3 : xcef 3 were prepared. The spectroscopic and optical properties in the visible (VIS) and UV range were instigated and discussed. 122 ISSN

137 II. EXPERIMENTS The glass samples were prepared by using Li 2 CO 3, Gd 2 O 3, H 3 BO 3 and CeF 3 in the composition range of 60Li 2 O:10Gd 2 O 3 :(30-x)B 2 O 3 : xcef 3 where x = 0.05, 0.10, 0.50, 1.00 and 1.50 mol% each batch of about 10 g. All raw materials were mixed, ground and then melted at 1,050 C in high alumina crucible in electrical furnace for 3 hours. The molten were quenched at room temperature on a stainless steel mold under an air atmosphere. The quenched glasses were annealed at 300 C for another 3 hours and cooled down to the room temperature. Densities of the glasses were measured using a digital balance (4-digit sensitive microbalance Denver, Pb214) and then molar volumes were calculated. The UV- VIS-NIR spectrophotometer (UV-3600 Shimadzu) was used to investigate the absorption spectra in the range from 200 to 2500 nm. Emission spectra were measured by exciting with the electromagnetic wave of 270 nm from a Xenon compact arc lamps using Cary Eclipse Fluorescence Spectrophotometer. All investigations were carried in room temperature. III. RESULTS AND DISCUSSION The variation of density and molar volume of glasses with the addition of Ce 3+ are shown in Fig. 1. and Fig. 2. It is observed from this figure that the density and molar volume of glasses increases with addition of Ce 3+. The similar results are reported by Ramtege et al. [7] in Pr 6 O 11 doped lithium borate glass system. The density of the glasses was significantly influenced by the molecular weight of CeF 3 ( g/mol) which is much higher than B 2 O 3 (69.62 g/mol) so density of the glasses increases (Fig. 1). Ce 3+ ions have large ionic radius as compared to other constituent. The added Ce 3+ ions expand the glass structure therefore molar volume of glasses increases (Fig. 2.). In Fig. 3. was observed that the position of the absorption peak shift to longer wavelength as the content of CeF 3 increases. Fig. 1. Density of Ce 3+ :LGBO glasses. Fig. 2. Molar volume of Ce 3+ :LGBO glasses. When the glass is excited by 270 nm, a broadening emission bands centering at 328 nm is observed possibly due to the allowed transitions from the 5d excited states of Ce 3+ ISSN

138 ions to its 4f ground state [3, 8, 9]. All emission bands are increase with increasing of Ce 3+ content as show in Fig. 4. Fig. 3. Absorption spectra of the Ce 3+ : LGBO glasses. Fig. 4. Emission spectra of the Ce 3+ : LGBO glasses. IV. CONCLUSION The Li 2 O-Gd 2 O 3 -B 2 O 3 glasses doped with Ce 3+ were prepared using melt quenching technique. Density, molar volume, absorption and photoluminescence spectra were investigated. The density and molar volume of glasses increases with addition of Ce 3+. The position of the absorption peak shift to longer wavelength as the content of CeF 3 increases. Emission band centering at 328 nm is observed possibly due to the allowed transitions from the 5d excited states of Ce 3+ ions to its 4f ground state. The emission intensity trends to increase with increasing concentration of the dopant. V. ACKNOWLEDGMENTS We would like to thank National Research Council of Thailand (NRCT) for partially support of this research. REFERENCES [1] D. D. Ramteke, R.S. Gedam, Spectrochimica Acta Part A: Molecular and Bio Molecular Spectroscopy, Vol. 133, 2014, pp [2] F. H. ElBatal, M. S. Selim, S. Y. Marzouk, M.A. Azooz, Physica B Vol. 398, 2007, pp [3] Li Hua Zheng, Xin Yuan Sun, Ri Hua Mao, Hao Hong Chen, Zhi Jun Zhang, Jing Tai Zhao, J. of Non-Crys. Solids, Vol 403, 2014, pp.1 4. [4] D.A. Haas, M. Bliss, S.M. Bowyer, J.D. Kephart, M.J. Schweiger, L.E. Smith, Nucl. Ins. Met Phys. Res., Sect. A Vol 652, 2011, pp ISSN

139 [5] Y. Arikawa, K. Yamanoi, T. Nakazato, E.S. Estacio, T. Shimizu, N. Sarukura, M. Nakai, T. Norimatsu, H. Azechi, T. Murata, S. Fujino, H. Yoshida, K. Kamada, Y. Usuki, T. Suyama, A. Yoshikawa, N. Sato, H. Kan, Rev. Sci. Instrum., Vol 80, 2009, pp [6] C. Jiang, P. Deng, J. Zhang, F. Gan, Phys. Let. A Vol. 323 (3-4), 2004, pp [7] D.D. Ramteke, H.C. Swart, R.S. Gedam, Physica B vol 480, 2016, [8] H. Lian, M. Zhang, J. Liu, Z.Ye, J. Yan, C. Shi, Chem. Phys. Let. Vol 395, 2004, pp [9] C. Dujardin, C. Pedrini, N. Garnier, A.N. Belsky, K. Lebbou, J.M. Ko, T. Fukuda, Opt. Mat. Vol 16, pp ISSN

140 FABRICATION OF POROUS AMORPHOUS SILICON CARBIDE FREE-STANDING MEMBRANES AND MULTILAYERS Cao Tuan Anh 1*, Luong Truc Quynh Ngan 2, Dao Tran Cao 2 and Nguyen Ngoc Hai 3 1 Tantrao University, Yenson District, Tuyenquang Province, Vietnam 2 Institute of Materials Science, 18 Hoang Quoc Viet, Hanoi, Vietnam 3 Uongbi High School, Uongbi District, Quangninh Province, Vietnam *: ctanh.iop@gmail.com Abstract. We present the results of fabrication of free-standing porous amophous SiC (asic) membranes and porous asic multilayer thin films. The free-standing porous asic membranes have been obtained after a two-step etching process of initial asic/si material. In the first step, the entire thickness of the asic thin film has been etched anodically in a dilute HF/H 2 O solution and this thin film has become a porous columnar layer. In the second step, the Si substrate layer has been removed from the asic/si material by chemical etching in a HF/HNO 3 solution. The porous asic multilayer thin films have been prepared by multistep galvanostatic anodic etching in a dilute HF/H 2 O solution. The number of layers, their thickness and porosity can be controlled by modulating the anodic current density and etching time. Keywords: Porous SiC, Anodic etching, HF, Mutilayer, Membranes. I. INTRODUCTION Silicon carbide (SiC) continues to gain attention from researchers around the world as they attempt to tailor it for new applications. SiC have many advantages over Si, including a relatively wide bandgap, excellent stability against chemical corrosion, high breakdown electric field, large thermal conductivity and a higher melting point. Like Si, if SiC is made to become porous at the nanoscale, it will have special properties that previously, in the bulk state, it does not have, such as enhanced photoluminescence [1], enhanced ability to sense certain chemicals [2] and enhanced diffusion of proteins to make protein sensors [3]. For this reason, porous SiC material has attracted the attention of numerous research groups. In addition, SiC multilayer structures can be used in the waveguides and microcavities, which are operating in hazardous environments [4]. However, according to our knowledge, until now, the production of porous SiC multilayer structure is not implemented. Free-standing porous asic membranes can be used in nanofiltration technique, bio sensors [3]. However, at present, the fabrication of free-standing porous asic membranes is very difficult [3, 5]. In our previous studies have shown that with the use of very low concentrations of HF and appropriate anode current density can control the etching rate and porosity of porous asic layer [6], in this study, the pore size was only several tens of nanometers and the pores grew down through the amorphous layer in a 126 ISSN

141 perpendicular columnar structure. The columnar porous asic thin film layer in the current report grew immediately below the sample surface, rather than below a skin and cap layer, as in the columnar porous structure in bulk crystalline SiC [7, 8]. This result will allow the fabrication of porous asic multilayer structures and free-standing porous asic membranes, in this report we will present the results of research on this issue. II. EXPERIMENTS The starting material was an undoped 3-µm-thick amorphous SiC (asic) thin film, which has been deposited by plasma-enhanced chemical vapor deposition (PE-CVD) on a single-crystalline Si(100) substrate. This starting material was fabricated at the DIMES Institute, Delft University of Technology, the Netherlands [9]. In this study, we have created a porous multilayers on the surface of asic thin film, using anodic etching, which was performed at constant current mode in a solution of HF/H 2 O with the HF concentration of 0.5 vol. %. The free-standing porous asic membranes have been created by two steps: the first step, asic thin films were etched at constant current mode in a solution of 0.5% HF/H 2 O. In this step, we created the porous layers have the thickness equal to the thickness of asic thin films; the second, the freestanding porous asic membranes were created by chemicaletching in HF/HNO 3 with the HF anh HNO 3 concentration of 50 vol. %. The reagents used were of the analytical reagents grade with purity as follows: HF 40 vol. %, HNO 3 65 vol. %. The morphology and thickness of porous layers were controlled by changing the anodic current density and time etching. In the experimental setup, the asic anode was placed parallel to the platinum grid cathode; the two electrodes were separated by 2 cm. Prior to anodization, aluminum was evaporated on the back of the sample to create an ohmic contact; this process allowed the asic sample to be used as an electrode. To ensure that only the asic surface was exposed to the electrolyte during anodic etching, the backside and edges of the samples were protected with a polystyrene layer, as done in our previous report [10]. The surface morphologies of the etched samples were studied with plan-view scanning electron microscopy (SEM). III. RESULTS AND DISCUSSION Our research on the mechanism of anodic etching asic in dilute HF solution shows that with appropriate anode current density, etching rate is constant over time. Furthermore, the porosity of the porous asic sample may be controlled by the change of anodic current density [6]. From these results we have been fabricated the multi-layer porous asic structures. Figure 1a is the cross-sectional SEM image of porous asic sample, which has two-layer structure, the first layer has been anodically etched for 11 min with a current density of 1.5 ma/cm 2, the second layer has been anodically etched for 20 min ISSN

142 with a current density of 2.0 ma/cm 2. Figure 1b is the cross-sectional SEM image of porous asic sample, which has four layers structure, the first and third layer have been anodically etched for 12 min with a current density of 1.0 ma/cm 2, the second and fourth layer have been anodically etched for 11 min with a current density of 1.5 ma/cm 2. All samples have been anodically etched in 0.5% HF/H 2 O electrolyte. Thus, porous asic multi-layer structures have been fabricated successfully, with the number of layers, thickness and porosity of the porous layer can be controlled. According to our understanding, this is the first time a multi-layer porous structure is created on SiC materials. These results open up the possibility of application of SiC porous materials in the fabrication of microcavities, waveguides similar to that of porous Si [11, 12]. In addition to the excellent properties of SiC, these devices can work in harsh environments. This new porous SiC structure could not only actualise SiC usage for nano-electronic devices but is also expected to open new applications as well. Fig. 1. The SEM images showing the cross-sectional view of porous asic multilayer thin films with two (a) and four layers, which have been anodically etched in 0.5% HF/H 2 O electrolyte. By using the two-step: the first step, the entire thickness of the asic thin film has been etched anodically in a dilute HF/H 2 O solution and this thin film has become a porous columnar layer; In the second step, the Si substrate layer has been removed from the asic/si material by chemical etching in a HF/HNO 3 solution etching as described in the abstract, we have been fabricated the free-standing porous asic membranes. Figure 2.A (orange part) is photograph of a free-standing porous asic membrane, which have been anodically etched in 0.5% HF/H 2 O electrolyte with a current density of 1.5 ma/cm 2. Figure 2a is the cross-sectional SEM image of this sample. Figure 2.b and 2.B are the SEM images of the top surface and the bottom of the sample, respectively. Figure 2.c and 2.C are the SEM images of the top surface and the bottom of the sample, which has been anodically etched in 0.5% HF/H 2 O electrolyte for 58 min with a current density of 2.0 ma/cm 2. The SEM images showed that the diameter of the pores of the free-standing porous asic membranes can be controlled by changing the electrochemical current density. With the controlling of the pore diameter, the free-standing porous asic membranes can be applied very well to the biosensor and nanofiltration [3]. 128 ISSN

143 Fig. 2. (a) The cross-sectional SEM image of a porous asic sample before removing the Si substrate, (A) photograph of a free-standing porous asic membrane (orange part), SEM images of b, c) top and (B, C) bottom surface of free-standing porous asic membranes, which have been anodically etched in 0.5% HF/H 2 O electrolyte with with different time etching and current densities. IV. CONCLUSION By using an aqueous diluted HF electrolyte and appropriate anode current density, the two and four layers structures of porous asic have been successfully made. In these structures, the thickness and porosity of the layers can be controlled by changing the anode current density and etching time. Just by doing this, porous asic thin films which have the thickness of porous asic layer with the thickness of asic original film were fabricated. Then by chemical etching to remove Si substrate, the free-standing porous asic membranes have been created. These novel types of porous asic structures not only significantly enriches the family of porous structures, but is also expected to exhibit many new electronic and optical properties, which will open up fresh opportunities for vast interesting applications in advanced nanodevice design and constructions V. ACKNOWLEDGMENTS The authors would like to thank Dr. H. T. M. Pham for supplying the SiC samples and BSc. Tran Minh Van for the power source. This work was financially supported by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under the project. REFERENCES [1] J. X. Fan, X. L. Wu, P. K. Chu, Progr Mat Sci, Vol. 51, 2006, pp ISSN

144 [2] E. J. Connolly, B. Timmer, H. T. M. Pham, J. Groeneweg, P. M. Sarro, W. Olthuis, P. J. French, Sensor Actuator, Vol. B 109, 2005, pp [3] A. J. Rosenbloom, S. Nie, Y. Ke, R. P. Devaty, W. J. Chokye, Mater. Sci. For., Vol , 2006, pp [4] J. Xu, J. Mei, D. Chen, S. Chen, W. Li, K. Chen, Diam. Relat. Mater., Vol. 14, 2005, pp [5] T. Omiya, A. Tanaka, and M. Shimomura, Jpn. J. Appl. Phys., Vol. 51, 2012, pp (1-5). [6] T. C. Anh., T. L. Quynh-Ngan, and T. D. Cao, AIP Advances, Vol. 4, 2014, pp (1-7). [7] J. H. Tan, Z. Chen, W. Y. Lu, Y. Cheng, H. He, Y. H. Liu, Y. J. Sun and G. J. Zhao, Nanoscale Research Letters, Vol. 9, 2014, 5 pp. [8] L. Wang, H. Shao, X. Hu, X.Xu, J. Mater. Sci. Technol., Vol. 29, 2013, pp [9] H. T. M. Pham, Ph.D. thesis (2004), Delft University of Technology, Delft, Netherlands. [10] D.T. Cao, C.T. Anh, N.T.T. Ha, H.T. Ha, B. Huy, P.T.M. Hoa, P.H. Duong, N.T.T. Ngan, N.X. Dai, J. Phys. Conf Ser, Vol. 187, 2009, pp [11] J. Xu, J. Mei, D. Chen, S. Chen, W. Li, K. Chen, Diam. Relat. Mater., 14, 2005, pp [12] T. C. Do, H. Bui, T. V. Nguyen, T. A. Nguyen, T. H. Nguyen and V. H. Pham, Adv. Nat. Sci.: Nanosci. Nanotechnol., Vol. 2, 2011, pp (5pp). 130 ISSN

145 A VAPOR SENSOR BASED ON NANO-POROUS SILICON MICROCAVITY FOR DETERMINATION OF FUEL MIXTURES Thuy Van Nguyen 1*, Van Dai Pham 1, Thi Cham Tran 1, The Anh Nguyen 1, Thuy Chi Do 2, Van Hoi Pham 1 and Huy Bui 1 1 Institute of Materials Science, Vietnam Academy Science and Technology 18 Hoang Quoc Viet Str., Cau Giay Dist., Hanoi, Vietnam. 2 Thai Nguyen University of Education, Luong Ngoc Quyen road, Thai Nguyen city, Vietnam *: vannt@ims.vast.ac.vn Abstract. In this study, we successfully demonstrated the rapid, sensitive, and reversible sensing of vapor sensor using nano-porous silicon microcavity (NPSM). We fabricated the NPSM on a highly doped p + -type silicon wafer (resistivity is of Ω.cm) and investigated its reflectance spectra before, during, and after exposure to different kinds of fuel mixtures such as bio-fuel (E5), commercial gasoline A92 and A95 added ethanol and methanol of different concentrations up to 30%. When the NPSM was exposed to organic fuel mixtures, the reflectance peak promptly shifted toward longer wavelengths. We determined that the red-shift in the reflectance spectrum could be attributed to the refractive index change of NPSM due to the interaction with vapor. The NPSM showed excellent sensing ability under the bio-fuel (E5), commercial gasoline A92 and A95 added ethanol and methanol of different concentrations. In addition, a slight hysteresis of the red-shift was observed during repeated exposure to fuel mixtures at different concentrations. After removing the fuel vapors, the reflectance spectrum promptly returned to its original state. Keywords: vapor sensor, nano-porous silicon microcavity (NPSMC), organic fuel. I. INTRODUCTION The discovery of biofuels from organic or food waste products has generated much interest because of their benefits brought to the green environment. Some of advantages of biofuels over fossil fuels that make the alternative fuel source an attractive option now and in the future such as lower emissions, renewable, biodegrable and safer. There have been various methods tried for reducing fossil fuel dependency and containing carbon footprints for a healthier and more eco-friendly future. Corn produced ethanol has been used for mixing with gasoline but there have been side effects like corrosion from ethanol. Besides, biofuels are also produced by mixing between the gasoline A92 with absolute alcohol (ethanol 99.5%), depending on the percentage of ethanol in products that have the type E5, E10, E85... (corresponding ratio of 5%, 10%... 85% ethanol in the mixture). If there are some organic solventsmixed with gasoline to produce biofuel, this damages the resin details in cars and motorbikes, so it is necessary to detect some organic solvents mixed with gasoline used. There are numerous methods that have been used for detecting organic solvents with high sensitivity and selectivity such as high-pressure liquid chromatography (HPLC), and gas chromatography (GC) in combination with mass spectrometry [1-4], but ISSN

146 these methods are bulky, slow and expensive. Thus, it is essential to develop low cost, simple and rapid methods for determination of organic solvents to detect their presence in biofuels. Recently, the vapor-phase photonic sensors based on porous silicon (PS) nanostructure have generated a great interest because of the interesting morphological and physical properties of the PS layer. PS is an ideal material for sensors due to tremendous advantages like, spongy skeleton, high surface area on the order of m 2 /cm 3, strong and fast interaction with liquid or vapor. Hence, in the past few years, many scientists focused on the characterization, drug delivery devices [5], chemical sensors, biosensors and other emerging and future nano scale applications using PS [6]. In this work, we present a novel technique for the detection of the presence of organic solvents in biofuels using a simple and low cost one dimensional (1D) nano scale Porous Silicon Microcavtiy (PSMC) vapor-phase sensor device. We fabricated the 1D- PSMC on a silicon substrate and investigated its reflectance spectra before, during, and after exposure the vapor mixture ofoganic solvents in different concentrations (from 5% to 30%) and some types of gasoline. II. EXPERIMENTS The spectral region of the optical response for 1D-PSMC sensors was evaluated by using Bragg s relation, where the thickness of defect layer was equal to λ/2 or λ, as follows: λ/4 = n H.d H = n L.d L. In this equation, λ is the wavelength corresponding to the center of the photonic band gap (PBG), n H ; n L and d H ; d L are the refractive index and the thickness of high and low index layers, respectively. The values n H.d H and n L.d L are called optical thickness of the layers. When a 1D-PSMC sensor is exposed to a vapor, the vapormolecules substitutes air in the pores and caused a red shift in the reflectance spectrum, which corresponds to a change in the optical thickness. Because its physical dimensions fix the thickness of the each layer in the stack, a shift in its reflectance spectrum must clearly be due to changes in the average refractive index [6]. The 1D-PSMC sensor was prepared by electrochemical etching method on a borondoped p+ -type single-crystal silicon wafer with a resistivity of Ω.cm in a 1:2 mixture of a 48% HF solution and pure ethanol, respectively. Further detail on porous silicon microcavity fabrication can be found in the reference [7]. After the electrochemical etching process, these structures were washed with 98% ethanol and dried in vacuum. To prevent the aging in porous silicon layers and to stabilize the PS structure from chemical reactions, the thermal oxidation of the structure is necessary. Besides, the surface of the asetched sample was covered by hydride (Si-Hx bonds), making it hardly to full interaction with bio-molecules in aqueous solution. Thus, we oxidized the prepared structure in ozone atmosphere for 45 minutes using the ozone generator (H01 BKOzone with a capacity of 500 mg/hour). 132 ISSN

147 Structural morphology of 1D-PSMC sensor was investigated using an ultraresolution field-emission scanning electron microscopy (FE-SEM S-4800). For the measurement of reflective spectra of the prepared sensor structures and the wavelength shift in the reflective spectra of sensor before, during and after exposure to the fuel vapors, we used a low-cost optical spectrometer (USB4000-VIS-NIR, Ocean Optics, USA) and a white source in wavelength range of nm from a Tungsten halogen lamp (HL LL, Tungsten Halogen Source, nm, hrs, 2800 K). The experimental configuration of measurement system is shown in Fig. 1. The white light collected by a Tungsten Halogen light Source and inserted in one branch of multimode fiber coupler R200-7-UV-VIS with core diameter of 200um for irradiating to sensors and the reflective spectrum of sensor output monitored by a spectrometer USB4000-VIS-NIR via the other branch of the fiber coupler. The collected light direction is perpendicular to the microcavity. In this schematic, valve 2 works as a controller of velocity of air stream though the flow meter, the test solvent chamber and the sample chamber. Valve 1 is only open to refresh the porous matrix after measurement. Fig. 1. Schematic configuration of the measurement for the kinds of fuel (bio-fuel (E5), A92, A95) added ethanol and methanol of different concentrations by using a vapor sensor based on the 1D-PSMC. Valve 1 and valve 2 are used to guide the airflow from a pump into the sample chamber. The arrows indicate the direction of the air or vapor flows. Figure 2a and Figure 2b show plan-view and cross-sectional images of fabricated multilayer 1D-PSMC sensor based on (HL) 4.5 (LL)(HL) 5 porous silicon multilayer structure, where 4.5 means four and half pairs of HL. This structure was obtained by applying the anodization current densities of 15 ma.cm -2 and 50 ma.cm -2 with an etching time of 5.56 sec. and 2.86 sec. for high and low refractive index layers, respectively. Figure 2c shows the reflective spectra of the porous-silicon microcavity before and after oxidization measured by spectrometer Ocean Optics USB4000-VIS-NIR. The blue shift of the resonant wavelength after oxidization is due to decrease of average effective refractive index the porous structure [8]. ISSN

148 (a) (b) Fig. 2. SEM plan-view (a) and cross-sectional view (b) of multilayer 1D-PSMC sensor. (c) Reflective spectra of multilayer 1D-PSMC sensor as-prepared (curve 1) and after oxidation in ozone ambience (curve 2). III. RESULTS AND DISCUSSION To testing the performance of the optical sensor for the determination of the fuels added ethanol or methanol, we studied the wavelength shift in the reflectance spectra with various conditions: 100% fuel, fuel added ethanol or methanol with different concentrations and fuel added mixture (ethanol + methanol) with different concentrations. The effective refractive index of the nano-psmc layer exposed fuel vapor would be increased due to the substitution of air with the vapor molecules in the pores and consequently the optical thickness of the layer is increased. As a result, the resonant wavelength shift would be dependent upon the refractive index value of the fuel s vapors. Table 1 presents resonant wavelength shift of sensor based on 1D-PSMC exposured in vapor mixtures (100% fuel and 95% fuel + 5% organic solvents (Ethanol, Methanol, Ethanol + Methanol)) with the airflow velocity of 0.84 ml.s -1. As shown in the table, during the exposure, the reflectance spectra consistently shifted toward the longer wavelengths and the variations in the reflectance spectra differed slightly depending on the type of fuel s vapors used. More specifically, when the 1D-PSMC was exposed to gasoline A95, Bio-fuel E5 and gasoline A92 vapors with unknown refractive index, the maximum reflectance peak promptly shifted toward longer wavelength by about 33.2, 36.0 and 40.6 nm, respectively. Therefore, this proves that the refractive index of commercial gasoline A92 is highest of the three types of fuel s used. Table 1. Resonant wavelength shift of sensor based on 1D-PSMC exposured in organic fuel mixture gas (100% organic fuels and 95% organic + 5% organic solvents (Ethanol, Methanol, Ethanol + Methanol)) with the airflow velocity of 0.84 ml.s -1. Mixture vapors of organic solvents and fuel Refractive index of organic solvents Resonant wavelength shift of sensor in some of fuels A95 E5 A92 100% fuel ISSN

149 Resonant wavelength shift (nm) The 4 th Academic Conference on Natural Science for Young Scientists, Master & 5 % Ethanol (99.7%) + 95% fuel (Ethanol) % (Ethanol + Methanol) + 95% fuel % Methanol (99.5%) + 95% fuel (Methanol) We also checked for variations of the optical thickness of 1D-PSMC during exposure to mixture vapors of organic solvents at 5% concentration in the types of fuel (gasoline A95, Bio-fuel E5 and gasoline A92). As a result, when a 1D-PSMC was exposed to mixture vapors with a high refractive index (n ethanol+fuels > n (1/2ethanol+1/2 methanol)+fuels > n methanol+fuels ), large variations of the maximum reflectance peak were observed, whereas when a 1D-PSMC was exposed to mixture vapors with a low refractive index, small variations of the maximum reflectance peak were observed. Figure 3 shows the dependence of the resonant wavelength shift on the vapor mixture of ethanol in the defferent concentrations and the types of fuelat the airflow velocity of 0.84 ml.s -1. As shown in this figure, with the start vapor mixture of 0% ethanol and 100% the types of fuel, the redshift in the reflectance spectra is reduced by about 9.5 nm, 12.1 nm and nm with gasoline A95, Biofuel E5 and gasoline A92, respectively in the given ethanol concentration range, which varied from 5 to 30 %. And, this figure also indicates that the shift values of the resonant wavelength were related to the variations of the refractive index of the mixture of solvent vapors used. In other words, when a 1D-PSMC was exposed to the vapor mixture of ethanol and fuels at a high concentrantion of ethanol, a small shift in its resonant wavelength shift was observed, whereas when a 1D-PSMC was exposed to the vapor mixture of ethanol and fuels at a low concentrantion of ethanol, a large shift in its resonant wavelength shift was observed. This cause is due tocompletely dissolved ethanol in fuels, which led tochange of the refractive index of mixture. A more detailed way, the more concentration of ethanol increases, the more the refractive index of fuel decreases, whereas the more concentration of ethanol decreases, the more the refractive index of fuel increases. These experimental results suggest that variations in the resonant wavelength shift depend on the concentration of desolved enthanol in fuels. Therefore, this result can be (1) (7) (1) Ethanol (A95) (4) Ethanol (E5) (7) Ethanol (A92) Concentration (%) Fig. 3. The dependence of the resonant wavelength shift on the vapor mixture of ethanol in different concentrations and types of fuels (A95, E5 and A92) at V=0.84ml.s -1. (4) ISSN

150 used in developing sensitive vapor sensors based on 1D-PSMC. Besides, this figure also shows that when the 1D-PSMC is exposed to gasoline A92 vapor, the resonant wavelength shift of sensor is equal to in case of the exposed 1D-PSMC to mixture of 5% ethanol and Bio-fuel E5 as the define of Bio-fuel E5. To check the sensitivity of 1D-PSMC sensor sample to the concentration of organic vapors which disoveled completely in fuel, we investigated the resonant wavelength shift during exposure to vapor mixture of organic solvents at different concentrations and biofuel E5, as shown in Figure 4a. When the concentration of organic solvent vapors in fuel increases, the resonant wavelength shift is reduced from nm at vapor mixture 0% organic solvents + 100% bio-fuel E5 to 20 nm, 21.6 nm and 23.9 nm with vapor mixture 30% organic solvents (Methanol,1/2ethanol+1/2 methanol and ethanol, respectively) + 70% bio-fuel E5. Fig. 4. The dependence of the resonant wavelength shift on the vapor mixture of (a) ethanol, methanol and (½ ethanol + ½ methanol) in different concentrations and Biofuel E5, (b) methanol and (½ ethanol + ½ methanol) in different concentrations and the types of fuel (A95, E5 and A92) at V=0.84ml.s -1. Figure 4b presents clearly the dependence of the resonant wavelength shift on the vapor mixture of organic solvents in different concentrations and the types of fuel. All experimental curves in the figures are followed the exponential function. The shift values of resonant wavelength come from refractive index changes in the concentration changes of organic solvents which are disoveled completely in types of fuel. Besides, this figure also indicates that the values of the resonant wavelength shift depend on refractive index of each organic solvents and types of fuel used. Therefore, this experimental result can be used to detect organic solvents in types of fuel. IV. CONCLUSION In conclusion, we successfully fabricated a 1D-PSMC with resonant wavelength in the visible region and use them as vapor-phase photonic sensors. We were able to demonstrate that the resonant wavelength shift comes from refractive index changes induced by the capillary condensation of the vapor mixture of organic solvents and types of 136 ISSN

151 fuel. The value of the resonant wavelength shift was found to be related to the refractive index of vapor adsorbed through capillary condensation, which depended on the concentration of the completely dissolved organic solvents in types of fuel. The photonic sensors are used for determination of organic solvents (ethanol and methanol) and mixture of organic solvent (ethanol+methanol) with concentration from 5% to 30% which are dissolved in types of fuel. Based on the experimental result we suppose that the enhanced sensors can distinguish each individual solvents in types of fuel with a small concentration below 5%. This sensor, to our knowledge, is suitable to detect the very small of solvents that damages the resin details in cars and motorbikes. V. ACKNOWLEDGMENTS This work financially supported by Vietnam Academy of Science and Technology under Project No.VAST03.06/ This work had been using the apparatus of National Key Laboratory for Electronic Materials and Devices in Institute of Materials Science. REFERENCES [1] K. Johasson, G. Jonsson-Pettersson, L. Gorton, G. Marko-Varga and E. Csoregi, J. Biotechnol. Vol. 31, 1993, pp [2] E. Falqué, E. Fernández Gomez, J. Chromatogr. Sci. Vol. 34, 1996, pp [3] M. D. Luque De Castro, J. Gonzalez-Rodriguez, P. Perez-Juan, Food, Rev. Int. Vol. 21, 2005, pp [4] M. Rocchia, A. M. Rossi, G. Zeppa, Sens. Actuators B. Vol.123, 2007, pp [5] J. Salonen, A. M. Kaukonen, J. Hirvonen, V. P. Lehto, J. Pharm. Sci. Vol. 97, 2008, pp [6] Han-Jung Kim, Young-You Kim, Ki-Won Lee, Horchhong Cheng and Dong Han Ha, Physica B. Vol.406, 2011, pp [7] Do, T. C., Bui, H., Nguyen, T.V., Nguyen, T.A., Nguyen, T. H., Pham, V. H., Adv. Nat. Sci.: Nanosci. Nanotechnol. Vol. 2, 2011, (5pp.) [8] Chapron, J., Alekseev, S. A., Lysenko, V., Zaitsev, V. N. Barbier, D., Sens. Actuators B. Vol. 120, 2007, pp ISSN

152 STUDY ON SYNTHESIS OF Fe-BTC MOF MATERIAL AT LOW TEMPERATURE AND ATMOSPHERIC PRESSURE Tran Dinh Tuan, Nguyen Thi Hoai Phuong, Ngo Hoang Giang, Nguyen Tien Hue, Do Huy Thanh and Ninh Duc Ha Institute of Chemical and Materials/Academy of Military Science and Technology, 17 Hoang Sam, Cau Giay, Hanoi *: Abstract: Fe-BTC, a mesoporous metal-organic framework (MOF), has high surface area. The structural characteristics of Fe-BTC make it a good candidate for potential applications in gas storage, separation, and heterogeneous catalysis. Mainly, this MOF is obtained by the hydrothermal synthesis in a Teflon-lined autoclave at high temperature (>150 0 C) under static conditions. However, this method has several disadvantages such as high temperature, high pressure, long time, and relatively low MOF yield. Therefore, development of a new method for synthesis of Fe-BTC is vitally important. Herein, Fe-BTC have been synthesized successfully at low-temperature (<100 0 C) and atmospheric pressure by reaction of iron(iii) nitrate and trimesic acid in water. With this method, higher MOF yield could be achieved, suggesting that this simple and energy saving method has the potential for application in practice. Keywords: Metal-organic frameworks (MOFs), Fe-BTC, low-temperature and atmospheric pressure. I. INTRODUCTION Metal-organic frameworks (MOFs) are crystalline porous material that consist of metal-carboxylate units, metal ions and organic linkers. These metal ions are coordinated to rigid organic molecules to form one, two or three-dimensional structures that can make the framework very porous [1]. The particular interest in MOF materials is due to the easy tunability of their pore size and shape from a micro to a mesoscale by changing the connectivity of the inorganic moiety and the nature of organic linkers. MOFs are well known for their various applications in gas store, separation, catalysis and drug delivery. Among the hundreds of known MOFs, there are several transition-metal MOFs that have shown highly hydrothermal and chemical stability. These include mesoporous iron (III) carboxylate (Fe-BTC), which has a rigid zeotype crystal structure, exhibits a high thermal stability (275 0 C) and a remarkable chemical resistance (to organic solvents or water under reflux). In addition, the presence of accessible coordinatively unsaturated metal sites allows the coordination of different species, making it an interesting candidate for many potential applications [1-3]. Fe-BTC is mainly prepared by a hydrothermal synthesis in a Teflon-lined autoclave at temperatures higher than 1500C under static conditions [4-7]. However, this hydrothermal method has several disadvantages, such as high synthesis temperature, long 138 ISSN

153 synthesis duration, and relatively low product yield in combination with the fact that synthesis is conducted at autogenously high pressures. Thus, from fundamental understanding and application s points of view, development of a new method for MOFs synthesis is very important. In this study, we develop a new method for synthesis of Fe-BTC at low temperature (<100 0 C) and atmospheric pressure by reaction of Iron (III) nitrate nonahydrate and trimesic acid under reflux. Due to the fact that our synthesis is conducted with agitation, the obtained Fe-BTC has merits like high yield and large BET specific surface area, accompanied with low synthesis temperature and short synthesis time, suggesting that this simple unappreciated method could be applied to MOFs syntheses in large scale. II. EXPERIMENTS 2.1. Materials All chemicals were used as received: Iron (III) nitrate nonahydrate (Fe(NO 3 ) 3.9H 2 O, 99%); benzene-1,3,5-tricarboxylic acid (H 3 BTC, 99%); hydrofluoric acid (HF); etanol (C 2 H 5 OH) and deionized water (H 2 O) Synthesis method Fe-BTC was prepared at low-temperature (<100 0 C) in atmospheric pressure, via the reaction of H 3 BTC with iron nitrate. Typically, Fe(NO 3 ) 3.9H 2 O (4.04 g, 10 mmol), H 3 BTC (1.89 g, 9 mmol), additional amounts HF and deionized water (6 ml) were mixed and charged into a three-neck round bottom flask (25 ml) equipped with a magnetic stirrer and a reflux condenser and kept at 95 0 C and a stirring speed of 300 rpm for 12 h. The reaction product was purified three times with a solvent extraction treatment with deionized water (350 ml) and ethanol (350 ml) at 70 0 C for 24 h, finally dried in a vacuum desiccator at C for 10 h Characterization - Xray diffraction measurements were conducted using standard powder diffraction analysis procedures. - SEM images were performed to identify the morphologies and particle size distribution of the crystals. - Functional groups were determined by IR spectra. - Specific surface area values of Fe-BTC were calculated by the Brunauer Emmett Teller (BET) method and the Langmuir method using adsorption of nitrogen adsorption isotherm. - The thermal stability of Fe-BTC was investigated by thermal gravimetric analysis (TGA). ISSN

154 III. RESULTS AND DISCUSSION 3.1. Structure and components of Fe-BTC Morphologies of Fe-BTC wich have been identified by FE-SEM micrographs are shown in Fig. 1. Fig. 1. FE - SEM images of Fe-BTC MOF. The particle size of synthesized Fe-BTC samples was around 2 10 µm. Fe-BTC was synthesized at low-temperature. Fe-BTC has octaheral shape and the particles are well dispersed. Composition and structure of the material were analyzed by X-ray diffraction (XRD) and infrared spectroscopy, results are shown in Fig. 2 and Fig. 3. Samples showed good crystallinity. Fig. 2 showed that the peak was very sharp. Characteristic peaks of the Fe-BTC are 2θ = 10,09 0 ; 10,26 0, 27,79 0 Fig. 2. XRD patterns of MOF Fe-BTC. The FT-IR spectrum of Fe-BTC (Fig. 3) is similar to the previous results [4, 6]. The FT-IR spectrum (Fig. 3) indicates that the purification method used here is particularly 140 ISSN

155 effective for removing residual H 3 BTC, as evidenced by the decrease of the C=O stretching vibration at cm -1, which is assigned to residual H 3 BTC [4], after final treatment with ethanol and deionized water Thermal properties of Fe-BTC Fig. 3. IR patterns of Fe-BTC. Fig. 4. Thermogravimetric analyses of Fe-BTC. The TGA plot of Fe-BTC shows a two-step weight loss between 25 and C (Fig. 4). The first one ( 34 wt.%) in a range from 25 to C is attributed to the desorption of the free water molecules inside the pores, and most likely, these water molecules interact with the iron trimers. The second weight loss ( 71 wt.%) in a range from 414 to C is related to the combustion of H 3 BTC. This clearly indicates that the overall thermal stability of the current Fe-BTC sample is similar to that of the samples synthesized by other ISSN

156 methods [5] and it should be activated at temperatures ranging from 89 to C prior to its application in adsorption and catalysis Surface area of Fe-BTC The porosity of the material is determined by measurement of surface area (BET). Fig. 5. Chart shows coordinates of BET Fe-BTC. S BET of sample Fe-BTC was 569 m 2 /g. The porosity of the as-prepared Fe-BTC was investigated using N 2 adsorption - desorption isotherms. IV. CONCLUSION Fe-BTC was synthesized at low temperature (<100 0 C) and atmospheric pressure for 12 h. The material has the particle size of about 2 10 µm and is crystalline in structure. The S BET of sample Fe-BTC was 569 m 2 /g. The Fe-BTC has potential for applications in adsorption and catalysis. REFERENCES [1] R.Macgillivray L. Published by John Wiley & Sons, Inc., Hoboken, New Jersey, [2] James, S.L. Chem. Soc. Rev., vol. 32, 2003, pp [3] O.M. Yaghi, M.O Keeffe, N.W. Ockwig, H. K. Chae, M. Eddaoudi, J. Kim. Nature, vol. 423, No. 6941, 2003, pp [4] P. Horcajada, S. Surble, C. Serre. Chemical Communications, No. 27, 2007, pp ISSN

157 [5] Y.K. Seo, J.W. Yoon, J.S. Lee. Microporous and Mesoporous Materials, vol. 157, 2012, pp [6] I. Ahmed, J. Jeon, N.A. Khan, S.H. Jhung. Crystal Growth & Design, vol. 12, 2012, pp [7] R. Canioni, C. Roch-Marchal, F. Secheresse. Journal of Materials Chemical, vol.21, No. 4, 2011, pp ISSN

158 TREATMENT FORSCIATICA AND DEGENERATIVE KNEE: APPLICATION OF LOW POWER SEMICONDUCTOR LASER Trinh Tran Hong Duyen *, Tran Minh Thai, Ngo Thi Thien Hoa, Nguyen Thi Hong Hanh, Dinh Thi Thu Hong, Nguyen Thi Huong Linh, Tran Trung Nghia, Nguyen Thanh Tam, Nguyen Quang Trung, Lam Thanh Son, Nguyen Duong Hung Laser Technology Laboratory, University of Technology, Vietnam National University HCM, 268, Ly Thuong Kiet Street, District 10, Ho Chi Minh City *: Abstract. The aim of our research isthe conservation of knee and scatic nerve, helping patients quickly off illness, returned to their normal working life.we carried out the low power semiconductor laser therapy for 44 patients with scatia and 66 patients with the degenerative knee. After the end of the treatment, the rate of the good result and the fair result is 81.82%, the rate of the average results is 18.18%. The results of our research prove that the effect of the simultaneous irradiation of two laser beams of 780 nm and 940 nm is high effective than the common treatments. Keywords: Sactia, degenerative knee, low power laser. I. INTRODUCTION Today, there are many causes of degenerative joints, it includes the joints in the spine, the joints in the limbs... Functional joints is connected the parts of the body, it helps the process of mobilization and withstand the various forces. Degenerative joints usually occurs at 3 position is spine, knee joint and hip joint. Degenerative spine can cause back pain, neck pain or causing sciatica and other conditions without causing by herniated disc.degenerative spine should be treated when cartilage (disc) injured put pressure on the spinal cord, nerve roots, it triggers neurological signs. Degenerative knee usually occurs in women over age 50, the common signs are pain when moving, while up and down stairs, difficult squatting, heard crunching in the joints... This situation is increasing, sometimes swollen joints. On the X-ray results show signs of growing spines and a narrow joint. For the treatment of patients with degenerative knee and sciatica, we have methods: acupuncture, reflexology, physiotherapy, nutritional medicine cartilage (glucosamine sulphate are many authors research treatment of osteoarthritis, there is work to strengthen cartilage regeneration, inhibition of the enzyme that destroys cartilage, decreasing the inflammatory joint pain, slow down osteoarthritis), pain medication, anti-inflammatory (paracetamol, aspegic, meloxicam)... These treatments is only effective temporarily, should avoid long periods and do not use these drugs have side effects such as stomach ulcers - the duodenum. Degenerative knee is treated with surgery when the joints hurt seriously, it makes deformity, joint misalignment, cracks cartilage injuries. On the other hand, patients have to rest, avoid heavy jobs. 144 ISSN

159 II. MATERIALS AND METHODS Clinical studies self-controlled, randomized. Because patients in this study were treated with western drugs, traditional medicine, acupuncture combined with stretchable spine, but treatment results are not as expectative. Therefore, treatment with low power semiconductor laser is expected method, before they are performing surgery. Application of low power semicondctor laser to treatsciatica: we have 44 patients and they are treated in the clinic of traditional medicine therapy (90, Nguyen Tri Phuong, Tan Chau district, An Giang province). A course of therapy is 21 days. Patients are treated two therapies. Table 1. Distribution of patients by age. Age < Patients Percentage Application of low power semicondctor laser to treatdegenerative knee: we have 66 patients, and they are treated in the clinic of the physiotherapy (528, Ngo Gia Tu, 9 Ward, District 5, HCM city). A course of therapy is 28 days. Patients are treated two courses. The evaluation of treatment outcomes are based on the X-ray result of patient before and after treatment. Table 2. Distribution of patients by age. Age > 60 Patients Percentage The level of painful is evaluated according to the VAS scale [1]: Table 3. The VAS scale and the overall evaluation of treatment outcomes. VASscore Level of painful Rating point Treatment result 0 No pain 0 good 1-2 Mild, annoying pain 1 fairly good 3-4 Nagging, uncomfortable. Trouble some pain Distressing, miserable pain 3 medium 7-8 Intense, dreadful, horrible pain Worst possible, unbearable, bad 5 excruciating pain III. RESULTS Application of low power semicondctor laser to treatsciatica: ISSN

160 We use the semiconductor laser works at a 940 nm wavelength impacts directly on acupuncture points to treatsciatica and the effect of two simultaneous wavelengths by semiconductor laser works at 780 nm and 940 nm wavelengths direct treatments on the lower back pain area. We use the semiconductor laser intravascular equipment that treating for patient. It enhances the blood circulation in the body to nourish the damaged disc and nerves around the painful area. Fig. 1. The semiconductor laser optoacupunture optotherapy equipment having 12 channels. Fig. 2. The semiconductor laser intravascular equipment. VAS score Table 4. The result of application of low power semicondctor laser to treat sciatica. Level of painful We have 44 patients Before treatment After the first therapy After the second therapy Number Percentage Number Percentage Number Percentage of of of patients patients patients Rating point 0 No pain Mild, annoying pain 3-4 Nagging, 2 uncomfortable. Trouble some pain Distressing, miserable pain 7-8 Intense, dreadful, horrible pain 9-10 Worst possible, unbearable, excruciating pain Rating point corresponding to distressing, miserable pain 0.48 corresponding to no pain corresponding to no pain After two therapies, level of painful at the sciatica has change: 43 patients have no pain (97.73%) and 01 patients have mild, annoying pain (2.27%). At the second therapy, the rating point is corresponding to no pain. This value is smaller times than the rating point before treatment. 146 ISSN

161 Application of low power semicondctor laser to treatdegenerative knee: We use theeffect of two simultaneous wavelengths by semiconductor laser works at 780 nm and 940 nm wavelengths direct treatment on the painful area. We use the semiconductor laser intravascular equipment that treating for patient, it enhances the blood circulation in the body to nourish knee and scatic nerve, nerves around the painful area. Table 5. The result of application of low power semicondctor laser to treatdegenerative knee. We have 66 patients VAS Level of painful Before treatment After the second therapy Rating score Number of Percentage Number of Percentage point patients patients 0 No pain Mild, annoying pain 3-4 Nagging, uncomfortable. Trouble some pain Distressing, miserable pain 7-8 Intense, dreadful, horrible pain 9-10 Worst possible, unbearable, excruciating pain Rating point 3.24 corresponding to 1.08 corresponding to mild, distressing, miserable pain annoying pain After two therapies, level of painful at the degenerative knee has change: 21 patients have no pain (31.82%); 25 patients have mild, annoying pain (37.38%); 14 patients have nagging, uncomfortable, rouble some pain (21.21%); 06 patients have distressing, miserable pain (9.09%). At the second therapy, the rating point is corresponding tomild, annoying pain. This value is smaller 3 times than the rating point before treatment. Before After Fig. 3. The X-ray results of Nguyen T.T. ISSN

162 Fig. 4. The X-ray results of Nguyen V.A. before treatment. Fig. 5. The X-ray results of Nguyen V.A. after treatment. IV. DISCUSSIONS Degenerative joint is a disorder in structure and function of the joint. Degenerative joint occurs with the participation of proinflammatory cytokines (Inteiarleukin-1- β, TNFα necrosis factor). There is also the nitric oxide (NO) participate in cartilage catabolism. These factors alter the biology and biochemistry of cartilage and bone under the cartilage: a substrate (proteoglycan) lost, degenerate collagen grid, activate enzymes to digest protein (metalloprotease), results the surface of cartilage gradually thin, fibrosis and patients have expressed pain and joint mobility restriction [2]. A.S. Al Rashoud [3] proceed to use continuous laser with laser beam 830 nm that treating for patients with degenerative knee. After 6 weeks, the results of laser group is higher than the control group s on clinical symptoms and statistics. Patrı cia Pereira Alfredo [4] proceeds to use pulse laser with laser beam 904 nm (4.3 ms) that treating for patients with degenerative joint. After the end of treatment, he realizedthat the level of painful in laser group has significant change (P ¼0.001), moving range (P ¼0.01), function of joint (P ¼0.001) and movement (P <0,001 ). He did not see improvements in the control group [4]. Gladson Ricardo Flor Bertolini [8] has evaluated the effects of two doses of lowlevel laser, at 830 nm, on pain reduction in animals subjected to sciatica. Eighteen rats were used, divided into three groups: GS (n=6), sciatica and simulated treatment; G4J (n=6), sciatica and treatment with 4 J/cm 2 ; and G8J (n=6), sciatica and irradiation with 8 J/cm 2. The right sciatic nerve was exposed and compressed using catgut thread. Five days of treatment were started on the third postoperative day. Pain was assessed by means of the paw elevation time during gait: before sciatica, before and after the first and second therapies, and the end of the fifth therapy. Low-level laser was effective in reducing the painful condition. Rafael Inácio Barbosa [9] has investigated, functional evaluation, the comparative effects of low-power laser (660 nm and 830 nm) on sciatic nerve regeneration 148 ISSN

163 following crushing injuries. Twenty seven Wistar rats subjected to sciatic nerve injury were divided into three groups: group sham, consisting of rats undergoing simulated irradiation; a group consisting of rats subjected to GaAlAs laser at 660 nm (10 J/cm 2, 30 mw and 0.06 cm 2 beam), and another one consisting of rats subjected to GaAlAs laser at 830 nm (10 J/cm 2, 30 mw and cm 2 ). Laser was applied to the lesion for 21 days. A sciatic functional index (SFI) was used for functional evaluation prior to surgery and on days 7, 14, and 21 after surgery. Differences in SFI were found between group 660 nm and the other ones at the 14 th day. One can observe that laser application at 660 nm with the parameters and methods utilised was effective in promoting early functional recovery, as indicated by the SFI, over the period evaluated. Tiina Karu [6] said the essentially effect of low power laser on biological systems is opto-biological reactions. When living tissues absorb photon energy of the laser beam, it happens the restructuring the process of the cell response. The first place receives photon isthe circuit respiration of cell. Kriuk CS [7] usedthe effect of two gas laser beam He-Ne and He-Cd that treating for 448 patients have unhealed wounds; after treatment, the process of wound healing is quickly. When using theeffect of two simultaneous wavelengths, it is capable of healing lesions to area of 100 cm 2 ; while treatment with one wavelength, the maximum healing is 50 cm 2 [6]. When using the effect of two simultaneous wavelengths, it will increase blood circulation to nourish the damaged. It enhances the blood circulation in the body to nourishknee and scatic nerve, nerves around the painful area. V. CONCLUSIONS After the end of treatment, the rate of the good result and the fair result is 81.82%, the rate of the average results is 18.18%. Using the therapy of low power semiconductor laser to treat for sciatica and the degenerative knee is rehabilitation therapy. This is a new treatment method has the following advantages: High effective treatment. Conservation perfectly functional lumbar spine. In the course of treatment is not occur complications and side effects that are harmful to the health of patients. Completely avoid the spread of infectious diseases. Technique treatment is simple, easy universalization. REFERENCES [1] Mary Ellen Wewers, Nancy K. Lowe, A Critical Review of Visual Analogue Scales in the Measurement of Clinical Phenomena, Pubmed, [2] Gary S. Firestein, KELLEY S Textbook of Rheumatology, Elsevier, pp ISSN

164 [3] A.S. Al Rashoud, R.J. Abboud, W. Wang, C. Wigderowitz, Efficacy of low-level laser therapy applied at acupuncture points in knee osteoarthritis: a randomised double-blind comparative trial, Elsevier Ltd on behalf of Chartered Society of Physiotherapy, [4] Patrı cia Pereira Alfredo, Jan Magnus Bjordal, Sı lvia Helena Dreyer, Sarah Ru bia, Ferreira Meneses, Giovana Zaguetti, Vanessa Ovanessian, Thiago Yukio Fukuda, Washington Steagall Junior, Rodrigo A lvaro, Branda o Lopes Martins, Raquel Aparecida Casarott, Efficacy of low level laser therapy associated with exercises in knee osteoar thritis: a randomized double-blind study, Clinical Rehabilitation, [5] Rana S Hinman, Paul McCrory, Marie Pirotta, Ian Relf, Kay M Crossley, Prasuna Reddy, Andrew Forbes, Anthony Harris, Ben R Metcalf, Mary Kyriakides, Kitty Novy and Kim L Bennell, Efficacy of acupuncture for chronic knee pain:protocol for a randomised controlled trial using a Zelen design, BMC Complementary and Alternative Medicine, [6] Tiina Karu, Photobiology of low-power laser effects, Health Physics Society, [7] Kryuk, A.S., Mostovnikov, V, Khokhlov, I.V, Serdyuchenko, N.S., The therapeutic efficiency of low-intensity laser light Minsk: Scinece and Technology, [8] Gladson Ricardo Flor Bertolini, Elisangela Lourdes Artifon,Taciane Stein da Silva, Daniela Martins Cunha, Priscila Regina Vigo, Low-level laser therapy, at 830 nm,for pain reduction in experimentalmodel of rats with sciatica, Arq. Neuropsiquiatr, 69 (2-B): , [9] Rafael Inácio Barbosa, Alexandre Marcio Marcolino, Rinaldo Roberto de Jesus Guirro, Nilton Mazzer, Cláudio Henrique Barbieri, Marisa de Cássia Registro Fonseca, Comparative effects of wavelengths of low-powerlaser in regeneration of sciatic nerve in ratsfollowing crushing lesion, Lasers Med. Sci., 25: , ISSN

165 SUPPORT TUBERCULOSIS TREATMENT USING LOW POWER SEMICONDUCTOR LASER Mai Huu Xuan 1, Tran Minh Thai 1, Ngo Thi Thien Hoa 2, Tran Van Be 3, Huynh Quang Linh 1 1 Laser Technology Laboratory 2 Division Rehabilitation Treatment Tan Chau, An Giang 3 Hospital Blood Transfusion and Hematology Ho Chi Minh City Abstract. In this paper we present a method to support tuberculosis treatment with low power semiconductor laser. The method used and 2 low-power semiconductor lasers with different wavelengths simultaneously creating the effects of 2 wavelengths, including optoacupuncture - optotherapy combined intravenous semiconductor lasers. The semiconductor laser used magnets that acting on acupuncture points according to traditional oriental acupuncture, laser optotherapy semiconductor moped impacts on vulnerable areas of the lung, the intravenous laser makes well circulatory system carrying blood and nutrients to the damaged area multiple, parallel that creates the increased immune helps the body absorb drugs good and strong resistance to anti-tb effectively. I. INTRODUCTION According to the "Report on Global TB Control 2010" of the World Health Organization (WHO), the prevalence of MDR-TB present at unprecedented high levels. In 2008, there are about 440,000 people with MDR-TB. The report also refers to the situation of XDR-TB, a form of tuberculosis is almost incurable. In 2010, 58 countries and territories have reported at least one case of XDR-TB. XDR-TB is a threat to public health may worsen the HIV/AIDS. This situation requires urgent investment countries to upgrade health facilities, strengthening of laboratory and diagnostic facility quickly, early treatment of TB Tuberculosis Diagnosis - The diagnosis of TB decisions now have to rely on the evidence determine the presence of TB bacilli (BK) where lesions by culture techniques or images of tissue typical of tuberculosis. - There are no clinical symptoms and X-ray specific to tuberculosis, because many other diseases have similar symptoms. - Found organize shit necrosis in lesions pathologist, also cannot be confirmed as TB, as it is facing in the granulomatous disease (Sacomdozo, taxes syphilis, fungus, dust lung Beryl, Atypical Mycobacteria form...). - By far one wishes to have specific serology Arif LA et al study found people with HLA - DR2, HLA-BW 15 (+) as the proportion of people with TB 8x (-) calculation. ISSN

166 - The tuberculosis diagnostic methods include: clinical, X-ray, microbiology, reaction. Mantoux and BCG, bronchoscopy, tissue diseases, cytology, blood tests and pleural fluid, try to diagnose treatment Method of Treatment Indications Drug Regimen: - Designated I: 2 S (E) HRZ / 6 HE hoac2 S (E) HRZ / 4 RH new TB - Specify II: 2 SHRZE / 1 HRZE / 5 H3 R3 E3 recurrent TB, failure appointed first, treatment later abandon the treatment, some may tuberculosis - Specify III: 2 HRZE / 4 HR or 2 HRZ / 4 HR Tuberculosis Children TB Program Vietnam stipulated 5 essential anti-tb drugs: Isoniazid (H); Rifampicin (R), pyrazinamide (Z), streptomycin (S) and Ethambutol (E) Traditional Method According to traditional medical treatment, acupuncture on the acupunture points of: - Tuberculosis of the lung: Fei Shu, Da Zhui, Kong Zui, Zu San Li (Sub: Kun Lun, Tai Yuan, Tai Xi, Ge Shu, Zhong Fu, Yin xi, Shen Men, Zhongwan, Tianshu, San yin jiao, Shenshu, Guan yuan) - in medicine Acupuncture - Shanghai. - Tuberculosis hemoptysis: Lie gue, Taiyuan, Chizexich, Zu san li (Shen men, Yunghi, Da ling) [9]. From the above facts we are forming the method to support the treatment of tuberculosis in community. II. OVERVIEW OF RESEARCH AND OCCUPATIONAL THERAPY WITH LOW POWER SEMICONDUCTOR LASER In recent years, appeared some studies of low power laser applications in tuberculosis treatment support. In [1] using a He-Ne laser intravascular gas at wavelengths 632,8 nm with 2 mw power supportive treatment in TB progresses faster and acute for young patients. In [2] presents the results of using intravascular nitrogen laser (wavelength 337 nm, 5 mw capacity) combined with western medicine in treating TB empyema treatment of tuberculosis. In [3] presented the results of using the semiconductor laser at a wavelength of 890 nm, support anti-tb drugs in the treatment of tuberculosis, using semiconductor laser working at a wavelength of 890nm with a capacity of (4-6) mw, frequency modulated screening Hz directly to the lesion in the lung from the patient's skin surface. In [4-6] using low-power semiconductor lasers for the treatment xref support TB and the results obtained earlier sputum conversion from 2-4 weeks. 152 ISSN

167 III. EXPRERIMENTS 3.1 The idea of therapy Using two wavelengths effects simultaneously, by two types of semiconductor laser working at two different wavelengths create, impact directly on the lesion in the lung; combined with the use of semiconductor laser rays lit up the grave impact classics in traditional oriental acupuncture; while closely coordinating with activated immune system by semiconductor laser intravenous treatment in support of tuberculosis in the community Choose the appropriate wavelength for the service support tuberculosis treatment From the simulation results of laser beam propagation work at different wavelengths with low power from the abdomen to the skin surface lungs, using the Monte Carlo method, is done in [1], we choose: - Semiconductor working at a wavelength of 780nm; - Semiconductor working at a wavelength of 940nm; creating the effect of two wavelengths simultaneously serving for the treatment. From the simulation results of laser beam propagation work at different wavelengths with low power from the skin to the tissue surface using the Monte Carlo method, performed in [2], we selected semiconductor lasers work 940 nm wavelength, making optical needle to perform optoacupuncture on classic acupuncture points in traditional Oriental acupuncture. From the research results in [3] we choose a wavelength of 650nm semiconductor laser to perform intravenous laser Mechanisms and Treatment Use of biological responses, by stimulating the biological effects bring, as a tool to implement supportive treatment of tuberculosis in the community As we all know, when a low power laser beam impact on living tissue with the power density in the range of ( ) W/cm 2 with show times from 10 seconds to tens of minutes will occur Brand stimulate biological response. Obviously the aforementioned biological response is an important tool to support implementation of TB treatment in the community with low power semiconductor laser. Researching results experimentally in [7] says: Effects of two wavelengths simultaneously, by two types of semiconductor laser working at a wavelength of 780 nm and 940 nm wavelength work in make up, make the defense response inflammatory, analgesic, regenerate,... happening faster and stronger than each wavelength. ISSN

168 Using two wavelengths effects simultaneously, by two types of semiconductor laser working at a wavelength of 780 nm and 940 nm wavelength work in creating, impacts directly on the lesions in the lungs, caused by tuberculosis. Using semiconductor laser optoacupuncture working at 940 nm wavelength with low power in traditional Oriental acupuncture in the treatment of tuberculosis Specifically, can the use of acupuncture stars here: - Tuberculosis of the lung [9] - Tuberculosis hemoptysis [9] Using low power semiconductor laser stimulation to increase the activation of the immune system to support the treatment of tuberculosis Using two wavelengths effects simultaneously, by two types of semiconductor laser working at a wavelength of 780nm and 940nm wavelength work in creating, directly impact on: the thymus, spleen and lymph nodes to increase activation immune system. - Use optoacupuncture semiconductor laser at a wavelength of 940 nm work impacts on the acupuncture points to increased activation of the immune system [8] - Use semiconductor laser intravenous to increased activation of the immune system [3]. - Using semiconductor laser intravenous working at a wavelength of 650 nm to provide sufficient blood for the lungs, the thymus. Equipment support treatment of tuberculosis in the community with low power semiconductor laser based on the rationale of treatment, discussed above, we offer models supporting TB treatment in communities with low power semiconductor laser For treatment, the need to use two kinds of following equipment: Fig 1. The semiconductor laser optoacupunture optotherapy equipment having 12 channels. Fig 2. The semiconductor laser intravascular equipment. 154 ISSN

169 Equipment optoacupuncture - phototherapy with low power semiconductor laser of 12 channels.semiconductor laser devices internal static opening. Parts of the device treatment consists of a channel, by semiconductor laser working at a wavelength of 650 nm undertaken with the following main parameters: Poweremission changes from (0-5) mw; Frequency modulated beam changes from (5-100) Hz. Modulation frequency is 50Hz frequency we see as broadly nutrition 3.4. Methodology and subjects in the treatment area Methods of clinical studies, there was no control group, self-control based on the following criteria: - Negative or positive sputum. - Health condition. - Increased weight. The total number of patients in the treatment area is 43 people, including: - 30 people with tuberculosis was identified superior hospital and for the local treatment with medicines people with tuberculosis and lung lesions entity. After a long time been treated in hospital, patients were stable and discharged from the hospital, after a while, these patients have difficulty breathing phenomena and health began to decline. - All 43 patients above are voluntarily participating in the research program of treatment with low power semiconductor laser of us. IV. RESULT AND DISCUSSION * Treatment Process Every day, patients must take medication before performing pulmonary tuberculosis treated with low power semiconductor lasers. - First date: patients treated with intravenous semiconductor laser, treatment time from 45 minutes to 60 minutes. - Second day: patients treated with guideline-optical devices - phototherapy with low power semiconductor laser with 12 channels of 60-minute duration and is divided into two stages: ISSN Fig. 3. Immune system. 155

170 + The first stage tuberculosis treatment with durations of 30 minutes. Stage follows: treatment to enhance the immune system of patients with duration of 30 minutes. + On the third and fourth day of treatment repeat as above. * Does the treatment once daily treatment. A course of treatment consists of 20 days of treatment. The evaluation results of treatment are made: - After 3 therapy for patients with tuberculosis in the community. - After 2 therapy for patients with tuberculosis, lung lesions and symptoms were dyspnea - after discharge. * Result: After 3 courses of treatment, 30 patients were completely healed pulmonary TB: - Negative sputum. - Health improved considerably. - Increase of at least 2 kg weight. Fig. 4. We use the semiconductor laser intravascular equipmenttreats for patient. After two treatments, the latter 13 patients completely recover. In the course of treatment does not cause complications and side effects that are harmful to the health of patients. V. CONCLUSION The adjuvant treatment of tuberculosis patients in communities with low power semiconductor laser provides following advantages: - Effective treatment of high, - Shorten the time significantly. This will avoid drug resistance. - The health of patients is significant recovery, - In the course of treatment does not occur complications and side effects that are harmful to the health of patient, - Treatment technique simple, widely popularizing. REFERENCES [1] Tran Minh Thai. Research and application of low-power semiconductor lasers for the treatment of tuberculosis in supporting the community. Ho Chi Minh City, [2] Tran Thi Ngoc Dung.Interaction of semiconductor laser working at near-infrared wavelengths with low power to living tissue. Dissertation, University of Technology - Vietnam National University HCMC, ISSN

171 [3] Tran Minh Thai et al. Semiconductor laser devices intravenous, Proceedings of Conference on Science and Technology 10th, Polytechnic University of Ho Chi Minh City., Sub-Committee: "Optical semiconductor laser motto" pages ( ) [4] T. Karu. Photobiological fundamentals of Low power laser therapy, IEEE. J. Quart. QE. 23, N10, [5] Kriuk C.S. and partner et al. Effective treatment with low power laser, Publisher of Science and Technology, Minck, [6] Mockbin C.B. and partner. Laser therapy moped. Technical Publishing, Mockba, [7] Phan Thi Thanh Thuy. Surveying the effect of semiconductor laser beam moped up drugs for topical anti-inflammatory to inflammatory foot mouse model. Master thesis, specialized "laser technique", [8] Tran Minh Thai et al. Research and application of low-power semiconductor lasers for the treatment of human disease and has had HIV. Proceedings of Conference on Science and Technology of Ho Chi Minh City University of Technology 10th.Subcommittee: Optical semiconductor laser magnets, pages ( ), [9] Le Quy Nguu. The word acupuncture acupuncture position, Traditional Medicine Association of HCMC, 2nd Edition, ISSN

172 APPLICATION OF LOW POWER SEMICONDCTOR LASER TO TREAT DEGENERATIVE LUMBAR SPINE HERNIATED DISC, OSTEOPHYTOSIS Trinh Tran Hong Duyen *, Tran Minh Thai, Ngo Thi Thien Hoa, Bui Chi Hung, Dinh Thi Thu Hong, Ngo Thi Tra Huong, Tran Thanh Vinh, Huynh Thanh Hoa, Tran Thi Ngoc Dung, Nguyen Dinh Quang, Nguyen Minh Chau Laser Technology Laboratory, University of Technology, Vietnam National University HCM, 268, Ly ThuongKiet Street, District 10, Ho Chi Minh City *: Abstract: The purpose of our method is the conservation of the lumbar spine, helping patients quickly off illness, returned to their normal working life.we carried out the low power semiconductor laser therapy for 200 patients with the degenerative lumbar spine osteophytosis and 80 patients with herniated discat lumbar spine.after the end of the treatment, the rate of the good result and the fair result is 85.71%, the rate of the average results is 14.29%. The results of our research prove that the effect of the simultaneous irradiation of two laser beams of 780 nm and 940 nm is high effective than the common treatments. Keywords: Lumbar spine, osteophytosis, herniated disk, the low power semiconductor laser. I. INTRODUCTION Oteoarthritis of lumbar spineis a chronic disease progresses gradually increasing pain, restrict movement, lumbar spine deformation without signs of inflammation. The basic injury of the disease is oteoarthritis of cartilage and spinal disc and changes in the bone under the cartilage, synovial membrane. Patients with pressure overload on articular cartilage and intervertebral disc for many years often lead to articular cartilage lesions, bone under cartilage, loss of elasticity of the discs, sclerotic ligaments, creating the symptoms and complications in osteoarthritis.the current treatments such as physiotherapy, medical therapy (use of analgesics, nonsteroidal anti-inflammatory drugs, muscle relaxant drugs, corticoid injections,...), surgical treatment.these treatments are only solve part of osteoarthritis but these treatments are not conservation of the lumbar spine, helping patients quickly off illness, returned to their normal working life. II. MATERIALS AND METHODS Clinical studies self-controlled, randomized. Because patients in this study were treated with western drugs, traditional medicine, acupuncture combined with stretchable spine, but treatment results are not as expected. Therefore, treatment with low power semiconductor laser is expected method, before they are performing surgery. 158 ISSN

173 Application of low power semicondctor laser to treat degenerative lumbar spine osteophytosis: we have 100 patients at 02 treatment facilities have used equipment low power semiconductor laser. A course of therapy is 20 days. Patients are treated two to three therapies. Table 1. Distribution of patients by age. Age Number of patients Percentage Application of low power semicondctor laser to treat degenerative lumbar spine herniated disc: we have 80 patients at 02 treatment facilities have used equipment low power semiconductor laser. A course of therapy is 21 days. Patients are treated two therapies. The evaluation of treatment outcomes are based on the results of MRI before and after the end of treatment. Table 2. Distribution of patients by age. Age Number of patients Percentage The level of painful is evaluated according to the VAS scale [4] and the overall evaluation of treatment outcomes: Table 3. The VAS scale and the overall evaluation of treatment outcomes. VAS Level of painful score 0 No pain 1-2 Mild, annoying pain 3-4 Nagging, uncomfortable. Trouble some pain 5-6 Distressing, miserable pain 3 ISSN Rating point Activity levels of the spine and the body - No restriction gestures: bending down, leaned back, tilt, turned his back. - The degree of bending: distance hand - ground < 20cm. - Normal movement - Limiting the part of movement: bending down, leaned back, tilt, turned his back. - The degree of bending: distance hand ground from 20 cm to 30cm. - Normal movement but, he only works for a short time - Limiting many movements: bending down, leaned back, tilt, turned his back. - The degree of bending: distance hand ground from 31cm to 40cm - Restricting the movement by pain - Limiting many movements: bending down, leaned back, tilt, turned his back. - The degree of bending: no result (> 60 Treatment result good fairly good medium 159

174 7-8 Intense, dreadful, horrible pain 9-10 Worst possible, unbearable, excruciating pain 4 5 cm) - The movements have difficult. - Restricting the movement totally: bending down, leaned back, tilt, turned his back. - The degree of bending: no result (> 60 cm) - The patient could not move, he just sits in a wheelchair or lies in bed. - None bending down, leaned back, tilt, turned his back. - The degree of bending: no result (> 60 cm) - The patient could not move, he just sits in a wheelchair or lies in bed. bad III. RESULTS We use the semiconductorlaser worksat a 940 nm wavelength impacts directly on acupuncture pointsto treatthe degenerative lumbar spine osteophytosis, herniated disc and theeffectof twosimultaneous wavelengths by semiconductor laser worksat 780nm and 940nm wavelengthsdirect treatments on the lower back pain area. We use the semiconductor laser intravascular equipment that treating for patient. It enhances the blood circulation in the body to nourish the damaged disc and nerves around the painful area. Fig. 1. The semiconductor laser optoacupunture optotherapy equipment having 12 channels. Fig. 2. The semiconductor laser intravascular equipment. Application of low power semicondctor laser to treat degenerative lumbar spine osteophytosis: 160 ISSN

175 Table 4. The result of application of low power semicondctor laser to treat degenerative lumbar spine osteophytosis. We have 100 patients VAS Level of painful Before treatment After the first therapy After the second score therapy Number Perce Number Percenta Number of Perce of ntage of ge patients ntage patients patients 0 No pain Mild, annoying pain Nagging, uncomfortable. Trouble some pain Distressing, miserable pain 7-8 Intense, dreadful, horrible pain 9-10 Worst possible, unbearable, excruciating pain Rating point 2.85 corresponding to nagging, uncomfortable corresponding to mild, annoying pain 0.04 corresponding to no pain Ratin g point After two therapies, level of painful at the degenerative lumbar spine osteophytosis has change: 96 patients have no pain (96%) and 04 patients have mild, annoying pain (4%). At the second therapy, the rating point is corresponding to no pain. This value is smaller times than the rating point before treatment. Application of low power semicondctor laser to treat degenerative lumbar spine herniated disc: Table 5. The result of application of low power semicondctor laser to treat degenerative lumbar spine herniated disc We have 80 patients VAS Level of painful Before treatment After two therapies Rating score Number of Percenta Number of Percentage point patients ge patients 0 No pain Mild, annoying pain 3-4 Nagging, uncomfortable. Trouble some pain Distressing, miserable pain 7-8 Intense, dreadful, horrible pain Worst possible, unbearable, excruciating pain Rating point corresponding to nagging,uncomfortable corresponding to mild, annoying pain 5 ISSN

176 After two therapies, level of painful at the degenerative lumbar spine herniated disc has change: 12 patients have no pain (15%) and 28 patients have mild, annoying pain (35%), 34 patients have nagging, uncomfortable, trouble some pain (42.5%), 06 patients have distressing, miserable pain (7.5%). At the second therapy, the rating point is corresponding tomild, annoying pain. This value is smaller 2.63 times than the rating point before treatment. Before treatment After treatment Doctor evaluates the level recovery of Tran T.D. is: Before treatment The hasdegenerative lumbar spine herniated disc. Bulge disc in the behind of L2/3 with d = 6 mm, pinched front cover membrane, narrow niches on either side, narrow hole conjugate sides, no pinched nerve roots sides. Bulge disc in the behind of L3/4 with d = 6mm,pinched front cover membrane, narrow niches on either side, narrow hole conjugate sides, pinched L3 nerve roots in both sides. After treatment Fig. 3. The MRI results of Tran T.D. (56 year olds). He has degenerative lumbar spine herniated disc. Bulge disc in the behind of L2/3 with d = 2 mm, pinched front cover membrane, narrow niches on either side, narrow hole conjugate sides, no pinched nerve roots sides. Bulge disc in the behind of L3/4 with d = 4mm,pinched front cover membrane, narrow niches on either side, narrow hole conjugate sides, pinched L3 nerve roots in both sides. Doctor evaluates the level recovery of Vo T.T. is: 162 ISSN

177 Fig. 4. The MRI results of Vo T.T. (60 year olds) before treatment. Fig. 5. The MRI results of Vo T.T. (60 year olds) after treatment. Before treatment The hasdegenerative lumbar spine herniated disc. Bulge disc in the behind of L1/2 and L4/5 with d = (6 7) mm, pinched front cover membrane, narrow niches on either side, narrow hole conjugate sides, slightly pinched nerve roots in both sides. After treatment The hasdegenerative lumbar spine herniated disc. Bulge disc in the behind of L1/2 and L4/5 with d = 4 mm, pinched front cover membrane, narrow niches on either side, narrow hole conjugate sides, slightly pinched nerve roots in both sides. ISSN

178 IV. DISCUSSIONS Tiina Karu [5] said the essentially effect of low power laser on biological systems is opto-biological reactions. When living tissues absorb photon energy of the laser beam, it happens the restructuring the process of the cell response. The first place receives photon isthe circuit respiration of cell. Kriuk CS [6] usedthe effect of two gas laser beam He-Ne and He-Cd that treating for 448 patients have unhealed wounds; after treatment, the process of wound healing is quickly. Milica Jovicic [1] used semiconductor laser beam 904nmthat treating for 66 patients with low back pain; after treatment, the VAS score at reduced to 3-4. Ali Gur [2] used laser beams 600 nm and 984 nm that treating for patients with low back pain, the results show that treating by laser beams combine with physical therapy making level of painful in patients are a significant reduction. Ryan C. Petering [3] proceed to treat for 546 patients with low back pain by low power laser with laser beams 632 nm and 904 nm, the results show that patients in the laser group have a significant reductionthan the control group s. These results show that using low-power laser therapy in clinical becomes more popular and has achieved many good results and high efficiency. When using theeffectof twosimultaneous wavelengths, it is capable of healing lesions to area of 100 cm 2 ; while treatment with one wavelength, the maximum healing is 50 cm 2 [5]. When using theeffectof twosimultaneous wavelengths, it will increase blood circulation to nourish the damaged. It enhances the blood circulation in the body to nourish the damaged disc and nerves around the painful area. V. CONCLUSIONS After the end of treatment, the rate ofthe good result and the fair result is 85.71%, the rate of the average results is 14.29%. Using the therapy of low power semiconductor laser to treat for the degenerative lumbar spine osteophytosis, herniated disc is rehabilitation therapy. This is a new treatment method has the following advantages: High effective treatment. Conservation perfectly functional lumbar spine. In the course of treatment is not occur complications and side effects that are harmful to the health of patients. Completely avoid the spread of infectious diseases. Technique treatment is simple, easy universalization. REFERENCES [1] Milica Jovicic, Ljubica Konstantinovic, Milica Lazovic, Vladimir Jovicic, "Clinical and functional evaluation of patients with acute low back pain and radiculopathy treated with different energy doses of low level laser therapy," Vojnosanit Pregl, [2] Ryan C. Petering and Charles Webb, "Treatment Options for Low Back Pain in Athletes," SPORTS HEALTH, ISSN

179 [3] Ali Gur, Mehmet Karakoc, Remzi Cevik, Kemal Nas, Aysegul Jale Sarac and Meral Karakoc, "Efficacy of Low Power Laser Therapy and Exercise on Pain and Functions in Chronic Low Back Pain," Lasers in Surgery and Medicine, [4] Mary Ellen Wewers, Nancy K. Lowe, "A Critical Review of Visual Analogue Scales in the Measurement of Clinical Phenomena," Pubmed, [5] Tiina Karu, "Photobiology of low-power laser effects," Health Physics Society, [6] Kryuk, A.S., Mostovnikov, V, Khokhlov, I.V, Serdyuchenko, N. S, "The therapeutic efficiency of low-intensity laser light," Minsk: Scinece and Technology, ISSN

180 CONDITIONS TO STRETCH PLASMID DNA MOLECULE IN OPTICAL TWEEZERS USING CW LASER GAUSSIAN BEAM Thai Dinh Trung 1, Mai Van Luu 1, Chu Van Lanh 1, Nguyen Van Thinh 2, Hoang Van Nam 3, Thai Doan Thanh 4, Ho Quang Quy 5* 1 Faculty of Physics and Technology, Vinh University, 182 Le Duan, Vinh City, Vietnam 2 Faculty of Natural Sciences, Baclieu University, Baclieu City, Vietnam 3 Hatinh University, Hatinh City, Vietnam 4 Hochiminh University of Food Industry, Tanphu, Ho Chi Minh City, Vietnam 5 Academy of Military Science and Technology, 17 Hoang Sam, Hanoi, Vietnam *: hoquangquy@gmail.com Abstract. We present the dynamics of trapped bead linking to plasmid DNA molecules in an optical tweezers. Using the finite difference Langevin equation (FDLE) the stretching processes of plasmid DNA molecule in CW regime are simulated. The conditions to stretch plasmid DNA molecule in stretching state are discussed. Keywords: Optical tweezers, DNA molecule, Brownian force, Elastic force, Extension. I. INTRODUCTION Up to now, there are many works interesting in the use of optical tweezers to trap the biological molecules, especially the DNA proteins [1-8]. In almost of the works, the optical tweezers were used to measure the extension force of all phage of DNA [1-14, 17-20], only. Lately, the equation of extension force of DNA molecule is modified [21] and the dynamics of polystyrene microsphere linking to λ-phage DNA molecule in optical tweezers are simulated [22]. In the last work, we have investigated the influence of all parameters as the peak intensity, duration and beam waist of the laser Gaussian beam as well as the initial position of trapped beat on the pulling and stability times. The results showed that, due to the contour length is much longer than the waist of trapping laser beam, so before doing a trapping process, the trapped bead must be pulled by certain ways into irradiated region, and in opposite, the λ-phage DNA molecule is not possible in full stretching state. There are questions that what is the DNA molecule could be stretched by optical tweezers, and what are the conditions to stretch it at the contour length? To answer above questions, in this paper using FDLE, we have simulated the dynamics of trapped bead, i.e., the polystyrene microsphere linking to plasmid DNA molecule in CW regime. The conditions to stretch plasmid DNA molecule are discussed. II. FINITE DIFFERENCE LANGEVIN EQUATION 2.1. The model of optical tweezers for DNA molecule 166 ISSN

181 Consider plasmid DNA molecule is embedded in a suitable fluid, i.e., the refractive indexes ratio m>1 [23, 24]. One end of DNA molecule is linked to a glass cover slip through a surface-anchored RNA (ribonucleic acid) polymerase, the opposite end is attached to polystyrene microsphere (trapped bead), which is captured or held under tension with optical tweezers, and its position is monitored by a CW laser Gaussian beam. Fig. 1. a) Cartoon of an often-used experimental geometry; b) The beginning set-up of trapped bead in tweezers [11, 13]. The often-used experimental geometry of DNA molecule with optical tweezers is shown in Fig. 1a. The arm of optical tweezers is to keep the DNA molecules in stretching state or detaching double-stranded DNA molecules. The attached bead plays the role linking DNA molecules to center of optical tweezers and glass cover slip. As shown in Fig. 1a, the bead is under acting of three forces: the elastic force of DNA molecules, Brownian force of fluid and optical force Finite difference Langevin equation The general Langevin equation (GLE) describing the dynamic of bead linking to DNA molecules in optical tweezers [25]: mρ t = γρ t + F gr,ρ ρ t F el ρ t + 2k B TγW ρ (t) (1) where m is the bead mass, γ = 6πηa is the friction coefficient, h is the viscosity of fluid, a is the radius of bead, W ρ (t) is the white noise at position ρ(t), F gr,ρ ρ t ISSN is the transverse gradient optical force, which depends on the intensity distribution of laser beam, radius of trapped bead, and polarizability of the bead in the fluid [21, 26, 32], F el ρ t is the elastic force, which depends on the extension of DNA molecule ρ i ρ 0 (Fig. 1b), k B =1.38x10-23 J/K is the Boltzmann constant, and T is absolute temperature (K) [21]. The term in the left of Eq. 1 is inertial. In the right of Eq. 1, the first term is friction, second is restoring, third is stretching and the last is white noise. Finite difference simulation of GLE are straight-forward: the continuous-time solution ρ t of an GLE is approximated by a discrete-time sequence ρ i, which is the solution of corresponding finite difference equation (FDE) evaluated at regular time steps t i =i t. If D t is sufficiently small, ρ i ρ(t i ), a FDE is obtained from the GLE as follows [21, 22, 25, 26]: 167

182 tρ i = ρ i 1 + F gr,ρ ρ i 1 +F el (ρ i 1 ) γ + 2k B T/γ( W 1 W 2 W 3 W 4 ) t (2) which is called as a finite different Langevin equation (FDLE), where W i, i=1,2,3,4 are random values of white noise at t i [15, 33]. The solution is obtained by solving the resulting FDLE recursively for ρ i, using the values ρ i-1 and ρ i-2 from previous iterations. We consider a polystyrene microsphere plays a role of trapped bead with radius a 0.05 μm [15, 27], average density of 1.35g/cm 3 [24, 28], and its mass of m 1.5x10-18 kg. The bead is embedded in water with viscosity η=0.001 Ns/m at temperature T of 300 K [15] so the friction coefficient is γ = 6πηa = 94.3x10 10 kg/s. Thus, the momentum relaxation time τ = m γ 0.17x10 8 s is much smaller than the time scales of typical experiments [29], consequently, it is often possible to drop the inertial term (i.e. set m=0). III. STRECHING PROCESS OF PLASMID DNA MOLECULE As an example for simulation, we consider a single plasmid DNA molecule with ionic condition of 10 mm Na + having a stable length of L b =47 nm and contour length of L=1.33 mm [18] is attached to a polystyrene microsphere playing a role of trapped bead with radius of a =0.25 mm, refractive index of n b =1.57, [25, 30] which is embedded water with refractive index of n f =1.326 [27, 31] viscosity η= Ns/m at temperature T of 300K [15]. The optical tweezers is using a CW laser Gaussian beam with wavelength of l=1.06 mm, which can be focused by lens so that its waist is constant and peak intensity can be changed. The trapped bead is placed at the beginning position, ρ 0 (mm) with a distance of L set L-L b ( m) on the left from the center of tweezers. That means ρ 0 =-L set L b -L as shown in Fig. 1b. Fig. 2. Stretching process of DNA molecule at initial position of ρ μm for the case of I 0 =10 2 W/cm 2, W 0 =1 μm. The dynamic, i.e., the position-time characteristic of polystyrene bead in optical tweezers describing the called stretching process of plasmid DNA molecule is simulated by Eq. (2) with time step of 1μs (Δt=1x10-9 s) for the case of CW laser Gaussian beam with 168 ISSN

183 peak intensity of I 0 =10 2 W/cm 2 and beam waist of W 0 =1 μm, and the bead s initial position ρ μm, is illustrated in Fig. 2. As is shown in Fig. 2, after a pulling time about T p 65 ms the trapped bead is kept stability in the called stable position of ρ st =-0.69 μm, and the maximum stretched length L st =ρ st - ρ 0 =0.64 μm. That means, at stable position the elastic force is balance with optical one and the maximum stretched length, will be constant (L st =0.64 μm=const) after the pulling time, T p 65 ms. To enhance the maximum stretched length, i.e., to pull the trapped bead more near to tweezers center, the peak intensity must be increased. That can be seen in Fig. 3, which describes the stretching processes of plasmid DNA molecule with different peak intensities. Fig. 3. Stretching processes of plasmid DNA molecule at initial position of ρ μm for different peak intensities I 0 =5x10 2 W/cm 2 (1), I 0 =10 3 W/cm 2 (2), I 0 =5x10 3 W/cm 2 (3) and I 0 =10 4 W/cm 2 (4), and W 0 =1 μm. The results show that with increasing of peak intensity, the maximum stretched length of plasmid DNA molecule increases, meanwhile the pulling time reduces with increasing of peak intensity. It is more clearly in Fig. 4, which illustrates the dependence of the maximum stretched length and pulling time on peak intensity. There are two asymptotes for pulling time lim l0 T p = 0, and maximum stretched length lim l0 L str = L. ISSN Fig. 4. Stretched length (a) and pulling time (b) vs. peak intensity when ρ 0 =1.33 μm=l. 169

184 Since the peak intensity of conditional laser beam has a certain limited value, i.e., I 0, the trapped never reaches the center of tweezers (it means ρ st <0 always), and the plasmid DNA molecule is never be in stretching state (it means L st <L always) when initial position is of ρ 0 =1.33 μm. From Fig. 4, we also see that, it is most sensible to choice the maximum stretched length of L str =1.2 μm 90%L, for which the plasmid DNA molecule could be seen to be in stretching state with reduced pulling time by increasing the peak intensity. Thus, now we find the peak intensity for that the trapped bead is stable in center of tweezers. For this purpose the stretching processes of plasmid DNA molecule at initial position of ρ 0 =1.2 μm for different peak intensities are simulated and illustrated in Fig. 5a. Fig. 5. a) Stretching process of plasmid DNA molecule at initial position of ρ 0 =1.2 μm (90% of contour length) using laser beam with beam waist W 0 =1 μm and different peak intensities I 0 =5x10 2 W/cm 2 (1), I 0 =10 3 W/cm 2 (2), I 0 =5x10 3 W/cm 2 (3) and I 0 =10 4 W/cm 2 (4).b) Stretching process of λ-phage DNA molecule at initial position of ρ 0 =14 μm (90% of contour length) using laser beam with W 0 =1 μm and peak intensities I 0 =10 6 W/cm 2. From Fig. 5a, we can see that in this case, to stretch a plasmid DNA molecule to state of stretched length of L str =1.2 μm, it is convenient to enhance the peak intensity of laser beam with W 0 =1μm to I 0 =1x10 6 W/cm 2, which is the obtainable value from a popular laser used for optical tweezers. Moreover, with this peak intensity the pulling time reduces to T p =0.03 ms. That means, to pull trapped bead into center tweezers and to stretch a plasmid DNA molecule in stretching state it must use a maximum laser energy of E = I 0 π ρ 0 2 T p, only, which is more smaller than that for a λ-phage DNA molecule, 1x10-2 J (Fig. 5b). IV. CONCLUSION The stretching processes of the plasmid DNA molecule with ionic condition of 10 mm Na + linking to a polystyrene microsphere placed at different initial positions in optical tweezers using CW laser Gaussian beam with given beam waist in scale of contour length and controllable peak intensity are investigated. The dependence of the pulling time, stable position of trapped bead and the maximum stretched length of DNA molecule on the peak intensity of laser beam is simulated, and the condition for that the trapped bead stable in center of tweezers is discussed. The results show that the pulling time and peak intensity (or maximum energy) of laser beam to trap a plasmid DNA molecule are reduced in comparison to that to trap a λ-phage one. 170 ISSN

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187 APPLICATION OF LOW POWER SEMICONDUCTOR LASER IN MINIMIZING HARMFUL SIDE EFFECTS AFTER CHEMOTHERAPY AND RADIATION THERAPY IN CANCERS Tran Minh Thai 1, Ngo Thi Thien Hoa 2, Tran Thien Hau 1*, Dang Nguyen Ngoc An 1, Le Quoc Dat 1 1 Laser Technology Laboratory, University of Technology, Vietnam National University Ho Chi Minh City, 268, Ly Thuong Kiet Str., Distr. 10, Ho Chi Minh City, Vietnam 2 The Department of Treatment and Rehabilitation in Tan Chau town, An Giang Province *: tthienhau@hcmut.edu.vn Abstract. Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body [1, 2]. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include a lump, abnormal bleeding, prolonged cough, unexplained weight loss and a change in bowel movements [3]. While these symptoms may indicate cancer, they may have other causes [3]. Over 100 cancers affect humans [2]. Many treatment options for cancer exist. The primary ones include surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy and palliative care. We practiced clinical research: total patients treated is twenty people who were radiotherapy and chemotherapy. The main contents of this treatment method, including: Used intravenous semiconductor laser working at 650 nm wavelength to increase blood flow with high quality to provide cancer patients. Incorporated to use the effect of the two simultaneous wavelengths by two semiconductor lasers work at two different wavelengths created, making the biological responses by biostimulation occurs faster and stronger, when the laser beams directly impact on the cells damaged by chemotherapy and radiotherapy in human. At the same time, combining with the use of optoacupuncture by semiconductor laser working at a 940 nm wavelength impacts directly on acupuncture points. Incorporated with strengthening the immune system for cancer patients in three methods: - Using the effect of the two simultaneous wavelengths by two semiconductor lasers work at two different wavelengths impacts on the thymus gland, spleen, and lymph nodes. - Using optoacupuncture by semiconductor laser working at a 940 nm wavelength to impact on acupuncture points. Using intravenous semiconductor laser to adjust specific immune system and not specific Improving liver function, to discharge of harmful substances out of your body, by three methods: - Using the effect of the two simultaneous wavelengths by semiconductor laser impact on liver tissue from abdomen skin. ISSN

188 - Using optoacupuncture by semiconductor laser works at a 940 nm wavelength impacts directly on acupuncture points to treat different types of hepatitis. - Using intravenous semiconductor laser to increase high quality blood flow to liver. Results showed that this treatment method has some initial advantages as follow to minimize harmful side effects after radiotherapy and chemotherapy in cancers, no complication and side effect occurred during treatment, to increase physical body in cancers after radiotherapy and chemotherapy to a significant level, simple technology and easy to widely popular, treatment form: outpatient, convenient for family care patients. Keywords: Cancer, chemotherapy, radiation, low power semiconductor laser, side effects I. INTRODUCTION Cancer treatment mostly surgery to remove the tumor, in combination with chemotherapy and radiation therapy. However, beside of the positive effect of chemotherapy and radiation therapy to destroy cancer cells, but it is still many side effects harm to healthy cells, neighborhood cells, especially blood cells is generated from bone marrow, hair cells, cells in the mouth, digestive organ, heart, lungs and the reproductive system, influence on the health of patients [6]. Frequently side effects after chemotherapy and radiotherapy of cancers are: Reduce resistance, Anemia, Thrombocytopenia, Mouth and Throat Problems, Nausea & Vomiting, Fatigue, Appetite Loss, Hair Loss (Alopecia), Sore Mouth, Diarrhea. Special attention to, the cancer increases the risk of thrombosis and the chemotherapy endangering the life [5, 6]. Actually the most of cancers only focused on treatment that do not focused on limited harmful side effects of chemotherapy and radiotherapy. This is the cause of exhaustion, the health reduce seriously. According to statistics, 30% of Cancers die from exhaustion before died of cancer cells. Therefore, the World Health Organization (WHO) is improving the quality of life considered as a permanent goal during cancer treatment. So, we suggested The program of application research of low power semiconductor laser in minimizing harmful side effects after radiotherapy and chemotherapy in Cancers. II. EXPERIMENTS The main contents of this treatment method, including: o Used intravenous semiconductor laser working at 650 nm wavelength to increase blood flow with high quality to provide cancer patients. o Incorporated to use the effect of the two simultaneous wavelengths by semiconductor laser works at 780 nm and 940 nm wavelengths created, making the biological responses by bio-stimulation occurs faster and stronger, when the laser beams directly impact on the cells damaged by chemotherapy and radiotherapy in human. At the same time, combining with the use of optoacupuncture by semiconductor laser 174 ISSN

189 working at a 940 nm wavelength impacts directly on acupuncture points in the Traditional Oriental acupuncture. o Incorporated with strengthening the immune system for cancer patients in three methods: Using the effect of the two simultaneous wavelengths with semiconductor laser of 780 nm and 940 nm wavelengths impacts on the thymus gland, spleen, and lymph nodes. Using optoacupuncture by semiconductor laser working at a 940 nm wavelength to impact on acupuncture points: Hegu, Quchi, Zusanli, Xuanzhong, Sanyinjiao, Da Zhui. Using intravenous semiconductor laser to adjust specific immune system and not specific Improving liver function, to discharge of harmful substances out of your body, by three methods: Using the effect of the two simultaneous wavelengths by semiconductor laser impact on liver tissue from abdomen skin. Using optoacupuncture by semiconductor laser works at a 940 nm wavelength impacts directly on acupuncture points to treat different types of hepatitis. Using intravenous semiconductor laser to increase high quality blood flow to liver. Treatment Equipment: The intravascular semiconductor laser equipment (Fig. 1) and the optoacupuncture - optotherapy equipment by low power semiconductor laser of 12 channels (Fig. 2). Laser Technology Laboratory researched and manufactured these equipments. Fig. 1. The intravascular semiconductor laser equipment. Fig. 2. The optoacupuncture - optotherapy equipment by low power semiconductor laser has 12 channels. III. RESULTS AND DISCUSSIONS Clinical treatment: Clinical research no control group, self-control before and after treatment with low power semiconductor laser, according to the following criteria on: ISSN

190 The clinical symptoms: condition of patient. The sub-clinical symptoms: - Concentration of erythrocytes, leukocytes. - Concentration of Liver enzymes such as: AST, ALT, GGT - Concentration of Triglycerides in blood. Treatment process: Patients were treated once a day. The first day, they are treated by the intravascular semiconductor laser with (45 60) minutes. This therapy increases fully blood flow to their body with high quality; especially to provide the injured areas, to spine, to liver, and to active immune system, The second day, they were treated with the optoacupuncture - optotherapy equipment by low power semiconductor laser 12 channels with 60 minutes, divided into 3 periods: The first period of 20 minutes: - Using the effect of two simultaneously wavelengths directly impact on the injured areas caused by radiotherapy and chemotherapy. - Using optoacupuncture by semiconductor laser on acupuncture points in Traditional Oriental acupuncture to treat the injured areas caused by radiotherapy and chemotherapy. The second period of 20 minutes: - Using the effect of two simultaneously wavelengths impact on the liver from abdominal skin surface. - Using optoacupuncture impacts on acupuncture points in Traditional Oriental acupuncture to enhance liver detoxification. The third period of 20 minutes: - Using the effect of two simultaneously wavelengths impact on the thymus, spleen and lymph nodes to activate the immune system. - Using optoacupuncture by semiconductor laser working at a 940 nm wavelength impacts on acupuncture points: Hegu, Quchi, Zusanli, Xuanzhong, Sanyinjiao, Da Zhui. The third and fourth day, this treatment cycle is repeated. A treatment course induces 20 treatment days. We will evaluate treatment results after three treatment courses. Patients in Clinical Research: Total patients treated is twenty people who were radiotherapy and chemotherapy. Distribution of cancer patients as follows: Nasopharyngeal cancer: 06 people, accounting for thirty percents (30%). Breast cancer: 06 people, accounting for thirty percents (30%) 176 ISSN

191 Lung cancer: 04 people, accounting for thirty percents (20%) Ovarian cancer: 04 people, accounting for thirty percents (20%) The average age of patients is years old. They treated to minimize harmful side effects after chemotherapy and radiation therapy in cancers, the same method, but in three different medical establishments. Treatment results Results of painful relief after treatment by VAS scale (Visual Analog Scale) Fig. 3. The painful expression on his face. Table 1. Assessing the level of painful according to VAS scale. VAS = 0 No pain 0 point 4 6 Distressing severe pain 0 2 Mild, annoying pain 1 point 6 8 Intense very severe pain 2 4 Discomforting 2 points 8 Unbearable pain moderate pain 10 3 points 4 points 5 points Table 2. The analgesic efficacy according to VAS scale after treatment with low power semiconductor laser in cancer patients after radiotherapy and chemotherapy. VAS (points ) Before treatment After treatment Mild, annoying pain Discomforting moderate pain Distressing severe pain Intense very severe pain Unbearable pain Average pain 63/20=3,15 28/20=1,40 In Table 2, we found that average pain after treatment is 1.40 points less than average pain before treatment 3.15 points ( 2.25 times). This difference was statistical significance. Results of haematological indices ISSN

192 Red blood cells (RBCs) indices: Table 3 showed average values of haematological indices in cancer patients after radiotherapy and chemotherapy (before treatment) and average values after three treatment therapies by low power semiconductor lasers. Table 3. The average values of hematological indices in cancer pantients before and after treatment by low power semiconductor laser. Total patients: 20 people Before treatment After treatment The average values of hematological indices The average values of hematological indices 2,905 M/ L 3,635 M/ L In Tab. 3, we found that after radiotherapy and chemotherapy the hematological values are very low, only 83% of the normal values ( ) M/ L. After they were treated by low power semiconductor laser, the hematological values is gradually rised and after the end of treatment the average hematological values achieved M/ L, greater than the lowest normal value is 3.86%. White blood cell (WBC) index: Tab.4 showed average values of white blood cell in cancer people after radiotherapy and chemotherapy, the above average values before and after three times treatment. Table 4. Average value of WBC indexes before and after treatment. Total patients: 20 people Before treatment After treatment The average value The average value 14,5 K/ L 9,35 K/ L In Table 4, we found that after radiotherapy and chemotherapy, the average value of WBC indexes in lung cancer is very high, it greater than the highest values of normal value (4-10) K/ l is 1.45 times. This value decreased after treatment, the average value of WBC indexes achieve 9.35 K/ l the normal value. This means that the lesions by radiotherapy and chemotherapy gradually rehabilitated with this therapy. Results of rehabilitation of liver function In liver cells, there are two types of enzymes: ALT (Alanine Aminotransferase) is also known as SGPT and AST (Aspartate Aminotransferase) is also known as SGOT, they have concentration very low in blood: less than 40 U/L (units in 1 liter). When liver cells are inflamed or gangrened, the enzymes from the liver cells are released into the blood. Therefore, increased liver enzymes is an indirect sign that the state of liver cells is destroyed. ALT is considered specific for hepatitis, while AST also increased in other diseases such as: myocardial infarction, myositis,... There is also the GGT enzyme (Gamma Glutamyltranspeptidase). When this enzyme increases also said that: liver disease by alcohol, hepatitis by chemicals. Normal values of above liver enzymes as follows: ALT (SGPT) 40U/L. 178 ISSN

193 AST (GOT) 40U/L. GGT: Men: (11 50) U/L and Women: (7 32) U/L. In Table 5, we presented the average value of liver enzymes AST, ALT and GGT after radiotherapy and chemotherapy in 20 cancer patients after treatment. Table 5. Average value of liver enzyme before and after treatment. Total patients: 20 people The average value of liver enzyme The average value of liver enzyme after before treatment treatment AST: 60,183 u/l AST: 38,657 u/l ALT: 62,13 u/l ALT: 37,51 u/l GGT: 76,32 u/l GGT: 50,48 u/l Normal values: AST 40u/L, ALT 40u/L, GGT 50u/L In Table 5 after radiotherapy and chemotherapy, we have the average value of liver enzyme higher than normal value, as: Liver enzymes of ALT higher normal value 1,505 times. Liver enzymes of AST higher normal value times. Liver enzymes of GGT higher normal value 1,526 times. Especially, AST enzyme characterizes for the liver damages, and GGT enzyme characterizes for liver damages by chemicals. After the end of treatment, the average value of liver enzymes reduced and achieved normal values. This is no small accomplishment. Because it helps the livers to complete detoxification Result of Triglyceride. We interested in levels of triglycerides in the blood of cancer patients after radiotherapy and chemotherapy. When concentration of triglycerides in the blood increases highly, it creates a high risk for the disease: heart, blood vessels, kidneys,... On Table 6 we presented average value of triglycerides in the blood of cancer patients after radiotherapy and chemotherapy. Table 6. Average values of triglycerides in the blood of cancer patients after radiotherapy and chemotherapy and after treatment. The average values of triglycerides in the blood of cancer patients after radiotherapy and chemotherapy 203,2mg/dl High risk for the disease: heart, blood vessels, kidneys, Total patients: 20 people The average values of triglycerides in the blood of cancer patients after treatment 143,3 mg/dl Normal value ISSN

194 In Table 6, we found that the average value of triglycerides in the blood of cancer patient after radiotherapy and chemotherapy achieved mg/dl. This value is higher than allowed normal value (150 mg/dl) is 1,355 times. According to [4] said, level of above triglycerides is very high in blood is "high risk" of heart disease, blood vessels, kidneys,... After the end of 03 treatment courses, the average values of triglyceride levels achieved 143,3 mg/dl - the normal value, reduced by 1,355 times with before treatment. This result shows that this method removes "high risk" for heart disease, blood vessels, kidneys,... in cancer patients after treatment with radiotherapy and chemotherapy. IV. CONCLUSIONS Treatments minimize harmful side effects after radiotherapy and chemotherapy in cancer patients with low power semiconductor laser is a new treatment method. This treatment method has some initial advantages as follow: To minimize harmful side effects after radiotherapy and chemotherapy in cancers. No complication and side effect occurred during treatment. To increase physical body in cancers after radiotherapy and chemotherapy to a significant level. Simple technology and easy to widely popular. Treatment form: outpatient, convenient for family care patients. REFERENCES [1] Cancer Fact sheet N 29. World Health Organization. February Retrieved 10 June [2] Defining Cancer. National Cancer Institute. Retrieved 10 June [3] Cancer - Signs and symptoms. NHS Choices. Retrieved 10 June [4] Nguyen Phu Khang. Clinical. Cardiovascular. Medicine Publisher. (2001). [5] A.P. Gautam et al. Low level laser therapy against radiation induced oral mucositis in elderly head and neck cancer patients-a randomized placebo controlled trial. Journal of Photochemistry and Photobiology B: Biology 144 (2015) p [6] Early Breast Cancer Trialists Collaborative Group (EBCTCG). Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and15-year survival: an overview of the randomised trials. Vol 366 December 17/24/31, ISSN

195 INITIAL RESULT OF THE TREATMENT OF FATTY LIVER BY LOW POWER SEMICONDUCTOR LASER Tran Minh Thai 1, Tran Thien Hau 1*, Ngo Thi Thien Hoa 2, Nguyen Pham Song Toan 1 1 Laser Technology Laboratory, University of Technology, Vietnam National University 268, Ly Thuong Kiet Str., Distr. 10, Ho Chi Minh City, Vietnam 2 The department of treatment and rehabilitation in Tan Chau town, An Giang Province *: tthienhau@hcmut.edu.vn Abstract. In this report, we present initial results to treat fatty liver by low power semiconductor laser with main contents consists of: Actively promoting process of destroying the fatty liver with inflammatory symptoms and preventing liver fibrosis and liver cirrhosis by the methods: - Using intravenous semiconductor laser to increase high quality blood flow to liver to rehabilitate liver damages. On the other hand, to treat hyperlipidermia And decreasing the fasting glycemic index in people with type 2 diabetes on normal value, or prediabetes value. - At the same time, using the effect of the two simultaneous wavelengths by two semiconductor lasers work at two different wavelengths making the biological responses by bio-stimulation occurs faster and stronger, when the laser beams directly impact on liver and on pancreas to treat the rehabilitation for liver disease and damages of pancreas. - Using optoacupuncture by semiconductor laser impacts directly on acupuncture points to treat different types of hepatitis and type 2 diabetes. The total number of patients in clinical research are 32 people with mild and moderate fatty liver. Clinical results showed that: - Treatment is highly effective for two types of fatty livers: fatty liver caused by the food contaminated with chemicals. And fatty liver by metabolic disturbances of hyperlipidemia ingredients. - No complications and side effects occurred during treatment. - Completely conserving the physiological functions of liver. - Simple technology and easy to widely popular. Keywords: fatty liver, low power semiconductor laser, intravenous semiconductor laser, liver diseases, hyperlipidemia. I. INTRODUCTION Normally, the liver has an accumulation of fat about 3-5% by weight of the liver. Fatty liver disease when fat makes up at least 5% of the liver. It is categorized into three levels of fatty liver [1]: Mild level: the accumulation of fat in the liver accounts for 5-10% weight of liver. ISSN

196 Medium level: the accumulation of fat in the liver accounts for 10-25% weight of liver. Serious level: the accumulation of fat in the liver accounts for over 25% weight of liver. Sometimes up to 40-50%. Most of people who have fatty liver in mild level often has no clearly symptoms and sometimes feel tiredness, anorexia, feel pain right upper quadrant. In people that have fatty liver in medium level and in serious level have symptoms as: anorexia, indigestion, bloating, nausea, liver enzymes (AST, ALT) increased over normal levels, cholesterol, bad cholesterol (LDL), triglycerides in the blood are increased, may have symptoms such as: yellow eyes, jaundice, abdominal distention,... The early detection of steatosis mainly based on ultrasound and CT - scanner method, combined with a blood test to confirm the diagnosis and classification. Fatty liver is not a pathology that only the clinical manifestation of many different diseases. Therefore, patients want to treat fatty liver, they must treat the cause of steatosis and no specific drugs that can cure this disease without treating the cause. In [1], Experts said there is currently no specific drugs that can cure this disease, if not treatment causes of it such as obesity, increased blood fats, alcohol,... In this context, we proposed: "Research and application low power semiconductor lasers for the treatment of fatty liver" with criteria: on the same an equipment can perform the treatment with good results fatty liver by many causes. II. EXPERIMENTS 2.1. The contents of the method Main contents consists of: Actively promoting process of destroying the fatty liver with inflammatory symptoms and preventing liver fibrosis and liver cirrhosis by the methods : - Using intravenous semiconductor laser works 650 wavelengths to increase high quality blood flow to liver to promote process of destroying of fat in the liver. On the other hand, to treat hyperlipidermia. According to [1] said, this is one of the main reason of non-alcoholic fatty liver. In [2] shows that, when using intravenous semiconductor laser works at a wavelength of 650 nm in the treatment of hyperlipidermia have high efficiency. At the same time, decreasing the fasting glycemic index in people with type 2 diabetes on normal value, or prediabetes value. According to [1] said, type 2 diabetes is one of the important reasons for causing fatty liver disease. In [3] shows that using intravenous semiconductor laser decreasing the fasting glycemic index in people with type 2 diabetes on normal value. 182 ISSN

197 - Using the effect of the two simultaneous wavelengths by semiconductor laser works at 780 nm and 940 nm wavelengths created, when the laser beams directly impact on liver tissue from abdomen skin to treat the rehabilitation for liver disease to make the biological responses by bio-stimulation occurs faster and stronger [4, 5] as: antiinflammatory response, anti- pain response, response of damage cell, response of regeneration, response of immune system, response of cardiovascular system, response of the endocrine system. - Combining using optoacupuncture by semiconductor laser works at a 940 nm wavelength impacts directly on acupuncture points to treat different types of hepatitis and type 2 diabetes Treatment Equipment The intravascular semiconductor laser equipment (Fig. 1) and the optoacupuncture - optotherapy equipment by low power semiconductor laser has 12 channels (Fig. 2). Laser Technology Laboratory researched and manufactured these equipments. Fig. 1. The intravascular semiconductor laser equipment. Fig. 2. The optoacupuncture - optotherapy equipment by low power semiconductor laser of 12 channels Treatment process Patients were treated once a day. Treatment process of fatty liver was carried out as following: The first day, patients are treated by the intravascular semiconductor laser with (45 60) minutes to provide fully blood to the liver with high quality and to treat hyperlipidermia. The second day, they were treated with the optoacupuncture - optotherapy equipment by low power semiconductor laser 12 channels with 30 minutes. Using two optotherapy heads which creates the effect of the two simultaneous wavelengths directly impact on liver tissue from abdomen skin to rehabilitate liver damages. Using ten optoacupuncture heads to impact on acupuncture points in Traditional Oriental acupuncture to treat different types of hepatitis. ISSN

198 The third day and the fourth day, this treatment cycle is repeated. A treatment course induces 20 treatment days. After two or three treatment courses, we will evaluate treatment results. III. RESULTS AND DISCUSSION 3.1. Treatment method Clinical research method no control group, self-control before and after treatment according to the following criteria: results of liver Ultrasound, test results of concentration of hyperlipidermia before and after the treatment The method to evaluate level of fatty liver We evaluated the level of fatty liver before and after treatment with semiconductor laser as follows: Mild fatty liver Results of liver ultrasound evaluated mild fatty liver The values of liver enzymes greater than the highest allowed values are 10% - Concentrations of hyperlipidermia greater than allowed values are 10%. Average fatty liver - Results of liver ultrasound evaluated average fatty liver - The values of liver enzymes greater than the highest allowed values are 20% - Concentrations of hyperlipidermia greater than allowed values are 20%. Severity Fatty liver - Results of liver ultrasound evaluated severity fatty liver - The values of liver enzymes greater than the highest allowed values are 30% - Concentrations of hyperlipidermia greater than allowed values are 25% Subjects in clinical research The total number of patients in clinical research are 32 people. They volunteer participate in the program research. Patients were divided into two groups: 20 cases of mild fatty liver (according to the above criteria); 12 cases of moderate fatty liver (according to the above criteria); 3.4. Clinical results For mild fatty liver group. In Tab. 1, we present: the average value of liver enzymes, the average value of hyperlipidemia ingredients and results of liver ultrasound before and after the end of two therapy treatment. Table 1. The average value of liver enzymes before and after treatment. Total patients: 20 people The average value of liver Before treatment After treatment enzymes AST enzyme 43,2 u/l 38,567u/L ALT enzyme 43,75u/L 37,51u/L GGT enzyme 53,75u/L 49,48 u/l The average value of Before treatment After treatment 184 ISSN

199 hyperlipidemia ingredients Toatal Cholesterol 225,7 mg/dl 174,3mg/dl Triglyceride 169,275mg/dl 130,725mg/dl Result of liver ultrasound Mild fatty liver Normal From Table 1, we found that, before treatment with low power semiconductor laser, the average value of: Concentration of liver enzyme is greater than the highest normal value respectively: 8%, 9.4% and 7.5%. Concentration of hyperlipidemia ingredients is greater than the highest normal value respectively: 12,85%, 12,85%. Result of liver ultrasound: mild fatty liver. After treatment, the average value of: Concentration of liver enzyme is less than the highest normal value respectively: 3.7%, 6.63%, 1.05%. Concentration of hyperlipidemia ingredients is less than the highest normal value respectively: 3,7%, 6,63%, 1,05%. No fatty liver. So, high treatment efficiency, 100% no steatosis. For average fatty liver group. On Table 2, we present: the average value of liver enzymes, the average value of hyperlipidemia ingredients and results of liver ultrasound before and after the end of two therapy treatment. Table 2. The average value of liver enzymes before and after treatment. Total patients: 20 people The average value of liver Before treatment After treatment enzymes AST enzyme 49,08u/L 38,33u/L ALT enzyme 49,17u/L 38,17u/L GGT enzyme 61,375u/L 47,71u/L The average value of Before treatment After treatment hyperlipidemia ingredients Toatal Cholesterol 230,83mg/dl 187,17md/dl Triglyceride 169,125mg/dl 130,875mg/dl Result of liver ultrasound average fatty liver Normal From Table 2, after treatment, the average value of: Concentration of liver enzyme is less than the highest normal value respectively: 4,36%, 4,79%, 4,8%. Concentration of hyperlipidemia ingredients is less than the highest normal value respectively: 5,725%, 14,01%. No fatty liver. So, high treatment efficiency, 100% no steatosis. ISSN

200 IV. CONCLUSION The above results show that, treatment method of fatty liver by low power semiconductor laser is a new treatment method. This treatment method has some advantages following as: - Treatment is highly effective for two types of fatty livers: - Fatty liver caused by the food contaminated with chemicals. This are shown by fatty liver patients have high liver enzymes particularly GGT enzymes. - Fatty liver by metabolic disturbances of hyperlipidemia ingredients. This are shown the fatty liver patients have high total cholesterol and high triglycerides. - No complications and side effects occurred during treatment. - Completely conserving the physiological functions of liver. - Simple technology and easy to widely popular. - Treatment form: outpatient, convenient for family care patients. REFERENCES [1] Dao Jian Feng, Fatty Liver Diseases, Shanghai Science Education Publisher, (2004). [2] Tran Minh Thai and et al, Scientific Research Project of level National University - Ho Chi Minh City, Project title: Application of low power semiconductor laser for treatmentof atherosclerosis of internal carotid artery in type 2 diabetes, University of Technology, [3] Tran Minh Thai and et al, Scientific Research Project of level National University - Ho Chi Minh City, Project title: "Research and application of low power semiconductor lasers for the treatment of type 2 diabetes", University of Technology, [4] Kriuk A. and et al, The treatment results of low power laser, Minsk Science and Technology Publisher, [5] Karu T., Photobiological fundamentals of low power laser therapy, IEEE of quant. Electronics, QE.23, N10, ISSN

201 APPLICATION OF LOW POWER SEMICONDUCTOR LASER TO THE TREATMENT OF ATHEROSCLEROSIS OF INTERNAL CAROTID ARTERY IN TYPE 2 DIABETES Tran Minh Thai 1, Ngo Thi Thien Hoa 2, Tran Thien Hau 1*, Ngo Van Trung 1, Phan Van To Ni 1 1 Laser Technology Laboratory HoChiMinh City University of Technology HoChiMinh city National University 2 The Department of Treatment and Rehabilitation in Tan Chau Town, An Giang Province, 268 Ly ThuongKiet Street, District 10, HoChiMinh City - Vietnam *: tthienhau@hcmut.edu.vn Abstract. In the recent years, the cerebro vascular accident (CVA) has a tendency to increase in Vietnam. CVA can cause sudden death or result in severe sequelae creating burdens to the patients families and the society. This paper present a method to treat atherosclerosis in the carotid arteries in type 2 diabetes people after CVA by combinating harmoniously between intravenous semiconductor laser with optoacupuncture - optotherapy equipment by low power semiconductor laser of 12 channels. The main content of treatment method including: - Using intravenous semiconductor laser working at a wavelength of 650 nm to supply fully blood with high quality for Pancreas to produce fully qualitative insulin. At the same time, to supply fully blood to Brain to rehabilate gradually brain damages caused by CVC, to treat atherosclerosis in the internal carotid artery and prevent formation of new atherosclerosis plaques in the internal carotid artery - Using the effect of the two simultaneous wavelengths with semiconductor laser of 780 nm and 940 nm wavelengths impacts on the pancreas to treat rehabilitation of liver lesions to enhance its function, a segment internal carotid artery outside the skull to enhance the elasticity of carotid artery segments and to expand diameter of internal carotid artery to enhance blood flow to the brain; specified areas such as motor area, awareness area, the language areas by acupuncture method in head in the traditional Oriental acupuncture. - Using opto-acupuncture by semiconductor laser of 940 nm wavelength impacts on classical acu-points in the traditional Oriental acupuncture to treat Pancreatitis, to improve liver function, to treat hemiplegia by CVC. This treatment method has many advantages: high treatment efficiency, no complication or harmful side effect occurred during treatment, conserving thephysiological functionsof carotid artery, simple technology and easy to transfer. Keywords: low power semiconductor laser, intravenous semiconductor laser, atherosclerosis, cerebral vascular accident, type 2 diabetes. I. INTRODUCTION ISSN

202 According to endocrine experts, the risks of CVA in people with type 2 diabetes are higher from 2 to 4 times than in nondiabetic people. The World Health Organization s (WHO) warning said that the two utmost causes of the CVA are respectively atherosclerosis of cerebral arteries and then high blood pressure; those two may go together or separately. The third cause is thrombosis caused by clot of blood from heart to brain [1, 3]. As reported in [2] by Nguyen Van Tao (1987), the two main lesions are cholesterol-rich plaques and artery plaques which often develop since the age of twenty. In [4], when operating the corpses of 1,000 healthy solders died in the battle, it found that 100% of those of age forty and older got atherosclerosis. According to [2], atherosclerosis often occur in: coronary artery, internal carotid artery, cerebral artery, abdominal artery,... The therapy with medicines, including aspirins and Statin drugs. This method is the simplest. But until now, we have not found therapeutic efficacy of it. Attention should be given to harmful side effects of it. o The therapy with coronary arty bypass surgery o The therapy of stent-widening o The therapy of vascular endothelium stripping surgery The above three therapies require high-tech, and carry out at well-equipped hospitals in big cities and many difficulties in clinical treatment for patients with type 2 diabetes. Owing to the above reasons, we propose a new method called: Treatment of atherosclerosis of internal carotid arteries in type 2 diabetes by low power semiconductor laser. In type 2 diabetes people, the above-mentioned three therapies for atherosclerosis are limit. Total patients in the treatment are 37 people. They are hemiplegic by CVC in cerebral infarction with type 2 diabetes. Results of Color Doppler Ultrasound said in their carotid atherosclerosis. They volunteer participate in the program study. II. EXPERIMENTS Clinical research no control group, self-control is based on: results of Color Doppler ultrasound of the carotid artery, results of fasting blood sugar level test measures before and after the end of treatment, results of the rehabilation of the sequalae to exercise and mentality after CVC. Divided into three groups: a treatment group with type 2 diabetes was treated to keep their blood sugar levels in normal value ((70-100) mg/dl) and prediabetes (> 100 mg/dl and < 126 mg/dl), one group with atherosclerosis, the other group with CVC. Using intravenous semiconductor laser working at a wavelength of 650 nm: - To supply fully blood with high quality for Pancreas to produce fully qualitative insulin. At the same time, to supply fully blood to Brain to rehabilate gradually brain damages caused by CVC. - To treat atherosclerosis in the internal carotid artery. At the same time, to prevent formation of new atherosclerosis plaques in the internal carotid artery 188 ISSN

203 Using the effect of the two simultaneous wavelengths with semiconductor laser of 780 nm and 940 nm wavelengths impacts on [5, 6]. - The pancreas to treat rehabilitation of liver lesions to enhance its function. - A segment internal carotid artery outside the skull to enhance the elasticity of carotid artery segments and to expand diameter of internal carotid artery to enhance blood flow to the brain; Specified areas such as motor area, awareness area, the language areas by acupuncture method in head in the traditional Oriental acupuncture (Fig. 1.) for patients can not taking MRI and CT scanner. A- Motor area (Rolando slot). B- Awareness area. C- Language area (Broca). D- Audition area. E- Visual area. F- Balance area. Fig. 1. The opto-acupuncture by low power semiconductor laser in head. Using opto-acupuncture by semiconductor laser of 940 nm wavelength impacts on classical acu-points in the traditional Oriental acupuncture to treat Pancreatitis, to improve liver function, to treat hemiplegia by CVC. Treatment equipments: the intravascular semiconductor laser (Fig. 2) and the optoacupuncture - optotherapy by low power semiconductor laser of 12 channels (Fig. 3). The two equipments are manufactured by the Laboratory of Laser Technology. Fig. 2. The intravascular semiconductor laser equipment. Fig. 3. The optoacupuncture - optotherapy equipment by low power semiconductor laser has 12 channels. ISSN

204 Treatment progress: Treatment had done once per day. The first day, the patients were treated with the intravenous semiconductor laser with (45 60) minutes. The second day, they were treated with the optoacupuncture - optotherapy equipment by low power semiconductor laser 12 channels with 60 minutes, divided into 3 periods: the first period of 20 minutes to treat hypoglycemia in type 2 diabetes, the second period of 20 minutes to treat atherosclerosis in internal carotid arteries. And the third period of 20 minutes to rehabilitate motor function after the CVA. This treatment cycle is repeated until at the end a therapy course. A treatment course consists of 20 days. After patients completed 02 treatment courses, we will evaluate treatment results. III. RESULTS AND DISCUSSION. The treatment results of type 2 diabetes people. The fasting blood glucose average index of 37 type 2 diabetes patients before and after completing two treatment courses with low power semiconductor lasers are presented in Tab.1 Table 1. The fasting blood glucose average index. Patients : 37 people The fasting blood glucose average index Before (mg/dl) After (mg/dl) According to the World Health Organization and American Diabetes Association Diabetes Normal According to QĐ No. 3280/QĐ-BYT of Ministry of Health Type 2 diabetes, blood sugar is not well controlled Good From Table 1, we found that: after two treatment courses, the fasting blood glucose average index of 37 patients with type 2 diabetes returned to normal values (70-100) mg/dl according to the criteria of the WHO and the ADA. Fasting blood sugar index of individuals. After two treatment courses, blood sugar index for each of one as: According to the criteria of the WHO and the ADA. The fasting blood glucose index ( mg/dl) - normal values: 32 people, accounting for 86.49%. The fasting blood glucose > 100 mg/dl and <126 md/dl - prediabetes: 05 people, accounting for 13.51%. According to Decision No.3280 /QĐ-BYT of Ministry of Health signed on 09/09/2011 on guidelines for diagnosis and treatment of type 2 diabetes. The fasting blood glucose index (79 - of 110) mg /dl are evaluated good: 36 people, accounting for 97.3%. The fasting the blood glucose index from ( ) mg / dl: 01 person 112 mg/dl, accounting for 2.7%. 190 ISSN

205 Treatment results of atherosclerosis plaques in the carotid artery by low semiconductor laser. Results of Color Doppler Ultrasound in the carotid artery of patients are presented in Tab. 2. Table 2. Results of Color Doppler Ultrasound in the carotid artery. Patients : 37 people No. Atherosclerosis plaques in Before Treatment After Treatment the carotid artery Number Percent % Number Percent % 1 Left side Right side Both sides From Tab. 2, we found that 37 patients with type 2 diabetes have atherosclerotic plaques in the internal carotid artery in left side are 06 patients, have them in right side are 06 patients, have them 25 patients in both sides. After 40 treatment times with this method, just only one patient had atherosclerotic plaques in right side, accounting for 2.7%. Above things shows that this method bring high efficiency. Treatment results of the rehabilation of motor function and intellectual resources after CVC in type 2 diabetes people with low power semiconductor laser. The level of hemiplegia is evaluated according to the Rankin. Treatment results according to Rankin is presented in Tab. 3. Table 3. Results of rehabilation of motor function according to the Rankin. Patients : 37 people Before Treatment After Treatment Level of hemiplegia Number Percent % Level of hemiplegia Number Percent % I I % II II % III % III IV % IV V % V From Tab. 3 show that before treatment the distribution of patients according to level of hemiplegia as: There are 02 patients in level III - medium sequelae, activities need help in life, accounting for 5.41%. There are 19 patients in level IV - serious sequelae, activities completely need server in life, accounting for 51.35%. There are 16 patients in level V - very severe sequelae with many complications, accounting for 43.24%. After Treatment, level of hemiplegia have completely changed so as: ISSN

206 There are 36 patients in level I - completely rehabilation, accounting for 97.30%. There are 01 patient in level II - mild sequelae, completely on your out in life, accounting for 2.7%. Progress of rehabilation of motor function and intellectual resources according to rate of Orgogozo. Treatment results through rate of Orgogozo is presented in Table 4. Table 4: Treatment results through rate of Orgogozo. Research patients: 37 people Before treatment After treatment Orgogozo points The number of patients Rate Orgogozo points The number of patients ,24% ,35% ,41% Rate 2,7% 97,30% Total number of Orgogozo point input is 2075 Total number of Orgogozo point output is: 3325 Average Orgogozo point input 29,05 Average Orgogozo point output 89,86% In Tab. 4, we found that: before treatment, the distribution of levels of hemiplegia sequelae as follows: there are 16 patients achieved 20 Orgogozo points - severe sequelae - accounted for 43.24%, 19 patients achieved 30 Orgogozo points - serious sequelae - accounted for 51.35%, 2 patients achieved 45 Orgogozo points - sequelae medium - accounting for 5.41%. After treatment, hemiplegia sequelae by CVC have completely changed so as: There are one patient 85 Orgogozo points - light sequelae, completely on your out in life, accounted for 2.70%. There are 36 patients achieved 90 Orgogozo points - completely recovery - accounting for 97.3%. 192 ISSN

207 General results: To evaluate treatment results, we are based on the levels of the treatment with excellent results, rather results, average results and bad results, the above criteria are as: Excellent results: After treatment, results of the fasting blood glucose test are ( mg/dl), results of Color Doppler Ultrasound in the carotid artery are negative, results of rehabilation of motor function after CVC: I level of hemiplegia in Rankin, Orgogozo points are 90 or higher. This results are 36 people, accounting for 97.3%. Rather results: After treatment, results of the fasting blood glucose test are ( ) mg/dl, results of Color Doppler Ultrasound in the carotid artery are positive, results of rehabilation of motor function after CVC: II level of hemiplegia in Rankin, Orgogozo points are (80 85) points. This result is one patient, accounting for 2,7%. Average results: After treatment, results of the fasting blood glucose test are > 126 mg/dl, results of Color Doppler Ultrasound in the carotid artery are positive, results of rehabilation of motor function after CVC: III level of hemiplegia in Rankin, Orgogozo points are (50 65) points. This result is no patient. Therefore, clinical treatment results for 37 patients as: 36 excellent results accounting for 92.30%, one rather results accounting for 2.7%, there is no average result. Excellent and rather results accounting for 100%. In which, excellent results than rather results are times. IV. CONCLUSION For this treatment method, patients in this clinical reasearch treated to keep their blood glucose test in normal value ((70-100) mg/dl) and in prediabetes value (> 100 mg/dl and < 126 mg/dl), together treated atherosclerosis, at the same time treated to rehabilate gradually hemiplegia sequelae and motor function after CVC. Treatment of internal carotid artery atherosclerosis in type 2 diabetes by low power semiconductor laser has the following advantages: high treatment efficiency, no complication or harmful side effect occurred during treatment, conserving thephysiological functionsof carotid artery, simple technology and easy to transfer. REFERENCES [1] Nguyen Van Dang, Cerebral vascular accident, Medicine Publisher, Ha Noi, [2] Nguyen Van Tao, The characteristics of atherosclerotic lesions were observed in a thousand cases of pathological anatomy at Military Hospital 108, PhD thesis, 108 Military Institute, [3] Pham Van Rao, Cerebral Vascular Accident with internal medicine recuperation, The Vietnam Medicine, Proceedings of the Scientific Works, 1992, pp ISSN

208 [4] Ho Huu Luong, Pham Viet Nga, Comments on cerebral vascular accident at the 103 Medicine Military Institute in 10 years ( ), The Study of Military Medicine, No.2, 1996, pp [5] Kriuk A. and et al, The treatment results of low power laser, Minsk Science and Technology Publisher, [6] Karu T., Photobiological fundamentals of low power laser therapy, IEEE of Quant. Electronics, QE.23, N10, ISSN

209 CLINICAL TREATMENT RESULT OF THE BENIGN PROSTATIC HYPERTROPHY OF THE OLD MEN BY USING LOW-POWER SEMICONDUCTOR LASER Tran Anh Tu 1*, Tran Minh Thai 2, Tran Thi Ngoc Dung 3, Ton Chi Nhan 4, Ngo Thi Thien Hoa 5, Nguyen Xuan Quang 6, Huynh Thanh Hoa 7 and Cao Tan Tieng 8 1,2,3 Laser Technology Lab, Institute of Technology, Vietnam National University of Hochiminh City, 268 Ly Thuong Kiet Street, District 10, Hochiminh City 4 Traditional Medicine Hospital Can Tho 5 Department of treatment and rehabilitation in Tan Chau town, An Giang Province 6 Khanh Hoa rehabilitation Hospital, Khanh Hoa Province 7 Department of treatment and rehabilitation, 25 XVNT, Hochiminh City; 8 Tue Tinh Duong, Dong Nai province *: tranatu@hcmut.edu.vn Abstract. The benign prostatic hypertrophy (BPH) is popular disease of the old men who is upper 45 year-old all over the world. Although BPH cannot kill ones, it decreases the life quality of the patients by two symptoms: blockage and excitation in urinating. Today, although there are many treatment techniques of BPH, there is no perfect technique because of their weakness. This paper studies the treatment of BPH with low power semiconductor laser. Keywords: Benign prostatic hyperplasia (BPH), low power semiconductor laser. I. INTRODUCTION BPH can be found in case of males, who are upper than 45 year-old [1-3]. Fraction of benign prostatic hypertrophic patients in some countries is as follows: France (35-47%), The U.S. (80%), China (20%) and Vietnam (40%) [2]. The main function of prostatic gland is to produce the liquid term of sterm. Sterm plays the role of transporting the antherozoid. Although prostatic gland is not the main part of excretion system, it still be very important. When the prostatic is hypertrophic, the blockage and excitation effect can be followed. Frequency of this effect is listed below [3]: Blockage effect: o Low urine current: 90% o Initializing urinating difficultly: 67% o Feeling of urine left after having urinated: 60% Excitation effect: o Night-urinating: 90% o Over-urinating: 83% ISSN

210 o Urine still drip after having urinated. Until now, the causes of prostatic hypertrophy still not be identified [1, 3]. However, one defined two causes of prostatic hypertrophy: testicle existence and the elder. In some country, when checking the body of death men upper 50 year-old, more than half of these caught the prostatic hypertrophy. For men upper 70 year-old, the fraction of sick is 75%. In 67 people upper than 80 year-old, the fraction is 95,5% [2]. Nowadays, although there is different methods for benign prostatic hypertrophy treatment as: Treatment with internal medicine, consisted of: Treatment with medicine. - Treatment with cultural medicine [4, 5]. Treatment with surgeon: - Open surgery. - Hormone surgery. Treatment with therapy: o Viba therapy. o - Stent in bladder. o High powered laser. But, there aren t the perfect treatment due to their failure: - Low treatment effect. - Reaction after treatment. They are the motivation of our study. II. EXPERIMENT 2.1. Principle of treatment - Solving quickly the blockage symptom and exciting caused by BPH. - Gradually curing the BPH and recovering to the normal state. - Maintaining the physiological function of prostate gland. - High safety of treatment including: no side-effect or catastrophe which is harmful to patients. - Being non-aigesic, and uncomfortable to patients when being in treatment. - Non-infection of dangerous illness. 196 ISSN

211 2.2. Mechanism of the treatment We use couple simultaneous wavelengths of two semi-conductive lasers (CSWTSL) working in different wave-lengths. When the low-powered laser beam interacts with the tissue with the power of ( ) W/cm 2 and time from 10 second to minutes will activate the biological responses. Through many of photoreactions and photo-generation, cells are changed diversingly, leading to the biological responses of the body. These responses include [6-8]: Anti-imflamation response; Anti-pain response; Anti-cell injury response; Regeneration response; Immunology response; Cardiovascular response; Endocrine response; ISSN Fig. 1. Postions of the treatment. Due to these responses, the biological excitation happens faster and stronger at the injury area and play important roles in treatment of BPH. Based on the result at [9], couple simultaneous wavelengths of two semi-conductive lasers at 780 nm and 940 nm have the effect of immunology response than having Profemid. In addition, this effect is better than decoupling one, and we suggest using this at these two position for treatment of BPH. First position: the concave of the pus, marked as A (Fig. 1, in the same position as Khu Gu) to solve the following problem: o Decreasing the hypertrophy of the middle part, lef part and right part for eliminating the blockage of the urethra top of bladder pathway. o Curing the inflammation of the bladder caused by the long-lasted blockage of urine. Second position: the central part of the asshole and testicle, marked as B (Fig 1, in the same position as Hui Yin) for: For decreasing the dysphasia of two sided parts and the rear part. The CSWTSL have good effect of the injury of 100 cm 2 [6, 7]. [1] shows that the hypertrophy of the center part is commonly found on the men in upper 50 years old. Using 940 nm semiconductive laser acupuncture directly to the crypts for eliminating the blockage and excitation. - Curing the urinating blockage, one can use these crypts: [10] o Guan Yuan, Zhong Ji, San Yin Jiao, Yin Ling Quan, Ci Liao. o Yin Ling Quan, Qi Hai, San Yin Jiao, Yin Gu, Da Ling. 197

212 - Uncontrollable urinating, one can use these crypts: o Qi Hai, Guan Yuan, Yin Ling Quan, Da Dun. o Ci Liao, Wei Yang, Zhong Ji, San Yin Jiao, Shen Zhu, Zhong Liao. - Activation of the immune system for serving the treatment, one can use these crypts: He Gu, Qu Chi, Zu San Li, Xuan Zhong, San Yin Jiao Organization involve in this study We collaborate with these institute for studying the clinical treatment using the low powered laser: Khanh Hoa rehabilitation Hospital, Khanh Hoa Province; Department of treatment and rehabilitation, 25 XVNT, Hochiminh City; Department of treatment and rehabilitation in Tan Chau town, An Giang Province; Tue Tinh Duong, Dong Nai province; 2.4. Research method of clinical treatment We use the clinical treatment without proof sample, self-proofing with the following criteria: The blockage symptom and excitation of the patient during treatment: We use the standard of American Urological Association for ranking the blockage symptom and excitation during the treatment process. Table 1. Standard of ranking the blockage symptom and excitation of BPH. Question Last month, is there leftover urine after urinating? Last month, did you need to urinate again after 2 hours? Last month, was the urination intermittent Last month, did you feel hard to suppress the urination? Last month, was your urine flow is weak? Last month, did you need to make effort to urinate? Last month, how many times at night did you walk up for urinate? Result: 0 7 point: low 8 19 point: medium point: serious No Less than 1/5 of time Less than ½ of time Equal ½ of time More than ½ of time Always Total Poin t 198 ISSN

213 Level of prostatic hypertrophy The level of BPH before treatment is evaluated via the first ultrasonic image. After clinical treatment, the patients are required to take ultrasonic image second time. Comparing these two images, we obtained the effective of the method. Treatment process and therapeutic procedure - Each day, patients have 50 minutes exposing laser in two periods: o Period 1: BPH treatment in 30 minutes o Period 2: activating immunological system in 20 minutes. - One therapeutic procedure includes 20 treatment times. Evaluation is made after one or two therapeutic process based on the hypertrophic volume of prostatic gland Participants There are 149 patients participating in this study. In that, the smallest hypertrophic volume of prostatic gland is 18.8 cm 3, and the largest one is 98 cm 3. These patients have had drug treatment but no improvement is observed. They participate voluntarily. Distribution of participants with age Table 2. Distribution of patient with age (149 people). Age Number Percentage From Table 2, we found that BPH begin occurring at in age. We note that in this age, patients in and is 85.91%, so this requires us to have suitable treatment, especially patients at this age usually are afraid of injury and surgery. Illness stage before treatment Before treatment, patients are evaluated about level of blockage symptom. Results show that 149 patients score at 20 to 31 points, ranked in serious cases. This motivates us quickly to carry out the treatment for the blockage and excitation before treatment for BPH. We observe that this symptom decreases during the treatment process which is just a few treatment times. After 10 to 20 treatment times, this symptom is eliminated. Table 3. Level of BPH before treatment (149 people). Low Medium High Extremely high Hypertrophic level (10 20) cm 3 (21 30) cm 3 (31 50) cm 3 (51 100) cm 3 Number % 16.78% 53.69% 27.52% Percentage 18.79% 81.21% ISSN

214 From Table 3, we find that patients who need to cure BPH in high and extremely high case are very large in number. III. RESULT AND DISCUSSION 3.1. Result of treatment of blockage symptom and excitation After finish the therapeutic procedure, all the patients are evaluated with the standard of AUS. Table 4. Level of the blockage symptom and excitation after treatment. Total patients: 149 Level Organization 0 point point point point 4 point Department of treatment and rehabilitation in Tan Chau town Khanh Hoa rehabilitation Hospital Department of treatment and rehabilitation, 25 XVNT, Hochiminh City Tue Tinh Duong, Dong Nai province Total Percentage 22,15 42,95 24,16 10,74 0 From Table 4, we find that the blockage symptom and excitation from 0 to 3 fall into low or very low cases. In other words, this symptom has been eliminated by the low powered laser Result of BPH treatment. After therapeutic procedure, patients take the ultrasonic images. We evaluate the image and find that our proposed treatment method archives very good results with various hypertropic level. - We find that despite the fact that various level of hypertropic volume, treatment by using low powered laser gives very good results. - BPH treatment method by using low powered laser can return the BPH volume to normal prostatic volume. - Treatment of BPH using low powered laser can return the protastic volume to normal one and conserve the functions and structure of prostatic gland. 200 ISSN

215 Institute Department of treatment and rehabilitation in Tan Chau town Table 5. Average decreasing volume after laser treatment. Hypertropic volume before treatment (cm 3 ) Hypertropic volume after treatment (cm 3 ) Hypertropic volume difference (cm 3 ) Khanh Hoa rehabilitation Hospital Department of treatment and rehabilitation, Hochiminh City Tue Tinh Duong, Dong Nai province Total Mean Table 5 shows that the mean decreasing volume of benign prostatic gland is cm 3. This number is not a small number and smaller than surgery Treatment time We find that there is dependent of treatment time on the BPH volume. The larger BPH volume is, the longer treatment time, but this is not linear. Mean time for treatment of 149 patients is days. This is not long time Side effect and catastrophy in and after treatment using low powered laser We find that there is no either side effect or catastrophy in and after treatment of all patient due to non-surgical properties of our method General evaluation. We rank the result into 4 catalogues. Excellent result - The blockage symptom and side effect decrease 90% or eliminated after treatment. - Prostatic volume decreases from 3.0 cm3 to tens cm3 (based on ultrasonic image). - No side effect or catastrophy. Good result - The blockage symptom and side effect decrease 80% or eliminated after treatment. - Prostatic volume decreases from 2.0 cm3 to 2.9 cm3 (based on ultrasonic image). - No side effect or catastrophy. Average result - The blockage symptom and side effect decrease 50-70% or eliminated after treatment. ISSN

216 - Prostatic volume decreases from 1.0 cm3 to 1.9 cm3 (based on ultrasonic image). - No side effect or catastrophy. Lower average result - The blockage symptom and side effect decrease lower than 50% or eliminated after treatment. - Prostatic volume decreases from 0.1 cm3 to 0.9 cm3 (based on ultrasonic image). - No side effect or catastrophy. Based on this catalogues, we evaluate the treatment results of 149 patients and show in Table 6. From Table 6, we find that: - Excellent results include 131 patients, which is 87.92%. - Good results include 7 patients, which is 4.07%. - Average results include 6 patients, which is 3.35%. - Lower average results include 5 patients, which is 3.35%. Therefore, overall patients having improvement is 96.55%. Excellent results is times larger than good result number. Table 6. Treatment result of BPH in human using low powered laser. Number of patients Excellent result Good result Average result Lower average result Department of treatment and rehabilitation in Tan Chau town Khanh Hoa rehabilitation Hospital Department of treatment and rehabilitation, HCM City Tue Tinh Duong, Dong Nai province Total Percentage We can conclude that the treatment methods of BPH using low powered laser is very efficient. IV. CONCLUSION From the result above, we conclude that: 202 ISSN

217 - The treatment of BPH using low powered laser have some precious properties. These are: o The blockage and excitation of BPH decrease quickly and leading to the elimination or decrement at 80-90% comparing to before treatment, after 10 to 20 treatment times. o This method is volume independent. o Mean decreasing volume of 149 patients is cm3. This decrement is not small, and just smaller than only surgery. o This is non-invasive method so there is no risk of infection such as HIV In addition, there is no hurt or uncomfortable feeling. o No side effect or catastrophy are harmful to patients. o Functions and structure of prostatic gland is well conserved. o Treatment method and equipment are simple, easy to broaden. - The treatment of BPH using low powered laser can perform at major institute from district to province. This is one of the most important differences with the surgical method. - Patient can be out when participating in the therapeutic procedure. - The cost of this method is much lower than other method. REFERENCES [1] Vương Quốc Trung (Trung Quốc), 270 câu hỏi phòng chữ bệnh tiền liệt tuyến (Bản dịch: Thái Phiên), NXB Phụ Nữ Hà Nội, [2] David M. Barrett, Những vấn đề cần biết về tuyến tiền liệt, NXB Y học Hà Nội, [3] Lê Quang Hồng, Hỏi đáp về bệnh tuyến tiền liệt, NXB Y học Hà Nội, [4] Lê Trung Chính, Báo cáo kết quả điều trị khối u phì đại lành tính tuyến tiền liệt của chế phẩm Tadimax, Y học Tp. Hồ Chí Minh, Hội nghị Khoa học Kỹ thuật lần thứ 4, Chuyên đề Y học cổ truyền, Tập 8, số 1, trang ( ), [5] Phạm Khánh Bền, Đánh giá tác dụng điều trị phì đại lành tính tuyến tiền liệt bằng viên nang trinh nữ hoàng cung, Y học Tp. Hồ Chí Minh, Hội nghị Khoa học Kỹ thuật lần thứ 22, Chuyên đề Y học cổ truyền, Tập 9, số 2, trang ( ), [6] Karu T., Photobiological fundamentals of low power laser therapy, IEEE J. Quart, Electronics, QE-23, NE 10, [7] Kriuk, C.S và cộng sự, Hiệu quả điều trị của laser công suất thấp, NXB Khoa học và Kỹ thuật, Minsk, ISSN

218 [8] Mockvin C.B và cộng sự, Trị liệu bằng laser công suất thấp, (Tuyển tập các công trình nghiên cứu), NXB Kỹ thuật Mockva, [9] Phan Thị Thanh Thúy, Khảo sát ảnh hưởng của chùm tia laser bán dẫn công suất thấp lên thuốc kháng viêm bôi ngoài da lên mô hình gây viêm chân chuột, Luận văn thạc sĩ, chuyên ngành Kỹ thuật laser, [10] Lê Quý Ngưu, Danh từ huyệt châm cứu, NXB Hội Y học cổ truyền Tp. HCM, ISSN

219 LASER CHARACTERISTICS OF A DIODE-PUMPED Nd:YAG LASER SYSTEM USED IN INDUSTRIAL APPLICATIONS Giang Manh Khoi*, Do Xuan Tien National Center for Technological Progress, 25 Le Thanh Tong, Hanoi, Vietnam *: khoigm02@yahoo.com Abstract. In this paper we report the laser characteristics of a diode side-pumped Nd:YAG laser, including laser power, pulse width, beam profile, laser beam quality. These are all important parameters that affect the use of laser equipment. The measured results show that maximum average laser power up to 75 W, 250 ns pulse width laser mode near TEM 00. This laser system has been used in an industrial laser machine to engrave and metal surface. Keywords: Solid-state laser, Nd:YAG laser, diode-laser pumping. I. INTRODUCTION Optical pumping of Nd:YAG lasers is of particular interest, because these laser have become widely accepted for industrial as well as medical use. A few years ago Nd:YAG lasers were mainly excited by using powerful discharge lamps, optical pumping with laser diodes is becoming more and more important. This is because powerful laser diodes are economically available nowadays and emit light at high optical power levels with a narrow spectral bandwidth which matches the absorption energy levels of the Nd:YAG crystal. The great advantage over the discharge lamp is that the emission of the laser diodes are almost completely absorbed by the Nd:YAG, whereas the relatively broad spectral emission of discharge lamps is absorbed only to a small extent. Hence, the efficiency of diode-pumped Nd:YAG laser is much larger than the lamp-pumped laser. And these diodepumped laser systems has been widely used in many applications. In this paper we report on a Q-switched Nd:YAG laser transversely pumped by laser diode arrays, with output pulsewidth of 250 ns and laser power of 70 W. The laser system are used in materials processing and engravingmetal surfaces. II. EXPERIMENTS A schematic arrangement of our diode-pumped Q-switched laser setup is shown in Fig. 1. The laser resonator consists of one dielectric flat output coupler (OC) and a dielectric high reflector (HR) with diameters of 25 mm. The laser crystal was a 3-mm dia. 90 mm long plane/parallel-cut Nd:Ya 3 Al 5 O 12 rod (1.1 at. % nominal doping level) with finely ground cylindrical surface and polished A/R coated end faces. ISSN

220 Fig. 1. The schematic diagram of diode-pumped Nd:YAG laser system. The laser rod is transversely pumped by 3 singlebar continuous-wave (CW) diode laser arrays with total optical pump power of 300 W and wavelength of 808 nm. A currentsource driver served as the semiconductor-laser electrical power supply with maximum current up to 24 A. Q-switch was achieved with an acousto-optic modulator. Q-switch pulse signal was controlled by RF driver. III. RESULTS AND DISCUSSION 3.1. Laser output power and pulse width We have measured the output parameters of the laser system. The results show that the laser produced maximum output power of 75 W in CW operation mode. The diagram of laser power vs current supply is shown in Fig. 2. We can see that the threshold current value is 8 A and optical pumping efficiency is about 20%. In Q-switch mode, the laser beam are modulated by an acousto-optic Q-switch with radio frequency (RF) variable in the 5-50 khz range. The Q-switched laser pulse width is 250 ns as shown in Fig. 3. Fig. 2. Laser output power vs. current supply. Fig. 3. Q-switched Laser pulse width (250 ns/div). 206 ISSN

221 3.2. Profile and quality factor of laser beam We verified single-transverse-mode operation of the Q-switch laser by measuring beam quality M 2 and contour plot of the Q-switch output pulse. Q-switch laser beam spot recorded by camera beam profiler was shown in Fig. 4. The experimental curve and Gaussian profile were suitable. It was advisable that we proved the Q-switch laser TEM 00 operation. Fig. 4. Laser beam spot and profile. Fig. 5. Setup of beam quality measurement. We have also measured the laser output beam quality (M 2 factor) with experimental setup as in Fig. 5. The M² value is an important measure of beam quality. It is widely used in the laser industry as a specification, and its method of measurement is defined in ISO standard. It is especially useful for determining the degree of beam divergence of real laser beams and the minimum focussed spot size. Fig. 6 is the result of M 2 ISSN Fig. 6. Result of M 2 measurement. 207

222 measurement in Thorlabs Beam v4.0 software. We have found the factor M 2 = 1.10 in axes X and Y, beam divergence of and beam waist diameter of 122 m. Fig. 7. Image of experimental setup. IV. CONCLUSION We have conducted experiments to measure the output parameters of a diode sidepumped AO Q-switch Nd:YAG laser. The measurement results shown that maximum laser power of 75 W, laser pulse width of 250 ns and beam quality M 2 = 1.1. These are important results to develop the laser in industrial applications. REFERENCES [1] E. Georgiou and V. Ladopoulos, Characteristics of a Diode-Pumped Q-Switched Nd : YAG Laser, with Continuously Variable Output Pulsewidth ns, Laser Physics, Vol. 8, No. 1, 1998, pp [2] Breck Hitz, J. J. Ewing, Jeff Hecht, Introduction to Laser Technology, IEEE Press, 2001 [3] H. Azer and S. Landre, Efficient diode-laser side-pumped TEM00 mode Nd:YAG laser, Optics Letters, Vol. 17, No. 24,pp ,1992. [4] Takada, Y. Akiyama, T. Takase, H. Yuasa, and A. Ono, Advanced Solid-State Lasers (Boston, OSA), , MB ISSN

223 BIOCHARACTERIZATION AND IDENTIFICATION OF LIGNIN DEGRADING FUNGUS CP36 ISOLATED FROM THE FORESTS OF NORTH CENTRAL VIETNAM Nguyen Thi Hong Lien 1, Nguyen Van Hieu 1, Pham Thi Bich Hop 1, Cao Van Son 2, Dang Van Son 2, Phan Thi Hong Thao 1 * 1 Soil Microbiology, Institute of Biotechnology, Vietnam Academy of Science and Technology 2 Reseach Institute of Pulp and Paper Industry pthongthaoibt@gmail.com Abstract. Wood-rotting fungi represent an important component of forest ecosystems. Among them, white-rot fungi are the most efficient lignin degraders. Biopulping is one of the solutions to reduce environmental pollution caused by conventional pulp and paper industry. Today, the isolation and screening of lignin degrading fungi capable for application in biopulping are of keen interest in Vietnam. Twenty seven strains of ligninolytic fungi were isolated from 65 rot wood samples collected from the forests of North Central Vietnam. The isolate CP36, which showed remarkably high ligninolytic activity (lignin peroxidase 17.3 nkat/g of fermented substrate and manganese peroxidase 15.8nkat/g of fermented substrate), was characterized and indentified. The fruiting bodies of strain CP36 were of 2 6 cm diameter and 0,1 0,4 cm thick, fan-shaped, arranged in imbricated rows like roof tiles, with thin and sharp margin and thin concentric zones. The cap was smooth and dark yellow to reddish-brown. The hymenium was porous and yellowish. Fungus CP36 could grow well on extract malt agar, Hansen and PDA media, at C and ph 4-7. The mycelial growth rate was µm/h. The colony was off-white, blossom, irregularly circular. The mycelia were thick and closely bound together. Beside lignin, this fungus could degrade other substrates such as casein, carboxymethyl cellulose and starch. The biological characteristics of strain CP36 suggest that it belongs to subdivision Basidiomycota. Based on morphological comparison and phylogenetic analysis of 5.8S rdna gene, the wild strain CP36 showed high similarity (99%) to the species Leiotrametes lactinea. Keywords: Leiotrametes lactinea, degradation of lignin, white-rot fungi. I. INTRODUCTION Fungi act as decomposers in the forest and play an important role in the ecosystem. Wood-rotting fungi can be classified into three major groups, the white-rot, brown-rot, and soft-rot fungi; among these, white-rot fungi are the most efficient lignin degraders. Most of the white-rot fungi are from the family of basidiomycetes and some of them belong to the ascomycetes. The lignin degrading enzymes produced by white-rot fungi include mainly lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) [13]. The ability to degrade lignin has prompted interest in using white-rot fungi for bioconversion of lignified tissues, such as biopulping and biobleaching. Biopulping, the treatment of plant materials with natural wood decaying fungi, was envisioned as a method ISSN

224 for saving energy and making a stronger paper product [1]. The process of biopulping is one of the solutions to reduce environmental pollution caused by conventional pulp and paper industry.the objectives of the present paper was to isolate lignin degrading white-rot fungi from natural habitats, to screen these ligninase positive isolates and to identify the most potential ligninase producing white-rot fungi. II. EXPERIMENTS Sixty five fungal samples were collected from the forestsof Ninh Binh, North Central Vietnam. The fruiting bodies were isolated from tree trunks and decayed wood, kept in plastic bags, documented and stored at 4 oc for further study. Isolation and selection of lignolyticfungi Plant tissue culture technique was employed for the isolation of fungi. Fresh fruiting bodies were thoroughly washed under running tap water, sterilized with 95% ethanol for 30 seconds under aseptic conditions and thoroughly washed with sterile water.fragments of basidiocarp were then placed in Petri plates containing a MEA (malt extract agar) medium with 440 µl/l guaiacol or 100 mg/l Remazol Brilliant Blue R (RBBR), andincubated at room temperature for 7 days.the plates were studied with respect to the presence of lignin oxidation enzymes,morphological characteristics and growth rate of the cultures. The presence of such extracellular enzmes can be visualized as reddish-brown zones (on guaiacol plates) or clear zones (on RBBR plates) appeared around the fungal colony. Pure cultures of selected fungi were preserved on potato dextrose agar (PDA) slantsat 4 C [4]. Morphological and growth characteristics analysis The fungi were grown on 2% extract malt agar at 28 C. After 2 7 days, the form, diameter and colour of fungi colony were described.morphologyof myceliawere directly observed at 400 magnification under an Olympus inverted microscope IX71. The growth rate of mycelia was determined according to the method described by Schwantes and Salttler [12]: V= ΔX/ΔT, where V: growth rate of mycelia (µm/h); ΔX: radius of colony (µm); ΔT: cultivation time (hours). Cultivation and enzyme extraction Flasks of 250-ml volume containing 50 ml of 2% malt extract broth were inoculated with 3 fungal disks (6 mm diameter) takenfrom a 4-day culture of the CP36 strain. The fungus was grown on a rotary shaker 120 rpm, at 30 C for 5 days to the density of 1x10 6 1x10 8 conidia/ml. Ligninase production was carried out inplastic jarscontaining 100 g sterile solid medium made of sugarcane bagasse soakedwith Kirk s medium (ph 4.5) to 60% w/w moisture. The flasks were inoculated with 5% w/w fungal and incubated in the dark, at room temperature, for 10 days. Extracellular lignolytic enzymes were recovered by adding 210 ISSN

225 60 ml phosphate buffer ph 5.0 to 3 g fermented substrate, and the flask was shaken at 120 rpm for 30 minutes. The filtratewas centrifuged at 10,000 rpm for 5 minutes, and the supernatant was used for enzyme activity assay [6]. LiP and MnP activity was assayed by method of Mercer et al. [7]. Laccase was measured by method of Cho et al. [3]. Macroscopic characteristics The macroscopic characteristics of studied isolates, such as size, shape and colour of the stipe and pileus; the location of pileus; position of the gills; attachement of stipes to pileus; margin and surface pattern; and the size and shape of pore tubes, were observed and described as guided in Djarwanto and Tachibana [4]. The placement of the isolates to subdivision Basidiomycota was madeaccording to Sharma [14]; Molecular Identification DNA extraction The genomic DNA of this strain was extracted using alkaline extraction method [11]. PCR and sequencing The genomic DNA of the macro fungi were then subjected to PCR to amplify the 5.8S rdna gene using two primers: ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') và ITS4 (5 -CAGGAGACT TGTACACGGTCCAG-3 ) [5]. The sequences of fungus were compared with similar sequences from GenBankusingBlast programfor identification of fungal species. III. RESULTS AND DISCUSSION Collection of fungi Therainforests of Ninh Binhhavediverseflora and fauna, and Cuc Phuong is one of the most important National Parks of Vietnam. Here, the average yearly rainfall is of about mm, humidity of % and temperature of20 23 C. Sixty five fungal samples were collected mainly from Cuc Phuong and its vicinity. Isolation and screening of ligninolytic fungi Ligninolytic enzymes (lignin peroxidase, manganese peroxidase and laccase) are remarkably non-specific regarding substrates. Therefore, guaiacol and Remazol Brilliant Blue B are widely used asindicators for most analyses of ligninase activity. On the agar medium supplemented with those indicators, the oxidation of guaiacol or RBBR by ligninolytic enzymes can be visualized as reddish-brown coloration around the fungal colony, or disappearance of blue colour, respectively [9]. Fig. 1 shows the screening of isolated ligninolytic fungi based on the oxidation of guaiacol and RBBRon MEA medium. As explained above, the activeisolates of the upper row formed reddish-brown coloration on guaiacol medium, while the others on the lower ISSN

226 row discoloured the blue RBBR. Using this method, the presence of extracellular ligninolytic enzymes could be concluded rather soon, usually one day after inoculation. Fig. 1. Screening of active ligninolytic fungi based on oxidation guaiacol (upper row) and RBBR (lower row). As shown in Fig. 1, fungus CP36 produced high activity of ligninolytic enzymes when compared with other isolates and the positive control. Therefore, fungus CP36 was selected for further study on its enzyme activity and identification. For determination of extracellular ligninolytic activity, fungus CP36 was cultivated onsugarcane bagasse (Fig. 2) for 10 days. At the harvest, lignin peroxidase was of17.3 nkat/g of fermented substrate and manganese peroxidase of15.8 nkat/g of fermented substrate. Morphologicalcharacterization of CP36 fungus The fruiting bodies of fungus CP36 wereof 2 6 cm diameter and 0,1 0,4 cm thick, fan-shaped, arranged in imbricated rows like roof tiles, with thin and sharp margin and thin concentric zones. The pileus was smooth and dark-yellow to reddish-brown. The hymenium was yellowish when fresh, porous, bearing 3-4 pores per mm (Fig. 3). Fig. 2. Cultivation of fungus CP36 on sugarcane bagasse. Fungus CP36 could grow well on malt extract agar, Hansen and PDA media thanks toabundant extracellular enzymes such as ligninase, cellulase, proteinase and amylase. The suitable temperature and ph for growth of fungus CP36 was at C and ph 4-7. This fungus fully colonized thepetri dish in 5 days at 30 C. The mycelial growth rate was ISSN

227 µm/h. The colony was off-white, blossom, and irregularly circular. The micrograph taken under light microscope showed thick mycelia closely bound together (Fig. 4). The generative hypha were septate, with thick wall and clamp connections. Fig. 4. Fungus CP36 grown on MEA: (a) colony and (b, c) hypha with clamp connections (red ovals). Molecular phylogenetic analysis of fungus CP36 The internal transcribed spacer (ITS) region of fungal rdna has been successfully used for species identification. The ITS-1 and 4 primers, which flank the region covering the sequence of 5.8S, 18S and 28S rdna, are often used as primers to amplify DNA sequences from a wide range of fungi [15]. Genomic DNA of CP36 was isolated for PCR amplification. The amplified PCR product exhibited a band with 800 bp in length (Fig. 5). Using Blast program in NCBI database, phylogenetic analysis was done by CLC DNA work bench 6.6 programme, similar sequences were retreated for comparison with CP36.The result of phylogenetic analysis is given in Figure 6. Blast search of the complete sequence of ITS region and the phylogenetic analysis showed that fungus CP36 was closest (99% similarity) to the species Leiotrametes lactinea, hence the isolated fungus CP36 was identified asleiotrametes lactinea. Leiotrametes sp. is a tropical species that belongs to the proposed novel genus Leiotrametes Welti & Courtec. nested in the Trametes clade of the core polyporoid group [16]. Berrin et al. [2] also reported on Leiotrametes lactinea (Accession No. JX082368) collected from tropical forests in French Guiana. Fig. 5.Gel electrophoresis of PCR products M: DNA marker; 36: ITS region of CP36. ISSN

228 The number ofavailable reports on the isolation and identification of lignin degrading white-rot fungi is rather limited. Metuku et al. (2011) selected the highest ligninolytic fungus and identified as Pycnoporus sp. DIS 343f [8]. Ortiz et al. (2013) studied the biodiversity of white-rot fungi in Chiloé National Park (Chile), and identified 12 different species in the subdivision Basidiomycota [10].Our report is the first step to study white-rot fungi of tropical rainforests of Vietnam and their potential application. Fig. 6. Phylogenetic tree of Leiotrametes lactinea CP36. IV. CONCLUSION A ligninolytic fungus CP36 isolated from the forests of North Central Vietnam was selected for having high activity of extracellular lignin peroxidase (17.3 nkat/g of fermented substrate) and manganese peroxidase (15.8nkat/g of fermented substrate).in present paper, this isolate was studied with respect to itscultivation, enzyme activity, biological characteristics and identification. The results of morphological and phylogenetic analyses suggest that CP36 is closest (99% homology) to the species Leiotrametes lactinea, hence it can be named as Leiotrametes lactinea CP36. This is ahighly potential fungus for use in pulp and paper industry, particularly in biopulping. V. ACKNOWLEDGMENTS The present research was financed by the grantđt.01.15/cnshcb from the Ministry of Industry and Trade. REFERENCES [1] M. Akhtar, R. Blanchette, G. Mayers, and T. Kirk, Wiley. New York, 1998, pp ISSN

229 [2] G. Berrin, D. Navarro, M. Couturier, C. Olivé, S. Grisel, M.Haon, S. Taussac, C. Lechat, R. Courtecuisse, A. Favel, M. Coutinho, and L. Lesage, Appl. Environ. Microbiol., Vol. 78, 2012, pp [3] N. Cho, N. Pashenova, P. Hop, and A. Leonowicz, Proceedings of The 4 th International Symposium on New horizon of Bioscience in forest products field, Vol. 19, 2003, pp [4] Djarwanto, and S. Tachibana, Pakis. J. Biol. Sci., Vol. 12, 2009, pp [5] M. Gardes, and T. Bruns, Mol. Ecol., 1993, Vol. 2, pp [6] H. Iqbal, M. Asgher, and H. Bhatti, BioRes., Vol. 6, 2011, pp [7] D. Mercer, M. Iqbal, P. Miller, and A. McCarthy, Appl. Environ. Microbiol, Vol. 62, 1996, pp [8] P. Metuku, S. Burra, H. Nidadavolu, S. Pabba, and M. Singaracharya, Cell Tissue Res., Vol. 11, 2011, pp [9] T. Nishida, Y. Kashino, A. Mimura, and Y. Takahara, Moku. Gakkai, Vol. 34, 1988, pp [10] R. Ortiz, J. Navarrete, C. Oviedo, M. Párraga, I. Carrasco, E. Vega, M. Ortiz, and R. Blanchette, A. van Leeuw. J. Microb, Vol. 104, 2013, pp [11] J. Sambrook J. and D. Russell, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, [12] H. Schwantes, and P. Salttler, Oberhess. Naturwiss. Zeitschr., Vol. 38, 1971, pp [13] K.Selvam, M.Priya, C.Sivaraj, and K.Arungandhi, Int.J. ChemTech Res, Vol. 4, 2012, pp [14] O. Sharma, Tata McGraw-Hill Publishing Company Limited, New Delhi, [15] A. Velegraki, E. Kambouris, G. Skiniotis, M. Savala, A. Mitroussia-iouva, and N. Legakis, J. Immunol. Med. Microbiol., Vol. 23, 1999, pp [16] S. Welti, P. Moreau, A. Favel, R. Courtecuisse, M. Haon, D. Navarro, S. Taussac, and L. Meessen, Fungal Divers. Vol. 55, 2012, pp ISSN

230 RESEARCH INTO THERMAL DECOMPOSITION OF A MIXTURE OF RDX AND INSENSITIVE Nguyen Mau Vuong*, Ngo Van Giao, Dang Van Duong Chemical Institute, Academy of Military Science and Technology, Ha Noi, Viet Nam *: tptnvuong@gmail.com Abstract. The article gives the initial results of the melting temperature, at which the mixture begin to decompose, and some kinetic parameters when changing the composition of mixture A (including RDX, xerezin, stearic acid, sudan) based on the Diffirential Thermal Analysis (DTA) method. The obtained data is the basis of safety principles in the application of this explosive mixture. I. INTRODUCTION Many substances such as paraffin, xerezin, wax, polystyrene, polyethylene... [1, 5, 7, 9-11] are used to reduce sensitivity to cyclotrimethylentrinitramin (RDX). One of the mixtures often used to reduce sensitivity to RDX is the mixture of 3 substances including xerezin, stearic acid, and sudan. Combinating the three substances results in good effects on covering, decreasing sensitivity and sharply displaying colours. The usage ofthe explosive mixture in Vietnam is usually at the step ofapplying the foreign results. There are no detailed studies on essence, interaction of components at high temperatures to assess the safety during storaging, transporting and using. The method used to determine the kinetic parameters of explosives under the effect of temperature is mainly Differential Themal Ananlysis (DTA) or Differential Scanning Calorimetry (DSC) [3, 4, 8]. II. EXPERIMENTS Device: The system of equipments DTA 404 EP produced by NETZSCH Group. The test sample: Explosive mixtures are made of the ratio of RDX and insensitive compound as Table 1. Test samples are directly in the air. Table 1. Component ratio (% by weight) of test samples. No. Sample s name RDX (%) INSC (%) 1 Sample No Sample No. 2 95,0 5,0 3 Sample No. 3 94,5 5,5 4 Sample No. 4 94,0 6,0 5 Sample No. 5 93,5 6,5 216 ISSN

231 INSC (insensitive compound) includes 60.0% xerezin; 38.8% stearic acid; 1.2% sudan. Procedure: - Measuring is done at atmospheric pressure. Each sample is tested 5 times with different heating speeds. - Calibrating equipment. - Scaling sample: Weight of each test sample is about 40 mg, accurately to 0.1 mg. Samples are placed in a cup of alumina oxide with 5 mm diameter and 50 mm height. - Taking sample into the cup and closing test chamber. - Turning on auto recording mode. - Starting heating from room temperature, heating speed is constant for each test changes respectively β1 = 5, β2 = 7, β3 = 9, β4 =11, β5 = 13 (K per minute), until decomposition occurs completely (approximately 400 o C), then stoping heating. The chamber s temperature decreases to the room temperature naturally. - The data are recorded and processed by DTA software (in syncing with DTA EP 404). - Repeating the above process with different samples. III. RESULTS AND DISCUSSION DTA curves of sample No. 1, 2, 3, 4, 5 are showed in Figures 1, 2, 3, 4, 5 respectively. Figures 1, 2, 3, 4, 5 show that the DTA curves of the samples have a small end othermic effect with the melting, and then the exothemic effect happens with the process of thermal decomposition. However, when incresing heating rate β, the starting point of exothemic effect occursslower and the end point of effect comes later. With an increase of the concentration of INSC, thermal effects have lower sharp forms, meaning that thermal decomposition time is longer. This confirms the role of INSC in reducing the process of thermal decomposition. Fig. 1. DTA curve of sample No. 1. Fig. 2. DTA curve of sample No. 2. ISSN

232 Fig. 3. DTA curve of sample No. 3. Fig. 4. DTA curve of sample No. 4. From DTA curves of samples, the system of equipment givesthe temperature of the beginning of melting, the beginning of decomposing and the peak as in Table 2, 3, 4, 5, 6. No. Heating speed, K per minute Table 2. Physical parameters of the sample No. 1. Starting melt temperature (T m ) o C Fig. 5. DTA curveof sample No. 5. Beginning to decompose temperature (T c ) K o C Peak temperature (T p ) K K ,1 205,1 493,6 220,6 502, ,4 205,4 498,3 225,3 504, ,7 205,7 501,1 228,1 507, ,5 205,5 502,2 229,2 508, ,8 205,8 503,9 230,9 509,8 No. Heating speed, K per minute Table 3. Physical parameters of the sample No. 2. Starting melt temperature (T m ) o C Beginning to decompose temperature (T c ) K o C Peak temperature (T p ) K K ,9 204,9 488,9 215,9 496, ,3 205,3 491,9 218,9 499, ,4 206,4 493,4 220,4 503, ,3 205,3 493,8 220,8 505, ,0 206,0 494,9 221,9 508,1 218 ISSN

233 No Heating speed, K per minute Table 4. Physical parameters of the sample No. 3. Starting melt temperature (T m ) o C Beginning to decompose temperature (T c ) K o C Peak temperature (T p ) K K ,7 204,7 490,4 217,4 493, ,3 205,3 491,9 218,9 497, ,3 205,3 492,4 219,4 501, ,5 205,5 492,9 219,9 504, ,4 205,4 493,1 220,1 503,4 No. Heating speed, K per minute Table 5. Physical parameters of thesample No. 4. Starting melt temperature (T m ) o C Beginning to decompose temperature (T c ) K o C Peak temperature (T p ) K K ,6 205,6 490,9 217,9 494, ,3 205,3 492,3 219,3 496, ,7 205,7 493,2 220,2 502, ,6 205,6 494,6 221,6 504, ,9 205,9 495,3 222,3 506,6 No. Heating speed, K per minute Table 6. Physical parameters of the sample No. 5. Starting melt temperature (T m ) o C Beginning to decompose temperature (T c ) K o C Peak temperature (T p ) K K ,8 204,8 491,4 218,4 496, ,8 204,8 492,2 219,2 497, ,9 204,9 492,8 219,8 500, ,5 205,5 494,8 221,8 506, ,3 205,3 495,0 222,0 508,2 Calculate Kinetic parameters: According to [3, 4], the thermal decomposition of solids is represented by the Equation (1) or (2) at determined temperature T. ISSN dx dt = k T.(1-x) n (1) dx dt = Z.(1-x)n.e -E/RT (2) In that: dx/dt: reaction rate (s -1 ), x: fraction reaction or conversion (dimensionless), n: reaction order, Z: pre-exponential factor or frequency (s -1 ), E: activation energy (J mol -1 ); 219

234 T: absolute temperature (K); R = 8,314 J mol -1 K -1 (gas constant), e = 2, , k T : specific rate constant at temperature T(s -1 ), determined by Arrhenius equation (3). k T = Z.e -E/RT (3) According to [3, 4], at the time the max rate, corresponding to the peak temperature (Tm), equation (2) may be considered approximately to the equation (4) or (5), β is the heating rate. Equation (4) or (5) the basis equation of K is singer method. d(ln β T2 ) m d( 1 = Tm ) -E (4) R ln β T2 = ln Z.R m E - E R.T m (5) Equation (5) has the form of y = ax + b. In that: y = ln β T m 2, x = 1000 T m, a = - E 1000.R, b = ln Z.R. From determined values of β, T m is collected, so the linear equation (5) was E calculated and established. We obtain the linear equations as in Figures 6, 7, 8, 9, 10. y R² = y = x x y R² = y = x x Fig. 6. Plot equation (5) of sample No. 1. Fig. 7. Plot equation (5) of sample No. 2. y R² = y = x x y R² = y = x x Fig. 8. Plot equation (5) of sample No. 3. Fig. 9. Plot equation (5) of sample No ISSN

235 From these data, the values of the activation energy (E), frequency (Z) and specific rate constant at temperature (T) are determined as in Table 7. Table 7. Kinetic parameters and specific rate constant of the mixture A. No. Sample s name Activation Frequency Z, energy E, KJ.mol -1 s -1 Specific rate constant k T, s -1 1 Sample No x ; T=( ) 2 Sample No x ,430 1,033; T=( ) 3 Sample No x ,388 0,902; T=(493,1 504,1) 4 Sample No x ; T=( ) 5 Sample No x ; T=( ) In Table 7, the activation energy E and frequency Z decrease with the increase in the dosage of insensitivities in the mix. According to the Arrhenius equation (3), the reaction rate constant is inversely proportional to the activation energy and directly proportional to the frequency. The reducing activation energy makes reaction rate increase. However, the decline of frequency Zis stronger than the decline of E which results in the decreased reaction rate constants k T. These results reflect the fact that whenthe concentration of INSC increase volumn of detonation (VoD) of explosivereduced. It can be explained based on the value of E and A found as follows: When concentration of INSC is increasing, explosive mixture more easilier decompose, but the frequency of contact between the molecules of explosives RDX decreases stronger to break the thicker inert cover. These make reaction rate of decomposition reduce. This is most evident in the value of frequency Z ( ) of RDX (when having no INSC) about 10 9 times higher than having 5% of INSC ( ). According to the documents [2, 6], the research results of the thermal decomposition of mixed RDX and other adhesives showed activation energy E are in the range of ( ) kj/mol and frequencies Z are in the range of (1.95x x10 22 ) s -1. So, the calculation results obtained in Table 7 is reliable. To consider decomposition rate of the mixture A in the experimental condition more clearly, based on results of reaction rate constants obtained from Table 7, we can calculate the half-life of these mixture according to the formula: The half-life of these mixtures was obtained as in Table 8. t1 2 Table 8. The half-life of these mixtures A. = ln2 k T (6) No. Sample s name Temperature Specific rate Average range, K constant, s -1 Half-life, s half-life, s 1 Sample No Sample No ,430 1,033 0, ISSN

236 3 Sample No ,1 504,1 0,388 0, Sample No Sample No Table 8 shows the average half-life increases with the increase of the content of insensitivities in the mixture. It means that when decomposition occurrs, the higher the content of insensitivities in the mix A the slower the decomposition process occurs. This is completely suitable with the results calculate de, A, k T above. IV. CONCLUSION In conclusion, when the dosage of insensitivities in mixture A is increased, temperature of the beginning of melting decreases very little. The obtained results confirm that the safe temperature range using explosive mixture A with insensitivities (xerezin, stearic acid, sudan) is ( ) K or (20 200) C. Besides, the result also shows that decomposition limits (in terms of energy) and decomposition rate are inversely proportional to the concentration of the INSC. REFERENCE [1] Charles L. Mader, Terry R. Gibbs, Lasl Explosive Property Data, University of California, 1980, p. 271, 274. [2] Gurdip Singh, S. Prem Felix, PramodSoni, Studies on Energetic Compounds Part 31. Thermolysis and Kinetic of RDX and some of its Plastic Bonded Explosives, Thermochimica Acta 426 (2005), [3] Homer E. Kissinger, Reaction Kinetics in Differential Thermal Analysis, Anal. Chem., 29, 1702 (1957). [4] Homer E. Kissinger, Variation of Peak Temperature with Heating Rate in Diffirent Thermal Analysis, Vol. 57, No. 4, October 1956, Journal of Research of National Bureau of Standard. [5] Military Explosive, Department of The Army, September, 1984, p. 2-22, p , p [6] Michael Robert Williams, Mariana ViolettaMatei, The Decomposition of some RDX and HMX Based Materials in the One-dimensional time to Explosion Apparatus. Part 1. Time to Explosion and Apparent Activation Energy, Propellants, Explosives, Pyrotechnics 31, No. 6 (2006). [7] Rudolf Meyer, Josel Kohler, Axel Homburg (2007), Explosives,Weinheim, p.217, 243. [8] Suresh Mathew, K. Krishnan, and K. N. Ninan, A DSC Study on the Effect of RDX and HMX on the Thermal Decomposition of Phase Stabilized Ammonium Nitrate, Propellants, Explosives, Pyrotechnics 23, p (1998) [9] К. Д. Алферов, Взрывчатые Вещества, Часть II, 1965, c102 [10] Е. Ю. Орлова, Химия и Технология Призантных Взрывчатых Веществ, 1981, c [11] OCTB , Вещества Взрывчатые. Гексоген Флегматизированный. Чехнические Условия. 222 ISSN

237 SYNTHESIS AND BIOLOGICAL ACTIVITY OF NOVEL DERIVATIVES OF MURRAYAFOLINE A Le Duc Anh 1, Truong Ngoc Hung 2, Nguyen Thi Nga 2, Le Mai Huong 2, Tran Thi Hong Ha 2, Tran Thi Thu Thuy 2, Nguyen Manh Cuong 2, Le Thi Thoa 1, Tran Minh Cong 1, Nguyen Trong Dan 3, Luu Van Chinh 2* 1 Chemistry-Materials Institute, IMST, 17-Hoang Sam, CauGiay, Hanoi, Vietnam 2 Institute of Natural Products Chemistry, VAST, 18-Hoang Quoc Viet, CauGiay, Hanoi, Vietnam 3 Vietnam Russia Tropical Centre, 2-Nguyen Van Huyen, CauGiay, Hanoi, Vietnam *: chinhluuvan@gmail.com Abstract: Seven new derivatives of murray afolinea 6a-g were prepared by a procedure in two steps. Among these, c ompounds 6a-f were synthesized from two consecutive N- alkylation reactions. Their cytotoxicity was evaluated on four human cancer cell lines Hep- G2, LU, RD and Fl. The result showed that compound 6f exhibited the strongest activity against Hep-G2, RD and FL cell line with IC 50 values of 1.71, 1.39 and 2.53 μg/ ml, respectively. Keywords: Murrayafoline A, N-alkylation, cytotoxicity, secondary amine, heterocyclic amine. I. INTRODUCTION A number of carbazole alkaloids were recognized to exhibit potential biological activities such as antifungal [1], anti-hiv [2], anticancer [3] activities. Especially, murrayafoline A, a natural carbazole is promising anticanceragentabundantly occurring in theroots of Glycosmisstenocarpa in Vietnam. This compound was reported to show significant growth suppression of the human leukemia cell line HL-60 [4], and prevent the heart diseases [5]. Recently, a series of the murrayafoline A derivatives were synthesized by the modification of NH group, and evaluated the potential anti-inflammatory activity [6]. In this regard, seven new conjugates of murayafoline A with secondary and heterocyclic amineswere designed, prepared and evaluated their cytotoxic activity against several human cancer cell lines. II. EXPERIMENTS All chemicals were purchased from Sigma-Aldrich (USA). Murrayafoline A was isolated from the roots of Glycosmisstenocarpa in Vietnam. Melting points were measured on a Buchi 530 (Switzerland) melting point apparatus and were uncorrected. NMR spectra were measured on Bruker Advance 500M operating at 500 MHz for 1 H and 125 MHz for 13 C. High resolution mass spectra were recorded on FTICR mass spectrometer. Synthesis of murrayafoline A derivatives 6a-f ISSN

238 Synthesis of 3-(1-methoxy-3-methyl-N-carbazolyl)propyl bromide 2 3-(1-methoxy-3-methyl-N-carbazolyl)propyl bromide (2) was prepared in 83% yield according to the known procedure as described by Thuyand coworkers [5]. General procedure for the synthesis of murrayafolinea derivatives 6a-f A mixture of an appropriate secondary amine (4a-f) (1 mmol), K 2 CO 3 (0.21 g, 1.5 mmol), 3-(1-methoxy-3-methyl-N-carbazolyl)propyl bromide (2) (0.42 g, 1.3 mmol) and (1-butyl)triethylammonium bromide (23.8 mg, 0.1 mmol) in dry dimethylformamide (DMF) (15 ml) was stirred at room temperature for overnight and solvent was removed under reduced pressure. The resulting mixture was dissolved in water (20 ml) and extracted with EtOAc (3 20 ml). The combined extract was dried over anhydrous sodium sulfate and solvent was removed under reduced pressure. Crude 5a-f were purified by column chromatography eluting with n-hexane:ethylacetate. 3-(1-methoxy-3-methyl-N-carbazolyl)-1-(4-methylpiperazinyl)propane 6a Yield 71% (colorless oil); 1 H-NMR (500 MHz, DMSO-d 6, ppm) δ 8.02 (d, J = 7.5 Hz, 1H, H-8), 7.54 (d, J = 7.5 Hz, 1H, H-5), 7.50 (s, 1H, H-4), 7.38 (t, J = 7.5 Hz, 1H, H- 7), 7,13 (t, J = 7.5 Hz, 1H, H-6), 6.84 (s, 1H, H-2), 4.55 (t, J = 6 Hz, 2H, 9-CH 2 -), 3.93 (s, 3H, 1-OCH 3 ) 2.45 (s, 3H, 3-CH 3 ), 2.32 (s broad, 8H, H-6', H-5', H-3', H-2'), 2.23 (t, J = 5 Hz, 2H, 1'-CH 2 -CH 2 -CH 2-9), 1.85 (m, 2H, 9- CH 2 -CH 2 -CH 2-1ꞌ), 2.17 (s, 3H, 4ꞌ-CH 3 ). 13 C- NMR (125 MHz, DMSO-d 6, ppm) δ (C-1), (C-8a), (C-3), (C-9a), (C-7), (C-4b), (C-4a), (C-6), (C-8), (C-4), (C- 2), (C-5), 55.6 (1-OCH 3 ), 54.7 (1'-CH 2 -CH 2 -CH 2-9), 54.5 (C-2', C-6ꞌ), 52.3 (C-3', C- 5ꞌ), 45.5 (4ꞌ-CH 3 ), 42.7 (9-CH 2 -CH 2 -CH 2-1ꞌ), 27.2 (9-CH 2 -CH 2 -CH 2-1ꞌ), 21.3 (3-CH 3 ).HR- MS (m/z): [M+H] +, calculated for C 22 H 30 N 3 O: HR-MS (calculated for C 22 H 30 N 3 O: [M+H] , Found: (methoxy-3-methyl-N-carbazolyl)-1-(4-ethylpiperazinyl)propane 6b Yield 73% (colorless oil); 1 H-NMR (500 MHz, DMSO-d 6, ppm) δ 8.02 (d, J = 8.0 Hz, 1H, H-8), 7.54 (d, J = 8.0 Hz, 1H, H-5), 7.50 (s, 1H, H-4), 7.38 (t, J = 8.0 Hz, 1H, H- 7), 7.12 (t, J = 8.0 Hz, 1H, H-6), 6.85 (s, 1H, H-2), 4.55 (t, J = 8Hz, 2H, 9-CH 2 -), 3.93 (s, 3H, 1-OCH 3 ), 2.45 (s, 3H, 3-CH 3 ), 2.29 (m, 10H, H-6', H-5', H-3', H-2', 4ꞌ-CH 2 CH 3 ), 2.23 (t, J = 7 Hz, 2H, 1'-CH 2 -CH 2 -CH 2-9), 1.85 (m, 2H, 1'-CH 2 -CH 2 -CH 2-9), 0.97 (t, J = 7 Hz, 3H, 3H, 4ꞌ-CH 2 CH 3 ). 13 C-NMR (125 MHz, DMSO-d 6, ppm) δ (C-1), (C-8a), (C-3), (C-9a), (C-7), (C-4b), (C-4a), (C-6), (C- 8), (C-4), (C-2), (C-5), 55.6 (1- OCH 3 ), 54.8 (1'-CH 2 -CH 2 -CH 2-9), 52.6 (C-2', C-6'), 52.4 (C-3', C-5'), 51.5 (4ꞌ-CH 2 CH 3 ), 42.7 (9-CH 2 -CH 2 -CH 2-1ꞌ), 27.2 (9-CH 2 - CH 2 -CH 2-1ꞌ), 21.3 (3-CH 3 ), 11.9 (4ꞌ-CH 2 CH 3 ).HR-MS (m/z): [m+h] +, calculated for C 23 H 32 N 3 O: (1-methoxy-3-methyl-N-carbazolyl)-1-(1-imidazolyl)propane 6c 224 ISSN

239 Yield 57% (pale yellow solids); mp: o C; 1 H-NMR (500 MHz, DMSO-d 6, ppm) δ 8.03 (d, J = 8 Hz,1H, H-8), 7,65 (s, 1H, H-2'), 7.50 (s, 1H, H-4), 7.44 (d, J = 8 Hz, 1H, H-5), 7.39 (t, J = 8 Hz, 1H, H-7), 7.20 (s, 1H, H-5'), 7.15 (t, J = 8 Hz, 1H, H-6), 6.95 (s, 1H, H-4'), 6.82 (s, 1H, H-2), 4.52 (t, J = 7.5 Hz, 2H, 9-CH 2 -), 4.06 (t, J = 6.5 Hz, 2H- 1'- CH 2 -), 3.84 (s, 3H, 1-OCH 3 ), 2.44 (s, 3H, 3-CH 3 ), 2.15 (m, 2H, 9-CH 2 -CH 2 -CH 2-1ꞌ). 13 C- NMR (125 MHz, DMSO-d 6, ppm) δ (C-1), (C-8a), (C-2'), (C-3), (C-4'), (C-9a), (C-7), (C-4b), (C-4a), (C-6), (C- 5'), (C-8), (C-4), (C-2), (C-5), 55.5 (1- OCH 3 ), 43.8 (1'- CH 2 -), 42.2 (9-CH 2 -), 31.4 (9-CH 2 -CH 2 -CH 2-1ꞌ), 21.3 (3-CH 3 ).HR-MS (m/z): [m+h] +, calculated for C 20 H 22 N 3 O: (1-methoxy-3-methyl-N-carbazolyl)-1-(2-methyl-5-nitro-1-imidazolyl)propane 6d Yield 53% (yellow solids); mp: o C; 1 H-NMR (500 MHz, DMSO-d 6, ppm) δ 8.35 (s, 1H, H-4'), 8.02 (d, J = 8 Hz, 1H, H-8), 7.53 (d, J = 8 Hz, 1H, H-5), 7.49 (s, 1H, H- 4), 7.41 (t, J = 8 Hz, 1H, H-7), 7.15 (t, J = 8 Hz, 1H, H-6), 6.82 (s, 1H, H-2), 4.57 (t, J = 7.5 Hz, 2H, 9-CH 2 -), 4.04 (t, J = 6 Hz, 2H, 1'-CH 2 -), 3.81 (s, 3H, 3-OCH 3 ), 2.44 (s, 3H, 3- CH 3 ), 2.23 (s, 3H, 1H, 2'-CH 3 ), 2.11 (m, 2H, 9-CH 2 -CH 2 -CH 2-1ꞌ). 13 C-NMR (125 MHz, DMSO-d 6, ppm) δ (C-1), (C-5'), (C-2'), (C-8a), (C-3), (C-7), (C-4b), (C-4a), (C-4'), (C-6), (C-8), (C-4), (C-2), (C-5), 55.3 (1- OCH 3 ), 44.0 (1'- CH 2 -), 41.7 (9-CH 2 -), 29.9 (9-CH 2 - CH 2 -CH 2-1ꞌ), 21.3 (3-CH 3 ), 12.4 (2'-CH 3 ).). HR-MS (m/z): [M+H] +,calculated for C 21 H 23 N 4 O 3 : (1-methoxy-3-methyl-N-carbazolyl)-1-(2-benzotriazolyl)propane 6e Yield 36% (white solids); mp: o C; 1 H-NMR (500 MHz, DMSO-d 6, ppm) δ 8.03 (d, J = 8 Hz, 1H, H-8), 7.93 (m, 2H, H-4ꞌ, H-7') 7.55 (d, J = 8 Hz, 1H, H-5), 7.49 (s, 1H, H-4), 7.44 (m, 2H, H-5ꞌ, H-6'), 7.39 (t, J = 8 Hz, 1H, H-7), 7.15 (t, J = 8 Hz, 1H, H-6), 6.77 (s, 1H, H-2), 4.80 (t, J = 7 Hz, 2H- 2'-CH 2 -), 4.67 (t, J = 7 Hz, 2H, 9-CH 2 -), 3.63 (s, 3H, 1-OCH 3 ), 2.48 (m, 2H, 9-CH 2 -CH 2 -CH 2-2ꞌ), 2.43 (s, 3H, 3-CH 3 ). 13 C-NMR (125 MHz, DMSO-d 6, ppm) δ (C-1), (C-8'), (C-8a), (C-3), (C-9a), (C-5ꞌ, C-6'), (C-7), (C-4b), (C-4a), (C-6), (C-8), (C-4ꞌ, C-7'), (C-4), (C-2), (C-5), 55.2 (1- OCH 3 ), 53.7 (2'- CH 2 -), 42.0 (9-CH 2 -), 30.1 (9-CH 2 -CH 2 -CH 2-2ꞌ), 21.2 (3-CH 3 ).HR-MS (m/z): [M+H] +,calculated for C 23 H 23 N 4 O: (1-methoxy-3-methyl-N-carbazolyl)-1-(benzotriazolyl)propane 6f Yield 54% (white solids); mp: o C; 1 H-NMR (500 MHz, DMSO-d 6, ppm) δ 8.09 (d, J = 8 Hz, 1H, H-4ꞌ), 8.02 (d, J = 8 Hz, 1H, H-8), 7.81 (d, J = 8 Hz, 1H, H-7') 7.54 (m, 2H, H-5, H-6'), 7.47 (s, 1H, H-4), 7.42 (t, J = 8 Hz, 1H, H-5'), 7.38 (t, J = 8 Hz, 1H, H- 7), 7.14 (t, J = 8 Hz, 1H, H-6), 6.74 (s, 1H, H-2), 4.81 (t, J = 6.5 Hz, 2H- 1'-CH 2 -), 4.58 (t, J = 7.5 Hz, 2H, 9-CH 2 -), 3.47 (s, 3H, 1-OCH 3 ), 2.40 (s, 3H, 3-CH 3 ), 2.35 (m, 2H, 9-CH 2 - CH 2 -CH 2-1ꞌ). 13 C-NMR (125 MHz, DMSO-d 6, ppm) δ (C-1), (C-9'), (C- 225 ISSN

240 8a), (C-8'), (C-3), (C-9a), (C-6'), (C-7), (C-4b), (C-5'), (C-4a), (C-6), (C-4'), (C-8), (C-4), (C-7'), (C-2), (C-5), 55.0 (1- OCH 3 ), 45.2 (1'- CH 2 -), 42.1 (9-CH 2 -), 29.9 (9-CH 2 - CH 2 -CH 2-1ꞌ), 21.2 (3-CH 3 ).HR-MS (m/z): [M+H] +,calculated for C 23 H 23 N 4 O: Synthesis of N-(4-(4-fluorophenyl)-6-isopropyl-5-((1-methoxy-3-methyl-9Hcarbazol-9-yl)methyl)pirimidin-2-yl)-N-methylmethanesulfonamide 6g Synthesis of N-(5-(bromomethyl)-4-(4-fluorophenyl)-6-isopropylpyrimidin-2-yl)- N-methylmethanesulfonamide 5 3-(1-methoxy-3-methyl-N-carbazolyl)propyl bromide (2) was prepared in 87% yield according to the known procedure as described by Danand coworkers [7] Synthesis of N-(4-(4-fluorophenyl)-6-isopropyl-5-((1-methoxy-3-methyl-9Hcarbazol-9-yl)methyl)pirimidin-2-yl)-N-methylmethanesulfonamide 6g A mixture of murrayafoline A (1) (0.211 g, 1 mmol), K 2 CO 3 (0.621 g, 4.5 mmol), N- (5-(bromomethyl)-4-(4-fluorophenyl)-6-isopropylpyrimidin-2-yl)-Nmethylmethanesulfonamide 5 (1.25 g, 3 mmol) and (1-butyl)triethylammonium bromide (35.7 mg, 0.15 mmol) in dry dimethylformamide (15 ml) was stirred at 80 o C for 24 h and solvent was removed under reduced pressure. The resulting mixture was dissolved in water (20 ml) and extracted with EtOAc (3 20 ml). The combined extract was dried over anhydrous sodium sulfate and solvent was removed under reduced pressure to afford crude 3g that was purified by column chromatography eluting with n-hexane:ethylacetate 2:1. Yield 43% (white solids); mp: o C; 1 H-NMR (500 MHz, DMSO-d 6, ppm) δ 7.95 (d, J = 8 Hz, 1H, H-8), 7.66 (dd, J 1 = 3 Hz, J 2 = 8 Hz, 2H, H-6'', H-2''), 7.23 (m, 3H, H-7, H-5'', H-3''), 7.06 (t, J = 8 Hz, 1H, H-6), 6.90 (d, J = 8 Hz, 1H, H-5), 6.06 (s, 2H, 5ꞌ- CH 2-9), 3.85 (s, 3H, 1-OCH 3 ), 3.51 (s, 3H, 2ꞌ-NCH 3 SO 2 CH 3 ), 3.42 (s, 3H, 2'- NCH 3 SO 2 CH 3 ), 3.32 (m, 1H, 6'-CH(CH 3 ) 2 ), 2.43 (s, 3H, 3-CH 3 ), 0.77 (d, J = 6.5 Hz, 6H, 6ꞌ-CH(CH 3 ) 2 ). 13 C-NMR (125 MHz, DMSO-d 6, ppm) δ (C-6'), (C-4'), (C-4'', J = Hz ), (C-2'), (C-1), (C-8a), (d, J CF = 3Hz, C- 1''), (d, J CF = 8.5 Hz, C-2'', C-6''), (C-3), (C-9a), (C-7), (C-4b), (C-4a), (C-5'), (C-6), (C-8), (d, J CF = 21.6 Hz, C-3'', C-5ꞌꞌ) (C-4), (C-2), (C-5), 55.6 ( 1-OCH 3 ), 42.8 (5ꞌ-CH 2-9), 41.5 (2ꞌ- NCH 3 SO 2 CH 3 ), 33.1 (2'-N-CH 3 SO 2 CH 3 ), 31.2 (6ꞌ-CH(CH 3 ) 2 ), 21.2 (-CH(CH 3 ) 2 ).HR-MS (m/z): [M+H] +, calculated for C 30 H 32 N 4 O 3 FS: Bioactive assays The biological activity wasundertaken in Institute of Natural Products Chemistry (VAST). The antifungal and antibacterial activities were assessed by microbroth dilution method [8] using fungi and bacteria: E. coli, P. aeruginosa, B. subtilis, S. aureus, A. niger, F. oxysporum, S.cerevisiae and C. albicans and the compounds were assumed to be inactive if the minimum inhibitory concentration (MIC) exceeded 50 μg/ml, the references 226 ISSN

241 were used for antimicrobial of murrayafoline A derivatives including: Streptomicin for Gram (+) bacteria.b. subtillis (ATCC 27212) and S. aureus, (ATCC12222) with MIC values of 7.19 and μg/ml respectively, Penicillin for Gram (-) bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC 25923) with MIC values of 6.52 and respectively, Nystatin for filamentous fungi A. niger (439), F. oxysporum (M42) and S. cerevisiae (SH 20), C. albicans (ATCC 7754) with MIC values of 5.78, 2.89 and 1.45, 2.89 μg/ml, respectively. The values shown for these compounds are the average of three determinations. The in vitro evaluation of cytotoxicity was performed according to the described protocol of Likhitwitayawid [9] and the murrayafolinea derivatives were judged to have no cytotoxicity when their IC 50 values > 20 μg/ml. III. RESULTS AND DISCUSSION The synthesis of 6a-gwas outlined in schemes 1. For the synthesis of conjugates 6a-f, 1-bromo-3-propyl substituent 2 was first prepared in 83 % yield [6] (scheme 1). In the next step, N-alkylation of the heterocyclic amines including methylpiperazine (3a), ehtylpiperazine (3b) imidazole (3c), 2-methyl-5-nitroimidazole (3d), benzotriazole (3e) with the intermediate 2 was carried out in (DMF) in the presence of a mixture of K 2 CO 3 and transfer catalyst to give corresponding 6a-f in 36-73% yields. In case of benzotriazole, a mixture of target compounds 6eand 6f was isolated in ratio of 3/2 due to the reaction of 2 with both tautomer conformations of benzotriazole. The confirmed structure of 6a-f agreed well with NMR data. For the synthesis of 6g, hydroxyl group of 4 was first converted into bromide group by the reaction of 6 with phosphorous tribromide in acetonitrile to afford 5 in 87% yield. Next, the intermediate 5 was used as the agent for N-alkylation of murrayafoline A at 80 o C to give target compound 6g in 43% yield. The confirmed structure of 6g agreed well with NMR and MS data. 3 4a 4b 5 1 OCH 3 9a i 9NH 8a 8 OCH 3 N Br ii 3a-f 3 4a 4b 5 1 OCH 3 9a 9N 8a 8 R R: N 3' 5' a 1' N 1' 2' N 3' 5' 1 N b CH 3 1' 2' N N N N H 3 C 1 2 6a-f F F 3'' 3'' H 3 CO 1 3 iii iv 6'' H H 1'' 2 2 6'' H 9a C OH C Br 1'' 2 4a 3' C N 9 4b 3' N N 5' 5' N 8a 5 N N CH CH H 3 3 C N N CH CH 3 H 3 C N N CH CH 8 1' 1' 3 SO 2 CH 3 CH SO 3 2 CH 3 CH 3 SO 2 CH 3 CH g Scheme 1. Preparation of murrayafolinea derivatives 6a-f. F c 4 O 2 N N N N N N N 2' 3' 9' 1' 8' d 4' 3' 2' 9' 4' 1' 8' 6' e 4' 6' f ISSN

242 Reagents and conditions: (i) 1,3-dibromopropane, THF, NaH, rt, 72h, 83%; (ii) secondary amines, K 2 CO 3, DMF, rt,24 h, 36-73%; (iii) PBr 3, CH 3 CN, 10 o C 72h, 87%; (iv) Murrayafoline A, K 2 CO 3, DMF, 80 o C, 24 h, 43%. The result of antimicrobial activity evaluation showed that no compound was found to exhibit the antifungal and antibacterial activities even the conjugates of murrayafoline A with benzotriazole and imidazole by propylene linker.cytotoxic activity evaluation of the synthesized compoundsagainst four human cancer cell lines Hep-G2, Lu, RD and FL were listed in Table 1. No Table 1: The cytotoxic activity of murrayafoline A (1) and 6a-g. Compounds IC 50 (μg/ml) a Hep-G2 b RD b LU b FL b 1 6a b c d e f g Murrayafoline A Ellipticine a IC 50 shown for these compounds are the average of three determinations. b Cell lines: Hep-G2 (liver heptatocellular carcinoma, ATCC-HB-8065), LU (lung adenocarcinoma, ACTT-HBT-57), RD (rhabdomyosarcoma, ATCC-CCL-136) and FL (HeLa derivative, human cervix carcinoma). Murrayafoline A derivatives (6a-g) exhibited cytotoxic activity against four human cancer cell lines Hep-G2, Lu, RD and FL with IC 50 values ranging from μg/ml and stronger than that of murrayafoline A(1) except 6c, 6d and 6g as shown in Table 1. The level of activity also indicated that the cytotoxicity of target compounds was varied along with the nature of substituents attached to murrayafoline A, compound 6f containing benzotriazole component with flexible propylene linker exhibited the strongest activity against RD, Hep-G2 and FL with IC 50 values of 1.39, 1.71 and 2.53 μg/ml, respectively and approximately three to four fold stronger activity against three cancer cell lines than that of murrayafoline A. However, compounds6f and its tautomer 6e showed no activity against LU cell line. The derivative 6d containing 2-methyl-3-nitroimidazole showed activity against all cancer cell lines tested with IC 50 from μg/ml and can be considered as the same to the activity of murrayafoline A. The conjugation of murrayafoline A with methyl and ethylpipezarine though propylene linker (6a and 6b) inhibited all tested cancer cell lines with IC 50 ranging from μg/ml and approximately two to three fold stronger than those of murrayafoline A. Compound 6g with bulky component connected to murrayafoline showed no activity. Finally, although no active murrayafoline A derivatives can be compared with reference (ellipticine) in terms 228 ISSN

243 of IC 50 value, this research may be useful orientation for the design, synthesis of active compounds ofmurrayafoline A to exploit new applications for treatment of cancer. IV. CONCLUSION Seven new conjugates of murrayafoline A with secondary and heterocyclic amines were successfully synthesized and elucidated their structures by spectroscopic methods such as NMR and HRMS. These derivatives were assessed antimicrobial activity and the activity against the Hep-G2, LU, RD and FL of the synthesized compounds6a-g was also evaluated and discussed. V. ACKNOWLEDGMENTS The present research was supported by Vietnam Academy of Science and Technology (VAST) via a project: VAST04.02/15-16 REFERENCES [1] N.M. Cuong, H. Wilhelm, A. Porzel, N. Arnold, L. Wessjohann, Nat. Prod. Res. Vol. 22, 2008, pp [2] K. Hirata, C. Ito, H. Furukawa, M. Itoqawa, L.M. Cosentino, K.H. Lee, Bioorg. Med. CXhem. Lett. Vol. 9, 1999, pp [3] M. Itogawa, Y. Kashiwada, C. Ito, H. Furukawa, Y. Tachbana, K.F. Bastow, K.H. Lee, J. Nat. Prod. Vol. 63, 2000, pp [4] C. Ito, M. Itogawa, K. Nakao, T. Murata, N. Kaneda, H. Furukawa, J. Nat. Med. Vol. 66, 2012, pp [5] S.H. Woo, M.J. Son, J.C. Kim, S.H. Jung, N.M. Quan, T.T.D. Huong, B.H. Tai. Y.H. Kim, patent KR B (2011). [6] T.T.T. Thuy, N.M. Cuong; T.Q. Toan, N.N. Thang, B.H. Tai, N. X. Nhiem; H. J. Hong, S. L. Kim, S. Legoupy, Y.S. Koh, Arch. Pharm. Res. Vol 36, 2013, pp [7] P.D. Nam, V.V. Truong, N.T. Dan, Vietnam Journal of Chemistry, Vol. 50, 2012, pp [8] A.K. Mishra, N.K. Kaushik, Eur. J. Med. Chem. Vol 42, 2007, [9] K. Likhitwitayawid, C.K. Angerhof, G.A Cordell, J.M. Pezzutto, N. Ruangrungsi. J. Nat. Prod.Vol 56, 1993, pp ISSN

244 EFFECT OF CATALYSt ON THE BURNING RATE OF ENERGY MATERIALS BASEd ON NC-NG-DINA Le Duy Binh 1*, Nguyen Viet Bac 2, Pham Van Toai 1 and Dam Duc Trung 1 1 Institute of Propellant and Explosives, 192 DucGiang, LongBien, Hanoi, Vietnam 2 Institute of Chemistry and Materials, 17 HoangSam, CauGiay, Hanoi, Vietnam *: binhld.12p7.pro@gmail.com Abstract. Effect of catalyst on the burning rate of energy materials (most of them are materials based on NC with a different plasticizer compound such as NG, DG, DNTEG, NGl, ) has been researched for a long time. However, the certain researches on energy materials based on NC-NG-DINA have not been mentioned. By experiment, the author gave the initial results of effect of content of lead (II) oxide catalyst (including its particle size), additives to formation of a carbon black matrix (C t, DNT) on the burning surface, on burning rate of energy materials based on NC-NG-DINA. The obtained data is the important basis for the choosing and using of these energy materials. Keywords: Energy materials, burning rate, particle size, content, PbO catalyst, carbon and DNT additives. I. INTRODUCTION Energy materials based on NC-NG-DINA with main ingredient nitrocellulose (NC), which makes up about 54 to 62 percent and it s seen as the substrate of composite materials. Nitroglycerine (NG) and diethanolnitroamine dinitrate (DINA) are energy plasticizers, the total content of them is about 25 to 45 percent. In addition, energy materials contain some different substances such as a chemical stabilizer (centralite), burning catalyst (PbO), burning catalyst additive (C t, technology carbon), burning stabilizer (CaCO 3 ) and technology additive (vaseline) [3]. Some researches mentioned the effect of burning catalyst additives on burning rate of energy materials. However, most of them are materials based on NC-NG or with a different energy plasticizer. For that, it has not evaluated all the properties of burning process for energy materials [6-8]. Recently, the researchers [1-5] published papers relating to energy materials based on complex (triple base or composite materials). However, these researches only mentioned of energy materials based on NC-NG-DG [1, 2, 4, 5], sometimes it s initial results of these energy materials [3]. Therefore, researching into effect of burning catalyst on burning rate of energy materials based on NC-NG-DINA can somehow explain the complex burning process of these materials. In the paper, the authors focused on the effect of catalyst content, particle size and content of additives to formation of a carbon black matrix (C t, DNT) on the burning 230 ISSN

245 surface and on burning rate of energy materials based on NC-NG-DINA. Moreover, the paper mentioned also burning surface based on burning production of these materials. II. EXPERIMENTS 2.1. Materials Nitrocellulose (NC, with content was equal 11,94 percent of nitrogen in NC) and ethanol (C 2 H 5 OH) were made in Vietnam; diethanolnitraminedinitrate (DINA) and nitroglycerine (NG) were synthesised by the researchers; dinitrotoluene (DNT), centralite, calcium carbonate (CaCO 3 ), vaseline, zinc-stearate were made in China; technology carbon (N220) was made in Korea; lead (II) oxide (PbO) was made in Germany Preparation of NC-NG-DINA based energy materials Preparing samples of materials used for measuring burning rate: Ingredients (given) were stirred in water (not including additives of PbO, C t ) at 55 o C, for 2,5 hours and 6/1 of module. After stirring semi-finished products was rolled on special equipment at temparature about 85 to 95 o C and then it was compressed to create cyclinder shape (7 mm of diameter) to measure burning rate and burning surface. Preparing samples of materials used for measuring burning surface: Based on rule of energy materials burning mechanism, it is divided by five zones (phases): hot zone, phase transition zone (K-phase), primary flame zone, dark zone, second flame zone and gas production zone. The energy materials bar (cyclinder shape, 7 mm of diameter, 10 mm of height) was held tight on metal copper bar (smooth) by thin special gum layer. The properties of thermal motion of copper is spread rapidly so K-phase zone was created by tangent surface between energy materials and copper bar. The method to make out burning surface was described on figure 1a and Fig. 1b. ISSN Fig. 1. The making out burning surface. a) Materials bar before burning; b) Materials bar after burning Measurement of burning rate Measurement of burning rate at constant pressure (p = 100 atm) in pressure bomb contained inert gas (nitrogen gas) according to standard of 06 TCN 888:2001. The method of measuring was described as following: 231

246 - The method of measuring: Energy materials bar after being compressed to create cyclinder shape, thin gumcovered to anti-fire and heat insulation and then this materials bar was reeved through by two emay lines (resistor line), distance of between the first line and the second line is 50 mm. The energy materials bar was burned in pressure bomb at constant pressure. The burning time of materials bar was calculated as following: The first, when the materials bar was burned, the heat was generated to cut the first resistor line (start line) after that cutting the second resistor line (stop line). The burning time was saved by stopwatch equipment, this equipment was about exact time 10-6 (s). The burning rate was calculated equal ratio between the distance and the burning time. - Equipments: Bomb, balanced bomb, manometer, gas bomb (nitrogen gas). The schematic diagram shown in Fig Measuring bomb; 6 2-Balanced bomb; 3-Loading bomb; Manometer; 5-Keeping of sample; 6-Firing equipment; Stopwatch equipment; Fig. 2. Equipment of burning rate measuring at constant pressure. - The different equipments: Equipment of time stopwatch (exact time was about 10-6 s), firing equipment Measurement of burning surface The materials bar was burrned (Fig. 1b) after that being scanned on scanning electron microscope (SEM) to observe burning surface of burning production. III. RESULTS AND DISCUSSION 3.1. Effect of burning catalyst content on burning rate of energy materials based on NC-NG-DINA There were some researches [2, 4-7] showed that rate of burning process increases if content of catalyst increases in determined range. The rate of burning process will decrease if it s content is small or overcome. In fact, most of known energy materials [6-8] are 232 ISSN

247 based on NC-NG or NC with a different plasticizer compound carrying another energy such as NC-DEGDN, NC-DNTEG, NC-DNDEG, Therefore, there was a difference between these materials and the energy materials based on NC-NG-DINA, therein including effect of catalyst content. Based on energy materials of NC-NG-DINA (samples), the author studied on effect of PbO on burning rate. The results were described on Table 1. Table 1. Effect of PbO on burning rate of energy materials based on NC-NG-DINA. Sample + PbO, % Burning rate (100 atm), mmps 0,0 15,83 ± 0,05 0,2 16,37 ± 0,05 0,4 16,83 ± 0,05 0,6 17,22 ± 0,05 0,8 17,39 ± 0,05 1,0 17,21 ± 0,05 1,2 16,92 ± 0,05 1,4 16,85 ± 0,05 Note: Ingredients of sample including 58,5 % NC; 28,0 % NG; 8,6 % DINA; 1,40 % centralite; 1,60 % CaCO 3 ; 0,40 % C t and 0,70 % vaseline. Particle size of PbO was under 3µm. Table 1 showed that burning rate increases from 15,83 to 17,39 mmps if content of PbO increases from 0,0 to 0,8 percent. The burning rate decreases from 17,39 to 16,85 mmps if content of PbO increases from 0,8 to 1,4 percent. Maximum burning rate is equal 17,39 mmps corresponding to 0,8 percent of PbO. Documents [1-7] showed that burning process of materials is not enough to form of a carbon black matrix on burning surface of burning production if catalyst content is small. However, a carbon black matrix is broken down if content of catalyst is so high. Studying showed that effect of catalyst content on burning rate of energy materials is similar. However, the mathematical value of them is very different. Energy materials based on NC-NG-DINA has a less the content of catalyst than energy materials has been researched (it was about three to dozens of percent). Effect of burning catalyst was shown by the formula Z = U p /U 0p (where U p and U 0p were burning rates corresponding to samples with or without a catalyst at the same burning pressure). Therefore, it could take out a relationship between content of catalyst and effect coefficient of burning catalyst Z (at 100 atm) for energy materials based on NC-NG-DINA and as shown in Fig. 3. Fig. 3 showed that coefficient of Z increases from 1,0 to 1,1 times if content of PbO increases from 0,0 to 0,8 percent and coefficient of Z decreases from 1,1 to 1,07 if content of PbO increases from 0,8 to 1,4 percent. Maximum coefficient of Z is about 1,1 ISSN

248 corresponding to 0,8 percent of PbO and thus the maximum burning rate increases about 10 percents compared to materials sample without a catalyst. Fig. 3. The ralationship between coefficient of Z and content of catalyst at 100 atm. From studying results showed that effect of PbO burning catalyst was used in energy materials containing additive of C t is not much. Experiments showed that this burning catalyst is less effective than it is single (without C t ) Effect of particle size of catalyst on burning rate of energy materials based on NC-NG- DINA According to documents [1, 2, 4, 6, 7] showed that the burning catalyst effects on burning process according to two ways: firstly, it will increase reaction at K-phase, secondly, NC will be decomposed to go the other way (most of them formed gas of NO 2 ). These reasons load to formation of a carbon black matrix on burning surface of burning production. Therefrom, the burning rate of energy materials will increase. It can see that burning process of energy materials on K-phase surface is heterogeneous reaction. Therefore, particle size and distribution area of particle size will effect much on effect of burning catalyst what it made out. Table 2. Effect of particle size of catalyst on burning rate of energy materials based on NC-NG-DINA. Sample + region of particle size, µm Burning rate (100 atm), mmps 20 16,43 ± 0, ,71 ± 0, ,06 ± 0, ,27 ± 0, ,39 ± 0, ISSN

249 Effect coefficient of catalyst, Z The 4 th Academic Conference on Natural Science for Young Scientists, Master & Note: Ingredients of sample including: 58,5 % NC; 28,0 % NG; 8,6 % DINA; 1,40 % centralite; 1,60 % CaCO 3 ; 0,8 % PbO; 0,40 % C t and 0,70 % vaseline. In fact, to study on effect of particle size region on burning rate, the author chose region of particle size including regions: under of 20 µm, 10 µm, 7 µm, 5 µm and 3 µm, where, ingredients of sample were kept constant. The studying results were shown in Table 2. From results shown in Table 2, it could take out the relationship between coefficient of Z and particle size of catalyst as shown in Fig. 4. 1,08 1,06 1,04 1,02 1 0, Region of particle size of catalyst ( ), µm Fig. 4. The relationship between coefficient of Z and particle size of catalyst at 100 atm. From data in Fig. 4 showed that effect of burning catalyst and particle size are ratio that meaning it increases effect of burning catalyst if it decreases the particle size and it decreases effect of burning catalyst if it increases the particle size. At region of particle size under 3 µm compares to the one under 20 µm coefficient of effect of catalyst is about 1,06 times that meaning the burning rate increases about 6 percents. Thus, effect of the particle size of PbO on burning rate is not much Effect of additives to form of a carbon black matrix (C t, DNT) on burning rate of energy materials based on NC-NG-DINA The burning surface of energy materials sample based on NC-NG-DINA Experiments showed that sample of energy materials based on NC-NG-DINA could still form of a carbon black matrix whose ingredients maily consist of materials left from the burning process. However, this carbon black matrix is unstable or not to exist if materials ingredient does not contain additives of catalyst or to form of a carbon black matrix. In fact, the author studied on burning surface of energy materials samples based on NC-NG-DINA with or without a burning catalyst and additive to form of a carbon black matrix at the same atmospheric pressure. These studying results were shown in Fig. 5a and Fig. 5b. ISSN

250 a. The burning surface of energy materials based on NC-NG-DINA without a burning catalyst and additive to form of a carbon black matrix Effect of C t additive on burning rate b. The burning surface of energy materials based on NC-NG-DINA with a burning catalyst and additive to form of a carbon black matrix. Fig. 5. The burning surface of energy materials based on NC-NG-DINA at atmospheric pressure. Burning process of energy materials is ingredients burning process including additive of C t. Most of burning production of the additive will form of a carbon black matrix on burning surface [1, 2]. Documents [1, 2, 6, 7] showed that energy materials have to have a big enough with ingredients such as DNT, DBP, triacetine and some the same another ingredients or they have to have initial ingredient of C t in case of without above ingredients in order to form of a carbon black matrix. In fact, the author studied on increasing ratio C t in ingredients of energy materials. The results of studying on effect of C t on burning rate shown in Table 3. Table 3. Effect of C t content on burning rate of energy materials based on NC-NG-DINA. Sample + C t, % Burning rate (100 atm), mmps 0,00 11,97 ± 0,05 0,30 15,65 ± 0,05 0,40 15,83 ± 0,05 0,50 16,17 ± 0,05 0,60 14,47 ± 0,05 Note: Ingredients of sample including: 58,5 % NC; 28,3 % NG; 8,9 % DINA; 1,50 % centralite; 1,70 % CaCO 3 and 0,70 % vaseline. Particle size of PbO was under 3µm. From data shown in Table 3 that it could take out the relationship between coefficient of Z and content of C t as shown in Fig ISSN

251 Effect coefficient of catalyst, Z The 4 th Academic Conference on Natural Science for Young Scientists, Master & 1,36 1,35 1,34 1,33 1,32 1,31 1,3 1,29 1,28 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 Content of Ckt addive, % Fig. 6. The relationship between coefficient of Z and content of C t at 100 atm. Figure 3 showed that coefficient of Z increases from 1,31 to 1,35 if content of C t additive increases from 0,3 to 0,5 percent and then coefficient of Z decreases from 1,35 to 1,29 if content of C t additive increases from 0,5 to 0,6 percent. Maximum coefficient of Z is about 1,35 corresponding to 0,5 percent of C t additive and thus maximum burning rate increases about 35 percents compared to materials sample without an additive. Thus, it only needs small content of C t (about 0,5 percent) that it will form of a carbon black matrix on surface of burning production. The effect of burning catalyst will decrease if content of C t additive is bigger than 0,5 percent of C t. It was explained that converging process of burning catalyst on a carbon black matrix prevented by excess of C t. It could see the addive of C t effect on burning rate better than catalyst of PbO. The burning rate of sample containning catalyst of PbO (including C t ) increases about 10 percents compared to about 35 percents for sample containning additive of C t Effect of DNT additive on burning rate DNT is extra plasticizer addive for energy materials. An appearance of DNT in energy materials will be decreased its heat of combustion. However, the burning rate of materials will be increased if content of DNT increases at determined region. In fact that to confirm the truth of problem, the author studied on effect of DNT content on burning rate of energy materials. The studying results were shown in Table 4. Table 4. The effect of DNT content on burning rate of energy materials based on NC-NG-DINA. Sample + DNT, % Burning rate (100atm), mmps 0,0 11,97 ± 0,05 1,5 12,28 ± 0,05 2,0 12,48 ± 0,05 2,5 12,18 ± 0,05 Note: Ingredients of sample including: 58,5 % NC; 28,5 % NG; 8,9 % DINA; 1,60 % centralite; 1,70 % CaCO 3 and 0,80 % vaseline. Particle size of PbO was under 3µm. The studying results showed that the burning rate increases from 12,28 to 12,48 mmps if the DNT increases from 1,5 to 2,5 percent of content. The burning rate increases 237 ISSN

252 about 4 percent compared to sample without DNT. Maximum burning rate is about 12,48 mmps corresponding to 2 percent of DNT content. IV. CONCLUSION The paper researched into: - The effect of burning catalyst content on burning rate of energy materials based on NC-NG-DINA. The result showed that maximum burning rate increases about 10 percents compared to materials sample without a catalyst. - The effect of particle size of catalyst on burning rate of energy materials based on NC- NG-DINA. The result showed that it increases effect of burning catalyst if it decreases the particle size and vice versa. Maximum burning rate increases about 6 percent (at region of particle size under 3 µm compared to the one under 20 µm). Thus, effect of the particle size of PbO on burning rate is not much. - The effect of additives to form of a carbon black matrix (C t, DNT) on burning rate of energy materials based on NC-NG-DINA. The result showed that the energy materials based on NC-NG-DINA could still form of a carbon black matrix whose ingredients maily consist of materials left from the burning process. However, this carbon black matrix is unstable or not to exist if materials ingredient does not contain additives of catalyst or to form of a carbon black matrix. The DNT and C t are good additives to form of a carbon black matrix for these energy materials. The studying results showed that the maximum burning rate increases about 4 percents or 35 percents (comparing to sample without DNT or C t ) corresponding to 2 percent of DNT or 0,4 percent of C t respectively. REFERENCES [1] Le Duy Binh, Pham Van Toai and Dam Duc Trung, Effect of some additives on the burning rate of ballistics propellant based on NC-NG-DG, Vietnam Journal of Chemistry, N o. 53 (5e1), p (2015). [2] Le Duy Binh, Dam Duc Trung and Le Anh Dung, Effect of some additives on the plasticizing and burning rate of propellant based on NC-NG-DG, Journal of Science and Technology, N o. 173, p.3-9 (2015). [3] Do Duc Tri, Ngo Van Giao, Pham Van Toai, Le Duy Binh, Hoang The Vu, Ha Duc Giang, Ngo The Hai and Pham Quang Hieu, Some results of research on improving the manufacture of propellant charge NDSI-2K used for PG-9 ammunition, Journal of Military Science and Technology, special number of PE 14, p (2014). [4] Pham Van Toai, Le Duy Binh, Nguyen Van Khuong and Dam Duc Trung, Researching into the synthesis oxide cobalt to be used for ballistics propellant in role catalyst, Journal of Military Science and Technology, N o. 36, p (2015). 238 ISSN

253 [5] Pham Van Toai, Ngo Van Giao, Le Duy Binh, Nguyen Van Khuong, Hoang The Vu and Do Duc Tri, Research, design and manufacture ballistics propellant based on nitroglycerine and diethyleneglycol dinitrate plasticizers and used it in antitank rocket of B41M, Journal of Military Science and Technology, Special number of PE 14, p (2014). [6] Ньен Чан Аунг, Влияние состава пороха на эффективность действия катализаторов горения, Автореферат диссертации кандидата химических наук, Москва, Российский химико-технологический университет имю Д.Ию Менделеева (2008). [7] Денисюк А. П., Демидова Л. А., Особенности влияния некоторых катализаторов на горение баллиститных порохов, Физика Горения и Взрыва, Т. 40, 3, С (2004). [8] Денисюк А. П., Шепелев Ю. Г., Русин Д. Л., Шумский И. В. Влияние гексогена и октогена на эффективность действия катализаторов горения баллиститных порохов, Физика Горения и Взрыва, Т. 37, 2. С (2001). ISSN

254 EXPRESSION AND CHARACTERIZATION OF RECOMBINANT L-ASPARAGINASE IN PICHIA PASTORIS Thi Hien Trang Nguyen, Van Van Vu, Huu Quan Nguyen, Dinh Thi Quyen and Thi Tuyen Do * Institute of Biotechnology, Vietnam Academy of Science and Technology 18 Hoang Quoc Viet, Cau Giay Distric, Hanoi, Vietnam *: nthientrang@ibt.ac.vn, dttuyen@ibt.ac.vn Tel ; Fax: Abstract. L-asparaginase is an anti-neoplastic agent against childhood acute lymphoblastic leukemia and Hodgkin s lymphoma. L-asparaginase derived from two different sources: Escherichia coli and Erwinia chrysanthemi. In this study, L-asparaginase from Erwinia chrysanthemi was expressed in Pichia pastoris X33 under the control of the AOX1 gene promoter. The expression level of recombinant L-asparaginase in optimized culture media was 33% of the total cellular protein. The recombinant L-asparaginase was purified from the culture supernatant by the affinity chromatography with probond TM resin and showed a protein band of about 45 kda on SDS-PAGE. The molecular mass of L-asparaginase on SDS PAGE confirmed that the protein was glycosylated. The purified recombinant L- asparaginase showed a specific activity of 5.4 U/mg. Optimum temperature and ph were at 45 C and ph 7-8, respectively. Keywords: Erwinia chrysanthemi, expression, L-asparaginase, leukemia, Pichia pastoris. I. INTRODUCTION L-Asparaginase (E.C ) is an amidohydrolase which catalyses the hydrolysis of amino acid asparagine into aspartic acid and ammonia. Based on biochemical and crystallographic data, the known asparaginase sequences can be divided into three families: bacterial-type asparaginases, plant-type asparaginases and Rhizobium etli asparaginase. L- asparaginse from bacteria was divided into two types: I and II, L-asparginas I is used to reduce the formation of acrylamide in fried and oven-cooked foods especially in potato chips [19, 20]. L-asparaginase II is used in treatment of acute lymphoblastic leukemia and non-hodgkin s [14]. E. coli periplasmic L-asparaginase (EcAII) and Erwinia chrysanthemi L-asparaginase (ErA) have been successfully used in leukemia treatment. However, their L-glutaminase side activity limits their use and causes severe side-effects as a result of L- glutamine deprivation [15]. Serious liver disorders, acute pancreatitis, hyperglycemia, immunosuppression and other dysfunctions are some of the side-effects in patients receiving L-asparaginase treatment. Besides, differences between E. coli and Er. chrysanthemi asparaginase in respect to toxicity and efficacy have been found [2]. Presently, L-asparaginase is produced throughout the world by submerged fermentation (SmF). This methodology has many disadvantages such as the low 240 ISSN

255 concentration product formation and consequent handling, reduction and disposal of large volumes of water during the downstream processing etc. An alternative solution to molecular cloning and genetic engineering are the promising key tools which has ability to produced recombinant L-asparaginase. Cloning and expression studies of L-asparaginase in E. coli, B. subtilliss and Pichia pastoris has been reported [1, 5, 7, 8, 17, 18]. The P. pastoris expression system has been widely used to produce a variety of heterologous proteins. The expressed protein, depending on its characteristics and the choice of expression vector, can accumulate intracellularly or be secreted to the growth medium. Hence, we studied the expression, purification and chracterization of recombinant L-asparaginase in P. pastoris X33. II. MATERIALS AND METHODS Chemicals and reagents Peptone and yeast extract were purchased from Bio Basic Inc. (New York, USA); Yeast nitrongen base (YNB), L-asparagin and Nesler s reagent (Sigma-Aldrich Co., St. Louis, USA). Restriction enzymes, Taq DNA polymerase, T4-DNA ligase, EasySelect TM Pichia Expression Kit. Medium YP medium (2% (w/v) peptone, 1% (w/v) yeast extract), MT1 medium (1.34% (w/v) YNB, 1% (v/v) methanol, 0.1 M phosphate buffer, ph 6), MT2 medium (2% (w/v) peptone, 1% (w/v) yeast extract, 0.34% (w/v)ynb, 1% (v/v) methanol, 1% (w/v) (NH 4 )- 2SO 4, 4x10-5 biotin, 0.1 M phosphatebuffer, ph 6) MT4 medium (2% (w/v) peptone, 1% (w/v) yeast extract, 1% (v/v) methanol, 0.1 M phosphate buffer, ph 6), MT5 medium (2% (w/v) peptone, 1% (w/v) yeast extract, 1.34% (w/v) YNB, 1% (v/v) methanol, 0.1 M phosphate buffer, ph 6) Strains and vectors Expression plasmid ppaspg harbored the gene aspg encoding the L-asparaginase II from Erwinia chrysanthemy (Laboratory of Enzyme technology, IBT, VAST). P. pastoris X33 (Invitrogen Crop., Carlsbad, USA) were used for expression of the L-asparaginase. Yeast transformation and screening ppzaspg linearzed with SacI was transformed into P. pastoris X33 according to the EasySelect TM Pichia Expression Kit. Transformants were screened on YPD (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose, 2% (w/v) agar) plates containing Zeocin TM at a final concentration of 1000 µg/ml. The presence of the gene L-asparaginase in the transformants was confirmed by PCR using yeast genomic DNA as template with primers (ppzasn-pml F/PzAsn_Xba R) and (AOX1 F/ AOX1 R) (Table 1) ISSN

256 Table 1: Primer sequences. Primers pzasn_pml F PzAsn_Xba R AOX1 F AOX1 R Sequences ATA TAT CAC GTG GGC CGA TAA ACT GCC GAA T ATA AAG TCT AGA GCG TAG GTA TGG AAG TAT TCT T GAC TGG TTC CAA TTG ACA AGC GCA AAT GGC ATT CTG ACA TCC Gene expression The transformant P. pastoris X33/L-asparaginase was cultivated in 3 ml of YP medium supplemented with 1% (w/v) glycerol and at 30 C with agitation at 200 rpm until an OD 600nm was 5-6. The cell pellet was harvested by centrifugation at 6000 rpm for 5 min. For AOX1 promoter-controlled expression of the L-asparaginase, the cell pellet was resuspended in 30 ml of YP medium dupplented with 1% (w/v) methanol, and every 24 h to maintain induction. Cultivation was performed at 30 C and 200 rpm. Non-recombinant P. pastoris host cells (X-33 strain) were similarly treated and analyzed as negative controls. The culture supernatant was collected periodically by centrifugation at rpm and 4 C for 10 min to estimate the L-asparaginase activity. Optimization of expression condition for recombinant L-asparaginase In order to enhance the expression level of recombinant proteins in P. pastoris, the growth conditions in shake flasks such as type of medium and induction time were optimized To identify the best medium for the high-level production of raspg, five different media (MT1, MT2, MT4, MT5, YP) were examined. In detail, a single colony of the recombinant strain was grown in 5 ml of YP containing glycerol medium overnight and inoculated into 50 ml of five medium. Methanol was added every 24 hours to induce expression of the recombinant protein. The culture was allowed to grow for 72 hours and the culture supernatant was collected for level expression analysis To identify the best expression time, P. pastoris cells culture was collected every 24 hours after methanol induction for enzyme activity analysis. Purification of recombinant L-asparaginase The culture supernatant P. pastoris X33 containing the L-asparaginase was applied to an affinity Ni 2+ - ProBond TM resin (Invitrogen) charged into a chromatoghraphy column. The purification of the recombinant His-tagged L-asparaginase was carried out according to ProBond TM Purification System (Invitrogen). The rl-aspg solution was used for characterization Temperature and ph optima The temperature and ph optima of L-asparginase were determined by measuring the activity as described above using 20 mm Tris-HCl buffer ph 8.6 in the temperature range 242 ISSN

257 of C; and 20 mm acetate buffer (ph 4-5), phosphate buffer (ph 7-8) and 20 mm Tris-HCl buffer (ph 8-9) at 37 C, respectively. Electrophoresis analysis and protein concentration The molecular mass of the L-asparginase was determined by 12.5% SDS polyacrylamide gel electrophoresis [9] with Biometra equipment (Göttingen, Germany). Proteins were visualized by staining with 0.1% (w/v) AgNO 3. Determination of enzyme activity Enzyme activity of recombinant L-asparaginase was measured by method introducing in previous study [12] in which the rate of ammonia formation will be detected by Nessler s reagent at 37 C. One unit of L-asparaginase activity was determined as the amount of enzyme that librates 1 µmol of ammonia from L-asparaginase per minute at 37 C. III. RESULTS AND DISCUSSION Construction of expression system Pichia pastoris X33/pPaspg For transformation into P. pastoris cells, the recombinant vector was linear (4581 bp) for integration into yeast genome (Fig. 1). Plasmid ppaspg was integrated into the P. pastoris X33 genome by crossing over at the AOX1 locus, disrupting the wide-type AOX1 gene of the host. Fig. 1. Lineared ppiczαaasn (1); marker (M). Fig. 2. DNA electrophoresis of PCR product of DNA extracted from recombinant P. pastoris X33 using AOX primers (A) and gene primer (B) M: DNA ladder 10 kb. The integration of aspg was confirmed by PCR with gene aspg primers showing one band corresponding to the gene aspg (~ 1kb) (Fig 2A) and with AOX1 primers, showing two PCR products of 1.5 kb band corresponding to the gene (~ 1kb) plus AOX1 (588 bp) and 2.2 kb band corresponding to the other AOX1 gene in the P. pastoris X33 genome (Fig. 2B). All the transformants having those bands could express the strange protein. Among 12 checked clonies, 10 clones (X1, X2, X3, X4, X6, X8, X9, X11, X12) was selected to express the L-asparaginase. ISSN

258 Expression of the recombinant L-asparaginase After induction of methanol for 72 hrs, six selected transformants (X1, X2, X3, X6, X11, X12) showed L-asparaginase activity. The P. pastoris X11 transpformant showing the highest production of the L-asparaginase (0.63 U/ml, Table 2) was used for enzyme production. On SDS-PAGE, four transformants showed two extra protein two band corresponding to 35 and 45 kda (Fig. 3, lane ), which was not appear in the supernatant of P. pastoris X33 (Fig.3, lane X33). This heterologous protein might be the L- asparaginase expressed in P. pastoris X33. The difference in molecular mass of raspg might be due to the glycosylation. Table 2. L-asparaginase activity of crude supernatant of recombinant P. pastoris X33. Fig. 3. SDS-PAGE of crude supernatant of recombinant P. pastoris X33; X33 (control) are that of P. pastoris X33 without asn gene respectively. M: Protein marker (1:X1, 2: X2, 3: X3, 6: X6, 11: X11, 12: X12). Optimization of expression condition for recombinant L-asparaginase Fig. 4. Effected of type media to protein expression (M: marker, 1: MT5, 2: MT4, 3: MT2, 4: MT1, 5: YP). Fig. 5. Effected of time course of induction to enzyme activity. 244 ISSN

259 The level expression of raspg in five mediums MT5 (lane 1) was 5.82%, MT4 (lane 2) was 17.22%, MT3 (lane 3) was 12.96%, MT1 (lane 4): no detected express and YP (lane 5) was 33.21% (Fig. 4). So, YP medium was found to be optimal for the expression of raspg, it is simple medium suitable for producing large fermentation. The enzyme activity had highest at 72h (4 U/mg), and decrease when the time increase, achived 0.67 U/mg at 120 h. So recombinant L-aspraginase from P. pastoris X11 achived highest produce at 72 h in YP medium. Purification raspg The recombinant L-asparaginase was purified from the culture supernatant of P. pastoris X33/pPaspg by affinity chromatography Ni 2+ -ProBond resin and showed only one protein band of about 45 kda on SDS-PAGE (Fig. 6, lane 7). The purified L-asparaginase had a specific activity of 5.73 U/mg. In fact, the molecular weight was higher than predicted value (~35 kda). This difference can be explained by the contribution of glycosylations to the molecular mass of protein molecule because of the adjacent O-linked (hydroxyl group of serine, threonine or hydroxylisine) and N-linked (group of asparagines carbohydrate chains) to heterologous proteins in P. pastoris [10]. Moreover, the raspg sequence was predicted to N-glycosylation in the asparagine position 85, O-glycosylated in the serine position 40 and in the thereonin position 47 by program NetNGlyc 4.0 and NetOGlyc 4.0. This was also observed when recombinant L-asparaginase was produced in P. pastoris GS115 possessing a molecule mass of ~45 kda [4] higher than expected of ~35 kda [3]. Temperature and ph optimum Fig. 6. Purification of raspg (Lane 1: Crude supernatant; Lane 2: Flow through; Lane 3: Wash; Lane 4 7: Elution fragments). Fig. 7. Effects of temperature (A) and ph (B) on recombinant L-asparaginase. ISSN

260 The recombinant L-asparaginase had optimum temperature of 45 ºC (Fig. 7A) had optimum ph of 7-8 (Fig. 7B). The result similar Giaro s study which showed that the optimum ph values were 7.2 and 9.0, optimum temperature at both ph, 7.2 and 9.0, was 46 ºC [6]. The optimal activity of the recombinant L-asparaginase from a thermotolerant strain E. coli KH027 in E. coli DH5α was 43 C and ph 6 [13]. It was 37 C and ph 7.5 for raspg from E. coli W3110 in E. coli BL21 (DE3) [11] and that of raspg from Withania somnifera in E. coli BL21 (DE3) was 37 C and ph 8 for [16]. IV. CONCLUSION We have successfully expressed gene coding for L-asparaginase li in recombinant P. pastoris X33, P. pastoris/ppaspg-x11 had high expression levels. L-asparaginase best expression at 72 h induction in YP media. L-asparaginase was purified and characterization. V. ACKNOWLEDGEMENTS This study was supported by Vietnam Academy of Science and Technology, with the project Study on the production of recombinant L-asparaginase to inhibit cancer cell lines and treatment of acute lymphoblastic leukemia. REFERENCES [1] Bonthron D.T., Gene, 91 (1) 1990 pp [2] Duval M., Suciu S., Ferster A., Rialland X., Nelken B., Lutz P., Benoit Y., Robert A., Manel A.M., Vilmer E., Otten J., Philippe N., Blood, 99 (8) 2002 pp [3] Ferrara M.A., Severino N.M.B., Mansure J.J., Martins A.S., Oliveira E.M.M., Siani A.C., N. P.J., Torres F.A.G., S. B.E.P., Enzyme and Microbial Technology, pp [4] Ferrara M.A., Severino N.M.B., Valente R.H., Perales J., Bon E.P.S., Enzyme and Microbial Technology., pp [5] Ferrara M.A., Severino N.M.B., Valente R.H., Perales J., Bon E.P.S., Enzyme and Microbial Technology, pp [6] Girao L.F.d.C., Rocha S.L.G.d., Teixeiri R.S., Ferrara M.A., Perales J., Bon E.P.d.S., The 5th Congress of the Brazilian Biotechnology Society (SBBIOTEC): Meeting abstracts, 2014, pp. 78. [7] Jia M., Xu M., He B., Rao Z., J Agric Food Chem, 61 (39) 2013 pp [8] Kotzia G.A., Labrou N.E., J Biotechnol, 127 (4) 2007 pp [9] Laemmli U.K., Nature, pp [10] Lin-Cereghino G.P., Lin-Cereghino J., Ilgen C., Cregg J.M., Curr. Opin. Biotech., pp ISSN

261 [11] Magdy M.Y., Mohammed A.A.-O., J Biochem Mol Biol Biophys, 3 (6) 2008 pp [12] Mashburn L.T., Wriston J.C.J., Arch Biochem Biophys, pp [13] Muharram M.M., Abulhamd A.T., Mounir M.S.-B., Afr J Microbiol Res, 8 (15) 2014 pp [14] Narta U.K., Kanwar S.S., Azmi W., Crit Rev Oncol Hematol, 61 (3) 2007 pp [15] Ollenschlager G., Roth E., Linkesch W., Jansen S., Simmel A., Modder B., Eur J Clin Invest, 18 (5) 1988 pp [16] Oza V.P., Parmar P.P., Patel D.H., Subramanian R.B., 3 Biotech, 1 (1) 2011 pp [17] Pokrovskaya M.V., Aleksandrova S.S., Pokrovsky V.S., Omeljanjuk N.M., Borisova A.A., Anisimova N.Y., Sokolov N.N., Protein Expr Purif, 82 (1) 2012 pp [18] Pourhossein M., Korbekandi H., Adv Biomed Res, pp 82. [19] Rosen J., Hellenas K.E., Analyst, 127 (7) 2002 pp [20] Tareke E., Rydberg P., Karlsson P., Eriksson S., Tornqvist M., J Agric Food Chem, 50 (17) 2002 pp ISSN

262 OPTIMAZATION OF CULTURE CONDITIONS FOR PRODUCTION OF PROTEASE BY LECANICILLIUM LECANII Nguyen Huu Quan *1 and Vu Van Hanh 2 1 Thai Nguyen University of Education, 20 Luong Ngoc Quyen Road, Thai Nguyen City, Vietnam 2 Institute of Biotechnology, VAST, 18 Hoang Quoc Viet Road, Distr. Caugiay, Hanoi, Vietnam *: nhquan.ibt@ibt.ac.vn Abstract. Proteases are enzyme catalyzing hydrolysis of peptide bonds in molecule proteins into smaller peptides and amino acids. Proteases have wide applications in many industries such as food processing, textiles, and detergents manufacturing. Protease is a factor supporting the process of killing insects by entomopathogenic fungi as Lecanicillium lecanii. In this study, eight L. lecanii strains have been used for the production of protease under liquid state fermentation. Results showed that L. lecanii 1037 strain showed maximal protease activity at 30 C, ph 6.5 after 144 hours of cultivation, shaking 200 rpm/min. The medium containing carbon and nitrogen sources were corn powder and soybean meal, respectively and supplemented with 1.5% casein for maximal protease production from L. lecanii strain L. lecanii strain 1037 showed maximal chitinase production was 5.26 U/ml and increase of 1.63 times compared with less than optimal. In conclusion, when using the culture from several cheap and available materials in Vietnam such as soybean meal, corn powder to culture L. lecanii strain then the protease activity generates higher than the original culture which has important implications in implementing the goal of creating a large amount of protease, thereby contributing to reducing costs of protease products on the market today. Keywords: Carbon sources, culture condition optimal, Lecanicillium lecanii, protease, nitrogen sources. I. INTRODUCTION Proteases are the group of enzymes which hydrolyze the peptide bonds in polypeptides, proteins, and other substrates into low-molecular-weight products such as: amino acids and peptides. Proteases include proteinase, peptidase and amidases. Proteinase cleaves protein molecular into proteose, peptone and amino acids. Peptidase cleaves peptone into amino acids; whereas amidases cleaves amino acids into amoniac. Proteases are produced by different organisms, such as bacterial, fungi, insects, plants and animals [19]. Proteases have wide applications in many industries such as food processing, textiles, detergents manufacturing, tanning, and soap manufacturing and account for about 60% of the total worldwide sale of enzymes [21]. Protease can also be used in agriculture to control plant pathogens and degrading the cuticle of various insects effectively [20]. In this study, we report the selection of L. lecanii strain, which is a potent 248 ISSN

263 protease producer and optimization of culture conditions required from cheap and available materials for enzyme production. II. MATERIALS AND METHODS 2.1. Strains Eight L. lecanii strains were used in this study from Laboratory of Functional Biocompounds, Institute of Biotechnology, VAST Determination of enzyme activity Protease activity was determined by Anson to improve method described previously [15]. Protease catalysed hydrolysis reaction of casein substrate which denatured from soluble products in trichloracetic acid (TCA). Phosphomolibdic acid and phosphoolframic acid in reagents Folin-Ciocalteau react with tyrosine (Tyr) and tryptophan (Trp) in the proteins to form a blue complex with maximum absorbance at 750 nm. One unit (U) of the protease activity was defined as the amount of enzyme that released 1 µmol of tyrosine per minute under the standard assay conditions. All measurements were carried out in triplicate with the resulting values being the mean of the cumulative data obtained Optimization of culture medium for protease production The culture conditions of L. lecanii strains were surveyed in 6 experiments, respectively. The survey factors in the previous experiment were used immediately for the subsequent experiment (Table 1). Table 1. The culture conditions of L. lecanii strain for protease biosynthesis. No The factors The level of change 1 Protease production 2 ph Culture Temperature substrate concentration Nitrogen sources MT medium, ph 5.0 at 30 C, additional 0.5% casein as substrate induction source. Enzyme was collected at different interval times from h (24 h apart) to determine protease activity. MT medium, following ph values from (1.0 apart) at 30 C, shaking 200 rmp/min to determine optimal ph. Enzyme was collected to determine protease activity after 144h culture. MT medium, ph 6.5, additional 0.5% casein, shaking 200 rmp/min at different temperature intervals at 28, 30 and 37 C. Enzyme was collected to determine protease activity after 144h culture. MT medium, ph 6.5 at 30 C and additional casein at different concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0%. Enzyme was collected to determine protease activity after 144h culture. MT medium, ph 6.5 at 30 C and additional 1.5% casein as substrate induction source. Peptone was replaced by one of various nitrogen sources (meat extract, soybean powder, casein, urea, NaNO 3, KNO 3, (NH 4 ) 2 SO 4, KNO 3 and NH 4 NO 3 ) of 0.5% concentration. Enzyme was ISSN

264 6 carbon sources collected to determine protease activity after 144h culture. MT medium, ph 6.5 at 30 C and additional 1.5% casein as substrate induction source and 0.5% soybean as nitrogen source. Yeast extract was replaced by one of various carbon sources (glucose, corn powder, corncobs, sugarcane bagasse, peanut shells, coffee shells, rice bran, CMC, dry tangerine peel, starch, cassava pulp) of 0,5% concentration. Enzyme was collected to determine protease activity after 144h culture. III. RESULTS AND DISCUSSION 3.1. Screening L. lecanii strains for protease production In nature, the ability of protease production from the fungal strains of the same species is different. Therefore, the process of screening and selection of L. lecanii strains capable for the highest protease biosynthesis are essential. Among eight L. lecanii strains (1035, 1036, 1037, 1038, 43H, 418 and 1185), the L. lecanii strain 1037 showed the highest protease production (2.2 U/ml), followed by the strain 1039 (2.14 U/ml), and strain 1185 showed the lowest protease production of 0.3 U/ml. The strain 1037 was selected for optimization of culture conditions for the protease production Protease production by L. lecanii 1037 Fermentation time is one of the important factors affecting the enzyme biosynthesis of microbial strains. The protease production by L. lecanii 1037 increased gradually from U/ml at 48 h of cultivation to U/ml at 144 h of cultivation. Then the protease production decreased to 1.48 U/ml at 240 h of cultivation (Fig. 1). Hamid et al. (2009) reported that the highest level of protease activity occurred at 72 h by A. niger strain when the survey of the fermentation period [8] whereas, A. oryzae strain produced the highest protease level at 48 h of growth [3]. Oyeleke et al. (2012) showed that the optimum period for production of protease from A. fumigate and A. flavus was at 144 h [16]. Thus, this result was coincident with report of Oyeleke et al. (2012) and somewhat different from those of previous studies. Fig. 1. Protease production course by the L. lecanii Fig. 2. Effect of the medium ph on the protease production by the L. lecanii ISSN

265 3.3. Effect of initial medium ph on protease production The productivity of the enzyme from fungal culture is dependent on ph medium. L. lecanii 1037 produced the highest protease level of U/ml in the optimized mineral medium at the initial ph of 6.5. At the initial ph 5.5, 6.0 and 7.0, the protease production was also quite high (70%, U/ml) in comparison to the optimum ph. At ph below 5.0 and above 7.5, the enzyme production decreased to 48-69% ( U/ml) (Fig. 2). As such, protease production from L. lecanii 1037 was appropriate at neutral ph. Some previous research found that, the protease production was the highest from A. niger at ph of [8], [9]; from A. flavus at ph of 5.0 [13]; from Bacillus cereus CA15 at ph of 8.0 [7]; from B. licheniformis MMS-1 at ph of 9.0 [12], and from Streptomyces corchorus ST36 at ph of 6.0 [6]. Thus, optimal ph for protease production from L. lecaniii 1037 was higher than Aspergillus genus, and lower than Bacillus and Streptomyces genus Effect of cultivation temperature on protease production L. lecanii 1037 produced the highest protease level of U/ml at 30 C of cultivation among three tested temperatures of 25, 30 and 37 C (Fig. 3). Optimum temperature for the protease production by A. flavus was also 30 C as reported by Chinnasamy et al. (2011) [4] whereas, optimum temperature for the protease production was at 35 C for A. oryzae [10]. The maximum protease production by Bacillus was obtained at a temperature of 50 C [2]. Fig. 3. Effect of the cultivation temperature on the protease production by strain Fig. 4. Effect of the casein concentration on the protease production by strain Effect of casein concentration on protease production Casein is a substrate for protease and showed the induction effect on the protease production. The addition of casein at the concentration from % to the MT medium increased the protease production gradually from to U/ml. The addition of more casein of 2 to 4% to the culture medium decreased the protease production gradually to U/ml (Fig. 4). The addition of 1.5% casein induced the maximum protease production by L. lecanii This result was different from some previous research. Corol el al. (2003) reported that the addition of 1.5% casein to Crapek Dox medium, the ISSN

266 protease production from A. niger was the highest [5] whereas, A. oryzae produced the highest protease level at 0.8% casein concentration of growth [1] Effect of nitrogen sources on protease production Among the examined nitrogen sources (Fig. 5), soybean powder was the best nitrogen source for the protease production by L. lecanii 1037 (5.276 U/ml) and higher peptone. However, meat extract and casein were appropriate nitrogen source for the protease production of 5.19 U/ml and U/ml, respectively. The protease production was the lowest of U/ml in medium containing urea as the nitrogen source. Fig. 5. Effect of nitrogen sources on the protease production by L. lecanii PT: pepton, CT: meat extract, CS: casein, BDT: soybean powder, UR: urea, Na: NaNO 3, KN: KNO 3, NO: (NH 4 ) 2 SO 4, NN: NH 4 NO 3. Fig. 6. Effect of carbon sources on the protease production by L. lecanii Glu: glucose, CNM: yeast extract, LN: corncobs, BM: sugarcane bagasse, VL: peanut shells, VCP: coffee shells, TC: rice bran, CMC: cacborxyl methyl cellulose, VQ: dry tangerine peel, TB: starch, BS: cassava pulp, BN: corn powder. Radha el al. (2011) showed that, the protease production by Aspergillus spp was the highest in medium containing soybean powder as the nitrogen source [17]. While, the potassium nitrate was the best nitrogen source for protease production by Aspergillus spp. [18] and A. flavus [13] whereas, A. niger IHG 9 produced the highest protease in medium containing beef meat [8]. Thus, this result was similar to the research of Radha el al. (2011) and different from previous research Effect of carbon sources on protease production Among the investigated carbon sources (glucose, yeast extract, corncobs, sugarcane bagasse, peanut shells, coffee shells, rice bran, cacborxyl methyl cellulose, dry tangerine peel, starch, cassava pulp, corn powder), corn powder was the most appropriate carbon source for the protease production (6.842 U/ml, 100%) by L. lecanii Other carbon sources reduced the enzyme production by about half in comparison to the corn powder as carbon source (Fig. 6), especially the protease production was reduced to 22% (1.472 U/ml) when L. lecanii 1037 was grown in the MT medium containing corn powder as the carbon source. 252 ISSN

267 The protease production from Aspergillus spp. was the highest in medium containing glucose and starch at concentrations of 1.5 and 1.0%, respectively [17]. While, the wheat bran and gelatin was the best carbon source for protease production by Aspergillus sp. [18]. Fikret et al. (2011) showed that, starch was the carbon source for the protease production by B. cereus CA15 [7] whereas, B. licheniformis BBRC produced the highest protease in medium containing maltose and lactose [14]. Thus, each microorganism strains appropriate for a different carbon sources Optimal medium From these results, we choose the medium from materials available in nature by keeping the mineral composition of MT medium, instead of yeast extract by corn powder and peptone by soybean powder, the concentration casein suspension of 1.5%. Fertility protease synthesis in substitute medium increases 1.63 times the MT medium. When using the culture from several cheap and available materials in Vietnam such as soybean powder and corn powder to culture L. lecanii strain, the protease activity generates higher than the original culture which has important implications in implementing the goal of creating a large amount of protease, thereby contributing to reducing costs of protease products on the market today. This result was similar to the research of Madhumithah et al. (2011) that A. niger produced the maximum protease production at the culture temperate of 35 C, with the initial ph of 6.0, cauliflower and cabbage as inducer [11]. Radha et al. (2011) also confirmed that the addition of 1.0% soybean powder as nitrogen source, 1.5% glucose and 1.0% starch as carbon source, the protease production from Aspergillus spp was the highest [17]. IV. CONCLUSION The highest production of protease from L. lecanii 1037 was selected from the survey results of 8 L. lecanii strains. L. lecanii 1037 produced the maximum protease at 30 C of culture temperature, with the initial ph of 6.5 and after 144 hours of cultivation. Corn powder and soybean powder showed the best carbon and nitrogen source for the protease production, respectively. The enzyme production was maximum in the culture medium MT containing 0.5% cornstarch powder as carbon source and 0.5% soybean as nitrogen source, 1.5% casein as inducer at ph of 6.5. V. ACKNOWLEDGEMENTS The study was supported by the Master Program of Development and Application of Biotechnology in Agriculture and Rural Development Towards 2020, Vietnam Ministry of Agriculture and Rural Development (Project: Research and production of conidial fungal Lecanicillium spp. for controlling aphids (Aphididae) damaging crop, ). ISSN

268 REFERENCES [1] Le Nguyen Doan Duy, Huynh Thi Phuong Thao, Nguyen Cong Ha, Journal of Science, Can Tho University, 33, (2014). [2] Vo Hong Thi, Nguyen Hoang My, Nguyen Pham Huyen, Journal of Natural Science and Technology, Vietnam National University, Hanoi, 28, (2012). [3] C. Chancharoonpong, P. C. Hsiehb, S. C. Sheub, International Journal of Bioscience, Biochemistry and Bioinformatics, 2, (2012). [4] M. Chinnasamy, G. Duraisamy, G. K. Dugganaboyana, R. Ganesan, K. Manokaran, U. Chandrasekar, Journal of Biological Sciences, 4, (2011). [5] G. Coral, B. Arikan, M. N. Ünaldi, H. Güvenmez, Ann. Microbiol., 53, (2003). [6] A. el-raheem, R, el-shanshoury, M. A. el-sayed, W. A. el-shouny, Acta Microbiol Pol., 43, (1994). [7] U. Fikret, P. Ilknur, K. Göksel, I. Y. Ebru, EurAsia. J. BioSci., 5, 1-9 (2011). [8] M. Hamid, U. H. Ikram, Pakistan J. Zool., 41, (2009). [9] G. J. Joel, R. Rebecca, J. G. Roseline, Int. J. Curr. Res., 3, (2011). [10] J. Karthic, K. G. Siddalingeshwara, P. SunilDutt, T. Pramod, T. Vishwanatha, Journal of Drug Delivery & Therapeutics, 4, (2014). [11] C. G. Madhumithah, R. Krithiga, S. Sundaram, C. S. Sasikumar, S. Guhathakurta, K. M. Cherian, World. J. Agric. Res., 7, (2011) [12] C. D. Mathew, R. M. S. Gunathilaka, International Journal for Biotechnology and Molecular Biology Research, 6, (2015) [13] C. Muthulakshmi, D. Gomathi, D. G. Kumar, G. Ravikumar, M. Kalaiselvi, C. Uma, Jordan Journal of Biological Sciences, 4, (2011). [14] Z. G. Nejad, S. Yaghmaei, R. H. Hosseini, Archive of SID, 22, (2009). [15] Nguyen Thi Thao, Quyen Dinh Thi, Journal of Biotechnology, 2, (2004) [16] S. B. Oyeleke, E. C. Egwim, S. H. Auta, J. Microbiol. Antimicrob, 2, (2010). [17] S. Radha, V. J. Nithya, R. H. Babu, A. Sridevi, N. B. L. Prasad, G. Narasimha, Arch. Appl. Sci. Res., 3, (2011). [18] S. Shivakumar, Arch. Appl. Sci. Res., 4, (2012) [19] C. R. Tremacoldi, N. K. Watanabe, E. C. Carmona, Wld. J. Microbiol. Biotech., 20, (2004). [20] M. H. Wafaa, L. K. Amany, and M. A. Amal, Plant Pathology Bulletin, 15, (2006). [21] R. Woods R, C. Burger, M. Bevan, and I. Andbeacham, J. Microbiol., 143, (2001). 254 ISSN

269 INVESTIGATION OF STABLE STRAIN OF STREPTOMYCES SP. KB1 FOR STABILITY OF BIOACTIVE SECONDARY METABOLITES PRODUCTION BY CONTINUOUS SUB-CULTURE Kittisak Chawawisit * and Monthon Lertcanawanichakul School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat 80161, Thailand Utilization of Natural Products Research Unit, Walailak University, Nakhon Si Thammarat 80161, Thailand *: chawawisit@gmail.com Abstract. Interesting strain of Streptomyces which produced bioactive secondary metabolites and showed broad spectrum of antimicrobial activity was identified and named as Streptomyces sp. KB1 based on the analysis of the 16S rdna sequence. Stable strain of Streptomyces sp. KB1 was investigated for bioactive secondary metabolites production by continuous sub-culture method. Population of stable strain that showed parental levels of anti-staphylococcus aureus TISTR 517 activity was continuously sub-cultured for 3-rounds and incubation time of each round as 5 days. Round I, II and Round III showed ratio of stable and non-stable strain as 1:2, 1:2 and 5:4, respectively. Moreover, stable strain of Round III, isolate no , also showed activity higher than parental levels. These preliminary results might be led to solving the instability of bioactive secondary metabolites production of Streptomyces sp. in the future. Keywords: Streptomyces sp. KB1, Bioactive secondary metabolites, Continuous sub-culture. I. INTRODUCTION Streptomyces is Gram-positive, free-living, saprophytic bacteria, widely distributed in soil, water and colonizing plants. Streptomyces sp. has genome size of about 10,000 kb and G + C content of 70 75% in their DNA [1-3]. Today, Streptomyces sp. is of considerable industrial importance because they produce important bioactive secondary metabolites such as antifungal, antiviral, antitumoral, anti-hypertensive, immuno-suppressive agent and mainly antibiotics [4]. Bioactive secondary metabolites are produced by a cell through the process of cellular metabolism. They are not necessary to the cells survival itself but are more so for that of the entire microorganism or produced when the cell is not operating under optimum conditions such as nutrient source depletion. Therefore, the most of bioactive secondary metabolites have been produced during the end or near the stationary phase of growth for killing or inhibiting the growth of the related microorganism in inappropriate environment [5]. Currently, bioactive secondary metabolites have been studied worldwide. However, there was a report about wild-type strains of Streptomyces sp. which isolated from nature have not stability in bioactive secondary metabolites production [6]. The instability of bioactive secondary metabolites production has been suggested to be associated with genetic instability. The most of Streptomyces sp. exhibit an extraordinarily high genetic instability 255 ISSN

270 rate in certain genes involved in their both primary metabolism and secondary metabolic pathways [7, 8]. For Streptomyces sp. KB1 was found that correlated with secondary metabolic pathways. It could produce the important bioactive secondary metabolites which showed broad spectrum of antimicrobial activity. Unfortunately, it has not the stability of production. Therefore, we aim for investigation of stable strain of Streptomyces sp. KB1 that has the stability of bioactive secondary metabolites production by means of continuously sub-culturing method under time-limited condition. II. MATERIALS AND METHODS Microorganisms, Media and Cultural conditions Producing strain: Streptomyces sp. KB1 was collected from the air sample at Aonang, Krabi province, Thailand, and isolated as pure culture on a half-formula of Luria Bertani (LB/2) agar medium (5 g/l Tryptone, 2.5 g/l Yeast extract, 5 g/l NaCl and 15 g/l Agar powder). The isolate KB1 was identified by 16S rdna gene sequence analysis at National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathum Thani province, Thailand, and submitted to GenBank database (NCBI, USA). Indicator bacteria: Staphylococcus aureus TISTR 517 was cultured in full-formula of LB agar medium (10 g/l Tryptone, 5 g/l Yeast extract, 10 g/l NaCl and 15 g/l Agar powder) at 37 C in static incubator for 24 hours. Phylogenetic Analysis Almost complete sequence (1,445 bp) of 16S rdna gene was determined and compared with those of other closely related taxa retrieved from the GenBank database. Phylogenetic tree was constructed by Neighbour-Joining plot. A sequence similarity search was done using GenBank BLASTN ( Sequences of closely related taxa were retrieved and aligned by means of MEGA 4 program. For the Neighbourjoining analysis, the distances between the sequences were calculated using Kimura's twoparameter model. Bootstrap analysis was performed to assess the confidence limits of the branching. Investigation of stable strain i) Starter, Round I, II and Round III preparation a) Starter preparation: 1 1 cm 2 of initial streak of 5-days culture of Streptomyces sp. KB1 was inoculated to 10 ml of LB/2 broth medium. Inoculum was incubated at 30 C, 200 rpm in shaking incubator for 5 days and assigned as starter or Round 0. b) Round I preparation: 1% of starter was sub-cultured to 10 ml of LB/2 broth medium in 6- screw cap test tubes and incubated at 30 C, 200 rpm in shaking incubator for 5 days. After incubation, 200 µl of culture broth of each tube was separated the cell sediment by using centrifugation at 12,000 rpm for 20 minutes and tested the anti- S. aureus 256 ISSN

271 TISTR 517 activity by using agar well diffusion method. Tube that showed activity was collected to prepare Round II. c) Round II and III preparation: Preparation of Round II and III sub-culturing of Streptomyces sp. KB1 is the same as sub-culturing Round I from Round 0. The flow chart of preparation of Round I, II, and Round III sub-culturing of Streptomyces sp. KB1 was illustrated in Fig. 1. ii) Anti- S. aureus TISTR 517 activity test Anti- S. aureus TISTR 517 activity was determined by means of agar well diffusion method as described by Ausubel et al. [9]. Fig. 1. Scheme of investigation of stability producing-strain of Streptomyces sp. KB1. III. RESULTS AND DISCUSSION After 16S rdna gene was amplified, the obtained sequence was subjected to similarity searches against public databases to infer possible phylogenetic relationship of the isolate KB1. The phylogenetic tree (Fig. 2) from representative strains of the related species indicated that the isolate KB1 should be placed in the genus Streptomyces. In the comparison of 16S rdna gene sequences, the isolate KB1 was mostly related with S. varsoviensis NRRL B-3589 T (98.89%), S. youssoufiensis X4 T (98.68%) and S. ramulosus NRRL B-2714 T (98.61%). While, Kitasatospora setae KM-6054 T was used as an outgroup. Based on the morphology and molecular phylogeny, the isolate KB1 was identified as genus Streptomyces and named as Streptomyces sp. KB1. The 16S rdna gene sequence of Streptomyces sp. KB1 has been deposited in the GenBank database under the accession number KF ISSN

272 Wild-type strain of Streptomyces sp. KB1 (starter or Round 0) was investigated for the strain instability by sub-culturing for 3-rounds (Round I, II and Round III). In our research, Round I, II and Round III of sub-culturing of Streptomyces sp. KB1 likened that they came from the same origin (starter or Round 0). Whereas, after sub-cultured the cultures from starter into the freshly prepared LB/2 broth medium for continuing growth to Round II and Round III. It was found that the producing strain of Streptomyces sp. KB1 was not stability for bioactive secondary metabolites. From our results it indicated that Streptomyces sp. KB1 had two populations including bioactive secondary metabolitesproducing population and bioactive secondary metabolites non-producing population. Both two populations were morphologically confirmed by streak plate method found that they are alike (data not show). These results accorded with research of Roth et al that reported the population of non-turimycin-producing strain of S. hygroscopicus overgrew more than turimycin-producing strain when it was continuously cultured under time-unlimited condition [6]. Fig. 2. The phylogram showing the position of the isolate KB1 with other Streptomyces based on 16S rdna gene. The scale bar represents substitutions per nucleotide position. The instability of bioactive secondary metabolites production was suggested that it has been associated with genetic instability [10]. Genetic instability is very common in Streptomyces sp. and can affect a variety of gene. It has been affected different phenotypically properties, often pleiotropically, including morphological differentiation and production of bioactive secondary metabolites [7-8, 10]. Genetic instability of most 258 ISSN

273 Streptomyces sp. came from three factors i) spontaneous mutation which had rate higher than 0.1% per spore ii) chemical- and physical- mutagenesis mutation and iii) -20 C storage mutation which were considered to associate between plasmid loss and chromosomal DNA depletion [7]. In nearly every well-characterized case of genetic instability in Streptomyces, the loss of gene function is due to depletion of the gene. In the case of S. glaucescens it is clear that the genetic instability involved with chromosomal DNA depletion. But, the location of the unstable genes is not clear. In addition, there is no physical evidence that the genes are plasmid encoded despite experiments to try to detect plasmid involvement [6, 8]. Therefore, the instability of bioactive secondary metabolites production of Streptomyces sp. KB1 was assumed that might be related with depletion of bioactive secondary metabolites gene on chromosome. These assumptions were supported by the work from Aigle et al (2014) which explained that the 8-9 Mb Streptomyces chromosome is linear, with a core containing essential genes (including bioactive secondary metabolites gene) and arms carrying conditionally adaptive genes [11]. Furthermore, Roth et al (1982) reported that occurrence of population of non-turimycinproducing strain during long continuous culture and they could not revert to turimycinproducing strain. This occurring was suggested that they had arisen by loss of genetic material on plasmid [6]. Contrast to our research, non-bioactive secondary metabolitesproducing strain from Round II of sub-culturing (the designate isolate 1-3-1) was subcultured into the freshly prepared LB/2 broth medium. It was found that could be reproduced bioactive secondary metabolites (Table 1). Table 1. Anti- S. aureus TISTR 517 activity of Streptomyces sp. KB1 variants. Round of sub-culturing of producingstrain Isolate designation Anti- S. aureus TISTR 517 activity Starter (Round 0) Round I * * Round II 1-3-1* * * +++ Round II * Round III Round III ISSN

274 Round III Round III of sub-culturing of non producing-strain No activity, +diameter of inhibition zone 15 mm, ++diameter of inhibition zone in the range mm, +++diameter of inhibition zone in the range mm, ++++diameter of inhibition zone 25 mm. * Isolate was selected for continuously subcultured into next Round. From these result indicated that (i) bioactive secondary metabolites gene was reversible loss or (ii) these bioactive secondary metabolites were produced by function of silent gene that was stimulated. Because of Streptomyces species has showed that single specie could carry more than 30 unexpressed or silent gene clusters for bioactive secondary metabolite biosynthesis [11]. Unexpressed or silent gene was stimulated by physical (such as UV radiation) and chemical (such as Ethidium Bromide) stimulation [7]. So, expression of silent gene is one of three factors of genetic instability. Whereas, stimulated agent was not used in our research. Therefore, the reversion from Round II (non-bioactive secondary metabolites-producing strain) to Round III (bioactive secondary metabolites-producing strain) might be came from reversible loss of bioactive secondary metabolites gene which accorded with research of Gravius et al that reported revertants of some Round III variant showed a morphology and oxytetracycline production same to Round I variant and the aforementioned variance related with genetic instability in the chromosome [8]. IV. CONCLUSION Streptomyces sp. KB1 is important bioactive secondary metabolites producer but it has both producing and non-producing strain which could be investigated by means of continuously sub-culturing method under time-limited condition. V. ACKNOWLEDGEMENTS The author would like to thank the laboratory officer of the faculty of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand, for facilities during this study. The present research was 260 ISSN

275 supported by Walailak University Fund and the institute of Research and Development of Walailak University under the grant number WU REFERENCES [1] A. Raja and P. Prabakarana, J. Drug Discov and Develop. Vol. 1, 2011, pp [2] M. Biswas, M.A. Rahman, H. Khatun, and M.A. Islam, Bangl Pharma J. Vol. 15, 2012, pp [3] T. Sharmin, M.A. Rahman, A. Sayeed, M. Anisuzzaman, and M.A. Islam, Bangl Pharma J. Vol 16, 2013, pp [4] R.E.L. Procopio, I.R. Silva, M.K. Martins, J.L. Azevedo, and J.M. Araujo, Braz J Infect Dis. Vol. 16, 2012, pp [5] A.K. Drummond, Doctoral dissertation. University of North Carolina Wilmington, Institute of Science, Adana [6] M. Roth, and D. Noack, J. Gen. Microbiol. Vol. 128, 1982, pp [7] K. Dharmalingam, and J. Cullum, J. Biosci. Vol. 21, 1996, pp [8] B. Gravius, T. Bezmalinovic, D. Hranueli, and J. Cullum, J. Appl. Environ. Microbiol. Vol. 59, 1993, pp [9] F. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.D. Seidman, J.A. Smith, and K. Struhl, Short protocols in molecular biology. New York. John Wiley & Sons Education, Inc [10] J.N. Volff, and J. Altenbuchner, J. Mol. Microbiol. Vol. 27, 1998, pp [11] B. Aigle, S. Lautru, D. Spiteller, J.S. Dickschat, G.L. Challis, P. Leblond, and J.L. Pernodet, J. Microbiol. Biotechnol. Vol. 41, 2014, pp ISSN

276 EFFECT OF COMPOSITION ON THE BURNING RATE OF PYROTECHNICS Doan Anh Phan*, Ngo Van Giao and Dang Van Duong Institute of Chemistry and Materials, Academy of Military Science and Technology, 17 Hoang Sam Str., Caugiay District, Hanoi, Vietnam * Abstract: Pyrotechnics is made of a multicomponent mixture of reoxidizers and oxidizers. Burning rate and activation energy of the burned pyrotechnics depends on its proportion of reoxidizers and oxidizer. This paper reveals effects of proportion of reoxidizers (Sb 2 S 3 ) and oxidizer(kclo 4 ) on burning rate and energy of pyrotechnics Sb 2 S 3 - KClO 4 - BaCrO 4. by DTA method. Keywords: burningrate, composition, antimony trisulfide, potassium perchlorate. I. INTRODUCTION Burning rate is one of the most important parameters of pyrotechnics. Proportion of mixture components and grainsizes most effect on burning rate. Decompostion temperature of of pyrotechnics is determined by methods of measuring burning time in standard tube [1-3] and analysing DTA, DSC, TG. Activation energy, rate and mechanism of reaction is also determined by thermal decompostion [4-11]. Each proportion of components of pyrotechnics such as Al-KClO 4, Mg-KClO 4,Zr-Ni/KClO 4,Mo/BaO 2, Mn/BaO 2 has their special decomposition temperature and burning rate. Burning rate is inverse proportion to activation energy, that is if burning rate increases then activation energy of recation decreases and oxidation-reduction reaction is easier [4-11]. Pyrotechnics which have average burning time and good quality are used widely in both civilian and military affairs. Materials making pyrotechnics with burning time of tens miliseconds to over ten seconds are abundant. Components of this pyrotechnics mainly include KClO 4, PbCrO 4, and Sb 2 S 3. KClO 4 is an oxidizer and also a conduction substance of reaction. Oxidizer PbCrO 4 is used to lengthen the burning time. Sb 2 S 3 is combustible substance, when content of Sb 2 S 3 rises the burning rate rises. Because pyrotechnics includes both deoxidizer and oxidizer and is not used in hermetic environments, decomposition reactions can happen in self-catalyzed air environments. This paper reveals some research results of the effects of the composition of KClO 4 and Sb 2 S 3 on activation energy and burning rate of pyrotechnics. II. EXPERIMENTS 2.1. Calculateinge and choosinge rate of pyrotechnic components 262 ISSN

277 Pyrotechnics is a mechanical mixture of ground substances. The burning process in devices is that happens in a hermetic emvironment, so it is necessary to choose components to guarantee the enough amount of oxygen for burning. Moreover, to lengthen the expiry date, components should not have moisture absorbability, do not corrode the materials of the devices, and do not react together in a nomal condition 5, 11. Table 1. Chemical-physical parameters of substances. Substance (- H 298 ), kcal/mol Amount of substance for 1 g oxygen as burning (g) KClO PbCrO Sb 2 S Condition for calculation is oxidation ballance is 0. According to [5, 11], for calculation, design pyrotechnics, we have to determine main components that decide burning process, other components are considered known ones. In determined pyrotechnics, Sb 2 S 3 is a fuel and a conductingburning rate substance, PbCrO 4 is main component that delays burning process, KClO 4 is burning catalyst. From Table 1: PbCrO 4 = 10.6 = 85.3% Sb 2 S 3 = 2.36 = 19% with supposition that the rate of KClO 4 is 5 %, the amount of Sb 2 S 3 that reacts to KClO 4 completely is: Sb 2 S 3 = 5x2.17 = 4.59% 2.36 Components of pyrotechnics includes (in 100 %): PbCrO 4 = 85 5 = 80 % KClO 4 = 5 % Sb 2 S 3 = 19-4 = 15 % This pyrotechnic component is not always optimum for any request, this component only plays the role of orientation, research needs to test to choose right rate. From theoretical calculation, the experiment on making pyrotechnic samples for measuring parameters: energy, burning rate, and calculation of temperature parameters. There are two cases: o The rate of Sb 2 S 3 remains the same, while the rate of KClO 4 changes; o The rate of KClO 4 remains the same, while the rate of Sb 2 S 3 changes; 2.2. Choose materials particle size ISSN

278 According to [1], particle size of material is chosen as: 2.3. Method of sample preparation KClO 4 : < 10 m PbCrO 4 <5 m Sb 2 S 3 < 20 m Chosing the pyrotechnics that has the following components: (3-7) % KClO 4, (78-84) % PbCrO 4 and (11-19) % Sb 2 S 3 ; binder nitrocellulose2,0 % (out of 100 %). Pyrotechnics is compressed in ( 3,5 x L20) tubes at pressure 1,800 kg/cm 2, pressure is held in 5 seconds, in condition of temperature of (28 2) o C, RH of (50 65) %. After that, samples are maintained in PP bag for 24 hours 2.4. Experimental methods - To identify burning heat(q c ) and (W o )of pyrotechnics by equipment PARR 1261 (USA) andequipment for meassuring the volume of gas. Carrying out as standard 06 TCN 889: To identify ignition temperature by specialized equipment, carring out as part 3.5 of standardtqsa795: To identify buring time by specialized equipment [2-4, 11] and time measurement device with accuracy 10-6 sec. - To measure burning rate [13] by using formula u = L/t, where: L- the leng of tube, mm; t buring time, s. - Standard test method for kinetic parameters [9] Experimental data handling Data handling follow Dixon standard, the number of experiement is more than 20. III. RESULTS AND DISCUSSIONS 3.1. Effects of the rate of KClO 4 on energy characteristicand burning rate of pyrotechnic Samples remain the rate of Sb 2 S 3, while changing the rate of KClO 4 ; Effect on enegry characteristic Results for measuring enegry characteristic are given at Table ISSN

279 Table 2. The rate of components and energy characteristic of samples. Components M1 M2 M3 M4 M5 M6 M7 PbCrO 4,% KClO 4,% Sb 2 S 3,% NC, % (out of 100 %) Burning heat (Q c ), (kcal/kg) Volume of gas (W o ), l/kg Ignition temperature, o C 361,2 357,8 352,2 349,4 348,2 347,2 346,8 The value of burning heat and volume heat fits in theory calculation. The dependence of burning heat and ignition temperature on the rate of KClO 4 is given at line Chart 1 and line Chart 2. Chart 1. The dependence of burning heat on the rate of KClO 4. Chart 2. The dependence of ignition temperature on the rate of KClO 4. It can be clearly seen from the two line chart that: - Burning heat rises Q = kcal/kg as the rate of KClO 4 rises from 2 % to 8 % (from kcal/kg to kcal/kg). - The volume of gas remains the same (results belong to measurement error) as the rate of KClO 4 rises from 2 % to 8 %. That value only rise when the rate of KClO 4 increases by 8 %. - Ignition temperatures decrease because thermal decomposition of KClO 4 is lower than that of PbCrO 4 (653 o C and 844 o C respectively). Therefore, a rise in the amount of KClO 4 and a decrease in the amount of PbCrO 4 lead to a decrease in the ignition temperature of pyrotechnics. The amount of free oxygen that is created from the ISSN

280 decomposition of KClO 4 and PbCrO 4 will take part in the reaction completely and creates side products such as PbSO 4, PbSO 3 which have big thermogenous, so the burning heat increases fast but the volume of gas remains the same. Mainly created gas is N 2 due to decomposition of nitrocellulose. When the amount of KClO 4 rises by 8 %, side products such as PbSO 4, PbSO 3 get maximum values and start excessing oxygen after buning. It means that the volume of gas start increasing The effect of the rate of components on burning rate Each pyrotechnics has definite burning rate at definite dentisy. Burning rate is an important parameter in research process and application for products. To determine the burning rate of pyrotechnics, this research use some samples in Table 2 to evaluate the effect of the rate of components on burning speed. Measured results in standard tube are shown in Table 3. Table 3. Burning time in standard tube of samlpes having different components. Samples Quantity M1 20 M3 20 M4 20 M5 20 M7 20 Burning time in standard tube(s) 4.797; 5.140; 4.916; 5.219; 5.072; 5.419; ; 4.800; 5.072; 5.181; 4.940; 4.739; ; 4.831; 5.321; 4.853; 4.939; ; 4.481; 4.792; 4.916; 4.786; 4.807; ; 5.056; 4.999; 4.697; 4.519; 4.456; ; 4.673;4.723; 4.909; 4.485; ; 4.462; 4.517; 4.533; 4.455; 4.691; ; 4.314; 4.331; 4.457; 4.921; 4.752; ; 4.556; 4.348; 4.532; 4.561; ,405 ; 4,338 ; 4,764 ; 4,929 ; 4,878; 4,691 ; 4,408 4,667 ; 4,686 ; 4,823 ; 4,974 ; 4,396 ; 4,304 ; 4,263 4,809 ; 4,621 ; 4,727 ; 4,753 ; 4,819 ; 4,834 5,149 ; 4,464 ; 5,075 ; 4,734 ; 4,585 ; 4,705 ; 4,773 4,651 ; 4,788 ; 4,303 ; 4,957 ; 4,522 ; 4,838 ; 4,744 4,546 ; 4,674 ; 4,798 ; 4,799 ; 4,538 ; 4,74 Average burning rate, mm/s ,15 3,10 From Table 3, to a chart is built to show the relationship between burning rate and the rate of KClO 4, as follows: 266 ISSN

281 Chart 3. The change burning rate of pyrotechnics according to the rate of KClO 4. It can be seen from Table 3 and Chart 3 that as the amount of KClO 4 increases, burning time decreases (burning rate rises). Therefore, for this pyrotechnics, KClO 4 is only oxidizer and catalyst for burning process. When the amount of KClO 4 increases, it reduces the burning process, so there exists a value of maximum burning rate The effect of the rate of Sb 2 S 3 on energy characteristic and burning rate of pyrotechnics Samples remain the rate of KClO 4, while changing the rate of Sb 2 S 3 ; Effect on energy characteristic y = x x R² = % KClO Measured results of energy characteristic are shown in Table 4: Table 4. The rate of components and energy characteristic of samples. Energy characteristic M8 M9 M10 M4 M11 M12 13 PbCrO 4,% KClO 4,% Sb 2 S 3,% NC, % (out of 100 %) Burning heat (Q c ), (kcal/kg) Volume of gas (W o ), l/kg Ignition temperature, o C 365,2 358,8 352,2 349,4 348,6 347,2 346,8 The value of burning heat and volume heat fits in theory calculation. The dependence of burning heat and ignition temperature on the rate of Sb 2 S 3 is given at line Chart 4 and line Chart 5. ISSN

282 It can be seen from Table 4, chart 4, and chart 5 that burning heat increase gradually when the rate of Sb 2 S 3 increase from 12 % to 18 % (from 189,2 kcal/kg to 214,5 kcal/kg). Chart 4. The dependence of burning rate on the rate of Sb 2 S 3. Chart 5. The dependence of ignition temp on the rate of Sb 2 S 3. The volume of gas decrease from 13.1 l/kg to 7.5 l/kg when the rate of Sb 2 S 3 increase from 12 % to 14 %. After that the volume of gas remain the same when the rate of Sb 2 S 3 increase from 14 % to 18 %. It is because when the rate of Sb 2 S 3 increase from 12 % tonear 14 %, initially the amount of excessive oxygen is large after burning reaction, then the amount of excessive oxygen decrease and leads to a decrease in the volume of gas. When the rate of Sb 2 S 3 increase from 14 % to 18 %, amount of oxygen created by decomposition of KClO 4 và PbCrO 4 will take part in burning reaction completely, so the volume of gas will not increase. The main created gas is N 2 due to the decomposition of nitrocellulose. As the rate of Sb 2 S 3 increase, the ignition temperaturedecrease because the thermal decomposition of Sb 2 S 3 is much lower than that of PbCrO 4 (550 o C and 844 o C respectively). Therefore, the rise in the amount of Sb 2 S 3 and the decrease in the amount of PbCrO 4 lead to a decrease in ignition temperature of pyrotechnics. When the rate of Sb 2 S 3 increase from 12 % to 18 %, burning heat will increase due to the amount of excessive oxygen, S is oxidized to S +6. Side product is PbSO 4 which has big thermogenous, so burning heat increase Definite burning rate of pyrotechnics Each pyrotechnics has definite burning speed at definite dentisy. Burning speed is important parameter in research process and application for products. To determine burning speed of pyrotechnics, research use some samples in table 4 to evaluate effect of the rate of components on burning speed. Measured results in standard tube are shown in Table ISSN

283 Table 5. Burning time in standard tube of sampleshaving different components. Samples Quantity M8 20 M10 20 M4 20 M11 20 M13 20 Burning time in standard tube(s) 6.540; 6.197; 6.316; 6.619; 6.452; 6.819;6.021; ; 6.472; 6.581; 6.340;6.139; 6.472; 6.640; ; 6.253; 6.339; ; 4.481; 4.792; 4.916; 4.786; 4.807; 4.831; ; 4.999; 4.697; 4.519; 5.140; 4.229; 5.066; ; 4.909; 4.761; ;4.462; 4.517; 4.533; 4.455;4.691; 4.418; ; 4.331; 4.457; 4.921; 4.752; 4.556; 4.393; ; 4.532; 4.561; ; 4.506; 4.300; 4.612; 4.154; 4.594; 4.498; ;4.929; 4.524; 4.492; 4.321; 4.412; 4.169; ; 4.940; 4.505; ; 4.540; 4.342; 4.181; 4.306; 4.120; 3.749; ; 3.739; 3.624; 4.106; 3.935; 3.862; 3.967; ; 3.841; 4.104; Average burning rate, mm/s From Table 5, a chart is builtto show the relationship between burning rate and the rate of Sb 2 S 3, as line Chart y = x x R² = % Sb 2 S Chart 6. The change in the burning rate of pyrotechnics according to the rate of Sb 2 S 3. It can be seen from Table 5 and Chart 6 that, at the same condition of technology and test, standard tubes catch fire easily and keep burning process stable. Burning time of standard tube decrease in the order of samples M8, M10, M4, M11, and M13. When the rate of Sb 2 S 3 increase, burning rate will increase as well due to complete burning process that makes both burning heat and burning rate rise. IV. RESEARCH THE DECOMPOSITION PROCESS OF PYROTECHNICS Using DTA method for research decomposition process of pyrotechnics. ISSN

284 - As temperature increases with the rate of 2.5 to 10 o C/minute, decompositon process do not determine peaks of decompositon temperature. - As temperature increases with the rate of 10 o C/minute at least,decomposition process determine peaks of decomposition temperature clearly. Table 6. Parameters of process of increase temperature of samples. Order 12,5 0 C/min 15 0 C/min 17,5 0 C/min 20 0 C/min 22,5 0 C/min M M M M M Chart 7. The temperature decomposition DTA of samples M4. Heating rate: [1]- 12,5 0 C min -1 ; [2] C min -1 ; [3]- 17,5 0 C min -1 ; [4] C min -1 ; [5]- 22,5 0 C min -1 It can be clearly seen from chart 7 that nitrocelluloce is decomposed at around 200 o C; KClO 4 is decomposed and releases O 2 and KClO 3 at 310 o C; KClO 3 is decomposed and releases O 2 and KCl at 610 o C; decomposed temperature of Sb 2 S 3 and PbCrO 4 are 550 o C and over 850 o C respectively. At the peak of around 410 o C, reactions happen strongly; and at small peak of C, reactions finish. It is explained by theory of solid-solid structure. According to that theory, before the time of burning, there is not dispersed process between solid crystals. As burn crystal of oxidations are dispersed, fuel starts to disperse into the crystal of oxidations and releases heat. This process is called ignition process. 270 ISSN

285 Using Kissinger method for treatment of datas andcalculation of active energy. Datas are given in Table 7. Table 7. The active energy of samples, kj/mol. Samples According to Kissinger method M M M M9 76,72 M12 61,95 It can be seen from Table 7 that in the field of test: As the rate of KClO 4 increases (from M1 to M4), E will decrease according to stability of the increase of burning rate. If amount of KClO 4 increases, E increases and burning rate decreases (from M5 to M7). As the rate of Sb 2 S 3 increases (from M9 to M12), E will decrease according to the stability of increase of burning rate, burning rate also increases so fast. V. CONCLUSIONS Can be concluded from the research oneffects of KClO 4 and Sb 2 S 3 on energy characteristic and burning rate: - As the rate of Sb 2 S 3 and KClO 4 increases, burning heat will increases and ignition temperature will decrease. - As the rate of KClO 4 increase, burning rate will increase and at determined value burning rate will be maximum. - As the rate of fuel increases, burning rate will increase as well. Basing on DTA, when changing of the rate of oxidation and fuel, research determines the value of active enegry which is so big to prove that burning process is stable. REFERENCES [1]. Doan Anh Phan, Ngo Van Giao, Research into effects of particle size of antimony trisulfide onburning rate of pyrotechnic system including potassium perchlorate,lead chromate, and nitrocellulose, The 3 rd Academic Conference on Natural Science for Master and PhD Students from Asean Countries, Phnom Penh, Cambodia, Proceedings, 2014, P [2]. P.P. Elischer, G. Cleal, M.Wilson, MRL-R-994, The development of a boron and oxide delay system, Department of Defence Materials research laboratories. ISSN

286 [3]. E. Eugene Kilmer N0LTR , A low gassing mixture igiter delay for various pyrotechnic compositions, explosions U.S. Naval Ordnance laboratory research department white OAK, Maryland. [4]. MIL-C A, Military specification composition, delay, [5]. John A. Conkling, Chemistry of pyrotechnics Basic Principles and Theory, Marcel Dekker, Inc., New York, [6]. Jinn-Shing Lee, Chung-King Hsu, Kuen-Shan Jaw, The thermal properties ofkcio 4 with Different particle size, Thermochimica Acta, 2001, P [7]. Шидловскии Машиностроение, Mockba [8]. К. К. А Д Р Е Е В, Термическое разложение игорение взрывчатых веществ, Издатьльство НАУКА Моква [9]. HUANG Ji-xian (2006), Research on the factors influencing on the burning rate of boron type delay compositons, Initiators & Pyrotechnics, 4, 2006, P [10]. Xu Jun Feng. Peng Jia Bing. Wang Xiu Zhi. Zhang Zhou Mei. Zhang Hua. Effect of the binding agents on the burning rate of the Tungsten delay compositions, Chinese Journal of Energetic Materials, Vol. 15, N 0. 2, 2007, p [11]. E , Standard test method for kinetic parameters by Differential Scanning Calorimetry Using isothermal Method, ISSN

287 REDUCING OF CARBON DIOXIDE POLLUTION BY THE BACKWARD DIFFUSION METHOD Vu Ba Dung Department of Physics, Hanoi University of Mining and Geology, Viet Nam Abstract. The diffusion or forward diffusion is main mechanism emission of carbon dioxide (CO 2 ) in the air, which is cause of the atmosphere pollution and green house effect. Forward diffusion is a process, in which the molecule flux goes from high concentration areas to low concentration areas and thus the carbon dioxide can emit easily in to the atmosphere from surface of earth (from plants, active volcanoes and forest fire. Backward diffusion is opposite to forward diffusion. The flux of backward diffusion goes to the increasing direction of concentration. In this paper, the backward diffusion of carbon dioxide is studied and a method of pollution control on carbon dioxide in the atmosphere is also presented. Keywords: Backward diffusion, Negative diffusivity, Carbon dioxide pollution. I. INTRODUCTION Diffusion (forward diffusion) is a fundamental process of nature and presenting in almost field of living. Fluxes of forward diffusion go along the direction of reducing concentration. Recently, some researching results show that the backward diffusion process have studied [1-8]. Backward diffusion is a process, in which the transport of molecule goes from low concentration areas to the high concentration areas. Although the backward diffusion process is opposite to basic diffusion laws (Fick s laws), this process is not contradictory with fundamental principle of thermodynamics. Studying of the backward diffusion is interesting and useful. If the backward diffusion of carbon dioxide can be taken place, this toxic gas not only can not emit in to atmosphere, which could also go back to their source on surface of the earth. So the backward diffusion can be applied to reducing of carbon dioxide pollution. II. THE BACKWARD DIFFUSION PROCESS The diffusion process takes place on the conditions that there are the differences of both the concentration and the thermal velocity between the two areas, which can be called General diffusion. Both the Fick s and the Onsager s equation can not describe this diffusion type. We can find out an equation that can describe the general diffusion process. We assume that: the moving of particles are the same as the ideal gas molecules; the thermal velocities in two areas I and II are different; the gradient of concentration is ISSN

288 constant (Fig. 1). The thermal velocity and characteristic length of particles in area I and II are (u 1, ) and (u 2, ). The mass flux from area I to II is: and the mass flux from area II to I is: the net mass flux along x is given by: 1 1 J 1 = u1c 1 = u 1(C+ ΔC) J 2 = u2c 2 = u 2(C+2ΔC) J = J1 - J 2 = u1c1 - u2c2 6 6 the difference of concentration is proportional to the gradient [9]: C ΔC = λ x substituting equation (1), (2) and (4) into (3) we have: suppose that u 1 is n times greater than u 2 (n 1) using equation (6) equation (5) becomes: Fig. 1. Model of the Backward diffusion. 1 1 C 2 C J = (u1 -u 2 )C+ u1λ - u2λ (5) 6 6 x 6 x (1) (2) (3) (4) u 1= nu 2= nu (6) in which J adv is advection flux: (n -1)u (2 - n)uλ C J = - = J adv + J 6 6 x gen (7) 274 ISSN

289 (n -1) J adv = uc 6 and J gen is the diffusion flux (or general diffusion flux): (2 - n)uλ C J dif = - 6 x the diffusivity D gen is determined by: (2 - n)uλ D gen = 6 We can use the expression [10]: C J =- t x substituting equation (14) into (16) we have: (8) (9) (10) (11) = t 6 x 2 C (2 - n)uλ C The properties of diffusion process and sign of diffusivity depends on the difference between the thermal velocity particles of low concentration and higher concentration area: when thermal velocity of low concentration area is double more than thermal velocity of high concentration area, the diffusivity (10) is negative and diffusion is Backward diffusion means flux diffusion goes from low concentration area to high concentration area. The equation (12) describe backward diffusion is called Backward diffusion equation. 2 (12) III. APPLYING TO THE REDUCING OF CARBON DIOXIDE POLLUTION We assume that: thermal velocity of low concentration area is tree time s greater than thermal velocity of high concentration area; the particle of diffusion is carbon dioxide. The diffusivity (12) becomes backward diffusivity of carbon dioxide in the air: uλ D = - (13) 6 CO 2 where u and are determined by: 8kT u= πm (14) kt λ= (15) 2πσ p 2 ISSN

290 D CO k T 3 p m (16) where m is mass of CO 2 (7.3x10-26 kg); is radius (5x10-10 m); p is pressure (10 5 N/m 2 ); T is temperature (300 K) and k = 1.38x10-23 J/K, so the backward diffusivity of carbon dioxide is: 5 2 DCO D ( cm / s ) 2 (17) with diffusivity (17) the general diffusion equation (12) becomes the backward diffusion equation of carbon dioxide 2 C C = -D0 2 t x (18) Solution of the equation (18) can be solved by the separation of variables approach [11, 12]. Supposing that the is along axis Ox between coordinates x = 0 and x = L = 1 m. Choosing the boundary and initial conditions C(0,t)= C(L,t)= C 0 (19) C(x,0)= f(x) (20) The solution C(x, t) of the equation (18) can be found as follows [11, 12]: in which b k is determined by: 2 2 CL -C0 k π kπ C(x,t)= x+c 0 + bkexp Dt sin x 2 L (21) k=1 L L 2 C -C kπ b = f(x)- x -C sin xdx L L L L L 0 k 0 0 (22) If initial condition is chosen: C(x,0)= f(x)= C0 C0 sin x (23) L the solution (21) becomes: in which b k is: k π kπ C(x,t)= C 0 + bkexp Dt sin x 2 k=1 L L (24) 2 2 C L 0 k 1 2C0 π kπ b k = sin x.sin xdx = L (25) L L 0 0 k ISSN

291 solution of the backward diffusion of carbon dioxide is: 2 π C(x,t)= C 0 +C0exp Dt sin x 2 L L If C 0 = 400 ppm, the solution (26) becomes: 2 4 C(x,t)= exp 3 10 t sin x (26) (27) The solution (27) showed that: the CO 2 concentration of high concentration area is increasing with increasing of time. However, every diffusion processes are limited. After periods of times, the diffusion processes will be finished at the time of τ, which mean the mass flux is equal to zero: 1 1 J = u1c1 - u2c 2 = where C 1 is the concentration of low concentration area and C 2 is the concentration of high concentration area. The equation (28) is rewritten: (28) u2c 2 = u1c 1 (29) The backward diffusion processes (u 1 = nu 2 ), condition of the end of diffusion process is: C 2= nc 1 (30) In the backward diffusion of carbon dioxide, the area of high concentration is in the co-ordinate x = L/2 and the area of low concentration is in the co-ordinate x = 0; L. So, condition of end of diffusion process (30) becomes: L C(, )= nc0 2 (31) substituting solution (26) into equation (31), we have: 2 π 2 exp D n 1 L (32) so, the time of the end of the backward diffusion τ is: 2 ln( n 1)L (33) 2 πd ISSN

292 Chart 1. The dependence of burning heat on the rate of KClO 4. For the backward diffusion of carbon dioxide, we have τ = 3.7x10 3 s. Graphs of the solution (27) are plotted in Fig. 2. Curves a and b are graphs of solution at the time of t 0 = 0 and t = τ =3.7x10 3 s. Equation (27) and graph in Fig. 2 showed that: the CO 2 concentration of high concentration area is increasing and the CO 2 concentration of low concentration reducing with the time increasing. IV. DYNAMICS SIMULATION OF CO 2 BACKWARD DIFFUSION Based on random walk theory, the backward diffusion process of carbon dioxide is simulated. The purpose of computer simulations is to show the presence of backward diffusion process. We simulated random walk of individual particles. A simulation of the molecule migration was executed in the two-dimension coordinate axes and in different two parts I and II. The walk probabilities of every molecule P jk from the occupied j th site to the nearest-neighbor free site k th are the same and equal to 0.25 in both the parts. The time of step random jumps of molecules is the same and which could be changed. However, the length of a random walk is chosen differently in part I and part II and which can be changed. In the first instance (t = 0), there are 22 and 44 molecules in the part I and part II. The place of all molecules in two parts is determined randomly. We chose the thermal velocity of the molecules in lower concentration (part I) are greater than four times the thermal velocity of the part II (high concentration part). To do this, we have chosen: - The minimum length of the molecule random walk step is d 0 = 0.2 mm. - The time of the both part I and part II are the same (τ 1 = τ 2 = τ =1 ms). - The length of the random walk step in two parts can be changed (d 1 = n 1 d 0 and d 2 = n 2 d 0 ). 278 ISSN

293 Fig. 3. Result of carbon dioxide backward diffusion simulation. The program of simulation is written by Visual Basic language on PC. Result is presented by the motion pictures on the monitor of PC. Fig. 3a shows the positions of particles in part I and part II at the initial time t = 0. Fig. 3b presents the pictures of the positions of molecules after the ten minutes, which shows that molecules of part I moves to part II and molecules of part II also moves to part I. However, the numbers of molecules of the part I moves to part II is greater than the numbers of molecules of the part II moves to part I. As result, there is a mass flux goes from low concentration area (part I) to high concentration area (part II). This mass flux is the backward diffusion flux. Witting program for simulation have difficult, so we cannot simulate exactly for carbon dioxide (such as thermal velocity, number molecules). However, this result is qualitatively corresponding to theory. V. CONCLUSION As the thermal velocity of molecules in low concentration area is double more than thermal velocity of high concentration area, the diffusion process of carbon dioxide is backward diffusion. In the backward diffusion, the CO 2 flux goes from a low concentration area to a high concentration area means the carbon dioxide concentration in the upper atmosphere will reduce. Thus, the backward diffusion can be applied to reducing of carbon dioxide in the atmosphere. REFERENCES [1] P. Agryrakis, A. Chumak, M. Maragakis, and N. Tsakiris, Phy. Rew B 80 (2009) [2] G. Giboa, N. Sochen, and Y. Zeevi, IEEE Transaction On Image Processing, 11 (2002) [3] Vu Ba Dung, Dinh Van Thien, Ta Thi Dung, and Hoang Dinh Chieu, Proc. Natl. Conf. Theor. Phys. 37 (2012), [4] Vu Ba Dung and Dinh Van Thien, Journal of Physics: Conference series (2014) 357. [5] V. Karpov, Phys. Rev. Let., 75, 2702 (1995). ISSN

294 [6] N. Christov, Europhys. Let., 36, 687 (1996). [7] G. Gilboa, N. Sochen and Y. Zeevi, IEEE Transactions on image processing, 11, 689 (2002). [8] P. Argyrakis, A. Chumak, M. Maragakis, and N. Tsakiris, Phys. Rev., B 80, (2009). [9] K. Olah, Per. Polyt. Chem. Eng., 49, 91 (2005). [10] A. Fick, Progg. Ann., 94, 50 (1855). [11] J. Crank, The Mathematics of Diffusion (Clarendon Press, Oxford, 2004) p.17. [12] G. Everstine, Anal. Solution of Partial Differential Equations, (Gaithersburg, Maryland, 2012) p ISSN

295 DEVELOPMENT OF ELECTROCHEMICAL DETECTOR COMBINE WITH DIGITAL MICROFLUDIC SYSTEM Sarunchana Wiboonsak 1, Chonticha Chunhakit 1, Paveena Nakbumpen 1, Kessararat Ugsornrat 1, Patiya Pasakorn 2, Thitima Maturos 2 and Adisorn Tuantranont 2 1 Department of Industrial Physics and Medical Instrumentation, King Mongkut s University of Technology North Bangkok, Bangkok, Thailand 2 Nanoelectronics and MEMS Laboratory, National Electronics and Computer Technology Center, Pathumthani, Thailand *: kessararat_u@hotmail.com Abstract. In this research, development of an electrochemical detector combined with digital microfluidics system was designed and fabricated for chemical and biomedical analysis with minimal reagent consumption. For design, the detector was designed at the end of T-junction electrowetting on dielectric (EWOD) as digital microfluidic. This EWOD was used for merging droplets to move the droplets to three electrodes as the detector for analysis. For fabrication, electrochemical detectors were fabricated by screen printing technique for low cost manufacturing process and the EWOD was fabricated by microfabrication. The detector consists of a screen-printing electrode: a carbon working electrode, carbon counter electrode and a silver/silver chloride reference electrode. The systems has been successfully fabricated and tested for rapid analysis. For analysis, the screen-printing electrochemical detectors were demonstrated for minimal reagent consumption which is potassium iodide. The concentration of the reagent were varied from 1, 2, 3, 5, 7 and 10 mm. The total analysis time including droplet mixing and measurement was less than 20 seconds. Keywords: Electrochemical detector, digital microfluidic system, screen printing. I. INTRODUCTION The electro wetting on dielectric have been no reports on an integrated with screen printed electrochemical detector device. Electrochemical technique offers high performance detection on miniaturizing electrode in microfluidic system. So, EWOD device is integrated with electrochemical detector, it can be detector that has very high performance analysis which is popular method for digital microfluidic technology. The screen printing method is very interesting to fabricate electrochemical detector. Electrochemical technique offers high performance detection on miniaturizing electrode in microfluidic system [1-4]. This research, we propose the design of electrochemical detector with the single plate EWOD device. The electrochemical detector is initially designed to focus on low cost by screen printing method, the system consist of T-junction EWOD configuration for merging buffer reagent and analyte droplet and three internal electrochemical detectors at the end of T-junction. The internal three electrodes consist of a carbon working electrode, and a carbon counter electrode, and a silver/silver cholride reference electrode [4-8]. ISSN

296 II. DESIGN The electrochemical EWOD digital microfluidics system consists of T-junction EWOD microchip for merging buffer reagent and analyte droplets and three screen-printed electrochemical electrodes as the detector at the end of T-junction. The system was design by L-edit software for a microfabrication and screen printing process as shown in Fig. 1. Fig. 1. (a) Layout of the electrochemical detector combines with digital microfluidics system, (b) Layout of electrochemical detector. III. EXPERIMENT The microchip was separate to two parts. The first part which is T-junction EWOD microchip as digital microfluidic microchip which was fabricated by microfabrication. The EWOD electrodes are designed with closely square electrode arrays. The gap between square electrodes and the width of square electrode are designed to be 100 µm and 1 mm, respectively. Cr/Al electrodes are selected as electrode material due to supporting low cost manufacturing process. For digital microfluidic part, the EWOD Cr/Al electrodes were fabricated on glass substrate. The pattern of electrodes that is drawn by L-edit program which is used for lithography process that creates a mask for sputtering. They consist of thirty control electrodes that are double in a linear array for T-junction EWOD on glass substrate. The surface of glass substrate was cleaned with acetone and methanol. The PCB dry film as photoresist was then laminated on glass substrate. Next, the photoresist was exposed to UV light under the designed photomask alighner. It was then developed and cleaned with deionized water. The glass substrate with photoresist pattern is shown in Fig. 2(a). For microfabrication, 100-nm thick Cr/Al electrodes are deposited by DC-sputtering process. Firstly, 30 nm thick chromium and then, 700 nm aluminum films were successively sputtered at an argon pressure of 10-2 mbar. Then, the metal film on photoresist was lifted off by ultrasonication in acetone for 20 minutes. After that, the dielectric layer, namely, a 100 nm-thick silicon dioxide layer, was deposited by RFsputtering. Then, a 20 nm super hydrophobic Teflon AF 1601 (Dupont, USA) layer was spin-coated at 1000 rpm for 5 minutes on the silicon dioxide layer. The glass chip was baked again at 100 o C for 30 minutes to cure Teflon AF. The super hydrophobic layer support to provide suitable contact angle and mobility of a droplet. The internal three electrode of electrochemical detector consists of carbon working electrode, carbon counter 282 ISSN

297 electrode and silver/silver chloride reference electrode on the end of T-junction EWOD on single plate substrate. The photograph of glass chip with EWOD Cr/Al electrode is shown in Fig. 2 (b). Fig. 2. (a) photograph of glass substrate with photoresist pattern (b) photograph of EWOD chip integrate with the electrochemical detectors consist of carbon working electrode, carbon counter electrode, and silver/silver chloride reference electrode on the end of T- junction EWOD. IV. EXPERIMENTAL RESULT A. Merging and transporting droplets for analysis Droplets of ionized water were successfully manipulated, mixed, and transported for electrochemical detection by EWOD processing. Fig. 3 shows the snapshots of deionized water at various steps on T-junction EWOD. In experiment, two droplets were initially placed on the center of four grounded electrode. The applied voltage is 300 V for transporting two droplets until merging and detection. Fig. 3. Snapshots of droplets merging at various steps on T-junction EWOD. ISSN

298 B. Electrochemical Analysis at the detectors For analysis, the electrochemical detector was demonstrated for rapid potassium iodide. For analysis with minimal reagent consumption. Microdroplet of buffer reagent and analyte droplet solution was mixed and detected by cyclic voltammegram with short processing time with seconds. The analyst droplet and buffer were mixed at the T-junction and moved to the end of T-juction for analysis. The CV measurement was performed using concentration window between 1 to 10 mm and the process were repeated with difference concentration from 1 to 10 mm. The concentration was determined from the product of initial potassium iodide concentration and the ratio of analyte s droplet volume and the final volume of the mixed droplet. The results of cyclic voltammograms of 1, 2, 3, 5, 7 and 10 mm potassium iodide 0.1 V/s scan rates. The relationship between the various current with different scan rate of potassium iodide is shown in Fig. 4. Fig. 4. Result of electrochemical analysis from three electrochemical detectors. The relationship between the various current with different scan rate. V. CONCLUSION A novel lab on a chip for biosensor has been successfully designed, fabricated and tested for analysis. The microchip consists of two parts, T-junction EWOD for merging reagent and three electrochemical detectors at the end of T-junction EWOD. The T- junction EWOD has been fabricated by microfabrication and the electrochemical detector has been fabricated by screen printing. In addition, the droplets were controlled by digital operation. Moreover, the screen-printed electrochemical detector was demonstrated for rapid potassium iodide. VI. ACKNOWLEDGMENTS This work was supported by Nanoelectronics and MEMS Laboratory, National Electronics and Computer Technology Center (NECTEC), National Science and Technology Development Agency (NSTDA) 284 ISSN

299 REFERENCES [1] W. Dai and Y.P. Zhao, International, Journal of Nonlinear Sciences and Numerical Simulation, 8(4), 2007, pp [2] J. Berthier et al., Sensors and Actuators A. 127, 2006, pp [3] J. Lienemann, A. Gremer and Korvink, IEEE Transactions on Computer-Aided Design of Integrated Circuits and Systems. 25(2), 2006, pp [4] H. Moon H, S.K. Cho, R.L. Garrell and C.J. Kim, Journal of Applied Physics. 92 (7), 2002, pp [5] A. Wisitsoraat, et al. Sensors, 9, 2009, pp [6] W. Joseph, Chemical Review. 108 (2), 2008, pp [7] P.H. John and A.W. Stephen, Trends in Analytical Chemistry, 16, 1997, pp [8] O.D. Renedo, M. A. Alonso-Lomillo and M. J. Martinez, Talanta, 73(2), 2007, pp ISSN

300 STRUCTURAL AND PHOTOPHYSICAL PROPERTIES OF CURCUMINOID AND METAL- CURCUMIN COMPLEXES DERIVED FROM TURMERIC (CURCUMA LONGA) Nguyen Thanh Binh 1*, Nguyen Dinh Cong 1, Nguyen Thi Lan Huong 1, Ngo Thi Nhan 1, Nguyen Thi Khanh Van 1, Le Xuan Hung 2, Vu Thi Bich 1,2 1 Institute of Physics, VAST, 10 DaoTan, BaDinh, Hanoi 2 Duy Tan University, Da Nang City, Vietnam *: tbnguyen@iop.vast.ac.vn Abstract. The structural properties of curcuminoid derived from turmeric (Curcuma longa) and metal- curcumin complexes (Zn-cur., Mg-cur. and Sn-cur.) were investigated by SEM images and vibrational (FTIR and Raman) spectroscopy. By comparison with commercial curcumin we have found that: i) a yellow poor soluble powder in water obtained via extraction and purification of turmeric is curcumin in the ketone form; ii) the metal ions used possess the ability to attract the ion pair electrons on oxygen atoms of the curcuminoid molecule to form a water-soluble complexes (Zn 2+ was little binding, but Mg 2+ and Sn 2+ ions could bind at least two curcumin molecules). All that results were confirmed by the study on photophysical properties using steady-state absorption and fluorescence spectroscopy and fluorescence lifetime in the ps domain using TCSPC technique. Keywords: Curcumin, metal- curcumin complex, FTIR and Raman, SEM, UV-Vis, TCSPC. I. INTRODUCTION Curcuminoid is a natural yellow-orange compound derived from turmeric (Curcuma longa). The principal compound of curcuminoid is curcumin and its properties have attracted great attention due to its medicinal activity since ancient times such as anticancer potential, antioxygenation, antibiosis and it may help in Alzheimer s Disease (AD). It has been observed that the incidence of Alzheimer s disease is significantly reduced among people that are known to regularly consume turmeric in their diet [1, 2]. Molecules of curcumin have a conjugated symmetrical structure with single (-C-C-) and double (-C=C-) bonds alternately. They have two benzene rings, two methoxy and particular two hydroxy groups. Curcumin can exist in at least two tautomeric forms: keto and enol. In the central part of the molecule, it can have a C=O and a C-OH group for the enol form and two C=O groups for the keto form. Curcumin is hydrophobic in nature and insoluble in water, that leads to poor bioavailability, limited absorption in body. To overcome this problem, one of several methods is the formation of metal- curcumin complexes. It is well know that curcumin forms strong complexes with most of the known metal ions to possess potential activities in the areas of biological, antibiotic, tumor inhibitor. The structural and physical properties of these complexes depend on the nature of the metal ions. Metalcurcumin complexes not only modify the physico-chemical properties of curcumin but 286 ISSN

301 they also affect the biological reactivity of the metal [3, 4]. Moreover, curcumin undergoes much faster degradation when exposed to sunlight. This photodegradation involves formation of the excited state of curcumin [4, 5]. Therefore, a deeper understanding of the structural and photophysical properties of curcuminoid, metal-curcumin complexes on molecular level is needed. In this work, the structural properties of curcuminoid and metal-curcumin complexes were investigated by SEM and vibrational (FTIR and Raman) techniques. The steady-state absorption and fluorescence spectroscopy, life time fluorescence in the picosecond time domain using TCSPC technique were performed to study the interactions between the ligand curcumin and the metal ions on the highly sensitive spectroscopic properties. II. EXPERIMENTS Materials. The water-poor soluble curcuminoid and the water-soluble metalcurcumin complexes were obtained by extraction and isolation from a rhizome of turmeric (Curcuma longa) and by a reaction of curcuminoid with metal ions (metal salts: ZnCl 2, SnCl 2, MgCl 2 ), as previously described [6, 7], respectively. Our obtained curcuminoid was a mixture of 51,1% curcumin I, 32,6% curcumin II (mono demethoxy curcumin) and 6,1% curcumin III (bis demethoxy curcumin). The yields were 1,25% of curcuminoid and 4,3% of manganese, 14,1% of zinc and 33,1% of tin complex with curcuminoids. The pure curcumin was purchased from Fluka Chemical Co. (Sigma- Aldrich Co.). Ethanol was used without further purification. Apparatus. FTIR spectra were recorded on a Nicolet in the cm -1 range in using the KBr pellet technique. The Raman spectra were scanned on a confocal Labram Raman Micro- spectrometer Dilor-Jobin Yvon-Spex, equipped with a detector CCD in the range of cm -1. For excitation, we used the red line (632.8 nm) of a He-Ne laser. The surface morphology of samples was obtained on a Field Emission Scanning Electron Microscope, S-4800, Hitachi. The UV-VIS absorption spectra were measured by an UV Shimadzu spectrophotometer. Steady-state fluorescence measurements were carried out with the Carry Eclipse Fluorescence System ( excitation = 420 nm). Fluorescence lifetimes were detected by Time-Correlated Single-Photon Counting (TCSPC) method. The used picosecond TCSPC home made setup has ~200 ps time resolution, an diode laser (~ 0,1 mw, laser pulse width ~ 10 ps, repetition frequency 4-50 MHz), excitation = 405 nm and an PMT - Hamamatsu 5783P, time 300 ps. The average fluorescence lifetimes for the decay curves were calculated from the decay times and the relative contribution of the components using the following equation. av (1) 1a1 2a2 where τ 1 and τ 2 are the first and second components of decay time of curcuminoid and a 1 and a 2 are the corresponding relative weightage of these components. ISSN

302 Determination of the quantum yield is generally accomplished by comparison of the wavelength integrated intensity of the unknown to that of the standard. We have calculated the fluorescence quantum yield with the following equation: I OD n 2 R Q = Q (2) R 2 I R OD nr where Q is the quantum yield, I is the integrated intensity, OD is the optical density, and n is the refractive index of solvent. The subscript R refers to the reference fluorophore of known quantum yield. In this expression it is assumed that the sample and reference are excited at the same wavelength, so that it is not necessary to correct for the different excitation intensities of different wavelengths. In our case, we used Rhodamin 6G as a reference fluorophore with the quantum yield of 0.95 [8]. To more understand the modulations in excited state behavior of curcuminoid and metal- curcumin complexes, we have calculated the radiative (k Fl ) and non-radiative (k NR ) decay rate constants using the following two equations [8]: k FL Q 1 FL and kfl knr av av (3) where k Fl and k NR are the radiative and non-radiative rate constants, respectively, and Q Fl is the fluorescence quantum yield of curcuminoid and metal- curcumin complexes. III. RESULTS AND DISCUSSION Vibrational spectra. A detailed study on the vibrational spectra of curcumin (Fluka Co.), curcuminoid and metal-complexes (Me: Mg, Sn, Zn) in solid state was reported earlier [9]. The Raman spectra in the range of cm -1 and the FTIR spectra in the range of cm -1 of curcuminoid display the global characteristic vibrational groups. Comparing curcumin of Fluka Co. with and curcuminoid we have seen that vibrational modes of these samples are almost the same. This confirms that the chemical structure of curcuminoid obtained from turmeric is comparable with curcumin and all spectra of obtained curcuminoid show only bands of the diketo form. That are a (OH) sharp band at 3519 cm -1 (IR) and some low intensity bands at 3006 cm -1 and 2918 cm -1, 2848 cm -1 which are assigned to the aromatic ν(c-h) and the methyl group motions, respectively. In the middle region, a strong band at 1623 cm -1 (IR) and 1625 cm -1 (R) which is (C=C) of the benzene ring and a shoulder at 1633 (IR) and 1638 (Raman) which is a characteristic band of C=O vibrations in the diketo form have been observed. Furthermore we have found a very strong band at 1600 cm -1 that corresponds to a mixing of (C=C) and (C=O) of the benzene ring. In the region of cm -1 except a highly mixing modes, one very clear band at 1429 cm -1 (IR and R) could be assigned to deformation vibrations of the two methyl groups. In both IR and Raman spectra we have seen the same vibrations at nearly the same frequencies (1270 cm -1 (R) /1250 cm -1 (IR), 1183 cm -1, 1322 cm -1 (R)/ 1314 cm ISSN

303 (IR) and 1206 cm -1 ) which are attributed to the in plane deformation vibrations of (CCH) of phenyl rings, skeletal in plane deformations, (C-O) and (C=C-H) of interring chain, respectively. The vibrations of the phenyl group are strongly mixed with skeletal ones in this region. For this reason one prominent band at 1152 cm -1 in both IR and Raman spectrum was assigned also to (C-O-C) vibration. Moreover, the (C-H) out of plane vibrations of the aromatic ring have seen at 1023, 810, 713 cm -1 (IR), 886 cm -1 (R) and the (C-O) vibrations at 960 and 853 cm -1. In the range of cm -1, except some torsion vibrations at the low frequency region, we could see deformation vibrations of both benzene rings and the out of plane vibrations of both OH groups which are at 460 cm -1 (R) and 466 cm -1 (IR). For metal-curcumin complexes (Zn-cur., Mg-cur. and Sn-cur.) beside the appearance of metal-oxygen at 966 cm -1 (R), from IR spectra, we have seen the ratio I 960 cm -1 /I 1023 cm -1 and I 810cm -1 /I 853 cm -1 1 in the case of curcumin (Fluka Co.) and curcuminoid, but in the case of metal-curcumin complexes, this ratio is enhanced (nearly 2 for Mg-cur. and Sncur. and it was lightly changed for the Zn-cur. complexes). We note that 960 cm -1 and 810 cm -1 are characteristic bands of C-O bond and 1023 and 853 cm -1 are characteristic bands of C-H. This indicates that the metal could break a double bond C=O of keto groups and bonding with oxygen. It is also shown that Zn 2+ was little binding, but Mg 2+ and Sn 2+ ions could bind at least two curcumin molecules. This conclusion was confirmed by investigations of the morphology of these samples by SEM. Surface morphology of curcuminoid. Fig. 1. show the surface morphology of all obtained curcuminoid and metal-cur. complexes (Mg-cur., Zn-cur. and Sn-cur. complexes). Curcuminoid were the rods or plaques in the dimension of a micron (a). These plaques become much more porous (Mg-cur. (b) and Sn-cur. complexes (d)) or small broken plaques (Zn-cur. complex (c)). This shows the curcuminoid readily binds the Mg 2+ and Sn 2+ ions than the Zn 2+ ion. Fig. 1. SEM image of the surface morphology of (a) curcuminoid, (b) Mg-cur., (c) Zn-cur. and (d) Sn-cur. Complexes. Steady-state Absorption and Fluorescence. Fig. 2 illustrates normalized absorption and fluorescence spectra of curcuminoid and its metal-cur. complexes in ethanol. We have seen that curcuminoid in ethanolic solution showed a broad characteristic UV-VIS absorption at around nm with maximum absorption band at wavelength 426 nm assigned to the band of π-π* electronic transition and shoulder near 438 nm, while the curcumin ligand showed a band at 421 nm and a shoulder near 440 nm. The metal-cur ISSN

304 complexes formed show maximum absorption shifted by ~5 nm (from 426 nm to 421 nm) which indicated the involvement of the carbonyl group of curcumin in metal complexation and the shoulders at 440 nm which attributed to a curcumin- metal ion charge transfer transition. The variation of the absorption band of curcumin in different complexes depends on the nature of metal ions [10]. The emission and absorption of the curcumin show exciton diffusion, and refers to the migration of excitons between molecules. Although the presence of exciton diffusion is well-known, the details of the phenomenon have been studied in a disordered solid. Specifically, the impact on this process of dispersion in molecular energy levels has been showed in some organic materials [4]. As for the fluorescence spectrum in the ethanol solution (Fig. 2), curcuminoid show a broad band at 535 nm and the metal-curcumin complexes show a broad band at nm (except at 570 nm for Zn-cur.). We have seen that while a 5 nm blue shift of metal-curcumin complexes appears in the UV-VIS absorption spectra of curcuminoids in ethanol (except for Zn-Cur complex), a red shift with the same magnitude in the fluorescence maximum emerges in this solvent. It is known that the cause of decreastion fluorescence intensity after linking with metal ions is involvement of non-radiative processes [4]. In other words, the fluorescence quantum yields, decreased due to induced non-radiative processes by metal ions. In our case, after linking with metal ions, the steady-state fluorescence quantum yield decreased (from 5,7% to 4,4% for Mg-Cur. and Zn-Cur. and until 1,9% for Sn-Cur.). Fig. 2. Normalized absorption (left) and fluorescence spectra (right) of metalcurcumin complexes in ethanol. Table 1. Absorption and fluorescence emission maxima, λ Abs and λ Fl ( exc.= 420 nm); fluorescence quantum yield, Φ Fl ; average fluorescence decay time, τ av ; radiative and non-radiative decay rates, k Fl and k NR of curcumin and of curcuminoid, metal-curcumin complexes (Mg-cur., Sn-cur. and Zn-cur.) in ethanol solution. Me ion λ Abs (nm) λ Fl (nm) 290 ISSN Φ Fl τ av (ps) k Fl (10 9 s -1 ) k NR (10 9 s -1 ) Curcumin Mg Sn Zn Fluorescence lifetime. Time resolved photoluminescence measurements of disordered molecular metal-cur. complexes show an emission spectrum that significantly a function of time following excitation by a 100 ps of semiconductor laser pulse and an

305 using the TCSPC technique (Fig. 3). The change in fluorescence lifetime helps to get a clear idea about the modulation of photophysical properties (radiative and non-radiative) of the investigated molecule. Using the TCSPC technique, all average fluorescence lifetimes ( av ) were calculated by Eq 1. It is well known that the competing non-radiative processes shortened the fluorescence lifetimes considerably. Our results show that the lifetimes of metal- cur. complexes (Mg-cur, Sn-cur and Zn- cur) found a decrement from 485 ps (curcuminoid) to 362 ps ((Zn-cur.) (Tab. 2). Moreover, similar results were also observed in the case that the non-radiative decay rate (k NR ) of metal-cur complexes increased up to 2, s -1 in comparision with curcuminoid (k NR =1, s -1 ). The results were shown the excited state electron transfer processes from the curcumin to the metal ions. That confirm metal ions used in our work could bind at less two curcumin molecules. These phenomena can be attributed to the diffusion of excitons through the disordered molecular metal-cur. complexes by means of Forester energy transfer between curcumin molecules. This site-to-site transfer rate is governed by the emission and absorption spectra of the exaction donating and the exaction accepting molecule. From Forester s original formulation, the rate of exciton transfer, F, is given as: F 1 R F R 6 Fig. 3. Fluorescence decays of curcumin and of a) curcuminoid, b) Mg-cur., c) Zn-cur. and d) Sn-cur. complexes in ethanol ( exc. = 420 nm). where R is the distance separating the donor and acceptor, τ is the intrinsic radiative lifetime of the donor, and R F is the Förster radius. As indicated by the rate expression, the Förster energy transfer occurs more readily when the acceptor molecule has lower energy than the donor. In other words, the Förster mechanism preferentially transfers excitons towards lower energy sites. Combining this observation with the assumption that the excitonic density of states, g ex (E), has a non-zero width (i.e. that there exists some dispersion in exciton energies in curcumin), we obtain a straightforward explanation of the observed time-resolved spectral change: diffusion by Förster energy transfer progressively drives the excitons in the curcumin molecule towards the lowest energy sites. The detail spectral properties have been summarized in Table 1. (4) IV. CONCLUSION In conclusion, we have successfully synthesized the water-poor soluble curcuminoid and the water-soluble metal- curcumin complexes (Zn-Cur., Mg-Cur. and Sn-Cur.). All ISSN

306 obtained results from vibrational (FTIR and Raman) spectroscopy, absorption and fluorescence spectra and SEM images show that the Mg 2+ Zn 2+ and Sn 2+ ions could bind at least two curcumin molecules. The fluorescence lifetime of metal- curcumin complexes decreases in comparison with that of curcuminoid and this fact demonstrates the increase of the symmetry of the curcumin molecule. V. ACKNOWLEDGMENTS This work was supported by the National Foundation for Science and Technology Development of Vietnam (NAFOSTED) under Grant ĐT.NCCB-ĐHUD.2012-G/07. REFERENCES [1] B. B. Aggarwal, A. Kumar, M. S. Aggarwal, and S. Shishodia, In: Preuss H, ed. Phytopharmaceuticals in Cancer Chemoprevention. Boca Raton: CRC Press; 2005, pp [2] Larry Baum and Alex Ng, Curcumin interaction with copper and iron suggests one possible mechanism of action in Alzheimer s disease animal models, J.Alzheimer's Disease, 6-4, 2004, pp [3] S. Daniel, J. I. Limson, A. Dairam, G. M. Watkins and S. Daya, J. Inorg. Biochem., 2004, 98, pp [4] Damayanti Bagchi, Siddhi Chaudhuri, Samim Sarda, Susobhan Choudhury, Nabarun Polley, Peter Lemmens and Samir Kumar Pal, RSC Adv.,2015, 5, pp [5] Pria. R. S., Balachandran. S, Joseph Daisy, Mohanan. P. V, Universal J. of Physics and Application, 2015, 3(1), pp. 6. [6] Tran Thanh Luong, Nguyen Duc Hai and Pham Nguyen Dong Yen, 2002 Reports and SRW of IMS, HCM-Brand. [7] Tran Thanh Luong, Nguyen Duc Hai, Pham Nguyen Dong Yen and Nguyen Thi Mai Hương, J. Med. Mat. 11-4, 2006, pp [8] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd Ed., Springer Science + Business Media, LLC, [9] Vu Thi Bich, Nguyen Thi Thuy, Nguyen Thanh Binh, Nguyen Thi Mai Huong, Pham Nguyen Dong Yen, and Tran Thanh Luong, Physics and Engineering of New Materials, Ed. D T Cat, A. Pucci and K. Wandelt, Springer-Verlag Berlin Heidelberg Pub. Germany, vol. 127, 2009, pp [10] Bachar Zebi, Zéphirin Mouloungui, and Virginie Noirot, Bioinorganic Chem.and App., Vol. 2010, 2010, Article ID , 8 pages. 292 ISSN

307 FABRICATION OF Fe 3 O 4 MAGNETIC FLUID BY HYDRO THERMAL FOR BIOMEDICAL APPLICATIONS Vuong Thi Kim Oanh 1, *, Tran Dai Lam 2, Do Hung Manh 1, Le Trong Lu 3, Phung Thi Thu 1, Pham Hong Nam 1 and Nguyen Xuan Phuc 1 1 Institute of Materials Science, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam 2 Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam. 3 Institute for Tropical Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam *: vuongoanhch19@gmail.com Abstract. The hydrothermal method is often used to synthesize nanoparticles due to its advantages like as simply prepared conditions, no toxicity with environment and uniformed particle size. In this work, Fe 3 O 4 particles with relatively high saturation magnetization were synthesized by hydrothermal method. The structure, morphology and magnetic properties of the samples depending on the temperature change of reaction were studied by modern techniques: X-ray diffraction patterns (XRD), field emission scanning electron microscope (FE-SEM), and vibrating sample magnetometer (VSM). The XRD results pointed out that all as-prepared samples had single phase structure with medium crystal size of about 15 nm. The FE-SEM images also showed that the sphere particles had size from 10 to 18 nm. The highest value of saturation magnetization obtained at room temperature was 68 emu/g with the synthesized sample at 180 o C. The Fe 3 O 4 nanoparticles coated with modified chitosan had high saturation magnetization, good uniformity and dispersion, and stability.the magnetic fluid having highest magnetic saturation was surveyed by magnetic heating effect. Maximum temperature reaches up to 46.7 o C at concentration of 2 mg/ml. The results suggest that the obtained Fe 3 O 4 nanoparticles will promise a huge application potential in biomedicine especially in hyperthermia cancer treatment. Keywords: Hydrothermal, Fe 3 O 4, modified chitosan, magnetic fluid, magnetic heating effect. I. INTRODUCTION Spinel ferrite nanomaterials have been attracting special attention owing to distinct properties appearing at nanometer scale as well as its extremely applied potential in many domains, for instance; information technology, environmental handling, catalyst, biomedicine [1, 2]. Principal applications of magnetic nanoparticles in biomedicine include: biomolecules extraction, targeted drug delivery, contract enhancement of magnetic resonance imaging (MRI) and thermal magnetic therapy [3, 4]. Fe 3 O 4 nanoparticles have received a lot of attention in biomedical applications due to biocompatibility and being non-toxic. In this paper, Fe 3 O 4 fabricated by hydrothermal ISSN

308 method was studied because of simply synthetic conditions, environmental friendliness and high saturation magnetization together with relatively uniformed nanoparticles size. Simultaneously, thanks to the biocompatible and biodegradable ability of modified chitosan, it was chosen to enclose Fe 3 O 4 nanoparticles for applications in biomedicine. The effect of reaction temperature on structure, morphology and magnetic property was surveyed, analyzed and discussed. In recent years, there were many different methods used to research on synthesis of Fe 3 O 4 nanoparticles such as co-precipitation, sol-gel, hydrothermal, gas phase preparation method and so on [5-7]. However, these methods have had limitation. For example, to obtain highly qualitative nanoparticles by co-precipitation method, synthesis conditions such as stirring and dropping velocity must be exactly constrained. Sol-gel and gas phase methods usually make cluster particles and it s hard to control the shape, the size and the uniform of particles. II. EXPERIMENTS 2.1. Chemicals: All used chemicals are commercial products of Sigma-Aldrich, including: FeCl 3.6H 2 O, FeCl 2.4H 2 O, NaOH, HCl, modified chitosan (CSBT) with high purification. FeCl 2.4H 2 O and FeCl 3.6H 2 O were dissolved by HCl 2M acid before preparing samples Preparation process: The mixture including 2 ml Fe 2+ (2M) and 4 ml Fe 3+ (2M) was contained in glass vessel and mixed well. Then, drop NaOH 2M graduadlly into above mixture, seeds began to be formed at this time. After that, the mixture was immediately transferred into a Teflon-lined stainless steel autoclave and heated in the range of o C. The autoclave was subsequently placed in a furnace in 2h at 120, 140, 160, 180 o C corresponding to M1, M2, M3, and M4 samples. The autoclave was naturally cooled to room temperature after the hydrothermal treatment and the precipitate was recovered by washing with distilled water (when ph =7, stop washing) to eliminate unwanted impurity. Final product was divided into two part; one was dried in an oven to research structural characterizations and magnetic properties, the other was functionalized by chitosan to synthesize magnetic fluid Coating of Fe 3 O 4 with modified chitosan process: 200 mg of washed Fe 3 O 4 was dispersed into 20 ml of distilled water. 1 g of modified chitosan (CSBT) was dissolved in 100 ml of CH 3 COOH 2-5%, then stirred in 2 h and purified to obtain CSBT solution. Two above solutions were mixed well by using a magnetic stirrer in nitrogen condition, after that an amount of NaOH 0.5 M was added to achieve the solution of ph=7, concurrently continued stirring in 30 minutes. The final product was homogeneous black ferrofluid. The structural characterizations were defined by a SIEMENS D5000 (X-ray diffraction pattern (XRD) with an unfiltered Cu K radiation (( = A o ) at room temperature. The crystal size was confirmed by X Pert High Score Plus commercial soft. 294 ISSN

309 Intensity (a.u.) (220) (222) (400) (422) (511) (311) (440) The 4 th Academic Conference on Natural Science for Young Scientists, Master & Morphology and size of nanoparticles were observed by the Field mission scanning electron microscope (Hitachi S-4800). The saturation magnetization at room temperature was determined through the highest magnetic field of 11 koe using a vibrating sample magnetometer (VSM). Fourier transform infrared spectroscopy (FT-IR Nicolet 6700) was used to analyze the bond between CSBT shell and Fe 3 O 4 core. Size distribution and stable evaluation of ferrofluid were measured by the Zetasizer (Nano ZS Malvern UK). The heat generating capacity of nanoparticle systems was measured in magnetic field at 70 Oe and frequency of f = 178 khz, final temperature was recorded after 1500s, provided by the commercial generator (RDO HFI 5KW). III. RESULTS AND DISCUSSION The X-ray diffraction pattern of samples at different temperatures was represented at Fig. 1. It can be observed from Fig. 1 that all diffraction peaks of samples coincide well with those of the standard Fe 3 O 4 powder data (spinel structure). This means that assynthesized products are single phase. M4 M3 M2 M theta (degree) Fig. 1. XRD patterns of Fe 3 O 4 samples synthesized at different temperatures: (M1): 120 o C, (M2): 140 o C, (M3): 160 o C, (M4): 180 o C. Average crystal size grows up when reaction temperature increases with values 7, 9, 13 and 18 nm, respectively (showed on the Table 1). All samples were then measured by using the FESEM machinery (Fig. 2). Figure 2 shows that Fe 3 O 4 nanoparticles are sphere shape and relatively uniform. The boundary of particles becomes increasingly clear and the size increases when rising temperature (Table 1). In addition, all results also indicated that there was a good agreement with results of X-ray diffraction (Fig. 1). ISSN

310 Fig. 2. SEM images of Fe 3 O 4 synthesized at different temperatures: (M1): 120 o C, (M2): 140 o C, (M3): 160 o C, (M4): 180 o C. Fig. 3. M(H) curves of Fe 3 O 4 samples at different temperatures: (M1): 120 o C, (M2): 140 o C, (M3): 160 o C, (M4): 180 o C. Figure 3 shows the hysteresis of Fe 3 O 4 samples at different reaction temperatures. Herein, the saturation magnetization (Ms) increases from 54 to 68 emu/g when the reaction temperature rises from 120 o C to 180 o C, respectively (Table 1). It can be deduced that with the increase of reaction temperature the Ms value tends to be higher which is in relation of the increase of particle size and crystal quality. The results are better than published results [8, 9]. Table 1: Average crystal size (D XRD ), average nanoparticle size (D FE-SEM ) and saturation magnetization (M s ) of four Fe 3 O 4 samples synthesized at different reaction temperatures. Sample Size particles (nm) D XRD (nm) D FE-SEM (nm) Ms (emu/g) M M ISSN

311 The 4 th Academic Conference on Natural Science for Young Scientists, Master & M M From above analyzed results, M3 and M4 samples were chosen to fabricate magnetic fluids. The information about the bond between CSBT and magnetic nanoparticles of two samples after washed by water to remove free molecules was confirmed by the FT-IR spectrum. M4 M3 Fe 3 O Wavenumber (cm -1 ) Fig. 4. FT-IR spectra of Fe 3 O 4 /CSBT at different temperatures: (M3): 160 o C; (M4): 180 o C and Fe 3 O 4. It can be explicitly seen from Fig. 4 that there is the appearance of feature vibrations related to functional groups of Fe 3 O 4 and CSBT shell. Typically, the peak of 689 cm -1 observed in the spectrum can be attributed to Fe-O vibration of Fe 3 O 4. Peak of 1045 cm -1 is assigned to vibration of C-O bond and peak 1637 cm -1 is vibration of COO - group in accordance with the vibration of NH 2. In addition, the vibration of OH and NH appear in the spectrum region from cm -1. The results showed that these peaks contained functional groups of CSBT. This demonstrated that Fe 3 O 4 nanoparticles were covered by the polymer. To evaluate the particle size as well as the stability of samples, they were measured by using the Zetasizer equipment, their results are indicated in Fig. 5 and Fig. 6. Figure 5 exhibits that two samples only appear a unique peak. This displayed that nanoparticles have relatively uniform size with average size around 150 nm. ISSN

312 Fig. 5. The particle size distribution pattern of M3 (left) and M4 (right). On the other hand, Zeta potential of all samples is well over 16 mv. Namely, the zeta voltage of M3 and M4 samples is 50.9 and 57.4 mv, respectively (Fig. 6). Following publications of Authors, almost all magnetic fluids with the zeta voltage in the range of mv are very stable [10, 11]. Fig. 6. Zeta potential of magnetic fluid samples M3 (left) and M4 (right). Obviously, M4 sample has the highest saturation magnetization and the best capacity for dispersion, it was therefore chosen to investigate the magnetic heating effect at different concentrations to the maximum temperature (T max ). Figure 7 illustrates magnetic heating curves of ferrofluids at different concentrations which were measured under a 70 Oe magnetic field amplitude and frequency of 178 khz. It can be observed that the increase of Fe 3 O 4 concentration (from 1-5 mg/ml) leads to the increase of maximum temperature.the maximum temperature achieves the highest value of 63,9 o C at concentration of 5 mg/ml. When the concentration decreases to 2 mg/ml, the temperature is 46,7 o C which is in the range of o C (this temperature range is desirable in application of hyperthermia cancer treatment). This result proved that magnetic fluid is good dispersion and uniform particles size [12]. 298 ISSN

313 Tempetature ( o C) mg/ml 4mg/ml 3mg/ml 2mg/ml 1mg/ml Time (s) Fig. 7. Magnetic heating curves measured for suspensions of (M4-180 o C) simple at the different Fe 3 O 4 concentration. IV. CONCLUSION In conclusion, we successfully synthesized Fe 3 O 4 nanoparticles with crystal monophase structure and strong magnetization by hydrothermal method. The obtained nanoparticles had spherical shape, controllable size in the range of nm with the highest saturation magnetization value reaching up to 68 emu/g. The maximum temperature is 46.7 o C at concentration of 2 mg/ml. This is an easy approach for synthesis and application of magnetic nanomaterials in biomedicine. V. ACKNOWLEDGMENTS This research was supported by DT.NCCB-DHUD.2012-G/08 of NAFFOSTED. REFERENCES [1] Meng X., et al., Magnetic CoPt nanoparticles as MRI contrast agent for transplanted neural stem cells detection. Nanoscale, (3): p [2] Le Trong Lu, et al., Facile synthesis of stable, water-soluble magnetic CoPt hollow nanostructures assisted by multi-thiol ligands, J. Mater. Chem., (33): p [3] Lee Jae-Hyun, et al., Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nat. Med., (1): p [4] Won Seok Seo, et al., FeCo/graphitic-shell nanocrystals as advanced magnetic-resonanceimaging and near-infrared agents. Nature Materials, : p ISSN

314 [5] Poedji Loekitowati Hariani, et al., Synthesis and Properties of Fe 3 O 4 Nanoparticles by Coprecipitation Method to Removal Procion Dye, International Journal of Environmental Science and Development, (3): p [6] T. J. Daou, et al., Hydrothermal Synthesis of Monodisperse Magnetite Nanoparticles. Chem. Mater., (18): p [7] Xu, Y., et al., Preparation and characterization of polyacrylic acid coated magnetite nanoparticles functionalized with amino acids. Thin Solid Films, : p [8] Ki Do Kim, S.S.K., Yong-Ho Choa, and Hee Taik Kim, Formation and Surface Modification of Fe3O4 Nanoparticles by Co-precipitation and Sol-gel Method., J. Ind. Eng. Chem., : p [9] Sh. Karamipour, M.S.S., N. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, : p [10] Hanaor, D., et al., Journal of the European Ceramic Society, (1): p [11] O'Brien, R.W., et al., Faraday Discussions of the Chemical Society, 90(0), 1990: p [12] Vuong Thi Kim Oanh., et al. Materials Chemistry and Physics, 163, 2015, p ISSN

315 PREPARATION AND PHOTOCATALYTIC ACTIVITY OF TiO 2 -SiO 2 -Fe 3 O 4 COMPOSITES FOR DECOMPOSITION OF POLY CHLORINATED BIPHENYLS (PCBs) IN WASTE TRANSFORMER OIL Nguyen Quoc Trung 1, Vu Duc Chinh 1,*, Vuong Thi Kim Oanh 1, Pham Nam Thang 1 and Vu Thi Hong Hanh 2 1 Institute of Materials Science, Vietnam Academy of Science and Technology, No 18 Hoang Quoc Viet road, Cau Giay district, Hanoi, Vietnam 2 Faculty of Physics, Thai Nguyen University of Education, Thai Nguyen, Vietnam *: chinhvd@ims.vast.vn Abstract. Poly chlorinated biphenyls (PCBs) are a class of chlorinated organic chemicals that are not easily destroyed in the environment. PCBs are dangerous for human and animals, because they are toxic. The treatment of PCBs by incineration is not effective because more toxic compounds are emitted in the environment. In this report, we investigated the photodegradation of PCBs by TiO 2 -SiO 2 -Fe 3 O 4 nanocomposites. TiO 2 -SiO 2 mixed oxides have been prepared by sol-gel technique from tetrabuthyl orthotitanate and tetraethyl orthosilicate. TiO 2 -SiO 2 -Fe 3 O 4 composites were prepared by heteroagglomeration. Their morphology, structure, and photocatalytic performance in the photodegradation of PCBs were characterized by scanning electron microscopy, X-ray diffraction spectroscopy, energydispersive X-ray analysis, and GC-microspectrophotometer. The results show that TiO 2 - SiO 2 -Fe 3 O 4 composites exhibit an enhanced photocatalytic performance in photodegradation of PCBs under light irradiation. Keywords: PCBs, Transformer oil, TiO 2 -SiO 2 -Fe 3 O 4, Photocatalytic. I. INTRODUCTION Poly chlorinated biphenyls (PCBs) which are widely used as heat transfer fluids, hydraulic fluids, solvent extenders, plasticizers, flame-retardants, and dielectric fluids are toxic compounds [1]. Due to their chemical stability and hazardous nature, they are categorized as persistent organic pollutants. PCB chronic toxicity includes damages to the heart, liver, kidney, central nervous, and reproduction systems [2, 3]. Although there are some reports on industrial and domestic waste treatment of PCBs, there is still a need for more effective and green process [4]. Advanced oxidation processes are some of the popular treatment methodologies for environmental pollutants and they have been widely investigated recently [5]. TiO 2 is one of the most widely used photocatalysts because it is stable, inexpensive and non-toxic [6, 7]. The photocatalytic activity of titanium dioxide can be improved by the addition of SiO 2 which increases the available surface area of the catalyst, allowing the increase in adsorption of organic molecules. The improved adsorption of pollutant molecules on the ISSN

316 surface of silica increases the photocatalytic activity of TiO 2 -SiO 2 mixed oxides as compared to pure TiO 2 [8]. Furthermore, the addition of SiO 2 increases the amount of adsorbed water and hydroxyl groups on its surface, which influences the photocatalytic activity of TiO 2 -SiO 2 mixed oxides [9, 10]. The methods used for the preparation of binary oxide nanomaterials include conventional solid state mixing, hydrothermal, coprecipitation and sol-gel processes [11-14]. Among these methods, the sol-gel technique is the most effective method for controlling textural and surface characteristics of nanomaterials to produce homogenous materials with high surface area [15-17]. Herein, we report the synthesis of TiO 2 -SiO 2 nanoparticles (NPs) from tetrabutyl orthotitanate (TBOT) and tetraethyl orthosilicate (TEOS) by sol-gel method. The prepared materials have been characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). Fe 3 O 4 is a very important ferromagnetic material which is used for many applications (pigment, recording materials, catalysis, magnetocaloric refrigeration, and drug-delivery carrier) due to its low cost, good hydrophilic and biocompatible properties [18]. In this work, TiO 2 -SiO 2 -Fe 3 O 4 is investigated as photocatalyst. Photodegradation of PCBs (under visible light) was used to characterize and evaluate the photocatalytic activity of the obtained sample. II. EXPERIMENTS Materials TBOT, TEOS, acetyl acetone, FeCl 3.6H 2 O, FeCl 2.4H 2 O are from Merck without further purification. Preparation of TiO 2 -SiO 2 nanoparticles TBOT and TEOS were used as precursors for TiO 2 and SiO 2 synthesis, respectively. Anhydrous ethanol was used as solvent, and ammonia was used as a catalyst for hydrolysis. In a separate flask, TBOT was added to a mixture of acetylacetone and anhydrous ethanol. Secondly, TEOS was mixed with the solution containing bidistilled water, ammonia water and anhydrous ethanol under stirring. The final step was to add simultaneously and slowly the Ti and Si sols into a beaker containing anhydrous ethanol under stirring. After finishing the operation mentioned above, the beaker was covered by a plastic film and aged for 24 hours. Then, acetone was added and stirred. The gel powder was washed sequentially with ethanol and doubly distilled water, dried in an oven at 100 o C and calcined at 600 o C for 2 hours. Preparation of Fe 3 O 4 nanoparticles The synthesis of Fe 3 O 4 magnetic NPs is based on the mixture of FeCl 3.6H 2 O and FeCl 2.4H 2 O salts in the molar ratio 2:1. In a typical reaction, 2 ml of 2M FeCl 2 and 4 ml of 302 ISSN

317 2M FeCl 3 were mixed with the magnetic stirrer. While vigorously stirring the reaction mixture, the solution of 2M NaOH was dropped into the mixture and black precipitate was produced immediately. This solution was placed in autoclave and heated at 120 o C-180 o C. The reaction time is 2 hours. Synthesis of TiO 2 -SiO 2 -Fe 3 O 4 nanocomposites In this procedure, iron-modified TiO 2 powders were prepared by grinding TiO 2 -SiO 2 in an agate mortar with iron oxide. The obtained powders were dried for 24 hours at the temperature of 80 o C and calcinated at 300 o C for 2 hours. Characterization methods XRD patterns of prepared samples were records with a D5000 X-ray diffractometer using Cu Kα (λ = nm) radiation. The average crystallite size of prepared particles was determined by the Scherrer equation. Morphology studies were carried out using Field Emission Scanning Electron Microscope (FE-SEM) S4800-Hitachi. The composition of the catalyst was determined by energy dispersive X-ray spectroscopy (EDS) with a Nova NanoSEM 450. Photocatalytic test The UV-Vis light was provided by halogen lamp without filter which was placed in front of the reactor. The distance of the photocatalyst from the UV light source was about 20 cm. The lamp yields a spectrum ranging from ultraviolet to infrared light ( nm). The temperature of photodegradation system was about 60 o C. After each irradiation, the concentration of PCB was determined by measuring GC-MS 2010 equipment. The percent of pollutant removal was determined from the following equation: % removal = (C 0 -C)/C 0 x 100 where C 0 represents the initial comcentration of the PCB and C is the final concentration after illumination by light. III. RESULTS AND DISCUSSION SEM micrographs and EDS spectra of TiO 2 -SiO 2 9% NPs calcined at 600 o C (Fig. 1) show the formation of aggregated secondary particles by the agglomeration of primary particles. Additionally, the TiO 2 -SiO 2 agglomerates possess a rough and porous surface, resulting in increased surface area. The chemical composition of the samples was analyzed by EDS. The results indicated that the chemical compositions of TiO 2 -SiO 2 9% were 7.6 at. % Si, confirming that TiO 2 and SiO 2 are well distributed each other. These compositions agree with theoretical calculations. SEM image for the Fe 3 O 4 shows that they are nanospheres (Fig. 2a). The particles size is ranging from 10 nm to 20 nm as per SEM image. XRD partern of the Fe 3 O 4 nanospheres (Fig. 2b) presents the characteristic peaks (at 2θ: 30.4, 35.7, 43.4, 53.8, 57.4, and 63 o ) of cubic spinel structure (JCPDS No ). ISSN

318 Fig. 1. SEM micrographs and EDX spectra of TiO 2 -SiO 2 9% NPs. Identified PCBs in PCB transformer oil included PCB-52, PCB-101, PCB-138, PCB- 153, PCB-180 and PCB-209. Because all PCBs are poorly soluble in water, prior studies on PCB degradation were carried out in alkanes and alcohols. Many organic solvents were (a) (b) Fig. 2. SEM micrographs (a) and XRD partterns (b) of Fe 3 O 4 a) 304 ISSN

319 b) c) Fig. 3. GC profiles of PCBs before (a), after 30 (b) and after 60 (c) min of irradiation. employed in previous studies such as hexane, isooctane, 2-propanol, and methanol [19-21]. Overall, photochemistry in both organic and aqueous media is predominantly dechlorination; nevertheless, depending on the solvent used, some solvent derivatives can also be found [22-26]. There are few reports that have employed ethanol for this purpose. It is reasonable to predict that the performance of ethanol is similar to other solvents. Some solvents, e.g. hexane and isooctane, are highly flammable. When degradation takes a long time for high PCB concentrations, fire hazard will be of concern. Moreover, reaction rate of PCB was found to be faster in hydroxylic solvents than in non-polar solvents [24]. When H 2 O 2 is added, the dominant mechanism of PCBs destruction is hydroxyl radicals. The highly reactive OH radical species produced in the process are expected to quickly react with PCBs. Hydroxyl radical is an extremely reactive and nonselective oxidant; thus, when produced in sufficient quantities, it can lead to complete oxidation of organic compounds to carbon dioxide, water, and inorganic ions [25]. Hydrogen peroxide is a very important factor for efficiency of the treatment process and its economic desirability. ISSN

320 The oil solution was irradiated with visible light in the presence of TiO 2 -SiO 2 -Fe 3 O 4 samples in suspension. TiO 2 -SiO 2 -Fe 3 O 4 was used as the photocatalyst under visible light. The NPs were dispersed in the fluid through stirring. Fig. 3 shows the GC profiles of photocatalytic degradation of overall PCBs before, after 30 and 60 min of irradiation. 71.6% and 77.8% of PCB degraded in 30 min and 60 min under visible light, respectively. These results demonstrate that the PCB degradation under visible light using TiO 2 -SiO 2 - Fe 3 O 4 was significant. Visible light weakly activates TiO 2 (because TiO 2 has a large band gap of 3.2 ev, which is sensitive only to UV with wavelength shorter than 387 nm) had the ability to activate Fe 3 O 4 to yield electron-hole pairs. The electron is transfered from the valence band to the conduction band in Fe 3 O 4, then to the conduction band of TiO 2, while the hole is remained in the valence band of Fe 3 O 4. As a result, a large amount of electrons and holes on the surface of the photocatalyst can be separated and participate in the photocatalytic reactions, leading to an enhancement of photocatalytic activity. IV. CONCLUSION TiO 2 -SiO 2 -Fe 3 O 4 NPs were successfully synthesized, and used efficiently as photocatalyst for removal of PCBs in waste transformer oil. SEM, EDX and XRD analysis were employed to investigate the morphological structure of the photocatalyst NPs. The nanocomposite displays an excellent photocatalytic degradation activity under the visible irradiation. V. ACKNOWLEDGMENTS Part of this work was done at the Key Laboratory of Electronic Materials and Devices, Institute of Materials Science. REFERENCES [1] Y. Fang, S.R. Al-Abed, Applied Catalysis B: Environmental. Vol. 78, 2008, pp [2] Y.H. Shih, C.K. Wang, Journal of Hazardous Materials. Vol. 165, 2009, pp [3] M.A. Manzano, J.A. Perales, D. Sales and J.M. Quiroga, Chemosphere. Vol. 57, 2004, pp [4] F.L. Leaes, A.P. Daniel, G.B. Mello, V. Battisti, J.S. Bogusz, T. Emanuelli, L.L.M. Fries and I. Costabeber, Food Chem Toxicol. Vol. 44, 2006, pp [5] M.M. Mahlambi, C.J. Ngila and B.B. Mamba, Journal of Nanomaterials. Vol. 2015, 2015, Article ID , 29 pages. [6] Y. Ou, J.D. Lin, H.M. Zou and D.W. Liao, J Mol. Catal A-Chem. Vol. 241, 2005, pp [7] D. Jiang, Y. Xu, B. Hou, D. Wu and Y.H. Sun, J Solid State Chem. Vol. 180, 2007, pp ISSN

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322 RESERVOIR OPTIMIZATION METHODS: DYNAMIC PROGRAMMING AND DIFFERENTIAL EVOLUTION Phan Thi Thu Phuong Faculty of Civil Engineering, Hanoi University of Civil Engineering, 55 Giai Phong Street, Hanoi, Vietnam *: Abstract: Reservoir optimization is a problem that has effect on many other aspects and is concerned in many countries. To operating a reservoir optimally, many methods have been studied and applied such as linear programming (LP), dynamic programming (DP) and genetic algorithm (GA). While LP is investigated years ago and mathematically proven, DP is first introduced in 1957 by Bellman then developed to solve sequential or multi-stage decision problem. Nandalal (2007) claimed that DP is well suited for reservoir operation problem with the state variable is storage, the decision variable is release and the stage is by time period. Traditional DP uses incremental for both state variables and decision variables. On the other hands, differential evolution (DE), a method in GA group, is based on Darwin s evolution theory. Although Kumar and Reddy (2006, 2007) have researched and applied DE into many reservoirs and shown that even though being a rule-of-thumb method but DE is often capable of achieving global optimal solution. In this report, we study DP and DE, then develop the corresponding programs to apply DE and DP to a specific objective which is Pleikrong reservoir, the biggest one in Sesan cascade in Vietnam, to achieve the maximize electricity production in dry season. Keywords: reservoirs, optimization, dynamic programming, differential evolution, Sesan cascade. I. INTRODUCTION Optimization problems occur in many subjects of life. One of the most concerned optimization problems in the many countries in the world is reservoir regulation optimization problems. Normally, reservoirs were built for many goals such as power generation, irrigation, navigation, water supply or flood control downstream, etc. A reservoir regulation optimization problem is finding the best regulation to reach one or some optimal goals within the characters of the reservoir and regulation conditions. The functions that describe the optimal goals are called objective functions. An optimal problem is clarified single objective or multi-objective depended on the number of the objective functions. The conditions that describe the states, the design and the operation of the reservoir are called the constraints. A set of values of decision variable that satisfy all the constraints and give the optimal value of the objective functions is the optimal solution of the problem. For example, objective function of a reservoir optimization problem could be the maximum of electricity. And the constraints of this problem could consist: 308 ISSN

323 o Equation of mass balance for water o Characteristic equations of the reservoir o Maximum water storage capacity of the reservoir o Minimum and maximum releases II. METHODS Many methods are studied to solve these problems such as Linear Programming (LP), Dynamic Programming (DP), Nonlinear Programming (NLP), Genetic Algorithm (GA), etc. This paper focuses on DP and Differential Evolution that are two recently widely used optimal methods. Dynamic Programming Dynamic Programming is a well-known method that is widely used to solve optimization problem. It was first introduced by Bellman in The core of DP is the optimality principle of Bellman about the optimization problem in recursive form: An optimal policy has the property that whatever the initial state and initial decisions are, the remaining decisions must constitute an optimal policy with regard to the state resulting from the first decisions [1]. This method is usually applied to problems that are sequential. DP discomposes an N-decision problem into N of separate, interrelated sub-problems, each sub-problem is a stage. Each stage is characterized by chosen state variables. The decision analysis from this stage to the next one until reaching the last one could be done through space or time by control the decision variables. V 1,n0 V 2,n V 12,n V 13,n V 1,n-1 V 2,n-1 V 12,n-1 V 13,n-1 V 0 V 14 V 1,1 V 2,1 V 12,1 V 13,1 V 1,0 V 2,0 V 12,0 V 13,0 Stages 10 days 20 days 120 days 130 days 140 days Fig. 1. Chart of the optimal trajectory in forward computational procedure. ISSN

324 Applying DP to a reservoir optimization problem, the state variables are the water levels or the corresponding volume of the reservoir, the control variables are the releases. The problem is finding the best set of releases in a period of time to get, for example, the maximum hydro power production under some constraints. The calculation time is now separated into N stages. At each stage, the states are the water levels in use or the states of the capacity of the reservoir. Possible releases and corresponding power productions are calculated to reach from this state of the previous stage to that state of the next stage. Then DP will find the set of releases to have maximum power production. Differential Evolution Differential Evolution is a method belonged to Genetic Algorithm group. It was introduced by Price and Storn in As all GAs methods, DE is based on the Darwin s theory of natural evolution. The set of vectors of the previous step are called parents. The set of vectors of the next step are called new generation. The parents are mutant and combined to create new vectors. These newborns are compared to their parents by their fit to the objective function. If the newborns give better values of objective function, then they are set as new generation. Otherwise, parents are kept as the next generation. However, while traditional GAs use low mutation rates and fixed step sizes, DE uses weighted differences between solution vectors to perturb the population to reach to the optimal solution faster than traditional GAs [5]. Applying DE to a reservoir optimization problem, the variables are the releases. The problem is finding the best set of releases in a period of time to get the maximum hydro power production under some constraints. The constraints could be: the inflows and the releases must be satisfied the equation of mass balance for water; the releases are lower than the maximum releases and upper the minimum releases as the design of the reservoir;... In this case, the calculation steps are: - Choose an initial set of releases which is initial variables - Change the previous releases, use them to create new releases new variables to prepare for the next step (these steps are called mutation and crossover) - Put these new releases to the objective function which is electricity production function - If the objective function gives a better value, then new releases are passed to the next step. Otherwise, keep the old releases (this step is called selection). - Repeat the calculation until finding the set of releases which gives the maximum value of objective function. These steps might be described as the chart below. 310 ISSN

325 Fig. 2. Chart of Differential Evolution. where: G: Generation (Current calculation step). G max : maximum step. NP: number of variables in a set. X i,g : Set of variables in step G (in reservoir optimal problem, X i,g is the set of releases). X best, G : the best set of variables of step G. F: a real constant factor, called mutant factor in Differential Evolution. V i,g, U i,g : new variables, changing from X i,g. f(x): objective function (in reservoir optimal problem, f(x) is function to find electricity production. As shown in [3, 4], the algorithm to find X best, the best set of variables, is simple. After choosing the initial set of variables, two operators, which are mutation and crossover, are applied on each individual variable to create new variable. Then each variable is compared to the old variable of the previous step. The best variable is selected for the next step. Repeating the algorithm until the optimal critical is satisfied, the set of best variables of all the steps is the X best that the algorithm needs to find [4]. III. RESULTS AND DISSCUSSION Now, DP and DE are applied to a Pleikrong reservoir in the dry season of Pleikrong reservoir is the biggest reservoir in the Sesan cascade that is located in the Highland of Vietnam. Its main purpose is producing electricity. The optimal problem for Pleikrong reservoir is set as below: ISSN

326 In the dry season of the Highland, 140 days from 11st February to 30th Jun (followed the operation rules in the decision No QD-TTg of the Govement of Vietnam signed in July 17), finding the Q i - average in 10 days of the releases to get the maximum of electricity production. In this case, Q i ( i=1,...,14) is the variable of the problem. The electricity production of Pleikrong plant at time period i of 10 days is calculated by the following formula [2]: E i = 9.8 * h i * Q i * k *24*10/1000 (MWh) (1) where: i is from 1 to 14, is 14 stages of 10 days; h i - water height at time period i, Q i - release at time period i, k overall generation efficiency. Fig. 3. Relationship between water levels and storage volumes of the Pleikrong. Then electricity production E is calculated as: E = E 1 + E E 14. (2) Electricity production E is the objective function of this optimization problem. The constraints are: 0 Q i the release is bounded by the minimum and the maximum releases The releases and the inflows are satisfied the equation of mass balance for water The data that is used in this paper was provided by the team of Institute of Mechanics of VAST in project of building the reservoir operation for Sesan cascade in dry season (under contract N 01/2011/QTVH - SESAN on June 02, 2011). 312 ISSN

327 Characteristics of reservoirs of Sesan River Plant Sesan Cascade Hydroelectric Diagram Upper Kon Tum Explaination Factoríes Table 1. Inflows to Pleikrong reservoir. Stages 11/Feb- 20/Feb 21/Feb- 02/Mar 03/Mar- 12/Mar 13/Mar- 22/Mar 23/Mar- 01/Apr 02/Apr- 11/Apr 12/Apr- 21/Apr Inflows (m3/s) Stages 22/Apr- 01/May 02/May- 11/May 12/May- 21/May 22/May- 31/May 01/Jun- 10/Jun 11/Jun- 20/Jun 21/Jun- 30/Jun Inflows (m3/s) Using DP and DE to solve the problem above, the results are: Table 2. Optimal releases for Pleikrong reservoir in dry season of 2010 by DP and DE. N 0 period Time of period Optimal releases (m 3 /s) by DE Optimal releases (m3/s) by DP 1 11/Feb-20/Feb /Feb-02/Mar /Mar-12/Mar /Mar-22/Mar /Mar-01/Apr /Apr-11/Apr /Apr-21/Apr /Apr-01/May /May-11/May /May-21/May /May-31/May /Jun-10/Jun /Jun-20/Jun /Jun-30/Jun Electricity producion (MWh) (MWh) Dams Fig. 4. Sesan Cascade. ISSN

328 The results of both methods show the trend that the reservoir operation tends to storage the water at the beginning of calculation time, and strongly release at the end. The power production by DE method is slightly higher than the production by DP because DE storages more at first and have large releases at few last time steps. And both of the power productions by these two methods are much higher than the real one which is about MWh. IV. CONCLUSION Management of a reservoir is a sophisticated problem in real life. Optimization is an approach to the objective as much as possible. In this work, two methods DP and DE are presented and applied to a real reservoir regulation optimization problem in Vietnam. Using optimal methods such as DP and DE might give the operation with higher power productions than operation in real time. The agreement of these two methods is the power production could be higher if the water is stored at the beginning then released with large amount at the end of the calculation time. REFERENCES [1] Bellman, R.E (1957), Dynamic Programming, Princeton, NJ: Princeton University Press. [2] Nandalal, K. D. W., Bogardi, J. J. (2007), Dynamic Programming Based Operation of Reservoir: Applicability and Limits, Cambridge University Press. [3] Reddy, M. J., Kumar, D. N., Optimal Reservoir Operation Using Multi-Objective Evolution Algorithm, Water Resources Management, (2006), pp [4] Reddy, M.J., Kumar, D.N. (2007), Multiobjective Diferential Evolution with Application to Reservoir System Optimization, Journal of Computing in Civil Engineering, pp [5] Storn R., and Price K., Differential Evolution - A Simple and Efficient Heuristic for Global Optimization over Continuous Spaces, Journal of Global Optimization, vol. 11, (1997), pp ISSN

329 TYPE-2 FUZZY CO-CLUSTERING ALGORITHM FOR COLOR IMAGE SEGMENTATION Van Nha Pham* and Duc Thao Nguyen Academy of Military Science and Technology, Hoang Sam, Cau Giay, Hanoi, Vietnam *: Abstract. Fuzzy co-clustering is a method that performs simultaneous fuzzy clustering of objects and features. Due to characteristics of co-clustering, co-clustering is used to treat various types of data which has complex characteristics such as multi-dimensional, multifeature and large size. In this paper, we have proposed a new co-clustering algorithm which is called Type-2 Fuzzy Co-Clustering algorithm (CoT2FCM) for color image segmentation. This is a combination of co-clustering and type-2 fuzzy logic for increased visibility uncertainty problems in the data clustering. Our experiments were conducted on the color image datasets in Berkeley s photo library. The experimental results demonstrated the strength and potential of CoT2FCM in color image segmentation in terms of performance evaluation criteria, accuracy, stability and efficiency, compared with some of the recent popular fuzzy co-clustering approaches. Keywords: Fuzzy clustering, fuzzy co-clustering, categorical multivariate data, color image segmentation, type-2 set, type-2 fuzzy clustering, type-2 fuzzy co-clustering. I. INTRODUCTION Clustering is an unsupervised learning technique of categorizing data into several groups such that data belonging to one group are highly similar to one another, while data of different groups are highly dissimilar. To extend the capability and quality of clustering, combined clustering with fuzzy logic is helping us get the uncertainly problem is better in data. Meanwhile, an object is not restricted to any clusters, which can belong to different clusters. J.C. Bezděk et al. [1] announced the fuzzy clustering algorithm FCM, opening up new research directions in the field of data mining by mounting fuzzy models with data to be clustered. [2-4] proposed image segmentation algorithm based on color space and spatial information. Type-2 fuzzy sets are studied and integrated with clustering techniques to solve the problem with complex models [5-8]. The scientists have studied type-2 fuzzy clustering by incorporating type-2 fuzzy logic with clustering [9, 10] to solve some complex problem which have large uncertainty that type-1 fuzzy clustering can not be done. In the data clustering, data is represented by data objects. Each data object can have one or more data elements called features or attributes. In fact, data objects often have multiple data elements, such as for document data, the data object is the text includes many words, phrases or keywords; pixel data object in the color image includes three color ISSN

330 components R, G and B. The technique that simultaneously clusters both object and feature is called co-clustering technique [11]. Co-clustering technique [12, 13] is an alternative clustering technique to clustering some data types which have complex data structure, such as web, multi-spectral images, hyper-spectral images, etc. To extend the applicability and quality of clustering, combining co-clustering with fuzzy logic is a need to handle better uncertainty in the data. This technique was called fuzzy co-clustering. K. Honda et al. [14] and K. Kummamuru et al. [15] have proposed fuzzy coclustering model for clustering multi-dimensional, multi-feature data types. The key idea behind HFCR [16] is the formulation of the dual-partitioning approach for fuzzy coclustering. [17] improved the recommender performance which is a combination of content-based and collaborative filtering approaches in a two-layer graph model getting use of web content and usage mining. W.C. Tjhi [18] have proposed fuzzy semi-supervised co-clustering algorithm for categorization of large web documents. In this paper, we combined the strength of fuzzy co-clustering and type-2 fuzzy sets. A novel type-2 fuzzy co-clustering algorithm (CoT2FCM) is proposed to solve limited issues of clustering on complex data models that the fuzzy clustering algorithms and fuzzy co-clustering algorithms can not be settled. The experimental results demonstrate the strength and potential of CoT2FCM in terms of performance evaluation criteria, accuracy, stability and efficiency, compared with some of the recent popular fuzzy co-clustering approaches. The rests of this paper are organized as follow. Section 2 provides the basic content of type-2 fuzzy set theory and type-1 fuzzy co-clustering algorithm. Section 3 introduces a new algorithm CoT2FCM. Section 4 presents some experimental results. Section 5 concludes the paper. II. BACKGROUND This section give a brieftly overview relate to type-2 fuzzy sets and fuzzy coclustering Type-2 Fuzzy Sets Definition 1: A type-2 fuzzy set is characterized by a type-2 membership function ( xu, ) where x X and u J x [0,1], i.e.: A (1) A {(( x, u), ( x, u)) x X, u J [0,1]} A x or ~ ~ (, ) / (, ), x [0,1] (2) A x u x u J x X u J x A in which 0 ( xu, ) 1 A, see [5, 6]. 316 ISSN

331 At each value of x, we say x=x, the 2-D plane whose axes are u and ( x ', u ) is called a vertical slice of ( xu, ). A secondary membership functionis a vertical slice of A ( xu, ) A. It is ( x x ', u ) A for x X, u J x ' [0,1], i.e. ~ ~ A A A x' x' (3) u Jx ' ( x x', u) ( x') f ( u) / u, J [0,1] 2.2. Fuzzy Co-Clustering algorithm Fuzzy co-clustering algorithm (CoFCM) is a clustering method that derived from simultaneous fuzzy clustering of objects and features. The process results in co-clusters, which reflect the interrelations between object clusters and feature clusters. CoFCM have been studied, improvements and applications in [21-29]. CoFCM algorithm assigns data objects to each cluster by using two fuzzy membership functions, that are the function of the partition or data object and the function of classification or the components of the data object. The objective function J CoFCM (U,V,P) is described as follows: C N K C N C K (4) J ( U, V, P) u v D T u log u T v log v CoFCM ci cj cij U ci ci V cj cj c 1 i 1 j 1 c 1 i 1 c 1 j 1 The components of the objective function (4) is determined by the following formulas: u ci C e f 1 K vcj Dcij ( ) T e j 1 K vfjdcij ( ) T j 1 U U and v cj K e q 1 N ucidcij ( ) T e i 1 N ucidciq ( ) T i 1 V V III. TYPE-2 CO-CLUSTERING ALGORITHM Type-2 fuzzy co-clustering algorithm (CoT2FCM) derived from combining the fuzzy co-clustering algorithm and type-2 fuzzy logic to solve limited issues in clustering with complex data models as multi-dimention, multi-feature, high size. CoT2FCM algorithm assigns data objects to each cluster by using two type-2 fuzzy membership functions, that are the function of the partition or data object and the function of classification or the components of the data object. Let X=={x 1, x 2,..., x i,..., x N } R K denotes an datasets including N data objects to be partitioned into C clusters, where x i represents K-feature data object. x ij denote the j th feature of the i th data object, P={p cj } with c=1, 2,.., C and j=1, 2,.., K be the set of feature cluster centers and D cij =Dist(x ij, p cj ) be the square of the Euclidean distance between x ij and p cj given by: 317 ISSN

332 D cij 2 2 d ( xij, pcjj ) ( xij pcj ) (5) Let u ci denotes the object membership of the i th data object to c th cluster, U={u ci } be the CxN object membership function matrix of datasets, v cj denotes the feature membership defined as the membership of feature j th to the c th cluster and V={v cj } be the corresponding CxK feature membership matrix of datasets. The CoT2FCM algorithm is an iterative optimization that minimizes the cost function defined as follows: C N K C N C K m m m m m m CoT 2FCM (,, ) ci cj cij U ci log ci V cj log cj c 1 i 1 j 1 c 1 i 1 c 1 j 1 (6) J U V P u v D T u u T v v To get optimal clustering results, the (6) reaches a minimum and J CoT2FCM is minimized subject to the following constraints: C c 1 K j 1 u 1, u 0,1, i 1, N ci ci v 1, v 0,1, c 1,C cj cj The constrained optimization problem of CoT2FCM can now be defined by applying the Lagrange multipliers λ i, and c and constraints (7) into (6) as shown below. C N K C N C K m m m m m m CoT 2FCM (,, ) ci cj cij U ci log ci V cj log cj c 1 i 1 j 1 c 1 i 1 c 1 j 1 J U V P u v D T u u T v v N C C K m m i uci c vcj i 1 c 1 c 1 j 1 ( 1) ( 1) where λ i, γ c are Lagrange coefficients, T U and T V are weights that indicate fuzzy level, m is fuzzy limit. Increasing T U and T V will increase the opacity of the cluster. To determine the value of the objective function (8), we have to find the formula for the membership functions u ci, v cj and distance D cij. Firstly, taking the partial derivative of J(U;V;P) in (8) with respect to U and setting the gradient to zero, we have, K J m vcj Dcij TU ( mlog uci 1) i 0 (9) U j 1 Subjecting u ci derived from (9) to the constraint in (7), we obtain the formula u ci as follows: (7) (8) 318 ISSN

333 u ci C e c 1 ( e K j 1 ( v K m cj j 1 D mt v U cij m cj D mt ) U cij ) (10) In a similar manner, we have feature membership function v cj and p cj as follows: v cj K e j 1 ( e N i 1 ( u N m ci i 1 D mt u V cij m ci D mt ) V cij ) (11) p cj N i 1 N u i 1 m ci u x m ci ij (11) Table 1. CoT2FCM Pseudo-code. CoT2FCM algorithm 1. Initialize the parameters T U, T V, m, maximum error limit ε and maximum number of iterations τ max. 2. Set iteration number τ=1. 7. Update v cj using (11) 3. Initialize u ci such that 0 u ci Update u ci using (10). 4. DO 9. Increase τ=τ Update p cj using (12) 10. WHILE (max( u ci [τ] u ci [τ 1] ) ε or τ=τ max ) 6. Update D cij using (5) 11. Output result. IV. EXPERIMENTAL RESULTS A. Validity indexes General fuzzy clustering, type-2 fuzzy co-clustering in particular are unsupervised learning process, the data objects are not labeled and anticipated structure. To determine the optimal number of clusters, we use indexes S [19] and CS [20]. To assess the quality of clustering, we use indexes PC and PE [21], Mean Squared Error (MSE) [21] and Image Quality Index (IQI) [22]. B. Experiments Table 1. Image segmentation results using FCM, CoFCM and CoT2FCM. No. Original image Results of FCM Results of CoFCM Results of CoT2FCM 1 2 PC u: PC u: PC u: PE u: PE u: PE u: MSE: MSE: MSE: 27.8 IQI: IQI: IQI: S: S: S: CS: CS: CS: PC u: PC u: PC u: PE u: PE u: PE u: ISSN

334 3 MSE: MSE: MSE: IQI: IQI: IQI: S: S: S: CS: CS: CS: PC u: PC u: PC u: PE u: PE u: PE u: MSE: MSE: MSE: IQI: IQI: IQI: S: S: S: CS: CS: CS: In this section, the experimental results are presented to demonstrate the effectiveness of the proposed algorithm in segmenting natural color images. A color image datasets to be analyzed was downloaded from Berkeley s photo library We have conducted experiments on three color images with number of cluster and fuzzy parameters were fixed, using three clustering methods FCM, CoFCM and CoT2FCM. The experimental results are shown in Table 1. This result, access indexes of CoT2FCM are better than FCMs and CoFCMs. As presented above, which clustering method achieves the Pcu higher (PEu lower), MSE lower and IQI higher, that clustering method is considered better. Table 2. Color image segmentation results using CoT2FCMon color image jpg. No. Type C K Pcu PEu MSE IQI S 320 ISSN CS x10-10 FCM CoFCM CoT2FCM FCM CoFCM CoT2FCM FCM CoFCM CoT2FCM FCM CoFCM CoT2FCM FCM CoFCM CoT2FCM FCM CoFCM CoT2FCM FCM CoFCM CoT2FCM In the next experiment, we have conducted clustering on a color image jpg with number of clusters from 3 to 9, using three difference clustering methods FCM, CoFCM and CoT2FCM. Clustering results are presented in Table 2. In this table, we can

335 see the results of indexes S and CS in three methods are reaching minimal first time in experiments with number of clusters is 4. This indicates that, all of three methods determine the optimum number of clusters is 4. PC u, PE u, MSE and IQI indexes of CoT2FCM in experimental with optimum number of clusters are better than FCM and CoFCM, respectively. In Fig. 1, in order from left to right, from top to bottom are the original image jpg and result images with number of cluster is from 2 to 10, using clustering method CoT2FCM. Result pixels in the same cluster is filled with a specified color. Original image C=2 C=3 C=4 C=5 C=6 C=7 C=8 C=9 C=10 Fig. 1. Image segmentation results using CoT2FCM on Original image " jpg" with number of clusters from 2 to 10. We have some comments after conducting experiments above. For each type of data, we used a set of corresponding parameters Tv, Tu and m. To find the appropriate parameters we have tested many times, then compare the results of the trials to determine the parameters when it is best to choose. When increasing the parameters Tv and Tu, the width of fuzzy area increases, the speed of convergence increases. When changing fuzzy weight m, the quality of clustering changes. For color image datasets in our tests, the fuzzy parameters are defined as follows: Tv=9.5x10 6, Tu=10.5, m=1.7. V. CONCLUSION In this paper, Type-2 Fuzzy Co-Clustering model which is combining between clustering pixel and pixel s color components and type-2 fuzzy logic used for color images segmentation. A new algorithm CoT2FCM was derived from CoFCM algortihm by the construction of a new formula objective function with type-2 fuzzy membership functions. The experiment was conducted on the data in Berkeley s sample photo gallery with the input parameters are adjusted to have more comprehensive information, assess effectiveness. Basically image segmentation quality of CoT2FCM better than CoFCM s and FCM s. ISSN

336 With results of research and experimentations above, we will apply CoT2FCM to solve complex problems, multi-dimensional data and large size. However, CoT2FCM still affected by the initial initialization and speed is quite slow when compared to other methods. In future, we will use some techniques to helping initiate a better, higher processing speed and solve the problem of online automatic identification. The next could be to apply the our prosal to several real problems like hyperspectral image classification, applications in medical diagnosis or environmental applications. REFERENCES [1] C. Bezdek, R. Ehrlich, W. Full, The fuzzy C-means clustering algorithm, Computers & Geosciences, Vol. 10 (2 3), 1984, pp [2] K. Bhoyar, O. Kakde, Colour Image Segmentation using Fast Fuzzy C-Means Algorithm, Electronic Letters on Computer Vision and Image Analysis, Vol. 9, 2010, pp [3] Z. Ding, J. Sun, Y. Zhang, FCM Image Segmentation Algorithm Based on Color Space and Spatial Information, International Journal of Computer and Communication Engineering, Vol.. 2(1), 2013, pp [4] D. Aneja, T.K. Rawat, Fuzzy Clustering Algorithms for Effective Medical Image Segmentation, I. J. Intelligent Systems and Applications, 2013, pp [5] N.N. Karnik, J.M. Mendel, Operations on type-2 fuzzy sets, Fuzzy Sets and Systems, Vol. 122(2), 2001, pp [6] J.M. Mendel, R.I. John, Type-2 fuzzy sets made simple, IEEE Transactions on Fuzzy Systems, Vol. 10(2), 2002, pp [7] L.A. Lucas, T.M. Centeno, M.R. Delgado, Land Cover Classification Based on General Type-2 Fuzzy Classifiers, International Journal of Fuzzy Systems, Vol. 10 (3), 2008, pp [8] J. Clairet, A. Bigand, O. Colot, Color Image Segmentation using Type-2 Fuzzy Sets, IEEE International Conference on E-Learning in Industrial Electronics, 2006, pp [9] O. Linda, M. Manic, General Type-2 Fuzzy C-Means Algorithm for Uncertain Fuzzy Clustering, IEEE Transactions on Fuzzy Systems, Vol. 20(5), 2012, pp [10] L.T. Ngo, D.D. Nguyen, Land cover classification using interval type-2 fuzzy clustering for multi-spectral satellite imagery, IEEE International Conference on Systems, Man, and Cybernetics (SMC), 2012, pp [11] I.S. Dhillon, S. Mallela, D.S. Modha, Information-theoretic co-clustering, Proc. of the 9th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, 2003, pp [12] I.S. Dhillon, Co-clustering of Documents and Words using bipartite spectral graph partitioning, Proc. of the seventh acm sigkdd international conference on knowledge discovery and datamining, 2001, pp [13] Y. Song, S. Pan, S. Liu, Constrained co-clustering for textual documents, Association for the Advancement of Artificial intelligence, ISSN

337 [14] C.H. Oh, K. Honda, H. Ichihashi, Fuzzy Clustering for Categorical Multivariate Data, Proc. of Joint Ninth IFSA World Congress and Twentieth NAFIPS International Conf., 2001, pp [15] K. Kummamuru, A. Dhawale, R. Krishnapuram, Fuzzy Co-clustering of Documents and Keywords, IEEE International Conf. on Fuzzy Systems, Vol. 2, 2003, pp [16] W.C. Tjhi, L. Chen, A heuristic-based fuzzy co-clustering algorithm for categorization, Fuzzy Sets and Systems, Vol. 159, 2008, pp [17] K. Honda, M. Muranishi, A. Notsu, H. Ichihashi, FCM-type Cluster Validation in Fuzzy Co- Clustering and Collaborative Filtering Applicability, IJCSNS International Journal of Computer Science and Network Security, Vol.13(1), 2013, pp [18] Yang Yan, Lihui Chen, William Chandra Tjhi, Fuzzy semi-supervised co-clustering for text documents, Fuzzy Sets and Systems, Vol. 215, 2013, [19] X.L. Xie, G. Beni, A validity measure for fuzzy clustering, IEEE Trans. Pattern Anal. Mach. Intell, Vol. 13, 1991, pp [20] H.L. Shieh, A Hybrid Fuzzy Clustering Method with a Robust Validity Index, International Journal of Fuzzy Systems, Vol. 16(1), 2014, pp [21] Z. Wang, A.C. Bovik, Mean squared error: love it or leave it? A new look at signal fidelity measures, IEEE signal processing magazine, 2009, pp [22] Z. Wang, A.C. Bovik, A universal image quality index, IEEE signal processing letters, Vol. 9(3), ISSN

338 APPLICATION OF GAMMA-GAMMA COINCIDENCE METHODS IN NEUTRON ACTIVATION ANALYSIS Truong Van Minh 1, Pham Dinh Khang 2, Nguyen Xuan Hai 2, Tran Minh Hung 1, Ho Huu Thang 2 and Nguyen An Son 3 1 Dong Nai University, 04 Le Quy Don, Bien Hoa, Dong Nai, Vietnam 2 Nuclear Research Institute, 01 Nguyen Tu Luc, Dalat, Vietnam 3 Da Lat University, 01 Phu Dong Thien Vuong, Dalat *: truongminhdnu@gmail.com Abstract. Neutron activation analysis (NAA) is a technique used for determination of trace elements composition. However, it is difficult to determine the qualitative and quantitative of elements which spectrum is overlaped by the influence of elements in sample. To overcome these problems, gamma-gamma coincidence method has been developed and used in several countries. In this paper, we have applied of coincidence spectrometer for activation analysis. The results of studies based on Standard Reference Material samples irradiated by thermal neutron beam at the 3 rd horizontal channel of the Dalat Nuclear Reactor. Keywords: Neutron activation analysis; coincidence spectrometer; gamma-gamma coincidence; trace element. I. INTRODUCTION The gamma-gamma coincidence method is mainly applied for experimental studies of the nuclear structure. Thanks to its ability to reduce the gamma background and the selection of events correlation in timing. In the previously works, these methods have been used for NAA [1, 3]. Y. Hatsukawa et al have been used a coincidence spectrometer with detector arrays in order to improve sensitivity of NAA. In this manner, 24 elements in the reference igneous rock samples were determined simultaneously without chemical separation method [1]. When quantifying of elements in environmental samples by the INAA method, gamma-ray measurements from trace elements become difficult in the presence of those from interfering elements. Usually chemical separation processes are required to eliminate interfering elements for the determination of trace elements in environmental samples. The gamma gamma coincidence spectrometer using two detectors was already applied to neutron activation analysis. Recently, In Dalat Nuclear Research Reactor (DNRR), a gamma-gamma coincidence spectrometer has been setup for NAA and experimental nuclear structure studies [2]. In this paper, we report a new quantification method with combination of neutron activation and gamma-gamma coincidence spectrometer, and its application to reference environmental samples. 324 ISSN

339 II. EXPERIMENTS 2.1. Sample preparation The sample was prepared from a standard material of Coal Fly Ash-1633b [4]; mg sample was sealed in a high purity polyethylene container and irradiated at central irradiation tube with neutron flux ~ n.cm -2.s -1 for 10 hours. After 60 days decayed, the sample was analyzed on the gamma-gamma coincidence spectrometer in normal mode and coincidence mode. The electronics configuration of spectrometer is shown in Fig Experimental measure ISSN Fig. 1. The experimental arrangement and electronics configuration. The coincidence spectrometer includes two HPGe detector, type GMX35 of 35% and 38% relative efficiency, and 1.9 kev energy resolution at 1332 kev. The sample place was centered on the axis of the two detectors at a distance of 4 cm between the sample and the end cap of each detector. The parameters of the spectrometer are setup by method described in Ref. 0. In the normal mode, the spectra were acquired for 0.8 hours and in gamma-gamma coincidence mode, for 48 hours. The collected data were saved and analyzed off-line by multi-parameters method Data analysis In the normal mode, the peak areas were measured by fitting with colegram software [5]. In the gamma-gamma coincidence mode, the gamma rays emitted form trace elements were analyzed using gated spectra obtained by putting gates on - matrix. The double fold - coincidence events were stored to a two-dimensional E -E matrix, which had channels. A gate spectrum was obtained, by gating on a -ray energy in one detector, from a second detector in coincidence with the first one. In order to correct for the chance coincidence in the gate spectrum, the spectrum gated at background region near the peak position was subtracted from the spectrum at the peak. The detection limits were evaluated, 325

340 assuming a -peak area was twice the standard deviation of the underlying background. The detection limits (for normal or coincidence mode) were calculated by Eq. 1 as follows 0: C DL p 3.29C 1 B P P t B t where: P/B is the peak-to-background ratio; P/t is the net counting rate in the peak; t is the counting time; C DL is the detection limit in units of concentration; C is the concentration or activity; ɳ P and ɳ B are standard deviations of peak and background. The concentration of Se in the sample (for normal or coincidence mode) was calculated by Eq. 2 as follows [7]: Np / tc W. D. C a Np / tc W.. DC. s where: N p is the net area of the sample (a) and standard (s); S = 1 exp(- t i ) is the saturation factor, t i is the irradiation time; D = exp(- t d ) is the factor decay time, t d is the decay time; C = [1 exp(- t c )]/( t c ) is the factor counting times, t d is the counting times; W is the weight of the sample and standard (g); ρ is the concentration of the element (mg/kg). (1) (2) III. RESULTS AND DISCUSSION 3.1. Results Fig. 2 shows the gated spectra in coincidence and in normal mode for the Coal Fly Ash-1633b sample highlighting 75 Se main gamma lines. Fig. 3 shows the gated spectra in coincidence, gated at 605 kev of isotope 134 Cs and gate at 889 kev of isotope 46 Sc. Fig. 2. The spectra of Coal Fly Ash in normal and coincidence modes, the coincidence is gated at 264 kev. 326 ISSN

341 Counts Counts The 4 th Academic Conference on Natural Science for Young Scientists, Master & Gate 605keV keV-Cs keV-Cs Gtae 889keV 1121keV-Sc The peak to background ratios, detection limits, and concentration of elements, which were gating of corresponding gamma energies, are shown in the Table 1 and Table 2. Isotope Energy (kev) Fig. 3. The spectra of Coal Fly Ash in coincidence modes, the coincidence are gated at 605 kev and gate at 889keV. Table 1. The peak to background ratios, detection limits of elements in Coal Fly Ash in normal and coincidence modes. Peak to background ratios E(keV) Improve peak to background ratios (times) Detection limit (mg/kg) Normal Coincidence Normal Coincidence Improve detection limit (times) Se Co Cs * Fe Sc Tb ISSN

342 3.2. Discussion Table 2. The Concentration of elements in Coal Fly Ash in normal and coincidence modes. Elements Concentration Concentration (mg/kg) (mg/kg) Normal Coincidence Certificate Se Co Cs Fe * Sc Tb Note: concentration of Fe is determined by percentage (%). Based on Fig. 2, the inferences of the isotopes 182 Ta and 181 Hf were evaluated by using 264 kev for gating to determine selenium in environmental sample. Fig. 3 shows the gated spectra by gate at 605 kev of isotope 134 Cs and gate at 889keV of isotope 46 Sc. In the results shown that the coincidence mode using the gated spectra is determined better the qualities of the isotopes in the sample than the normal mode. In normal mode, it observed only a few isotopes in the sample. This mainly reason could not reject the Compton background, so gamma-rays from trace elements with low intensities could not observed in these projected spectra. Another main note in the results is the detection limit in coincidence mode. In this research, it is the largest improved about 5.92 times and the peak to background ratios is the largest improved about times. In the case of using peaks for gating, the interference of other isotopes was nearly solved. IV. CONCLUSION The gamma-gamma coincidence method has been shown to give accurate determination of trace elements in environmental samples. The advantages of this method are improved the detection limit of elements, and the interference of other gamma rays at the energy of interest were solved without the need for radiochemical separation. This study has broadly the application of the gamma-gamma coincidence spectrometer in DNRR of Dalat Nuclear Reserach Institute. The determination of content of elements in the samples are necessary for all analytical samples that this method was completed. The content of each elements contained in the specimen was calculated. 328 ISSN

343 V. ACKNOWLEDGMENTS The authors would like to express their gratitude to the leadership of Nuclear Reserach Institute created the best condition for us to implement our experiments on gamma gamma coincidence spectrometer in the 3 rd horizontal channel of the Dalat Nuclear Research Reactor. REFERENCES [1] Hatsukawa Y, Oshima M, Hayakawa T, Toh Y, Nuclear Instruments and Methods in Physics Research A. Vol. 482, 2002, pp [2] Khang P. D, Tan V. H., Hai N. X., Dien N. N., Nucl. Instr. and Meth. A. Vol. 631, 2011, p [3] Nguyen Xuan Hai, Truong Van Minh, Pham Dinh Khang, Ho Huu Thang, Nguyen Ngoc Anh, J. Radioanal Nucl Chem. Vol. 304, 2015, pp [4] Coal Fly Ash, Certificate of analysis [Internet], Standard Reference Material 1633b. [5] Presentation of the colegram software [Internet]. Available from: [6] Gedcke AD, How counting statistics controls detection limits and peak precision [Internet], AN59 Application Note, ORTEC. Available from: Detection-Limits-Peak-Precision.pdf [7] F.D. Corte, The k 0 -Standardization Method: A move to the optimization of Neutron Activation Analysis, PhD Thesis, GENT University, (1987) 464. ISSN

344 A METHOD TO OPTIMISE THE TOP QUARK MASS IN DI-LEPTON DECAY CHANNEL Phan Thi Hong Ngoc 1*, Kaven Yau Wong 2 and Markus Cristinziani 3 1 Long An Teacher Training College, Long An province, Vietnam 2 ERC TopCoup member, Physics Institute, Bonn University, Germany 3 ERC TopCoup Leader, Physics Institute, Bonn University, Germany *: phanthihongngoc.cdsp@longan.edu.vn Abstract. Top quark is the most massive elementary particle in the Standard Model (SM). The top quark mass has a special role in electroweak symmetry breaking, linking to physics beyond the SM and limiting the allowed Higgs boson mass via radiative corrections. Due to the short lifetime, the top quark always decays before hadronisation, which is produced in abundance at the Large Hadron Collider (LHC) and allows many precise measurements. The aim of the study is to reduce the total uncertainty of the top quark mass measurements by applying the calibration curve method. Although several decay modes of the top-quark pair ( ) events have been used for topquark mass measurements, this study presents the decaying into di-lepton channel with the data collected by the ATLAS detector of 7 TeV total energy at the LHC. The final results including statistic (stat.) and systematic (syst.) uncertainties were m top = (stat.) 1.4 (syst.) GeV for m T2 variable and m top = (stat.) 1.3 (syst.) GeV for m lb variable, consistent with the recent ATLAS top-quark mass measurements in di-leptonic decay channel released. Keywords: top quark mass, optimize the total uncertainty of the measurement, the calibration curve method, repeated Gaussian fit, dilepton decay channel. I. INTRODUCTION The top quark is the most massive elementary particle in the Standard Model (SM) of particle physics. This gives the top quark mass an interesting parameter of the SM with a special role in electroweak symmetry breaking, linking to physics beyond the SM and limiting the allowed Higgs boson mass via radiative corrections [1]. The top quark decays before hadronisation and gives us access to a bare quark due to its short lifetime ( s) [2]. Top quark events are background in many processes beyond the SM. It is produced in abundance at the Large Hadron Collider (LHC), which allows many precise measurements. The top quark mass has been measured by several methods in different channels with the data collected at the Tevatron and the Large Hadron Collider (LHC) by the CMS and ATLAS collaboration. The researches have focused on increasing the precision of the top quark mass by reducing the total uncertainty of its measurements. 330 ISSN

345 II. EXPERIMENTS This analysis focused on measuring the top quark mass in the topquark pair ( ) decaying into the dilepton decay channel, using 4.7 fb -1 intergrated luminosity of 7 TeV total energy data collected by the ATLAS detector at the LHC [3-6]. Fig. 1. The decaying into di-lepton channel. The dilepton decay channel has the smallest branching fraction in all decay channels (Fig. 1). However, this channel has a very clear and distinctive signature in the detector, only diluted by the missing transverse energy attributed to two undetected particles, neutrinos. In addition, the background of this process from gluon-multijet production in the event is small, leading to a smaller effect of the systematic uncertainty related to jets such as jet calibration on the measurement. The cleanest samples of decaying in dilepton channel events were selected by requiring exactly two oppositely charged leptons including eclectron electron (ee), muon-muon ( ) and electron-muon (e ), at least two b-jets, and large missing transverse momentum (E Tmiss > 60 GeV) due to the two neutrinos escaping the detector, which leads to a challenge to a top quark mass measurement in this channel. In order to circumvent the missing transverse momentum problem, the orthogonal dimensional decomposition (m T2 ) of the stransverse momentum is applied [7-9]. The m T2 variable is given in the following formula: with The m T2 variables are used in events with two invisible particles in the final state and represent a lower boundary for the parent particle mass, and can therefore be interesting to measure the top quark mass in the dilepton decay channel. In addition, the invariant mass of the lepton b-jet system (m lb ) variable is used to measure the top quark mass, where only lepton and b-jet system are considered. The m lb is defined by the formula: Monte-Carlo (MC) simulation is used to determine the acceptance of the signal events as well as the contributions of background processes, provided by GEANT4 ISSN

346 simulation [10] and the MC11 production campaign of the ATLAS Production Group [11]. The luminosity of MC samples are re-weighted in order to make the MC expectation compatible with the collected data. The MC samples used in the study can be ordered in three groups: signal samples, background samples and systematics variation samples. There were different analysis strategies to measure the top quark mass. The calibration curve method was used in this study. Observables sensitive to the top quark mass, m T2 and m lb, were chosen to be the estimated mean from a fit function on each distribution. The MC simulation samples at different top quark mass points in the range from 165 GeV to 180 GeV as the templates were applied to measure the selected observables (Fig. 2). Fig. 2. Distributions of the m T2per (left) and m lb (right) variables for different top mass points of MC samples after selecting events. A reasonable fit on the distribution was evaluated by its acceptable goodness of fit value and its visible association to the distribution. The function selected only focuses on fitting a part of the distributions containing the mean of the distribution. In this study, the observable for each distribution was determined by using a repeated Gaussian function to fit the distributions until a stable fit result was found. By this way, the fit was repeated many time with setting a new fit range in every fit, which was defined as [central bin binning range, central bin + binning range] (*binning size), where central bin is the bin including the mean of the Gaussian fit of the distribution and binning range is determined by the number of bins chosen. The binning size chosen in the analysis was 5 GeV. The binning range was chosen by scanning the numbers of bins in steps of 1 bin. The optimal binning range was selected at the binning range of 9 bins, based on the smallest total uncertainty for the measurement. The variables distributions were fitted by using an Gaussian function as in Fig ISSN

347 Fig. 3. An example of the repeated Gaussian fit, the first repeated fit (left) and the second repeated fit (right). The selected observable for the measurement is the mean parameter of the fit function ( ). The mean parameter values of the fit function from these distributions are then collected to make the calibration curve corresponding to each variable. The calibration curve showing the relation of the observed value and the top mass is a first degree polynomial function (a linear function). From the calibration curve, the top quark mass value is determined by the formula: where: p n is the calibration parameter corresponding to the n th degree term of coeffcient. A calibration curve created from different mass variations of MC samples and a mass extraction are illustrated in Fig. 4. The central value of GeV of the top mass sample is used as a test mass in order to guarantee the accuracy of the measurement. Measurements are always affected by uncertainties, including statistical and systematic. Statistical ones are due to the random fluctuations coming from the finite set of observations. On the other hand, systematic ones result from uncertainties related to the nature of the measurement apparatus, assumptions made by the experimenter or the model used to make inferences based on the observed data. Fig. 4. An example of calibration curve and top mass extraction. The systematic uncertainties were the most effect in the measurements [12-14]. They were estimated by varying the quantities associated with different systematic sources in the signal and background MC samples. The systematic sources as well as their estimation in the dilepton decay channel include objects systematics as jets, electrons, muons and ISSN

348 issing transverse momentum, signal and background normalisation systematics, systematics from the method calibration and production modelling. III. RESULTS AND DISCUSSION The repeated Gaussian fit was applied in data and and yields the fit values estimated in Fig. 5. Note that the top quark mass values were extracted through the calibration curve method by the variables independently and thus the most optimal estimator of the analysis was identified. The final results of top quark mass measurement with two variables are displayed as below. For m T2 : m top = (stat.) 1.4 (syst.) GeV For m lb : m top = (stat.) 1.3 (syst.) GeV The measurements were dominated by the systematic uncertainties, which mostly came from contributed systematics related to jet objects such as jets energy scale (JES) and b-jet energy scale (b-jes). The JES and b-jes of the m T2 variable shared the same weight of 25% in the total uncertainty, while 28% and 16% were the weight values of JES and b- JES for the m lb. Fig. 5. Fits in data of the m T2 (left) and m lb (right) distributions. Between the two variables used, m lb yields the result with the smaller total uncertainty because this variable did not require a full event reconstruction. In a comparison to the ATLAS result in summer 2012, which was given m top = (stat.) 3.0 (syst.) GeV using the mean of the m T2 variable in the electron muon channel [15], both the statistical and systematic uncertainties of the analysis were reduced by approximately a factor of two. The results aslo gave the compatibility of the top mass with the ATLAS measurement in 2013, with m top = (stat.) 1.5 (syst.) GeV [16]. In addition, the study also highlights the optimisation in event selections which can be applied for further reducing the uncertainty of the top-quark mass measurements. 334 ISSN

349 IV. CONCLUSION Reducing the total uncertainty of the top quark mass measurements was the main purpose of the study. The calibration curve method was applied in the analysis with two different variables, m T2 and m lb. The study measured the top mass and optimized its total uncertainty in the decaying into di-lepton channel with the data collected by the ATLAS detector of 7 TeV total energy at the LHC. The more effective estimator was m lb. It yielded the result with the smaller total uncertainty of 1.5 GeV. Particularly, the data at 8 TeV total energy collected in 2012 has been studied and promises more improvements to reduce the uncertainty values of the top quark mass measurements. In order to improve the accuracy of the measurements further, more studies can be considered. V. ACKNOWLEDGMENTS The study was remarkably supported by Dr. Markus Cristinziani and Dr. Kaven Yau Wong in ERC TopCoup group, Physics Institute, Bonn University, Germany. It is also much obliged to the financial support of the Mekong 1000 project of Vietnamese government. REFERENCES [2] A. Quadt, Top quark physics at hadron colliders, Eur. Phys. J. C48, 2006, doi: /epjc/s , url: [3] ATLAS Production Group, Data Periods, url: [4] 2011 pp Collisions, url: [5] 2011 Data Periods for pp running, url: [1] LEP Working Group for Higgs boson searches and ALEPH and DELPHI and L3 and OPAL Collaborations, R. Barate et al., Search for the Standard Model Higgs Boson at LEP, Phys.Lett. B CERN-EP , 2003, doi: /S (03) , arxiv:hepex/ [6] ATLAS Production Group, Top group s MC11(a,b,c) Samples For 2011 Data Analyses, url: [7] P. Konar et al., Superpartner Mass Measurement Technique using 1D Orthogonal Decompositions of the Cambridge Transverse Mass Variable M_T2, Phys.Rev.Lett. 105, 2010, , doi: /PhysRevLett , arxiv: [hep-ph]. ISSN

350 [8] CMS Collaboration, Mass determination in the t t system with kinematic endpoints, CMS PAS TOP , CERN, [9] M. Burns, K. Kong, K. T. Matchev et al., Using Subsystem MT2 for Complete Mass Determinations in Decay Chains with Missing Energy at Hadron Colliders, JHEP , 2009, doi: / /2009/03/143, arxiv: [10] S. Agostinelli and others, GEANT4: A simulation toolkit, Nucl. Instru. Meth. A , [11] ATLAS Production Group, Top group s MC11(a,b,c) Samples For 2011 Data Analyses, url: [12] A. K. Sinervo, Definition and Treatment of Systematic Uncertainties in High Energy Physics and Astrophysics, 2003, url: [13] ATLAS Collaboration, Jet energy scale and its systematic uncertainty in proton-proton collisions at = 7 TeV with ATLAS 2011 data, ATLAS-CONF , CERN, [14] Top Systematic Uncertainties 2011 rel , url: [15] ATLAS Collaboration, Top quark mass measurement in the e channel using the m T2 variable at ATLAS, ATLAS-CONF , CERN, June [16] ATLAS Collaboration, Measurement of the Top Quark Mass in Dileptonic Top Quark Pair Decays with = 7 TeV ATLAS data, ATLAS-CONF , CERN, July ISSN

351 PROPAGATION DYNAMICS OF LASER PULSES IN A THREE-LEVELV-TYPE ATOMIC MEDIUM UNDER ELECTROMAGNETICALLY INDUCED TRANSPARENCY Hoang Minh Dong 1, 2, Le Van Doai 1, Pham Van Trong 3, Mai Van Luu 1, Dinh Xuan Khoa 1, Vu Ngoc Sau 1 and Nguyen Huy Bang 1, * 1 Vinh University, 182 Le Duan Street, Vinh City, Vietnam 2 The Central Region Transport College, Nghi Lien, Vinh City, Vietnam 3 Hong Duc University, Thanh Hoa City, Vietnam *: bangnh@vinhuni.edu.vn Abstract: In the framework of semiclassical theory, dynamics of a single pair of probe and coupling laser pulses co-propagating in an electromagnetically induced transparency (EIT) medium is considered by numerically solving the Maxwell-Bloch equations. Influences of the pulse durations, intensity and pulse area of the coupling laser on the probe laser pulse are investigated. We see the conditions in which the probe pulse undistorted. Keywords: pulse propagation; coherent optical effects. I. INTRODUCTION Electromagnetically induced transparency is a quantum interference effect that permits propagation of light through an opaque atomic medium with a significant reduction of absorption. The effect was proposed theoretically in 1989 [1] and experimentally verified in 1991 [2]. It manifests itself a narrow transparent window within an absorption profile and a steeper dispersion of the refractive index, so the EIT has attracted a tremendous interest over the last years concerning to potential applications, e.g., all optical switching [3], slow-light [4], quantum information [5], nonlinear optics at low light level [6], enhancement of Kerr nonlinearity [7-8]. In addition to applications from the point of view of the atomic system and its optical response, pulse propagation under EIT conditions was also widely studied, such as propagation of soliton-like pulses in a three-level system [9], pulse propagation in prepared energy medium [10], adiabatic propagation of short laser pulses [11], dynamical control of pulse propagation [12-15], formation and propagation of ultraslow soliton [16], pulse propagation in a medium of lambda-type atoms [17]. Several reviews on progress in EIT effects and related applications are available [18-20] giving a deeper insight into the topic and providing lists of original references. Recently, the effects of pulse area and intensity of the coupling light on propagation of the probe pulse under EIT conditions in a three-level -type atomic medium have been proposed in terms of the probability amplitudes [21]. It is shown that, when the pulse duration is as short as sub-ps so that pulse area of the coupling light becomes small, there ISSN

352 is no EIT effect. As the pulse duration becomes longer and accordingly the pulse area become larger, EIT is established. In this work, we study the propagation dynamics of a single pair of short laser pulses in a three-level V-type atomic medium. The laser pulses are assumed to be short and the intensity of the coupling field is much stronger than the probe field. Unlike the case of the three-level -type atomic medium in Ref. [21], here we use density matrix method to account population relaxation of excited states which influences on the pulse propagation dynamics. We seek EIT conditions for undistortion of the probe pulse propagation. II. THEORETICAL MODEL We consider a V-type three-level scheme excited by two probe and coupling laser fields as shown in Fig. 1. Here, 1 is the ground state, whereas 2 and 3 are the excited states. The dipole allowed transitions are between states 1 and 2, and 3, the transition 2 3 is dipole forbidden. We denote 21 and 31 being the decay rate of the states 2 and 3, respectively. A weak probe laser pulse (with frequency p and field amplitude E p ) drives the transition 1 3 at Rabi frequency Ω p, whereas an intense coupling laser pulse (with frequency c and field amplitude frequency Ω c. E c ) excites the transition 1 2 at Rabi Fig. 1. Scheme of a three-level V-type atomic system. In the framework of the semi-classical theory, the density matrix elements ρ ij of the three-level atomic system interacting with two laser fields under the dipole and rotating wave approximations are given by [8]: i * i i * i c 21 c 12 p 31 p 13, (1a) i i 2 2, (1b) * c 12 c ISSN

353 where, c c i i 2 2, (1c) * p 13 p 31 i i i ( ) 2 2, (1d) 21 c c p 23 i i i ( ) 2 2, (1e) 31 p p c 32 i * i i 2 2, (1f) 32 p c c 31 p , (1g) ij is the population when i = j and the coherence when i 21 j; p p 31, are the probe and coupling frequency detunings, respectively; ij is represented with the decay rates from state i to j as [8]: ij 1 2 Ek Ei El E j ij ik jl, (2) In order to study the dynamics of laser pulses propagating in the medium, the Maxwell wave equation under the slowly varying envelope approximation is given by: 1 i m E( z, t) Pm ( z, t) z c t 2 c 0 m. (3) where m denotes for p(probe) or c (coupling), P ( z, t) is macroscopic polarization of the medium which is given by m m P ( z, t) Nd ( z, t) e m n1 n1 where, N is the density of the particles. i( t k z), with n = 2 or 3. (4) Substituting Eq. (4) into Eq. (3) we obtain the coupled Bloch-Maxwell equations for the Rabi frequencies as follows: 1 p( z, t) 2 i p 31( z, t) z c t 1 c( z, t) 2 i c 21( z, t) z c t, (5a). (5b) ISSN

354 here, mnd m 2 c 0 n1 2 is the propagation constant and n = 2 or 3, and 0 is the vacuum permittivity. It is convenient to transform Eqs. (1) and (5) in the local frame where z and t z / c, with c is the speed of light in vacuum. In this frame Eqs. (1) will the same with the substitution t and z, while Eqs. (5) are rewritten as: p (, ) 2 i (, ), (6a) p 31 c (, ) 2 i (, ). (6b) c 21 In the following numerical calculations, we apply the V-type three-level scheme to 87 Rb atomic medium, where MHz. III. RESULTS AND DISCUSSIONS In order to study dynamics of the probe pulse, we solve numerically the coupled Bloch-Maxwell equations (6) by using combination of the fourth-order Runge-Kutta and finite difference methods. The propagation length is represented in units of optical depth,. The temporal width of both probe and coupling pulses is assumed identically and has p Gaussian shape, namely, f 4ln ISSN , e, (7) where, 0 is the pulse temporal width, which is assumed to be identical for both probe and coupling lasers. In Fig. 2, we choose 0 and THz, and plot the temporal shape of p c the probe laser pulse p (, ) at different values of optical depths p 0, 3, 6, and 9 ps 1 with pulse durations ps (Fig. 2a and 2e), 1 ps (Fig. 2b and 2f), 10 ps (Fig. 2c and 2g), 100 ps (Fig. 2d and 2h). The coupling parameter is given as c0 2 THz (Fig. 2a-2d), c0 20 THz (Fig. 2e-2h). It is apparently to see that for a small or moderate coupling pulse area ( c0 0 20), the probe laser pulse is almost broken up into several sub-pulses. The number of oscillations at the trailing edge of the pulse increases as the propagation distance (or optical depth) increases. The reason for these oscillations at the trailing edge of the pulse is explained as follows. When the pulse duration (ps) is shorter than the lifetime of the excited state 2 (resulted a small pulse area) the EIT effect has not established, i.e. the pulse is absorbed by the medium. In this case, we simply observe few oscillations at the trailing edge as in Figs. 2a and 2b. When the pulse duration increases to p

355 100ps, thus the pulse area also becomes larger ( c ), the EIT starts to establish, as in Fig. 2d. If one increases the peak intensity of the coupling laser pulse, the EIT effect can occurs at shorter pulse durations. Specially, when 0 100ps and the coupling laser intensity 0 20 THz c, as in Fig. 2h, the probe pulse almost undistorted, which is similar to a soliton. ISSN

356 In order to see influence of frequency detunings of both fields on propagation of the probe pulse, we compare the temporal shape of the probe laser pulse p (, ) at different optical depths p 0, 3, 6 and 1 9ps for two cases: (a) symmetric detuning 0.5 THz, and (b) asymmetric detuning 0.5 THz. The other p Fig. 2. Temporal variation of the probe laser field p (, ) at p 0, 3, 6 c parameters using in this simulation are chosen as same as in Fig. 2g. It can be cleanly seen differences between the two cases, especially in the trailing edge of the probe pulse. The oscillations in the trailing edge appear in both cases, however, in the asymmetric detuning case oscillates larger (Fig. 3b). This is due to two-photon Raman resonance condition leading to EIT which is always fulfilled for symmetric detuning. p c and 9 ps -1 when duration ps [(a) and (e)], 1 ps [(b) and (f)], 10 ps [(c) and (g)], 100 ps [(d) and (h)]. Fig. 3. Temporal variation of the probe laser pulse p (, ) at p 0, 3, 6and 1 9 ps under the symmetric detuning (a) p c 0.5 THz detuning (b) 0.5 THz. p c, and asymmetric 342 ISSN

357 IV. CONCLUSION We have developed density-matrix formalism for investigation of pulse propagation in a three-level V-type atomic medium by using numerical method. Influences of the pulse durations, intensity, and pulse area of the coupling laser pulse are investigated. We found conditions at which the medium becomes transparency for the probe pulse i.e., the solitonlike pulse is observed. Furthermore, the influence of probe and coupling detuning on pulse propagation under the conditions of EIT is also considered. It is found that a proper EIT is established for the symmetric frequency detuning of the both fields. V. ACKNOWLEDGMENT The financial supports from Vietnam s Ministry of Education and Training under the grants coded as 08/2012/HĐ-HTQTSP and B are acknowledged. REFERENCES [1] A. Imamoglu, S.E. Harris, Opt. Lett. 14 (1989) [2] K. J. Boller, A. Imamoglu, S.E. Harris, Phys. Rev. Lett. 66 (1991) [3] B.S.Ham, J. Mod. Opt. 49 (2002) [4] L. V. Hau, S. E. Harris, Z. Dutton, C.H. Bejroozi, Nature 397 (1999) 594. [5] M. Fleischhauer and M. D. Lukin, Phys. Rev. Lett. 84 (2000) [6] S. E. Harris, L.V. Hau, Phys. Rev. Lett. 82 (1999) [7] L. V. Doai, D. X. Khoa, and N. H. Bang, Phys. Scr. 90 (2015) [8] D. X. Khoa, L. V. Doai, D. H. Son, and N. H. Bang, J. Opt. Soc. Am. B31, N6 (2014) [9] F. T. Hioe and R. Grobe, Phys. Rev. Lett. 73, (1994) [10] S. E. Harris and Z. F. Luo, Phys. Rev. A52 (1995) 928. [11] V. G. Arkhipkin, I.V. Timofeev, Quant. Electro. 30 (2000) 180. [12] M. Kiffner, T. N. Dey, Phys. Rev. A79 (2009) [13] R. Yu, J. Li, P. Huang, A. Zheng, X. Yang, Phys. Lett. A373 (2009) [14] E. Paspalakis, N. J. Kylstra and P. L. Knight, Phys. Rev. A65 (2002) [15] E. Ignesti, R. Buffa, L. Fini, E. Sali, M. V. Tognetti, S. Cavalieri, Opt. Comm. 285 (2012) [16] G. Huang, K. Jiang, M. G. Payne and L. Deng, Phys. Rev. E73 (2006) [17] P. R. Bermanand C. H. Raymond Ooi, Phys. Rev. A86 (2012), [18] M. Fleischhauer, A. Imamoglu and J.P. Marangos, Rev. Mod. Phys. 77 (2005) ISSN

358 [19] A. Joshi, M. Xiao, Prog. Opt. 49 (2006) 97. [20] J.P. Marangos, J. Mod. Opt. 45 (1998) 471. [21] G. Buica, T. Nakajima, Opt. Commun. 332 (2014) 59. [22] J.H. Eberly, Quant. Semiclass. Opt. 7 (1995) 373. [23] M.D. Crisp, Phys. Rev. A1 (1970) [24] S.E. Harris, Phys. Rev. Lett. 70 (1993) ISSN

359 A STUDY OF CO-PROCESSING WASTES CONTAMINATED OILS IN CEMENT KINLS Hoang Quoc Trong 1 and Pham Gia Dien 2 1 Center for High Technology Development, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 2 Institute of Chemistry, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi *: trong@htd.vast.vn Abstract: Cement industry consumes a big amount of fossil fuels. To produce 1 ton of clinker, it needs average 130kg coal. The cement plants can use 30% of industrial wastes amount for substitution of conventional fuels. Industrial wastes disposal causes environmental pollution, badly affects the life of communities and ecosystems etc... Industrial wastes are processed primarily by incineration and landfil with high costs. The kinds of wastes comtaminated oils are also hazardous industrial wastes with the high calorific value such as waste oils, cleaning cloth contaminated with oils... They can be used to substitute coals and oils in the cement plants. The demand of co-processing wastes comtaminated oils (using wastes as alternative fuels) in cement kilns is more increasing. Wastes to energy in cement kilns brings co-benefits for both the cement and industrial wastes treatment industries. Keywords: wastes contaminated oils, co-processing, cement kinls. I. INTRODUCTION Cement kiln is a system to convert raw materials into clinker in the cement production stablishments, and may include parts of pre-burning, pre-calcification. Industrial wastes (IW) co-processing in cement kilns means a combination of the cement production process for handling hazardous waste, including hazardous wastes used as fuels, alternative materials in cement production or be incinerated by temperatures in the kilns. Many IW kinds have high calorific value as waste oils, waste tires, waste plastics etc can be used to substitute fossil fuels in cement industry. In which, the wastes comtaminated oils (WCO) are hazardous IW can used as alternative fuels (AF) [1-3]. The rotary kiln used in cement manufacturing is able to burn a wide range of materials due to the long exposure time at high temperatures, intrinsic ability of clinker to absorb and lock contaminants into the clinker and the alkalinity of the kiln environment. However, the cement kilns only use conventional fuels as coals, oils, but not use AF or coprocessing WCO [1, 3]. The IW use becomes more popular in the world due to increasing fossil fuel prices, limited fossil fuel resources and environmental concerns [3]. Vietnam has now 2 cement plants using IW as AF. The Vietnam Holcim and Thanh Cong Cement Plants have used IW and biomass from agricultural wastes (husk, cashew nut shell...) to substitute from 15 to 22% of conventional fuels [1, 2]. ISSN

360 WCO including hazardous wastes, that are also co-processed in cement kilns. They are recently considered as potential AF resources for cement industry. It has meanings as brings co-benefits to cement plants in processing wastes and using AF. II. DATA AND METHODOLOGY 2.1. Cement manufacturing process There are three main steps in the cement manufacturing [1]: Preparing raw material, that includes mixing/homogenizing, grinding and preheating (drying). Burning of raw meal to form clinker in the kiln. The components of the raw meal react at high temperature (900-1,450 0 C) in the precalciner and in the rotary kiln to give clinker. Finish grinding of clinker and mixing with additives is done after cooling. The basic chemistry of cement manufacturing process called as calcinations, begins with the decomposition of calcium carbonate (CaCO 3 ) at about 900 C to leave calcium oxide (CaO, lime) and liberate CO 2. This is followed by the clinkering process in which the calcium oxide reacts at high temperature typically 1,350 to 1,450 0 C with silica, alumina and ferrous oxide to form the silicates, aluminates and ferrites respectively which forms the clinker. This clinker is then ground together with gypsum and other additives to produce cement. Fuels are required to generate thermal energy during the process of calcination in preheater tower and during the clinkerization process in kiln, mainly to be coals Advantages in co-processing WCO in cement kilns For Cement kilns: Fig. 1. Cement manufacturing process of Vietnam cement plants. Cement kilns have a number of characteristics which make them ideal installations, in which WCO can be completely destroyed and burnt in safety, such as: 346 ISSN

361 High temperature in kiln from 1,350 to 1,450 0 C and long residence time from 5 to 10 seconds in kiln and more 3 seconds in precalciner can destroyed 99,99% IW including persistent organic pollutants [1, 2]. Alkaline environment: WCO composition can contains the compounds of chlorine or sulphur etc, then while burning process, acid gases such as hydrogen chloride and sulphur dioxide are also disposed, they are absorbed and neutralised by the freshly formed lime and other alkaline materials within the kiln [1, 3]. For wastes contaminated oils (WCO): Their calorific values are higher than other IW [1]. Their ash can provide important constituents which contribute towards building the clinker phases in the same way as coal ash does [1, 3]. They are generally cheaper than the fossil fuels because most of the AF are generated from wastes which only require some processing cost. The significant advantage of co-processing WCO is the preservation of non-renewable energy sources and the reduction of WCO disposal sites and greenhouse gas emissions by substitution of fossil fuels and IW treatment limits [1] Disadvantages in co-processing WCO in cement kilns Solid wastes such as cloths, boxes contaminated oils is usually not same on dimension and unsorted. Therefore, it needs to be sorted and pretreated before used [1, 2]. The burning systems in cement plants are installed for using conventional fuels (Fossil fuels). So, those systems must be upgraded in conformity with WCO usage Identification of Destruction and Removal Efficiency To identify effective in co-processing WCO in cement kilns, the Destruction and Removal Efficiency (DRE) was one tool used. DRE used for each Principal Organic Hazardous Constituent (POHC) from the following equation: DRE = [(W in W out )/W in ] 100 (%) where: W in : Mass feed rate of one POHC in a waste feedstream; and W out : Mass emission rate of the same POHC present in exhaust emissions prior to release to the atmostphere. Tetrachloroethyle was a kind of POHC to be choiced to calculate DRE Choice of WCO used in cement kilns The specific criteria of waste choice for cement kilns must be high calorific value, set by the cement plants according to their own needs [3]. Besides, the other concern is the impact on the quality of clinker, the environment problems etc... WCO are generally a mixture of various wastes and therefore consistency in their composition cannot be guaranteed. There is a need for ensuring the chemical contents of the AF that meets regulatory requirements for environmental protection. The following properties, that are expected to be considered as physical state of the fuel (solid, liquid, gaseous); Physical properties (scrap size, density, ISSN

362 homogeneity); Chlorine content - less than 3% and Sulfur content - less than 3%; Nontoxicity; Composition and content of ash and content of volatiles; Calorific value - over 2,000 kcal/kg; Heavy metals content - less than 2500 ppm [out of which: mercury (Hg) less than 30 ppm, and total cadmium (Cd), thallium (Tl) and mercury (Hg) less than 100 ppm]; Grinding properties; Proportioning technology; Hazardous WCO (solid, liquid, sludge) of non-volatile matter is mixed with raw materials and additives to provide through regular material load; The emissions released; The cement quality and its compatibility with the environment must not decrease; Economically viable; and availability [1] Methods of experiment The methods of experiment was performed through the following stages [1]: Stage 1: Set up the experimental plan The two most popular kinds are the cleaning cloth contaminated with oils (solid wastes) and waste oils (liquid waste), that selected for experimental works at plants. Their volumes is from 2 to 6 tons for each experimental time. A total of experiment is 6 time for each waste. Cement plants for experiment were But Son and Hoang Thach. Experimental time was in from February 2012 to Demcember Stage 2: Experimental preparation The size of cleaning cloth contaminated with oils was not uniform and their components were complex. It has to be preprocessed to become smaller. The waste oils can be used directly. Stage 3: Experimental operation The production lines is equipped with automatic system in checking and processing; measuring information; adjusting and controlling the entire chain activities to optimize the production technology (multi-fuels control systems EFCS/ProcessExpert) [1]. The experimental process followed throught three steps: Quantitative and supply WCO into the kiln >> Control humidity, hazardous components >>> Control gas emissions and safety. Potential feed points for supplying conventional and AF fuels to the kiln system are: 1/ the main burner at the rotary kiln outlet end; 2/ a feed chute at the transition chamber at the rotary kiln inlet end (for lump fuel) (Direct part is hotdics); 3/ secondary burners to the riser duct; 4/ precalciner burners to the precalciner; 5/ a feed chute to the precalciner/preheater (for lump fuel); 6/ a mid kiln valve in the case of long wet and dry kilns (for lump fuel). III. RESULTS AND DISCUSSION 3.1. Techniques of waste use 348 ISSN

363 Based on the technical characteristics of cement kilns and regulations in handling hazardous waste, the use of the WCO must meet requirements such as the burning temperature zone over 1,100 0 C, residence time over 2 seconds, can feed into kilns and assuring the quality of cement products. The suitable point for feeding and burning identifies is hotdisc. This system is nearby the rotary kiln inlet point integrated in the heater exchange tower. Its temperature zone is always from 1,200 to 1,450 0 C, enough to destruct and remove absolutely POHC. Its technical diagram is as follows: Fig. 2. Tech. diagram of cement kiln. Fig. 3. Tech. diagram of using IW in cement kiln. In the above figure, WCO was feeded into kiln via the feed chute to hotdics. The hot gas stream was blowed from the rotary kiln outlet end to burn WCO with a mixture of other materials and fuels (coals). Ash of WCO became material of clinker. Waste oils can be feeded via the main burner at the rotary kiln outlet end or via secondary burners to the riser duct Effects of usage of WCO to cement product quality Through the experimental works at the both cement plants, the results of cement quality were as follows: Table 1. Result of cement product quality. Charateristics Unit Result Vietnam Standard 6260:2009 Compressive strength (3 rd dates) N/mm ,4 Min 18 Setting time min ISSN

364 Finess % Max 10 Le chatelier/soundness mm Max 10 As shown in Tab 1, all charateristics of cement product are in comply with cement standard PCB40 (Vietnam Standard 6260:2009) Results of DRE Through the experimental works, the DRE results of Tetrachloroethyle as followed: Table 2. Summarized results of DRE in 6 experimental time. Waste name Cleaning cloth contaminated with oils Appx. DRE Value (%) 1 st 2 nd 3 rd 4 th 5 th 6 th Waste oils From Tab. 2, DRE results showed its values from to %. This thing proves that, the cement kilns is capable to completely destruct WCO. The waste processing effective of cement kilns are always higher than and less polluted than of incinerators due to its temperature and burning residence time Effects of usage of WCO to environment Through the experimental works at the both cement plants, the cement product quality as followed: Table 3. Result of environmental quality. Parameters Unit Result Vietnam s limit CO mg/m NO 2 mg/m SO 2 mg/m VOCs mg/m As shown in Table 3, the result shows that the kiln is well in compliance with the Vietnamese emission limit. The concentration of paramters was under current emission limit value as specified in the Vietnam National Technical Regulations QCVN 41: 2011/BTNMT. However, the dioxins and furans (PCDD/Fs), the most important pollution parameters, have not been measured in those experimantal times due to no lab has enough capacity to analize them in Vietnam. 350 ISSN

365 3.5. Socio - economic benefits of co-processing WCO in cement kilns Substitution ability of WCO: 1 ton of cleaning cloth contaminated with oils can substitute from 0.55 to 0.67 ton dust coal and 1 ton of waste oils can substitute from 0.55 to 0.67 ton dust coal; Can substitute from 15 to 30% of conventional fossil fuel volumes; Minimize environmental disputes in society IW pollution; Saving non-renewable resources as coal, oil; Limit emissions of greenhouse gases (GHG) and ozone depleting gases (ODS); Suitable to orientation for environmental protection and sustainable development of Vietnam and the international conventions to which Vietnam has acceded to or ratified (UNFCCC 1992, Montreal Protocol 1987, Vienna Convention 1985 etc). IV. CONCLUSION The study of co-processing WOC in cement kilns have identified the ability of processing and using wastes as AF for the cement industry, especially for Vicem. That is the techniques of feeding, pretreatment of WCO; effects of usage of WCO to environment and product quality. Through experimental works at cement plants, the substitution ability of WCO was identified as followed: 1 ton of cleaning cloth contaminated with oils can substitute from 0.55 to 0.67 ton dust coal and 1 ton of waste oils can substitute from 0.55 to 0.67 ton dust coal and able to substitute from 15 to 30% of conventional fossil fuel volumes; The research has proved that, the co-processing WCO brings the co-benefits for both the cement and industrial wastes treatment, saving alternative energy resources, reducing greenhouse gas emissions, protecting the environment. That really is a great potential, opening up opportunities in green growth and sustainable development for Vicem. REFERENCES [1] Centre for Medicine and Biochemistry Technology, The research on using waste as alternative fuels in cement industry, [2] Vietnam Holcim Cement Co., Ltd, Report on the management of hazardous waste to the Ministry of Natural Resources and Environment, [3] Madlool, N.A., Saidur, R., Hossain, M.S., Rahim, N.A., A Critical Review on Energy Use and Savings in the Cement Industries, Renewable and Sustainable Energy Reviews 15 (4), 2011, pp ISSN

366 SCREENING FOR HAEMATOCOCCUS STRAINS CONTAINING ASTAXANTHIN ISOLATED FROM SOME PLACES IN VIETNAM Luu Thi Tam 1, Le Ha Thu 2, Le Thi Thom 1, Nguyen Cam Ha 1, Hoang Thi Huong Quynh 1, Pham Van Nhat 1, Hoang Thi Lan Anh 1 and Dang Diem Hong 1* 1 Institute of Biotechnology, Vietnam Academy of Science and Technology, Ha Noi, Vietnam 2 Da Lat University, Da Lat, Lam Dong, Vietnam Tel: / ; *: ddhong60vn@yahoo.com Abstract. Astaxanthin (3,3 -dihydroxy-β, β-carotene-4, 4 -dione) is a high value carotenoid and strong biological antioxidant, which has importance applications in the nutraceutical, cosmetic, food and feed industries. Although some bacteria, fungi and microalgae are able to biosynthesize astaxanthin, the unicellular alga Haematococcus pluvialis is regarded as the best source of natural astaxanthin. Nowadays, commercial astaxanthin production from this alga is mainly focused on due to their fast growth rates and high yield production. In order to find out the potential strain for astaxanthin production in Vietnam, we isolated some local strains/species and used rapid screening methods in microalgae based on cell density and astaxanthin content. In this study, we isolated successfully nine strains of Haematococuss spp. including Haematococcus sp. HB, TN4, TN8, TĐ08, TĐ01, LC, LC05, LC06 and LĐ which were contained astaxanthin from freshwater lakes in Vietnam. Among them, three strains of HB, LC and LĐ were defined as Haematococcus pluvialis. These strains are capable of well growing and accumulating high astaxanthin content. Keywords: Astaxanthin, Haematococcus pluvialis, isolated, green microalgae, screening. I. INTRODUCTION Astaxanthin (3,3 -dihydroxy-β, β-carotene-4, 4 -dione) known as the king of antioxidants is a red ketocarotenoid with extraordinary antioxidant capability and thus has wide applications in the nutraceutical, cosmetic, food and feed industries [1] and a huge market value more than $200M per year [2-4]. As astaxanthin content up to 1 5% of cell dry weight [5, 1], the unicellular green alga Haematococcus pluvialis is widely considered the best biological source for natural astaxanthin [1]. To date, it is well known that the accumulation of astaxanthin in Haematococcus pluvialis is associated preferentially with a morphological transformation of green motile vegetative cells to deep-red nonmotile cysts. So, the two-stage culture system is adopted in the successful strategy for the commercial production of H. pluvialis: green stage for cell growth and induction stage for astaxanthin accumulation [6, 7]. However, this alga exhibits some unfavorable characteristics as follow: slow growth rate and its complex life cycle, exhibiting motile and non-motile cells, solitary and/or grouped in palmella stages [8]. Thus, it is too difficult for its cultivation on large scale. 352 ISSN

367 In Vietnam, astaxanthin is paid more attention, especially in aquaculture and pharmaceutics. Haematococcus pluvialis, the richest natural source of astaxanthin is used widely all over the world. So, in this paper, we present results on isolation and screening of Haematococcus strains in Vietnam based on their s growth rate and astaxanthin content. II. MATERIALS AND METHODS 2.1. Microalga and culture conditions The strains of Haematococcus were isolated from freshwater lakes belong to Lao Cai, Thai Nguyen, Hoa Binh, Tam Đao (Vinh Phuc) and Lam Đong provinces and then deposited at the Department of Algal Biotechnology, Institute of Biotechnology belonging to Vietnam Academy of Science and Technology, Vietnam. These strains were isolated using method of single - cell isolation by micropipette as discribed in report of Anderson, 2005 [9]. Then, they were spread on agar plate with additional antibiotics (ampicillin, streptomycin, kanamycin and gentamicin with concentration 250, 50, 100 and 50 mg/l, respectively). The agar plate is then incubated at the proper temperature and light conditions until colony formation occurs. To select isolated algal colonies, a micropipette tip is driven into the agar to pick up the selected cells, and the cells are then discharged into dilute liquid culture medium. Algal cells were grown photoautotrophically in 250 ml Erlenmeyer flasks contained the liquid C/RM medium under culture conditions as: light intensity 35 µmol m -2 s -1 provided by cool white fluorescent lamps and light: dark period as 12 h : 12 h at temperature 25 ±2ºC as described by Hong et al [10]. The algal cells in culture flask and colonies were induced to accumulate astaxanthin under the light intensity of 250 µmol m -2 s -1 at 28 o C Analytical methods Genetic identification: The genomic DNA of the strains Haematococcus sp. was extracted by following the procedure described in report of Hong et al. (2011) [11]. Amplification of 18S rrna partial gene by PCR, cloning, and sequencing: The oligonucleotide primers used in amplifying the 18S rrna gene were designed based on published sequences of Haematococcus spp. in GenBank, namely, 18F 5 - GAGAGGGAGCTTGAGAAATG-3 and 18R 5 -CGCAGATTCACATCCTGAGCTAGT -3'. The PCR mixture (20 µl) contained 2 µl of 10 x PCR buffer (Takara, Shiga, Japan), 1.5 µl deoxyribonucleotide triphosphate (2.5 mm each; Takara), 1 µl each primer (10 µm), 1 µl genomic DNA ( ng), 0.3 µl Taq DNA polymerase (5 U µl -1 ; Takara), and distilled water (up to 20 µl). The DNA amplification was carried out in a GeneAmp PCR System 9700 (Applied Biosystems [ABI], Foster City, CA, USA) with the following program: 3 min of denaturation at 95 o C, and 30 cycles of 94 o C for 30 s, 55 o C for 30 s, 72 o C for 1 min, with a final extension at 72 o C for 5 min. The expected length of PCR product was ~ 1100 bp. The PCR products were transformed into Escherichia coli DH5α ISSN

368 T1 by using TOPO cloning kit (Invitrogen, Carlsbad, CA, USA), then the DNA plasmids were sequenced using an autosequencer - ABI PRISM 3100 Avant genetic Analyzer (USA). Sequence analysis: Sequences were edited and manipulated using MEGA3 software [12]. For sequence comparison, multiple sequence alignments were performed using each of the sequences gathered from the present study with 18S rrna sequences of 4 sequences Haematococcus retrieved from DNA Data Bank of Japan (DDBJ) European Molecular Biology Laboratory (EMBL) GenBank, using the Clustal X [13]. Cell growth: Cell growth was determined by dry weight and cell density. Cells (usually from a 15 ml culture broth) were harvested and washed with sterile distilled water by centrifugation at 5,000 g for 5 min using Sorvall Legend-RT 1900W centrifuge (Kendro, Langenfeld, Germany) and then dried at 105 o C and measured after attaining constant weight. Cell density was determined by counting the individual cells with a Burker-Turk chamber (Hirschmann, Laborgera te Hilgenberg, Germany). Pigment analysis: Pigment analysis by spectrophotometrically were performed as described by Strickland & Parsons, 1977 [14]. Astaxanthin accumulation rates were calculated by equation as below: mg pigment m -3 (μg L -1 ) = C/ V where: V - volume of filtered sample in liter; C (total carotenoid) = 4.0 E ; E = extinction values, at wavelengths indicated by the subscripts. Remark: It is assumed here that the total carotenoid extracted from the cell consisted mostly of astaxanthin and absorbance of 480 nm, which was originally designed for the measurement of carotenoid, can be applied accordingly in the case of H. pluvialis microalga. Astaxanthin content (%, w/w): C = C t /m t. Where C t (mg L -1 ) was the astaxanthin concentration after induction and m t (mg L -1 ) was the dry weight after induction [15]. Statistical analysis The data were subjected to statistical analysis, utilizing a factorial design analyzed by one-way analysis of variance (ANOVA). Differences were considered significant when P value < III. RESULTS AND DICUSSION 3.1. Biological characteristics of Haematococcus strains isolated from Vietnam In this study, we identified and characterized of Haematococcus strains which were isolated from fresh water lakes in Vietnam. These strains are designated as Haematococcus sp. HB, TN4, TN8, LC, LC05, LC06, TĐ01, TĐ08, LĐ in which HB, TN, LC, TĐ, LĐ were the acronym of provinces Hoa Binh, Thai Nguyen, Lao Cai, Tam Dao and Lam 354 ISSN

369 Dong. The morphological of the strains were investigated (Fig. 1 and Fig. 2), and taxonomical position was analyzed based on the sequences of 18S rrna gene (Fig. 3). Using method of single-cell isolation by micropipette combined with antibiotic treatment, we isolated successfully nine algal strains with many features like genus Haematococcus as cells solitary, ovoid to ellipsoidal, with broadly rounded ends; protoplast separated from cell wall to which it is connected by thin branched or unbranched cytoplasmic strands, two equal flagella. According to Droop, 1954 [16], cell morphology of H. pluvialis falls into one of the two forms: vegetative cell and cyst depending on culture conditions. Among all isolated strains, three strains HB, LC and LĐ have two form: vegetative cell (cells are green with spherical or ellipsoid shapes a diameter is approximately μm. The cells are enclosed by cell walls and are motile with two flagellate (a)) and cyst (cells are red with spherical shape a diameter is approximately μm (b)). Thus, based on morphological characteristics, we identified these strains was H. pluvialis. Fig. 1. Cell morphology of some strains Haematococcus isolated from fresh water lakes in Viet Nam. (a): vegetative cell form; (b): cyst cell form. Bar, 10 µm. Fig. 2. Colonies of some strain Haematococcus sp. on C agar plate after 10 days of astaxanthin induction. We had conducted screening Haematococcus strains which were capable of synthesize astaxanthin on the C agar plate. Obtained results were shown in Fig. 2. There 355 ISSN

370 were significant differences in the colonies color among Haematococcus strains after 10 days of induction. Based on the red color level of the colonies, we got three strains Haematococcus sp. HB, LC and LĐ that can synthesize astaxanthin. Phylogenetic relationship of Haematococcus species based on the completely aligned 18S rrna gene sequences were shown in Fig. 3. We used 18S rrna gene sequences of four Haematococcus strains published in Genbank for reconstructing the phylogenetic tree. The genetic homogeneous coefficient matrix (Table 1) and phylogenetic tree have shown that Haematococcus sp. HB, LĐ and LC has the highest homogeneous coefficient with H. pluvialis having accession number AF (100, 99.9 and 99.6%, respectively), followed by H. pluvialis HPU70590 (99.9, 99.6 and 98.7 %, respectively). Table 1. Genetic similarity (percent identity) and divergence matrix of some strains/species of Haematococcus. Fig. 3. Phylogenetic relationship of Haematococcus species based on the completely aligned 18S rrna gene sequences. 356 ISSN

371 Thus, based on morphological characteristics and genetic homogeneous coefficient of 18S rrna sequences and distribution on the phylogenetic tree, the obtained results allowed us to conclude that Haematococcus sp. HB, LC and LĐ were identified as Haematococcus pluvialis Screening of microalga under batch culture: Evaluation of growth and astaxanthin content Three strains of H. pluvialis HB, LC and LĐ were selected to evaluate the growth and astaxanthin accumulation potential. Our obtained results were shown in Fig. 4 and Fig. 5. Obtained results in Fig. 4 have indicated that HB strain was the best in three examined strains expressed by maximal cell density (60 x 10 4 cells. ml -1 ); dry cell weight (0.65 g. L -1 ) after 15 days of cultivation. For LĐ strain, when cultivated in RM medium, the growth parameters, i.e., maximum cell density and cell dry weight were lower than that in HB strain, but clearly higher than in LC strain. However, there was no significant difference in the cell density between LĐ and HB strains. Fig. 4. Cell density and dry cell weight of different H. pluvialis strains in RM medium. Fig. 5. Astaxanthin content and yield of different H. pluvialis strains in RM medium. As shown in Fig. 5, all cells changed to cyst form and cell number stopped increasing in the initial phase of photoinduction. The maximum astaxanthin content (2.8% DCW) was obtained at HB strain in this experimental condition. This astaxanthin content was around 1.12 and 1.56 times higher than that obtained in the case of strains LĐ and LC, respectively. However, maximum astaxanthin yield (37 mg L -1 ) was obtained at LĐ strain. Thus, two strains HB and LĐ were selected as potential astaxanthin production sources in Vietnam. The images were illustrated successfully cultivation of the microalga H. pluvialis isolated from Vietnam in different scales as in Erlenmeyer flasks, 1.5 and 10 litre plastic bottle shown in Fig. 6. ISSN

372 A D B Fig. 6. Cultivation of microalgae H. pluvialis isolated from Vietnam at different scale: Erlenmeyer flasks 250 ml (A) and 500 ml (B), plastic bottle 1.5 litre (C) và 10 litre (D). C IV. CONCLUSION We isolated successfully nine strains of Haematococcus in fresh water lakes of Hoa Binh, Thai Nguyen, Tam Dao (Vinh Phuc), Lao Cai and Lam Dong provinces in Vietnam. Among them, three strains of HB, LC and LĐ were identified as Haematococcus pluvialis and these strains can synthesize astaxanthin. Three strains have been evaluated in Erlenmeyer flasks about growth and astaxanthin accumulation potential. HB and LĐ strains are chosen the best strains based on well growth ability and higher astaxanthin content. These strains of H. pluvialis are the potential source for astaxanthin production in Vietnam. V. ACKNOWLEDGEMENTS This study was supported by a Grant-in-Aid for Scientific Research of Ministry of Agriculture and Rural Development ( ) for project of application of Biotechnology in Vietnam aquaculture. We are grateful for use the facilities of National Key Laboratory, IBT; Department of Algal Biotechnology, Institute of Biotechnology, VAST; Da Lat University, Da Lat, Lam Dong, Vietnam. REFERENCES [1] R.T. Lorenz, G.R. Cysewski, Trends Biotechnol. Vol. 18, 2000, pp ISSN