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1 12-15 November / 2013






7 We warmly welcome you to the 6th International Bioengineering Congress (BEC2013) and 19th International Biomedical Science and Technology Symposium (BIOMED2013) in Izmir, Turkey between November, The Congress aims to gather researchers from the entire spectrum of the multi-disciplinary fields of bioengineering with a special emphasis on human welfare. The BIOMED Symposium series is held every year by a different research group in Turkey aiming to bring scientists in both basic and applied research in biomedical science and technology. These two meetings will be held jointly to gather researchers from multidisciplinary fields to encourage the exchange of new information, support interchange of opinions and establish effective communication between them. The official language of the Congress and Symposium is English. We would like to invite you and your colleagues to participate to these events. We look forward to the scientific exchange, and the profile you will bring to our meetings. Prof. Dr. Fazilet VARDAR SUKAN Chair of the BEC2013. Prof. Dr. S. İsmet DELİLOĞLU GÜRHAN Chair of the BIOMED2013 i

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9 Local Organising Committee Fazilet Vardar Sukan, Chair Özlem Yeşil Çeliktaş, Vice-Chair Hülya Yılmaz Temel, Secretariat Arzu Yıldırım Ege University Aylin Şendemir Ürkmez Ege University Aynur Gürel Ege University Bahattin Tanyolaç Ege University Duygu Ayyıldız Tamiş Ege University Erdinç İkizoğlu Ege University Eyüp Bilgi Ege University Fikret Ceyhun Ertuğrul Ege University Gaye Öngen Ege University İrem Deniz Ege University İlknur Alpak Ege University Mehmet Özgün Özen Ege University Nuri Azbar Ege University Pınar Sözer Bahadır Ege University Seçil Erden Ege University S. İsmet Deliloğlu Gürhan Ege University Seda Nemli Ege University Şeyma Çoğan Ege University International Organising Committee Ali Demirci PennState University Aylin Şendemir Ürkmez Ege University Aynur Gürel Ege University Bahattin Tanyolaç Ege University Erdinç İkizoğlu Ege University Fernando Rodríguez González Universitat Autònoma de Barcelona Francisco Miguel Portela da Gama University of Minho Gaye Öngen Ege University Mohamed Bououdina University of Bahrain Nuri Azbar Ege University Rein Ulijn The University of Strathclyde S. İsmet Deliloğlu Gürhan Ege University Suphi Öncel Ege University Yannis Missirlis University of Patras iii

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12 PROGRAM Nov. 12, 2013, Tuesday 10:00-12:00 12:00-13:00 13:00-13:30 Registration Lunch Opening Ceremony Candeğer Yılmaz, Süheyda Atalay, Fazilet Vardar Sukan, S. İsmet Deliloğlu Gürhan :30 PS1 14:30-15:00 15:00-17:30 Plenary Session Colin Webb "Bioprocessing and The Bioengineer" Coffee Break Session 1 Session 2 Bioprocess Engineering Chair Persons: Alvin Nienow, Ali Demirci Biological and Genetic Enginnering Chair Persons: Yannis Missirlis, Bahattin Tanyolaç 15:00 15:30 Alvin Nienow I-BPE1 The Use of the Microscale Parallel Bioreactor Platform. 'ambr' for Clone Selection; and its Physical Characterisation Yannis Missirlis I-BGE1 Regulation of gene expression in cells and bacteria by dynamic mechanical conditioning 15:30-16:00 Tajalli Keshavarz I-BPE2 Exploiting Microbial Gossip Bahattin Tanyolaç I-BGE2 mirna in Lifesciences 16:00-16:30 Coffee Break 16:30-17:00 Fazilet Vardar Sukan I-BPE3 Trends in Knowledge-Based Bioeconomy Erhan Pişkin I-BGE3 Detection of Pathogenic Bacteria: Probes, Platforms and Detectors 17:00-17:15 Emrah Özcan O-BPE1 Comparison of Pullulan Production Performances of Air-Lift and Bubble Column Bioreactors and Optimization of Process Parameters in Air-Lift Bioreactor Elif Özdemir Kaynak O-BGE1 Improved Methods for DNA Isolation from Ancient Skeletal Material 17:15-17:30 Ali Özhan Aytekin O-BPE2 Improved Bacterial Cellulose Production Using Newly Developed Medium Hülya Yılmaz Temel O-BGE2 Genome Based SNP Mapping in Lentil Genome 17:30 18:30 18:30 Poster Session (Posters in all topics will be on display throughout the congress) Welcome Cocktail Nov. 13, 2013, Wednesday 09:00-10:00 PS2 10:00-10:30 10:30-12:30 10:30-11:00 11:00-11:30 11:30-12:00 12:00-12:15 12:15-12:30 12:30-14:00 14:00-15:45 14:00-14:30 14:30-14:45 14:45-15:00 15:00-15:15 15:15-15:30 15:30-15:45 Stephen Mayfield I-BGE4 Yusuf Baran I-BGE5 İhsan Gürsel I-BGE6 Yıldız Uludağ O-BGE3 Mehmet İnan 0-BGE4 Héloïse Imbault I-NBA1 Mert Döşkaya I-NBA2 Emel Ö. Çetin Uyanıkgil O-NBA1 Sinem Şimşek O-NBA2 Güner Ekiz O-NBA3 Production of Biofuels and Bio-products in Micro-Algae Molecular Mechanisms of Drug Resistance in Hematological Malignancy Applications of DNA Nanoparticles in Immunotherapy Fast, Simple and Sensitive Electrochemical Detection of Bisphenol A Using Novel Electrode Arrays Development of Pichia pastoris Host Strains for Recombinant Protein Production Human Vaccine Adjuvants Development: Experience from the Veterinary Field Novel Vaccine Development Strategies; Antigen Discovery Cytotoxic Activity of Anthocyanin Rich Black Carrot Extract on Neuro 2A Cell Line Phytochemical and Bioactivity Studies on the Roots of Nerium oleander Two Quinone Derivatives from the Fungus Septofusidium berolinense and Their Biological Activities Plenary Session Rein Ulijn "Minimalistic Bioinspired Nanotechnology using Peptides" Coffee Break Session 3 Session 4 Biological and Genetic Enginnering Chair Persons: Stephen Mayfield, Meltem Conk Dalay Session 5 Novel Bioactive Agents for Human Welfare Chair Persons: Héloïse Imbault, Mert Döşkaya Lunch Nesrin Hasırcı I-BIM1 Franco Simini I-BIM2 Hossein Hosseinkhani I-BIM3 Barış Oğuz Gürses O-BIM1 Mert Duman O-BIM2 Christian Manzke I-BPE4 Neslihan Oezden O-BPE3 İrem Deniz O-BPE4 Sena Yaman O-BPE5 Burcu Akdağ O-BPE6 Bioinspired Materials Chair Persons: Rein Ulijn, Aylin Şendemir Ürkmez Bioinspired Composites in Medical Applications Technology Transfer of Biomedical Equipment in Uruguay: Lessons Learned Innovation Technology to Engineering 3D in vitro intelligent living systems for biological applications Ionic Polymer Metal Composites for Biomimetic and Biomedical Applications Fe3O4 Reinforced Polycaprolactone Nanofibrous Scaffolds Session 6 Bioprocess Engineering Chair Persons: Tajalli Keshavarz, Fazilet Vardar Sukan New developments in flexible manufacturing of mabs Automated Process Control for Animal Cell Culture Aeration-Enhanced Bioethanol Production Production of Recombinant Thermostable Glucose Isomerase by Pichia Pastoris Investigation of Thermostable Glucose Isomerase Production by Sucrose Utilizing Escherichia Coli vi

13 15:45-16:00 16:00-17:30 16:00-16:30 16:30-17:00 17:00-17:15 17:15-17:30 17:30-19:00 19:00 Glyn Stacey I-NBA3 Fernando R. Gonzales I-NBA4 Ece Yıldız O-NBA4 Mehmet Özgün Özen O-NBA5 Session 5 (continued) Novel Bioactive Agents for Human Welfare Chair Persons: Erdal Bedir, Mert Döşkaya Points to Consider in the Selections of Cell Lines for Vaccine and Biotherapeutics Manufacture Obtaining a vaccine against African swine fever is not an utopia Comparison of Extraction Methods of Echinacea purpurea and Determination of Cytotoxic Activity of Obtained Extracts Rapid Test Kit Design with Monoclonal IgM Antibodies Coffee Break Poster Session Dinner Barbaros Çetin I-NFT1 Özlem Yeşil Çeliktaş I-NFT2 Aslıhan Kazan O-NFT1 Gizem Özan O-NFT2 Session 7 Novel Fluidic Technologies Chair Persons: Barbaros Çetin, Özlem Yeşil-Çeliktaş Microfludics and Lab-on-a-chip Technology for Biomedical Applications: An overview A Perspective on Enzyme Catalyzed Reactions in a Microfluidic System A Novel Approach for Activity and Stability Enhancement of Protease Subcritical Water Extraction of Bioactive Compounds from Black Tea Processing Waste Nov. 14, 2013, Thursday 09:00-12:30 Session 8 Session 9 Bioeconomy for Future Chair Persons: Mohamed Bououdina, Erdinç İkizoğlu Bioprocess Engineering Chair Persons: Murat Elibol, Esin Hameş Kocabaş 09:00-09:30 Mohamed Bououdina I-BEF1 Green Biosynthesis of Nanoparticles: Status and Future Challenges Ali Demirci I-BPE5 Biofilm Reactors by Using Plastic Composite Supports (PCS) for Production of Value Added Products by Microbial Fermentation 09:30 09:45 09:45-10:00 Fatma Meliz Tunç O-BEF1 Fazilet Gürer O-BEF2 Microalgae Based Green Diesel Production Utilization of Olive Pomace by a Green Technology Duygu Ayyıldız Tamiş O-BPE7 Burcu Gökbudak O-BPE8 Photoheterotrophic Adaptation of Microalgae and Enhanced Lipid Production Under Different Conditions Human Growth Hormone Production by Metabolically Engineered Bacillus subtilis : Feeding Strategy Development 10:00-10:15 Kübra Karaosmanoğlu O-BEF3 Hydrolysis of Hazelnut Husk Using Commercial Enzymes for the Production of Fermentable Sugars Suphi S. Öncel O-BPE9 Hydrogen Production by Mutant Strains of Chlamydomonas reinhardtii 10:15 11:00 Coffee Break 11:00-12:30 Session 10 11:00-11:30 11:30-11:45 11:45-12:15 12:15-12:30 12:30-14:00 14:00-16:00 16:00-16:30 16:30-17:30 19:00 Russell D. Jamison I-CBR1 Cansel Öğütçü I-CBR2 Fazilet Vardar Sukan I-CBR3 Commercialization of Bio-based Research Chair Persons: Russell D. Jamison, Fazilet Vardar Sukan Universities as Engines of Wealth Creation: Rethinking the Commercialization Model R&D Strategies in Biomaterial Production Horizon 2020 Strategies in Bio-based Research and Innovation Discussion on Horizon 2020 Collaboration Possibilities Lunch Panel Precompetitive Research in Biomedical Technologies Moderator: Fazilet Vardar Sukan Panelists: Cemil Arıkan Russell D. Jamison Stephen Mayfield Murat Özgören Coffee Break Award Ceremony and Closing Remarks Dinner vii

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15 INVITED SPEAKERS Alvin NIENOW Department of Biochemical Engineering, University of Birmingham Ali DEMİRCİ Department of Agricultural and Biological Engineering, PennState University Barbaros ÇETİN Department of Mechanical Engineering, Bilkent University Colin WEBB Department of Chemical Engineering, Manchester University Erhan PİŞKİN Department of Chemical Engineering, Hacettepe University Fernando RODRIGUEZ GONZALEZ Universitat Autonoma de Barcelona, CreSA, Immunology Unit Franco SIMINI Department of Biomedical Engineering, University of Republic Hossein HOSSEINKHANI Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology İhsan GÜRSEL Department of Molecular Biology and Genetics, Bilkent University Héloïse Imbault SEPPIC 9

16 Mert DÖŞKAYA Faculty of Medicine, Ege University Mohamed BOUOUDINA Nanotechnology Centre, University of Bahrain Nesrin HASIRCI Department of Chemical Engineering, Hacettepe University Rein ULIJN Department of Chemical Engineering, The University of Strathclyde Stephen MAYFIELD San Diego Center for Algae Biotechnology, University of California Tajalli KESHAVARZ Department of Molecular and Applied Biosciences, University of Westminster Yannis MISSIRLIS Department of Mechanical and Aeronautical Engineering, Patras University Yusuf BARAN Department of Molecular Biology and Genetics, İzmir Institute of Technology 10


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19 PS1 Bioprocessing and The Bioengineer Colin Webb University of Manchester, UK Keywords: Bioprocessing, Biorefinery, Bioengineering, Industrial Biotechnology From a chemical engineering perspective, we have recently moved from a century, in which the process industries were dominated by chemistry, by catalysts and by finite, non-renewable, resources. The century that we are now growing into will by contrast, surely, be dominated by biotechnology, by cells and by sustainable, renewable, resources. It is almost 100 years since, at the University of Manchester, Chaim Weizmann laboured in his laboratory to perfect the acetone-butanol-ethanol fermentation as the first example of large scale Industrial Bioprocessing. We now look to Industrial Biotechnology to supply a wealth of different products and it is technically feasible to produce the full range of functional molecules required to meet our chemical, fuel and material needs. The biorefinery now exists as a concept (see Fig. 1), set to challenge the dominance of the petrorefinery in supplying our everyday needs from feedstocks that we can rely on being available well beyond the limited supplies of petroleum. Our challenge is to engineer these biorefinery processes to compete economically. To this end, our appreciation of the role that the biochemical engineer can play in the development of bioprocess fundamentals is vital. Fig. 1. The biorefinery concept 13

20 I-BPE1 The Use of the Microscale Parallel Bioreactor Platform, ambr TM, for Clone Selection; and its Physical Characterisation Alvin W Nienow 1,2 1 EmeritusProfessor of biochemical Engineering, University of Birmingham UK 2 Visiting Professor of Biochemical Engineering, Loughborough University, UK Keywords, CHO cell culture, transitional flow, CFD, specific power input, mass transfer. Abstract: There is a growing body of evidence that the ambr TM workstation TAP Biosystems performs well in terms of helping to select appropriate clones for scale-up studies. However, the physical characteristics of this microscale bioreactor system have not been reported and the present paper shows that these are quite different from those that exist in larger scale stirred bioreactors 1. For example, the flow regime in the ambr TM vessel is transitional rather than turbulent and the sparged air/oxygen superficial gas velocity is relatively very low whilst the specific power input is much higher (~ 400 W/m 3 ) when compared to that used at larger scales (typically ~ 20 W/m 3 ). This specific power input is necessary in order to achieve k La values sufficiently high to satisfy the oxygen demand of the cells and give control of do 2. There is also a much higher contribution to mass transfer from surface aeration. In line with other studies, the present one also shows that the culture of CHO cells in a 15 ml ambr TM bioreactor gave similar cell growth and productivity to that achieved in a 5 L stirred bioreactor whilst the results from shake flasks were significantly different. The observation that performance does not deteriorate at the high specific power input used also fits in well with other recent work 2 in bioreactors using Rushton turbines at 1 kw/m 3. Given the differences in physical characteristics between the ambr TM and larger stirred bioreactors, it is suggested that this similarity in biological performance is due to their similar control capabilities and the equivalence of the stress parameters across the scales when compared with shake flasks. Acknowledgements: Lonza Biologics (Slough, UK) for providing the model CHO-cell line; the Technology and Strategy Board (UK) for their financial support. [1] A. W. Nienow, C. D. Rielly, K. M. Brosnan, N. Bargh, K. Lee, K. Coopman and C. J. Hewitt. (2013), Biochem. Eng., J., 76; [2] A. W. Nienow. W. H. Scott, C. J. Hewitt, C. R. Thomas, G. Lewis, A. Amanullah, R. Kiss and S. J. Meier (2013) Chem. Eng. Res. Des., (Trans. I Chem Eng. Part A), DOI: /j.cherd

21 I-BPE2 Exploring Microbial Gossip Maryam Safari 1, Elham EsmailiShiraziFard 1, Tajalli Keshavarz 1 1 University of Westminster, London, UK Keywords: Applications, Microbial communication, Quorum sensing Introduction: Life, as we perceive it, depends on communication between life units. While in animals and plants, hormones are involved in communication, hormone-like molecules are in action in the microbial world. Many of the requirements of cells for their continued survival materialize through communication channels. The microbes sense each other and respond accordingly through molecular talk; their response is displayed in the form of molecules that affect them and their environment subsequently. The earliest report on microbial communication appeared in 1970 [1] describing communicationbased luminescence originated by Vibrio fischeri in a relationship with Hawaiian squid Euprymna scolopes. As V. fischeri grows on E. scolopes, the concentration increases of a signal compound, 3- oxo-hexanoyl-homoserine lactone, produced by it; this signal molecule activates luminescence. This process was named 20 years later as quorum sensing [2, 3]. The quorum sensing mechanism is based on the concentration of the sensing compounds (quorum sensing molecules, QS) that reaches a threshold when a microbial critical mass is achieved. Under these concentrations, the genetic machinery of the specific microbial community responds by altering expression. Many Gram negative bacteria use homoserine lactones as QS molecules [1], but QS molecules in Gram positive bacteria are normally small post translational peptides. Staphylococcus aureus virulence determination is regulated by QS [4]. Examples of bacterial cellular processes in response to quorum sensing include production of virulence factors e.g. in Pseudomonas aeruginosa and Burkholderia cepacia [5], de-repression of a phenotype [6], and conjugal transfer, e.g. in Agrobacterium tumefaciens [7]. Investigation of QS in bacteria has achieved momentum due to their application as therapeutics or sensing tools. Majority of QS research has been carried out in bacteria, but this process is not limited to bacteria. QS has been studied in yeasts; repression of hypha formation in Candida albicans where it is reported to be due to the QS molecule, farnesol [8]. In recent years, QS has been reported in filamentous fungi too. There is notable potential in biotechnology and pharmaceutical industries as well as agriculture and medicine for exploitation of fungal QS. In this context the role γ-heptalactone as QS molecule in Aspergillus nidulans [9] and of involvement of QS in lovastatin production in Aspergillus terreus [10] has been reported. [1] Nealson, K.H.,Platt, T. and, Hastings, J.W. (1970) J. Bacteriol. 104: [2] Fuqua, W.C.,Winans, S.C.and, Greenberg, E.P.(1994) J. Bacteriol. 176: [3] Greenberg, E.P. (2003) J. Clin. Invest. 112: [4] Bassler, B.L. (1999) Curr. Opin. Microbiol. 2: [5] Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L., Nealson, K. H., Oppenheimer, N. J. (1981) Biochemistry 20: [6] von Bodman S. B., Majerczak, D. R., Coplin, D. L. (1998) PNAS 95: [7] Piper, K. R., Vonbodman, S. B., Farrand, S. K. (1993) Nature 362: [8] Hornby, J. M., Jensen, E. C., Lisec, A. D., Tasto, J. J., Jahnke, B., Shoemaker, R., Dussault, P., Nickerson, K. W. (2001) Appl Environ Microb 67: [9] Williams, H.E., Steele, J.C.P., Clements, M.O., Keshavarz, T. (2012) Appl. Microbiol. biotechnol. 96: [10] Raina, S., De Vizio, D., Elina, D., Palonen, K., Odell, M., Brandt, A.M., Soini, J.T., Keshavarz, T. (2012) Process Biochem 47:

22 I-BPE3 Trends in Knowledge-Based Bioeconomy Fazilet Vardar Sukan 1 Department of Bioengineering, Faculty of Engineering, Ege University, Bornova- Izmir, Turkey Keywords: Bioeconomy; biobased products; competitiveness Introduction: Improved industrial sustainability through biotechnology addresses many global environmental concerns and is economically competitive in a growing number of industrial sectors. By 2030, the products of biotechnology will have an estimated one-third share, worth 300bn, of industrial production. Biotechnology is expected to help meet the most urgent global challenges by facilitating the development of a sustainable economy built on bio-based industrial processes. Bio-economy is the sustainable production and conversion of biomass, for a range of food, health, fibre and industrial products and energy, through the application of biotechnologies in biorefineries which use non-food plants and trees for industrial purposes, thus decreasing the risk of conflicts between food and non-food production in agriculture and forestry (Figure 1). Existing bio-refineries at pilot scale in Europe has been mapped in an EU study, indicating many facilities already available and others currently being established. Discussion & Conclusions: Bio-based products are not one uniform product group, but a range of products with different characteristics and uses such as; a range of intermediate products, product components, and ready-made products, e.g. bio-plastics, bio-lubricants, bio-fibres for textiles, composite materials for construction and automotive, chemical building blocks, enzymes, and amino acids. There are already several bio-based products on the market in Europe; for instance, the chemical industry currently uses 8-10% renewable raw materials producing various chemical substances. In other market segments, the market shares for bio-based products are still very low. Figure 1. The impact of biotechnological applications versus traditional technologies. Europe has a few small companies specialised in bio-based products and several major chemical companies developing applications in this area. The aim is not simply to replace fossil raw materials, but to provide bio-based products with specific innovative properties that have advantages over conventional ones. Industry, government and the public are the main stakeholders for the development of biobased products whose production is affected directly or indirectly by a large number of legal acts and public policies and consumer perceptions at EU, national or even local level. Increased public awareness will increase the demand for bio-based products and biotechnological processes are expected to provide environmental, economical and social benefits for creating a more sustainable society in future years. 16

23 I-BGE1 Regulation of gene expression in cells and bacteria by dynamic mechanical conditioning Stergios Dermenoudis, Maria Katsikogianni and Yannis Missirlis * Laboratory of Biomechanics & Biomedical Engineering Dept. of Mechanical Engineering University of Patras, Patras, Greece Keywords: Bacteria, cells, gene expression, mechanotransduction Introduction (Bold): The quest to elucidate the various aspects of cell responses while in culture contacting appropriate biomaterials becomes more realistic if the relevant mechanical stimuli are incorporated in the experimental design. The structural transformations of the cells in such conditions are manifested by their cytoskeletal protein network turnover as well as of other cellular elements. In this talk examples of both bacteria (st.epidermidis) and cells (endothelial cells) under specific mechanical stimuli will be presented showing the regulation of certain gene expressions. In particular, the ica operon, responsible for slime production, genes of bacteria, and the genes responsible for actin-tubulin production in endothelial cells will be presented and discussed. The basis for such manifestations may be underlined by selective gene expressions. 17

24 I-BGE2 mirna in Lifesciences Bahattin Tanyolac Ege University, department of Bioengineering, Bornova-Izmir Turkey Keywords (Bold): gene regulation, mirna, next generation Sequencing mirnas are a class of small nonprotein-coding genesthat regulate the expression of genes posttranscriptionallyvia sequence-specific interaction with the 3 UTR oftarget mrnas, resulting in inhibition of translation and/or mrna degradation [1,2]. A large number of studieshave established that mirnas play essential roles in biologicalprocesses, such as development [3,4], cell proliferation[5], apoptosis [6], stress response, andtumorigenesis [7,8]. Here we present mirna sequences expressed under abioticstress conditions of heat, cold, CuCl, KNO3, Zeatin, ABA, Sucrose and Control with 3 replications. In total 24 mirna library were constructed and each library was sequenced in Next Generation Sequencing systems (SOLID). It was obtained 3 Million reads. Among the mirna, 1100 mirna detected to be new mirna by running against mirbase. They also were annotated to figure out their functions. Discussion & Conclusions: mirnas are an important molecules regulating gene expression.the advent of high throughput sequencing methodologies offers unprecedented opportunities to generate comprehensive sequencing data for the identification and quantification of known and novel mirnas. Further investigation of the molecular mechanisms through which mirnas regulate gene expression will provide important parameters for target identification and thereby the prediction of biological outcomes of mirnaexpression. Fig. 1. A model for mirna function [1]. Pasquinelli AE: MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet 2012, 13: [2]. Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116: [3]. Alvarez-Garcia I, Miska EA: MicroRNA functions in animal development and human disease. Development 2005, 132: [4]. Zhao Y, Srivastava D: A developmental view of microrna function. Trends BiochemSci 2007, 32: [5]. Enerly E, Steinfeld I, Kleivi K, Leivonen SK, Aure MR, Russnes HG, Rønneberg JA, Johnsen H, Navon R, Rødland E, Mäkelä R, Naume B, Perälä M, Kallioniemi O, Kristensen VN, Yakhini Z, Børresen-Dale AL: mirnamrna integrated analysis reveals roles for mirnas in primary breast tumors. PLoS One 2011, 6:e [6]. Hwang HW, Mendell JT: MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br J Cancer 2006, 94: [7]. Leung AK, Sharp PA: MicroRNA functions in stress responses. Mol Cell 2010, 40: [8]. Ventura A, Jacks T: MicroRNAs and cancer: short RNAs go a long way. Cell 2009, 136:

25 I-BGE3 Detection of Pathogenic Bacteria: Probes, Platforms and Detectors Erhan Piskin Hacettepe University and Biyomedtek, Ankara, Turkey Keywords: Detection, Pathogenic bacteria, Probes, Platforms, Readers Introduction: According to WHO (World Health Organization) more than 2.2 million deaths occur annually due to food and water-borne illnesses mostly caused by pathogenic bacteria, and 1.9 million among them are children. Food and water are the most important resources for humans, but unfortunately for microorganisms as well. Processed meat, poultry, vegetables and milk products are among the most probable carriers of potent food-borne pathogens, including Escherichia coli, Salmonella, Staphylococcus, Campylobacter, Listeria, and many others. The infectious dose of these pathogens can be very low (~10 bacteria) and emergence of drug-resistant strains makes the problem much severe. The scenery that we see today is only modern daily life incidences. Highly pathogenic bacteria can also be used as biological warfare agents not only they are so common, but they can be easily distributed via food and water, and unfortunately living creatures - human, animals, etc. as a result of very intense mobility traffic worldwide. Monitoring food and water quality has therefore been argued as the most important priority towards national and international health and safety issues with global emphasis on rapid and early detection of pathogen contamination especially in food and water. Detection: Current pathogen detection methods include: (i) microbiological techniques (conventional culturing); (ii) nucleic-acid based (e.g., PCR) and (iii) immunological (e.g., ELISA) [1]. Biobased sensing (the so-called also biosensors and related array platforms for multiple detections) has shown tremendous promise to overcome these limitations and is being aggressively studied to provide rapid, reliable and sensitive detection platforms longer which is time-consuming and laborious. While immunological or nucleic acid-based techniques require skill personnel, extensive sample preparation, and appropriate laboratory infrastructure with expensive equipment. Table or figure (maximum 300 dpi resolution) can be VI. added. International Figure captions Bioengineering should Congress be below Human the Welfare figures; table heads should appear above the tables. Insert figures and tables after they are cited in the Presenter Author Photograph Fig. 1. Detection of pathogenic bacteria. provide rapid, reliable and sensitive detection. Biosensors are analytical devices which translate a specific bio-recognition event into a measurable signal which are typically composed of: (i) a sensor platform carrying the bio-recognition entity (the socalled bioprobe) in immobilized form and usually nanostructured to increase sensitivity, (ii) a transduction platform that generates a measurable signal in the event of target and bio-ligand interaction and (iii) an amplifier which amplifies and process the signal to give a quantitative estimate of target within the medium. Biosensors work mainly on two basic detection technology: (i) using labels (including fluorescent, luminescent, enzymes, electrochemically active labels, etc.), (ii) with label free (QCM, SPR, elipsometer, Raman and Mass spectrometers, etc.). The main objective is to develop enhanced detection technologies with high levels of reliability, sensitivity, and selectivity with short assay times. Almost all of technologies mentioned above have been applied for detection of pathogens by using bacteriophages with different extent and success. Acknowledgements: Supported by EU-FP7-IAPP- NanobacterphageSERS project. 19

26 PS2 Minimalistic Bioinspired Nanotechnology using Peptides Rein V Ulijn and co-workers. WestCHEM, Department of Pure & Applied Chemistry, The University of Strathclyde, Glasgow, G1 1XL, United Kingdom. Keywords: Biocatalysis, Bionanotechnology, Enzyme, Hydrogel, Peptide Introduction: It is our vision to develop new molecular technologies that are inspired by the materials and systems found in living systems, but that are much simpler. In order to achieve this, each component is as simple as possible but retains its function. Aromatic peptide amphiphiles: The talk will firstly focus on the use of simple self-assembling systems based on aromatic peptide amphiphiles. We will demonstrate the control of (i) nanoscale morphology through peptide design (Figure 1) [1], (ii) functionality by using co-assembly approaches [2], (iii) mechanical properties and its use as tissue mimics, (iv) responsiveness to salts [3]. Applications will be discussed in cell culture and in the interfacing of biology [3] with electronics. Biocatalytic self-assembly: In the second part of the talk we will discuss our recent results in the the use of enzymatic reactions to control and direct selfassembly processes [4]. We will show how this process can be used for dynamic templating of nanoparticle formation. We will also demonstrate our progress in the synthesis of mimics of cytoskeleton. We will demonstrate the first examples of peptide nanofibers that display dynamic instability (figure 2) [5]. Catalytic peptides: We have developed methodology to allow for the discovery of short peptides that are able to catalyse the hydrolysis of amides and esters. These oligopeptides (undecamers) are selected from M13 phage libraries by taking advantage of localized catalytic gelation. Fig. 1. Nanostructures from aromatic peptide amphiphiles. Fig. 2. Nanopeptides with dynamic instability. Acknowledgements: Our work is funded by ERC, AFOSR, EPSRC, BBSRC and Leverhulme Trust. [1] Smith et al., (2008), Advanced Materials.; Chem. Soc. Rev.; [2] Fleming et al., (2013), Chem. Commun.; [3] Roy et al., (2012) Chem. Eur. J.; [4] Williams et al., (2009), Nature Nanotechnology.; Das et al., (2010), Nature Chemistry.; [5] Debnath et al., (2013) J. Am. Chem. Soc., in review. : 20 Hughes, Soft Matter, 2010, 2011, 2012; Roy et al., Chem. Eur. J., 2012; Birchall et al., Chem. Sci., 2011; Xu et al., Nanoscale 2010; Hirst et al., Nature Chemistry, 2010; Zelzer and Ulijn,

27 I-BGE4 Production of Biofuels and Bio-Products in Micro-Algae Stephen Mayfield California Center for Algae Biotechnology and Department of Biology, University of California San Diego Keywords: Algae, Biofuels, Biotechnology, Genetic Engineering Author Photograph Introduction: Fuel, food, and all biological products are simply different forms of chemical energy, and as such are closely related. All of these products are ultimately derived from photosynthesis, the process by which sunlight energy is converted to chemical energy. Over the last 100 years we have exploited cheap fossil fuels to drive unprecedented economic and agricultural growth, but in so doing we have released sequestered CO 2 into the atmosphere, which is now beginning to impact our climate. In addition, fossil fuel reserves are finite, and we are now starting to see the initial signs of depletion of these reserves, including the rising cost of fuel and food. Together these factors have provided the impetus behind the development of new renewable energy sources that can supplant fossil fuels while greatly reducing carbon emissions into the atmosphere. Eukaryotic algae offer tremendous potential for the large scale production of biofuels and bio-products as algae require only sunlight as an energy source and sequester CO 2 during the production of biomass, and algae can be much more efficient then terrestrial plants in fixing CO 2 and producing biomass. Using designed for purpose photosynthetic organisms we have the opportunity to develop production platforms for fuel and food that have unmatched efficiencies. Discussion & Conclusions: We are developing the genetic and synthetic biology tools to enable the production of designer algae as a bio-fuels and productivities. and bio-products platform. The challenges, potential, and some early successes of synthetic biology in algae for the production of high value products will be discussed. Acknowledgements: This work was supported by a grant from the US Department of Energy and the California Energy Commission 21

28 I-BGE5 Molecular Mechanisms of Drug Resistance in Hematological Malignancy Yusuf Baran 1* 1 Izmir Institute of Technology, Izmir, Turkey Keywords: Drug Resistance, Hematological Malignancies, Leukemia, Genetics, Sphingolipids Introduction: Chemotherapy is the most widely used treatment strategy for cancer which is the highest second reason for humanbeing deaths after heart related diseases. However, cellular resistance mechanisms developed by cancer cells and tissues in the beginning or proceeding times to applied anticancer agents is a significant problem preventing succesfull therapy. Resistance developed by cancer cells to structurally and functionally different cytotoxic agents is called as multi drug resistance. The resistance can be observed in the beginning of the treatment or during the treatment known as intrinsic or acquired resistance, respectively. The resistance phenotype is associated with the tumor cells that gain a crossresistance to large range of drugs that are structurally and functionally different [1-4]. Drug resistance mechanisms have different molecular genetics and biochemical reasons depending on the applied drug and the type of cancer. Secondary genetic alterations and disorders in cancer cells may also result in drug resistance. That is why it has vital importance to study and consider all signaling pathways, in multidrug resistance of cancer. Discussion & Conclusions: Multidrug resistance raises via many unrelated mechanisms, such as overexpression of energy-dependent efflux proteins, decrease in uptake of the agents, increase or alteration in drug targets, alterations in cell cycle checkpoints, inactivation of the agents, compartmentalization of the agents, inhibition of apoptosis, increases in DNA repair mechanisms, problems related with drug metabolism and aberrant metabolism of bioactive sphingolipids. Exact elucidation of resistance mechanisms and molecular and biochemical approaches to overcome multidrug resistance have been a major goal in cancer research. In this review, the mechanisms contributing multidrug resistance in cancer chemotherapy and also the approaches for reversing the resistance will be reviewed [2-7]. [1] Baran Y, Gunduz U. (2007). Journal of Turkish Internal Medicine, 3(2): [2] Baran Y, Ural AU, Gunduz U. (2007) Hematology, 12(6): [3] Baran Y, Salas A, Senkal CE, Bielawski J, Gunduz U, Obeid LM, Ogretmen B. (2007). Journal of Biological Chemistry, 282(15): [4] Ekiz HA, Baran Y. (2010) International Journal of Cancer 127(7): [5] Salas A, Ponnusamy S, Senkal CE, Meyers MA, Selvam SP, Saddoughi SA, Apohan E, Sentelle RD, Smith C, Gault CR, Obeid LM, El-Shewy HM, Oaks J, Ramasamy S, Marcucci G, Baran Y, Mahajan S, Fernandes D, Stuart RK, Perrotti D, Ogretmen B. (2011) Blood 117(22): [6] Ekiz HA, Baran Y. (2011) Anti-Cancer Agents in Medicinal Chemistry 11: [1] 7- Baran Y, Bielawski J, Ogretmen B, Gunduz U. (2011) Journal of Cancer Research and Clinical Oncology, 137(10):

29 I-BGE6 Applications Of DNA Nanoparticles In Immunotherapy İhsan Gürsel * Department of Molecular Biology and Genetics, Bilkent University, Ankara, TURKEY Keywords: Introduction: Exosomes are naturally occurring, membranous nanovesicles of nm in diameter. They arise from the endocytic cellular pathway through three stages: (i) formation of endocytic vesicles ii) multivesicular bodies (MVBs) formation in the cytosol and (iii) fusion of the MVBs with the plasma membrane to release their nanovesicular cargoes [1,2]. As well as functioning as natural vectors of intercellular signaling within a given tissue or between different cells and tissues, exosomes could be exploited for the delivery of therapeutic cargoes [3]. To explore the therapeutic potential, we investigated whether cell line-derived exosomes could be loaded with ss/ds DNA or RNA. The nano-delivery vehicle is expected to provide better protection of the cargo, more effective delivery to immune sites while facilitating better internalization that lead to enhanced activity. Exosomes were isolated from RAW264.7 supernatants by i) differential centrifugation, ii) filtration and iii) ultracentrifugation. Purified exosomes were loaded with Cy5- labeled K3 and D35 CpG oligodeoxynucleotides via dehydration-rehydration method and labeled with lipophilic dye, SP-DiOC(18). SP-DiOC labeled, CpG ODN loaded exosomes were incubated with RAW cells at 10:1 ratio (Exosome:Cell) for 24h for cytokine production assays or incubated for 2-8 hours for confocal studies. For exosome internalization studies, cells were analyzed by FACS or by confocal microscopy. Exosome treated cell supernatants were used for cytokine ELISA assays. Different CpG DNA loaded SP-DiOC labeled exosomes were (i.e. Exo(K3-Cy5)SP-DiOC or Exo(D35-Cy5)SP-DiOC) analyzed by FACS to assess the level of cellular internalization. Data revealed that K3-loaded exosomes treated cells were 80% positive for both Cy5 and SP-DiOC signals. RAW cells were found to be 100% positive for Cy5 and SP- DiOC when D35-loaded exosomes were used. Co-localization of exosomes and ODN within the cells were confirmed by confocal microscopy (Fig.1). Images indicated that both exosomes and ODNs were internalized inside the cell compartments.supernatants from CpG ODN loaded nanovesicle treated cells were used to detect TNFα, and IL6 production by ELISA. Data demonstrated that dose dependently both K3 and D35 type CpG ODN containing exosomes were more stimulatory than their free counterparts (Fig.2). This data implicated that K and D-type CpGODNs can be effectively encapsulated into exosomes upon dehydration/rehydration strategy. Encapsulation of these particles into exosomes enhanced the immunostimulatory activity of CpG ODNs as evidenced by their increased IL-6 and TNF-α secretion by RAW264.7 cells. Fig.1 Confocal microscope images showing internalization of CpGODN loaded exosomes. Fig.2. TNFα and IL-6 production from RAW264.7 cells following stimulation with different concentrations of free and exosome loaded K-type CpG ODNs. Collectively, our findings strongly demonstrated that exosomeloaded DNA led to formation of effective nanovesicle drug delivery system. This technique promises to offer biocompatible and personalized therapeutic approach, overcoming the risk of pre-mature elimination due to recognition of synthetic particles as non-self upon in vivo administration. Acknowledgements: This work was supported by TUBITAK SBAG grant #: 111S316. [1] Trajkovic K, Hsu C, Chiantia S, Rajendran L, et al Science 319: [2] Chaput N, Théry C Semin Immunopathol. 33: [3] Lakhal S, Wood MJ Bioessays 33:

30 I-BIM1 Composites in Medical Applications Nesrin Hasirci Middle East Technical University, Chemistry Department; Graduate Departments of Biotechnology, Biomedical Engineering, Micro&Nano Technology, Polymer Science&Technology; BIOMATEN-Center of Excellence in Biomaterials and Tissue Engineering, İnönü Bulvarı, Ankara / TURKEY graph Keywords: Bioactivity, Biocompatibility, Ceramics, Composites, Nanoparticles Introduction: Composites are materials made of more than one component with different physical or chemical properties where one component is the main matrix and the others are embedded in it. The components form a system which has more useful structural or functional properties compared to any of the constituent alone. Biological tissues are examples for natural composites having polymeric structures, cells, proteins and inorganic particles in a very good harmony [1,2]. By mimicking these structures in the early 1980s hydroxyapatite was used as the bioactive and reinforcing phase in high density polyethylene to produce a bone substitute. Polymers used in the preparation of scaffolds for tissue engineering applications may not have enough mechanical strength, and addition of inorganic particles make them stronger, or addition of bioactive agents make them more compatible with the biological environment. Therefore, composites play a critical role in providing suitable active media for cell attachment and proliferation. In the bone structure calcium phosphate is present and therefore, nano or micro size calcium containing particles will mimic the biological media and increase the healing process of bone defects, such as osteochondral defects [3]. Bioactive silicate glasses or silisium containing zeolite particles were also used in scaffold preparation and showed high bioactivity and high ability to bond to both soft and hard connective tissues. Class A bioactive materials such as bioglasses are osteogenetic and osteoconductive materials while Class B materials (such as HAp) exhibit only osteo conductivity. Addition of fibers such as glass, carbon, kevlar, boron, cellulose (wood/paper fiber and straw) and high strength polymers such as aramid are used to reinforce the polymeric matrix and impedes crack propagation. Incorporating of carbon nanotubes (CNTs) into the scaffold structure can also provide electrical conductivity as well as mechanical strength. Addition of zeolites into scaffolds after modifying with antibacterial ions or agents prevent biofilm formation and enhance the healing process. Presence of cells, antibiotics, drugs, growth factors, hormones or proteins in the composite matrix increase biological efficiency of the material [4]. Discussion & Conclusions: Composites of are preferable materials in medical applications as implants as well as medical devices since they present the enhanced properties of all components. Their interactions with cells can be modified in both directions, either improving cell attachment or decreasing cell affinity. [1] U Aydemir Sezer, E Aksoy, C Durucan, N Hasirci, (2012) Polymer Composites 33, [2] S Ucar, P Yilgor, V Hasirci, N Hasirci (2013) J. Appl. Polm. Sci., 130, [3] T Endogan, K Serbetci, N Hasirci, (2009) J. Appl. Polm. Sci., 113, [4] M Fedel, T Endogan, N Hasirci, D Maniglio, A Morelli, F Chiellini, A Motta (2012) J Bioact Compat Polym, 27,

31 I-BIM2 Technology Transfer of Biomedical Equipment in Uruguay: Lessons Learned Franco SIMINI Núcleo de Ingeniería Biomédica de las Facultades de Medicina e Ingeniería Universidad de la República Montevideo URUGUAY Keywords: Biomedical Engineering, Prototypes, Spin Off Companies, Technology Transfer. Introduction: Technology is applied knowledge to produce goods and services while Technology Transfer (TT) is the concession of knowledge from inventor to industry. Intellectual Property is not transferable but its use may be negotiated. Invention is the result of creativity, while innovation implies a market for the invention and dissemination and adoption happens when the innovation makes its way well into society. The functions of University are Teaching, Research and also Development [1]. Spin off firms or other agreements with Industry are ways to output research. Specifically, nib is an academic group in the University Hospital to detect equipment needs derived from clinical practice and research. With an interdisciplinary approach original solutions are sought, designed and tested. We consider TT cases taken from nib experience: 1) A clinically successful prototype having been produced to report on fetal heart rate variability, a spin off firm was created by graduates. The University bought the first units, but no other clients were interested and the initial money was used to keep the firm operating. This was the first failure: there are no clients for a no-firm. 2) Recent graduates with clinical devices to offer are accepted in a firm incubator, received business mentoring and had no running expenses. The firm was not attractive because the partners were employed in large Engineering firms. The lesson: firms must have personal commitment. 3) A clinical prototype reached a mature product, and funding was obtained for TT. We contacted a firm with no biomedical experience, but solid engineering reputation in elevator controls. An academic staff worked in the firm to help adopt the prototype. Sales boosted. The lesson is academic staff must get involved in firms to reach market. 4) Case 3 showed a limitation: the product saturated market, and it was decided to explore the Region. But no CE marking interrupted conversations to sell the device abroad. The lesson is certification is necessary to open markets. 5) A biomedical monitor sensors firm asked nib for support to better production. After 2 years of one nib academic staff in the firm, quality of procedures were improved and received certification, thus increasing sales. The lesson is presence of Academia in Industry is beneficial. Discussion & Conclusions: We found that the detection of clinical needs in a University Hospital turned into prototypes, followed by an agreement with established firms willing to adopt inventions, may lead to innovations, provided ample cooperation of academic staff with industry is allowed. Additionally we found that University should not expect royalties until a considerable benefit is reached by the licensed company. : [1] Simini, F. - "XXI century biomedical engineering in Latin America: top quality or disappear". Physics in Medicine and Biology; 39(1):240,

32 I-BIM3 Innovation Technology to Engineer 3D In Vitro Intelligent Living Systems for Biological Applications Hossein Hosseinkhani 1 1 Graduate Institute for Biomedical Engineering, National Taiwan University of Science and Technology (Taiwan Tech), Taipei, Taiwan (*Corresponding Author): Keywords (Bold): Biomaterials, Drug Delivery, Peptide, Self-assembled Systems ter Author Photograph Introduction: The ability to generate threedimensional (3D) in vitro living systems that can mimic organ and tissue structure and function is of benefit for a variety of biological applications from basic biology to drug discovery, and will have great impact on the future of science to use human organs and tissues not only as new therapeutic approaches but also as intelligent biological tools for many applications such as early detection of newly formed diseases, next generation of diagnostic tools, and an alternative energy source called "bioenergy" devices. The goal of this project is to merge biomaterials science, nanotechnology, and biological principles to generate 3D in vitro living organs to mimic organ/tissues in order to partially reduce the amount of in vitro and in vivo animal testing, clinical trials, and to solve the above problems and to jump from Lab Bench to the Market. We are not able to mimic these cellular microenvironment interactions in common tissue culture dishes; i.e. 2D in vitro culture systems [1]. Therefore, such an engineered design would be necessary to address the above challenges. 3D in vitro models should have similar structure as Natural extracellular matrix (ECM) ECM does [2]. These similarities are in terms of biological, chemical, and physical composition. Discussion & Conclusions: The present work overviews our several experimental data on the basis of biomaterials for biological applications sepcifically in regenerative medicine therapy based on drug delivery technology. Acknowledgements: This study was funded by the National Science Council of Taiwan through research grant NSC B MY3. (Bold, 10pt) [1] H. Hosseinkhani, P.D. Hong, D.S. Yu. (2013). Chemical Reviews, 113, [2] H. Hosseinkhani, Y. Hiraoka, C.H. Li, Y.R. Chen, D.S. Yu, P.D. Hong, K.L. Ou (2013). ACS Chemical Neuroscience, 4,

33 I-NBA1 Human vaccine adjuvants development: experience from the veterinary field Héloïse Imbault*,1, Laurent Dupuis 1 1 SEPPIC SA, 22 Terrasse Bellini, Paris La Défense, Puteaux Cedex, Keywords: adjuvant, emulsion, vaccine Introduction: Vaccine adjuvants are known for nearly one century but their needs and uses are stimulated by the decrease of the immunogenic properties of the modern antigen [1]. Development of new adjuvant technologies for human vaccines is long and costly, especially in the immunotherapy field. The veterinary field offers a large amount of data and experience. Discussion & Conclusions: Adjuvants can be classified in distinct families according to their chemical nature: emulsions, polymers, mineral salts, plant extracts, particulates and microbial derivatives. All families present benefits and drawbacks when associated to antigens in vaccine formulation. Adjuvant selection in a vaccine formulation process is one of the key points ensuring the success of vaccination. The vaccine adjuvant system, through its physicochemical design is tailored to the antigen and the vaccine protocol with the aim of finding the best compromise between efficacy, safety and cost effectiveness of the vaccine. In the veterinary field, the choice of the adjuvant formula is made taking count of critical criteria such as antigen type, target animals, specificity and kinetic of the immune response targeted (table 1). Veterinary adjuvant technologies are a treasure trove of information for human vaccine development. They are used widely in different vaccine types, including live vaccines, on a large amount of animals and disease models. Even though they are often injected, they can be used orally and by mucosal route for mass vaccination. Emulsion technology such as Incomplete Freund Adjuvant, MF59 (Novartis) or AS03 (GlaxoSmithKline), is also widely used in the veterinary field. It is possible to customize this type of adjuvant technology and formulate special emulsions to better guide the immune response. In a Coccidiosis trial, a specific adjuvant with a special oil was used to target the cellular immune response for instance. The results from the veterinary assays are not yet applicable into the human field. However they may be a good predictor of what could be used in the future in human vaccine development. Table 1. Adjuvant used in vaccines according to the species. Species Typical of Adjuvants Bovine/Cattle/Dairy/Small ruminants Avian Fish Swine Emulsion (W/O, W/O/W) Emulsion (W/O, W/OW/), Mineral salts, Polymers Emulsion (W/O), Mineral salts Emulsion (O/W, W/O/W), Mineral salts, Polymers : [1] Bowerstock T.L., Martin S Advance Drug Delivery 38:

34 I-NBA2 Novel Vaccine Development Strategies; Antigen discovery Mert Döşkaya*, 1 1 Ege University Medical School, Department of Parasitology, Center for Vaccine Development, 35100, Bornova/İzmir, Turkey Keywords: Antigen discovery, Vaccine, Vaccinology The existing traditional vaccine development strategies are not likely to generate new and safe vaccines against major global diseases such as AIDS, tuberculosis, and malaria [1]. Although novel vaccination strategies (DNA, recombinant protein, and peptide vaccines) as well as adjuvants are developed, the selection method(s) of antigens to be used in new vaccines has not evolved yet. To date almost all of the vaccine studies aimed to use one of the most antigenic protein which is frequently expressed on the surface of the microorganism. Several studies have been conducted by our group against Toxoplasma gondii using such strategies which has conferred limited protection [2, 3]. In the era of modern vaccinology, the initial step of the vaccine development pipeline is the discovery of the antigen to be used in vaccination study. Reverse vaccinology is a novel approach to discover antigens [4]. Using this approach, a new three valant meningitis B vaccine has been commercialized named Bexsero [5]. Another high-throughput vaccine antigen discovery method is using proteome microarrays to profile the humoral immune response to infection [6]. This method has been applied to several pathogens as well as Toxoplasma gondii [7]. Using the discovered antigens of T. gondii, a multivalant adjuvanted recombinant protein vaccine against T. gondii is being developed by our study group to be used in animal studies. Overall, in the new vaccine development era, antigens to be used in vaccine development have to be carefully selected by means of antigen discovery methods. Acknowledgements: The research was supported by the grant given by Scientific and Technical Research Council of Turkey (TUBITAK) (Project no: SBAG 110S200). [1] Koff W.C., Burton D.R., Johnson P.R. et al Accelerating next-generation vaccine development for global disease prevention. Science. 31;340(6136): [2] Döşkaya M., Kalantari-Dehaghi, M., Walsh, C.M. et al GRA1 protein vaccine confers better immune response compared to codon-optimized GRA1 DNA vaccine, Vaccine, 25(10), [3] Polat C., Gülçe İz S., Döşkaya M. et al Türkiye de İzole Edilen İki Farklı Toxoplasma gondii Suşundan Üretilen Adjuvanlı Takizoit Eriyik Protein Aşılarının Uyardığı İmmün Yanıtların Karşılaştırılması. Mikrobiyoloji Bülteni, 47(1), [4] Sette A, Rappuoli R Reverse vaccinology: developing vaccines in the era of genomics. Immunity. 33(4): [5] Serruto D., Bottomley M.J., Ram S. et al The new multicomponent vaccine against meningococcal serogroup B, 4CMenB: immunological, functional and structural characterization of the antigens. Vaccine. 30 Suppl 2:B [6] Davies D.H., Liang X., Hernandez J.E. et al Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery. PNAS, 102, [7] Liang L., Döskaya M., Juarez S. et al Identification of potential serodiagnostic and subunit vaccine antigens by antibody profiling of toxoplasmosis cases in Turkey. Mol Cell Proteomics. 10(7): M

35 I-BPE4 New developments in flexible manufacturing of mabs Christian Manzke 1 1 Sartorius Stedim Biotech, Göttingen, Germany : 29

36 I-NBA3 Critical issues for suitability of stem cell lines for clinical use Glyn Stacey 1 1 UK Stem Cell Bank, Stevenage, UK : 30

37 I-NBA4 Obtaining a vaccine against African swine fever is not an utopia P López-Monteagudo, 1, A Lacasta 1, F Accensi 1, JM Rodríguez, 2 ML Salas 2, Fernando Rodriguez 1 * 1 Centre de Recerca en Sanitat Animal-CReSA, UAB-IRTA, Campus de la UAB, Bellaterra, Barcelona, Spain 2 Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Madrid Presenter Author Photograph Keywords (Bold): ASFV, CD8 T-cells, DNA immunization, live attenuated vaccines Introduction: African swine fever (ASF) is a hemorrhagic disease which primarily affects domestic pigs, causing highly socio-economic losses in the affected countries. There is no effective vaccine neither treatment against ASF and nowadays the control of the disease is based in an early diagnostic and the culling of the infected or exposed animals, The complexity of the pathogen and the lack of a serious investment in the vaccine development has fed, in our opinion, the false perception that the development of an ASFV-vaccine is an impossible goal to achieve. There is no vaccine available African Swine Fever External membrane Capsid Internal membrane Internal membrane Matrix Nucleoid Results: During our talk we will present the results obtained in our laboratory at CReSA ( in Barcelona, fruit of the collaborative effort made between several Research groups. Our presentation will be divided in two parts, albeit both of them are intimately related. In first instance we focused our efforts on DNA vaccines as an immunization strategy [1], with two specific purposes: identifying potential protective viral antigens and deeper understanding the mechanisms involved in protection against ASF. More recently we have extended our work aiming to obtain a live attenuated vaccine against ASF by genetically modifying the viral genome. Fig. 1. The epidemiological situation of ASF, the complex structure and live cycle of the ASF virus and the real drama: the absence of an available vaccine Discussion & Conclusions: So far, the data obtained in our laboratory have been promising enough to allow us to ensure that getting an effective and safe vaccine against ASFV it is not a utopia. Obtaining the vaccine is just a matter of time, investment, and interest Acknowledgements: To Paco Ruiz Gonzalvo, our mentor. This work has been funded by the Spanish Government [1] Argilaguet JM, Pérez-Martín E, Nofrarías M, Gallardo C, Accensi F, Lacasta A, Mora M, Ballester M, ygalindo-cardiel I, López-Soria S, Escribano JM, Reche PA, Rodríguez F. PLoS One.(2012) 7(9):e doi: /journal.pone

38 I-NTF1 Microfluidics and Lab-on-a-chip Technology for Bioengineering: Applications and Challenges Barbaros Çetin 1 1 İhsan Doğramacı Bilkent University, Ankara, Turkey Keywords (Bold): microfluidics, lab-on-a-chip The miniaturization trend of electronic components since 1970 s and the development of advanced fabrication techniques for micro and nano-scale devices since 1980 s led to the usage of devices having the dimensions of micrometers and nanometers in many fields. This trend has helped nanotechnology became a new area of science at the intersection of chemistry, physics, biology and engineering. This intersection eliminated the boundaries between these disciplines. The elimination of these boundaries has posed many challenges and new directions for organization of education and research. One of the important challenges is the rapid development of biochips, miniaturized analysis systems or lab-on-a-chip (LOC) devices which are microfluidic platforms on which one can handle chemical and biological analyses, point-of-care testing, clinical and forensic analysis, molecular diagnostics and medical diagnostics for biological, biomedical and chemical applications. LOC devices can perform the same specialized functions as their room-sized counterparts. Chips can perform clinical diagnoses, scan DNA, run electrophoretic separa-tions, act as microreactors, detect cancer cells and identify bacteria and viruses [1]. On a single chip, hundreds of different reactions and/or analyses can be performed at the same time through hundreds of parallel microchannels. Originally it was thought that the most significant benefit of these LOC devices would have been the analytical improvements associated with the scaling down of the size. Further developments revealed other significant advantages such as, (i) very small amount of sample (in the nano to picoliter range, opening the door to the possibility of analyzing components from single cells), (ii) small amount of reagents, (iii) very short reaction and analysis time compared to room-sized counterparts, (iv) reduced manufacturing costs, (v) increased automation, (vi) high throughput, (vii) high portability, (viii) opportunity for massively parallel chemical analyses either on the same or multiple samples [2]. In LOC systems, the key operations to accomplish bioengineering applications can be listed as, separation, focusing, filtering, concentration, trapping, sorting, detection, sorting, counting, washing, lysis of bio-particles, and PCR-like reactions. The combination of these operations led to the complete analysis system for a certain bioengineering application. In this study, some important microfluidics based bioengineering applications and the challenges regarding the design, fabrication and integration of these systems are discussed. The recent research projects within the Bilkent University Micro-fluidics and Lab-on-a-chip Research Group are also presented. (Bold, 10pt) [1] D. Li. (2004). Electrokinetics in Microfluidics, pp Elsevier Academic Press. [2] D. Erickson, D. Li. (2004). Analytica Chimica Acta, 507,

39 I-NTF2 A PERSPECTIVE ON ENZYME CATALYZED REACTIONS IN A MICROFLUIDIC SYSTEM Ozlem Yesil-Celiktas Department of Bioengineering, Faculty of Engineering, Ege University, Bornova- Izmir, Turkey Keywords : Sol-gel; microreactor; microfluidics; enzyme activity; immobilization Introduction : Microreactors, which can be prepared by microfabrication techniques, constitute reaction apparatus with small dimensions, large surface to volume ratios and well defined reaction times. Microfluidic reaction devices take advantage of microfluidics or nanofluidics that enables use of micro- or nanoliter volumes of reactant solutions and offer many advantages over conventional scale reactors, including significant improvements in energy efficiency, heat and mass transfer, therefore reaction speed and yield, safety, reliability and scalability. Furthermore, microchannel reaction systems are ideal for many chemical and biological processes such as enzymatic reactions. Regarding biocatalysis, many potential applications for miniaturized bioreactors require only small volumes of enzymes leading to cut downs on resources to manageable levels which are of prime importance in regards to high cost novel biocatalysts. Discussion & Conclusions: Various enzymes such as trypsin, glucose oxidase, glycosyl transferases, cucumisin, p-nitrobenzylesterase, glucose 6- phosphate dehydrogenase and cellulase have been immobilized using sol-gel technique. Entrapment by sol gel has emerged as a relatively mild route for the immobilization of enzymes [1] which are entrapped in the gel network rather than being adsorbed on to surfaces of various organic and inorganic materials. It is also worth to mention that the sol-gel technique has several advantages over the conventional entrapment methods, including homogeneous distribution of the enzyme, high purity, biocompatibility, thermal and chemical stability of the matrix, low processing temperature, easy control of the morphology and the amount of entrapped enzymes [2]. Considering cellulase [3], the monoliths were fabricated using two different silica precursors and catalytic reactions were conducted in a continuous flow microreactor with a capacity of 35 µl and the performance of the system in the hydrolysis of carboxymethyl-cellulose was evaluated by applying various flow rates. The immobilized cellulase retained 100% of its initial activity after 24 h, whereas a 1.2 fold increase in activity and shrinkage of 1.7% were recorded after 3 days of aging at 4 C. In the microfluidic system, the highest conversion rate achieved was 28.82% with a flow rate of 5 µl/min exhibiting 4.0 fold better yield than the batch-wise using same volume/enzyme ratios which can be attributed to a favourable enzyme substrate affinity. Consequently, the applied technique not only provided shorter preparation times but also sustained the stability of immobilized cellulase. Such a versatile system is a good example of hydrolytic reaction occurring in the silica gel derived microchannels, where the network provides a support for encapsulation of enzymes. This might serve as a first step in the development of silica-based matrices for the encapsulation of further enzymes required for a reaction system. [1] Y.D. Kim, C.B. Park, D.S. Clark, Biotechnol. Bioeng. 73 (2001) [2] K. Han, Z. Wu, J. Lee, I. Ahn, J.W. Park, B.R. Min, K. Lee, Biochem. Eng. J. 22 (2005) [3] O. Yesil-Celiktas, S. Cumana, I. Smirnova, Chem. Eng. J 234 (2013)

40 I-BEF1 Green Bio-Synthesis of NPs Status and Future Challenges M. Bououdina *,1, A. Ayeshamariam 2, L. Hazeem 3, S. Al-Thawadi 4 1 Nanotechnoloy Centre, University of Bahrain, Kingdom of Bahrain 2 Department of Physics, Khadir Mohideen College, Adirampattinam, India 3 Department of Biology, College of Science, University of Bahrain, Bahrain 4 Department of Biology, College of Science, University of Bahrain, Bahrain (*Corresponding Author) Keywords: Algae; Bacteria; Bio-Synthesis; Nanoparticles; Environment. Nanoparticles (NPs) have attract great attention due to their unique properties compared to their bulk counterparts due to size and shape effects, high surface area and quantum confinement. Moreover, NPs production has increased greatly in recent years and the potential of their release to the environment has therefore increased. Their consequent effect on ecosystem health became an increasing concern that needs to be tackled. It became crucial to determine the fate and behavior of manufactured nanomaterial in the environment. NPs arrive on land have the possibility to contaminate soil, transferred into the surface and ground water, and interact with biota [1]. The physiochemical properties of NPs may affect greatly their interactions with organisms, although their lethal effect has been found mainly due to size or surface area [2]. The paper will be divided into three main parts. Part I was dedicated to Bio-synthesis of Ni and NiO NPs via three methods using leaves of Azadirachtaindica (neem) trees and Guavas (Koyya): (i) boiling method; (ii) through UVirradiation; and (iii) through sun light irradiation. Structure (XRD), microstructure (SEM, TEM), optical and magnetic characterizations confirm the formation of NiO for Guavas (Koyya) and Ni for Azadirachtaindica (neem). Cancer tests were carried and will be presented as well. Then Bacteria which produce extracellular stable metallic silver NPs were successfully enriched from cultivated soil, in the presence of AgNO 3 (100 mm). The toxic Ag + ion was reduced to non-toxic Ag 0 NPs through bacterial nitrate reductase. Inhibitory rate (%) Lag Log Stationary Growth phase 100 nm ZnO vs IR% 10mg/L 100 nm ZnO vs IR% 50mg/L 100 nm ZnO vs IR% 90mg/L Fig. 1. Inhibitory rate (%) using 100 nm ZnO during different growth stages of Chlorella sp. The last part was dedicated to investigate the biotoxicity effect of some representatives of metalbased NPs, namely ZnO, TiO 2 and Fe 3O 4. Examinations following OECD toxicity test protocol using the microalgae Chlorella sp. as a model organism, were carried out. [1] Nel A, Xia T, Madler L, Li N. (2006). Science 311: [2] Klaine SJ, Alvarez PJJ, Batley GE, Fernandes TE, handy RD, Lyon DY, Mahendra S, McLaughlin MJ, Lead JR. (2008). Environmental Toxicology and Chemistry 27(90): a 34

41 I-BPE5 Biofilm reactors by using plastic composite supports (PCS) for production of value added products by microbial fermentation Ali Demirci Department of Agricultural and Biological Engineering Pennsylvania State University, University Park, PA, 16802, USA URL: Keywords: Biofilm, bioproducts, fermentation, bioreactors, PCS, solid supports. One way to enhance the productivity of fermentation for value-added product productions is to increase the biomass in the bioreactors. Biofilms, which are natural forms of cell immobilizations, can generate increased volumetric productivity rates by maintaining high biomass concentrations in bioreactors [1]. Biofilm reactors show many advantages over suspended cell reactors, especially in their higher biomass density and operation stability. Biofilm reactors can retain 5 to 10 times more biomass per unit volume when compared to suspended reactors, which contributes to the increase of production rates, reduction of washing out risk when conducting a continuous fermentation at high dilution rate, and elimination of the need for reinoculation during repeated-batch fermentation. In order to forms biofilms, solid supports are generally used. In our lab, a special solid support called Plastic Composite Support (PCS) was developed, which provides not only ideal physical support structure, but also slowreleased nutrients during fermentation. PCS is an extrusion product of polyprolylene and agricultural products customarily made for specific microorganism. Given their potential for development into a repeated batch and continuous fermentations, their exceptional stability and need for lower nutrient requirements, biofilm reactors have great potential for applications in the large scale production of many products such as alcohols, organic acids, antibiotics, and enzymes [2]. Fig. 1. Formation of biofilms. Fig. 2. (A) Diagram of the PCS biofilm reactor, and (B) biofilm of microbial cellulose formed on the PCS. Conclusions : In this paper, the basic principles of biofilm reactors and successful examples of biofilm reactors for productions of value added products by microbial fermentation will be summarized. [1] Demirci A, Pongtharangkul T, Pometto LA III (2007) Applications of biofilm reactors for production of valueadded products by microbial fermentation. In: Biofilms in the food environment. Blackwell Publishing, Ames, IA, pp [2] Ercan, D. and A. Demirci. (2013). Current and future trends for biofilm reactors for fermentation processes Critical Reviews in Biotechnology. In-press. 35

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45 O-BPE1 Comparison of pullulan production performances of air-lift and bubble column bioreactors and optimization of process parameters in air-lift bioreactor Emrah Özcan*,1, Sayıt Sargın 1, Yekta Göksungur 2 1 Ege University, Department of Bioengineering, Izmir, Turkey 2 Ege University, Department of Food Engineering, Izmir, Turkey Keywords : A. pullulans, air-lift bioreactor, bubble column bioreactor, pullulan, RSM Introduction: The bio-based polymer pullulan is an important polysaccharide which is used in the food, medicine and biomedical industries [1, 2]. The production of pullulan from synthetic medium by Aureobasidium pullulans DSM-2404 in air-lift and bubble column bioreactor was investigated. The kinetics of polysaccharide, pullulan and biomass production was determined. Performances of pullulan production in air-lift and bubble column bioreactors were compared and response surface methodology (RSM) was used to investigate the effects of three factors (initial sugar concentration, aeration rate and incubation time) on the concentration of pullulan in an air-lift bioreactor. Methods: Cells for inoculation of the culture medium were obtained from cultures grown on potato dextrose agar slants at 28 C for 48 h. Two loops of A. pullulans cells were transferred to 250ml conical flasks containing 50ml of culture medium. The flasks were incubated at 28 C for 48 h in a rotary shaker incubator at 200 rpm. These cultures were used to inoculate the air-lift and bubble column bioreactor containing production medium at a level of 5% (v/v). To precipitate polysaccharide, absolute ethanol was added (2:1) at 4 C for overnight to cellfree broth. Pullulan content of polysaccharide was obtained by reducing sugar assay using pullulanase. Results : Maximum effective yield (g pullulan produced g -1 initial sugar) obtained in air-lift bioreactor (Y P/S=0,402) was found to be higher than that obtained in bubble column bioreactor (Y P/S=0,343). Results of the statistical analysis of the effects of three factors on the concentration of pullulan in air-lift bioreactor showed that the fit of the model was good in all cases. Initial sugar concentration, aeration rate and incubation time had a strong linear effect on pullulan concentration. Moreover, pullulan concentration was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions with the exception that the interaction between initial sugar concentration and incubation time was insignificant (P > 0.05). Maximum pullulan concentration of g/l was obtained at the optimum levels of process variables (initial sugar concentration 95.2 g/l, aeration rate 1.93 vvm, and incubation time 5.36 days). Discussion & Conclusions: It is clearly seen that pneumatic bioreactors, especially air-lift bioreactors, are more effective than mechanically agitated bioreactors as compared to many earlier published reports. This study is also the first detailed work about the use of response surface methodology for the optimization of fermentation parameters in pullulan production using air-lift bioreactor. : [1] Cheng KC, Demirci A, Catchmark JM (2011) Appl Microbiol Biotechnol, 92:29 44 [2] Singh RS, Saini GK, Kennedy JF (2008) 73:

46 O-BPE2 Improved bacterial cellulose production using newly developed medium Fatih Çakar, A.Özhan Aytekin*, Fikrettin Şahin Genetic and Bioengineering Department, Engineering and Architecture Faculty, Yeditepe University, Ataşehir, İstanbul, Turkey Keywords: Bacterial cellulose, Gluconacetobacter xylinus, medium optimization, static culture Introduction: Cellulose, is the most abundant polymers in biosphere, is composed of glucose monomers with β-1,4 glucosidic bounds. Bacterial cellulose (BC) has unique properties such as high crystallinity, high degree of polymerization, high tensile strength and high purity. Because of the unique properties, BC has found a multitude of applications in various fields of industry such as foods, biomedical, textile and biotechnology areas. In this study, a new bacterial cellulose production medium was investigated by one-factor-at-a-time method using Gluconacetobacter xylinus. Methods: G. xylinus FC01 strain used in this study was previously isolated and identified by 16S rrna sequence analysis in our laboratory. HS, Yamanaka, Zhou and molasses media [1] were used for reference medium. Microbial cultures were incubated for 14 days at 30 C under static and agitated (200 rpm) conditions in glucose, ethanol, yeast extract, peptone and acetic acid at different concentrations. After the incubation period, BC was collected and cell was removed [2]. Results: Inoculant was prepared in M1A0P6 medium that was consisted of glucose (10 g/l), yeast extract (10 g/l), peptone (7 g/l), acetic acid (1.5 ml/l) and ph was adjusted to 6.0. Ethanol tolerant G.xylinus strains are proved to be productive cellulose producer. Newly prepated production medium was slightly different from inoculants in which it contains ethanol (0.5 %) and ph was 5.0 that stimulate the growth of cellulose production. Even if HS, Zhou and Yamanaka also contains different amounts of carbon and nitrogen sources, M1A05P5 medium showed the highest production rate for G.xylinus (Table 1). Molasses is prepared in two different ways and molasses that was prepared by harsh method (H2SO4 treatment) showed better yield than defined media but it was still lower than our newly developed medium. Table 1. Cellulose and biomass production in different medium under static culture Medium Cellulose (g/l) Biomass (g/l) M1A05P HS Zhou Yamanaka Molasses*(Physica l treated) Molasses* (H 2SO 4 treated) *Molasses concentration was adjusted to 10 ml molasses / 50 ml production medium. Discussion & Conclusion: The operation of static culture is easier to proceed than agitated culture. The results showed that newly developed medium was the best for bacterial cellulose production for G. Xylinus. [1] L. L. Zhou, D. P. Sun, L. Y. Hu, Y. W. Li, J. Z. Yang. (2007). Journal of Industrial Microbiology & Biotechnology, 34 (7), [2] D. R. Ruka, G. P. Simon, K. M. Dean. (2012). Carbohydrate Polymers, 89 (2),

47 O-BGE1 Improved methods for DNA isolation from ancient skeletal material Elif Ozdemir-Kaynak 1,2*, Seref Akay 1, Ozlem Yesil-Celiktas 1 1 Department of Bioengineering, Faculty of Engineering, Ege University, Bornova-Izmir, Turkey 2 Department of Bioengineering, Faculty of Engineering, Canakkale Onsekiz Mart University, Canakkale, Turkey Keywords: Ancient DNA extraction, human bone, silica column, microwave DNA extraction Introduction: Isolation of nucelic acid residuals from old skeletal material is of great significance in the identification of human remains, but is difficult because most of them require considerable amounts of starting bone, increasing the need of disposable material, reagents and time [1,2]. Although noticeable different methodologies have been developed for DNA extraction from variety of human tissues or evidentiary materials, the methods commonly used, especially those using phenol-chloroform, are unable to eliminate the effects of co-extracting inhibitors that reduces amplification efficiency and not always sufficient [1-3]. Thus, efficient protocols for isolating DNA from damaged skeletal materials are still rare and most of them needs to be improved [1]. Methods: Three different isolation protocols were applied where the starting material was prepared as either bone powder or bone slices and incubated with the lysis/decalcification buffers overnight and subsequently processed with phenol-chloroform extraction and silica column extraction. Furthermore, samples were pretreated using microwave assisted extraction and DNA was isolated by silica column. Results: Effects of pretreatment methods were investigated and best results were obtained using bone powder, decalcification buffer and silica column extraction. Also microwave assisted extraction followed by silica column proved to be highly effective yielding a good quality DNA by NanoDropND-1000 and Qubit Quantification Platform. Results indicated that improved silica column extraction significantly increased the DNA quantity (0.205µg/ml) compared to that of phenolchloroform (0.0496µg/ml). Discussion & Conclusions: As for the pretreatment approach, microwave assisted DNA extraction followed by silica column was proved to be a superior alternative with an incubation time of 1min, thereby providing a rapid method for DNA isolation from ancient bones. Acknowledgements: Access to the Molecular Genetics and Cancer Molecular Biology Laboratories of Bioengineering Department of Ege University is highly appreciated [1] Caputo M., Irisarri M., Alechine E., Corach D., 2013, Forensic Sciences International: Genetics, 7, [2] Cattaneo C., Smillie D.M., Gelsthorpe K., Piccinini A., Gelsthorpe A.R., Sokol R.J., 1995, Forensic Science International, 74, , [3] DeSalle R., Grimaldi D., 1994, Current Opinion in Genetics and Development, 4,

48 O-BPE2 Genome based SNP mapping in lentil genome Hulya Yilmaz Temel*,1, Deniz Gol 2, Abdullah Kahriman 3, Bahattin Tanyolac 1 1 Ege University Deparment Of Bioengineering, Izmir, Turkey 2 Ministry Of Agriculture, Food Control Analysis, Izmir, Turkey 3 Harran University, Department Of Field Crops, Sanlıurfa, Turkey Keywords: Lentil, mapping, RIL, SNP Introduction: The development of markers and scoring across populations by using traditional methods has been a high-cost process because it is labor-intensive and time-consuming. Genotypingby-sequencing (GBS) is a new approach for detecting SNPs in large segregating populations and combining it with scoring data. The system reduces sample handling, needs fewer PCR and purification steps and barcoding is inexpensive and it allows fast and direct study of diversity and mapping of molecular marker or trait. Lentil is an important crop throughout Western Asia and Northern Africa, the Indian subcontinent and North America. It is a diploid (2n=2x=14 chromosomes), self-pollinating annual crop, with a haploid genome size of 4,063 Mbp. Lentil is an important source of dietary protein in both the human diet and in animal feed, and it also helps in the management of soil fertility. Methods: In this study, 101 individuals developed from a Precoz X WA cross, with singleseed descent until the F7 generation are genotyped and SNPs are detected by GBS technology and linkage map is constructed by using JoinMap4 software. The maximum number of markers in one linkage group was 86, the minimum was just 4. Discussion & Conclusions: This is the first study in lentil about GBS technology. The large amount of data generated in this study can potentially be used for genetic characterization, high-density linkage map analyses, map-based cloning, comparative genomics research, the detection of genetic variation among landraces and individuals in a population, genome-wide analyses of molecular variation and genome-based QTL analysis. [1] Elshire, R. J., Glaubitz J. C., Sun Q., Poland J. A., Kawamoto K., Buckler E. S., Mitchell S. E. (2011). Plosone. 6. [2] Lu, F., Lipka A. E., Glaubitz J., Elshire R., Cherney J. H., Casler M. D., Buckler E. S., Costich D. E. (2013). Plosone, 9. Results: Five hundred and nineteen out of 838 (%61) were mapped on 12 linkage groups. The 519 markers across cm per 1.03 cm. Linkage group sizes were ranging from 92.9 cm to 11.3 cm with a mean length of 44.9 cm and 43 markers per linkage group. 42

49 O-BGE3 Fast, simple and sensitive electrochemical detection of bisphenol a using novel electrode arrays Zehra Ölçer 1,2, Canan Doğan 2, Mahmut Ş. Sağıroğlu 1, Yıldız Uludağ*,1 1 TÜBİTAK - UEKAE - BILGEM, Gebze/Kocaeli, Turkey 2 Department of Chemistry, Gebze Institute of Technology, Gebze/Kocaeli, Turkey 3 TÜBİTAK- MAM - Chemistry Institute, Gebze/Kocaeli, Turkey Keywords: Array, Bisphenol A, Electrochemical detection, Electrode Introduction: Bisphenol A (BPA) has been widely used as a major component in the production of polycarbonate and epoxy resin that are used to manufacture plastic food / beverage containers and cans. The migration of BPA from the packaging to the food or beverage and further consumption by humans is considered as risk. In drinking water the maximum allowed concentration of BPA is 500 ng / ml according to EU regulations. Current BPA detection technologies include GC-MS, HPLC and fluorescence detection that require sample pretreatment, expert user, expensive reagents / columns and long assay time. Electrochemical detection of BPA could be considered as a fast, easy to use and cheaper alternative for BPA testing. According to the literature one disadvantage of electrochemical detection is its higher detection limit. In the current study, by utilising newly designed electrode array, this statement has been put into testing. Methods: Three electrode arrays were designed and later fabricated by the evaporation of Ti / Au (20 nm / 200 nm) layer on glass slides using Fine Metal Masks. Design 1 consists of 8 Au electrodes of different sizes (d: 1.5, 2, 3, 4 mm) that each has its own Au counter and quasi-reference electrode. Design 2 consists of 8 Au electrodes of different sizes (d: 1.5, 2, 3, 4 mm) that all share the same Au counter and quasi-reference electrodes. Design 3 consists of 8 Au electrodes of 1.5 mm diameter that all share the same Au counter and quasi-reference electrodes. Results and Discussion: Initially cyclic voltammetry tests were performed to assess the electrochemical performance of the arrays. The results show that measured diffusion coefficient and surface density was higher when smaller electrodes have been utilised. In addition, individual [design 1] and shared electrode designs [design 2] behaved similarly in terms of surface density and diffusion coefficient. The arrays with shared reference / counter electrodes (design 2 and 3) has been found more advantageous with respect to design 1 electrode arrays, since design 2 and 3 did not cause a potential peak shift when scan rate was increased. Later, a BPA detection assay has been performed using the design 2 and 3 electrodes. The current response obtained from the chronoamperometry results after the addition of 500 ng/ml BPA were used to assess the current density on different sized electrodes. The current density of 2 mm diameter sized electrodes showed 34 % lower, 3 and 4 mm diameter sized electrodes showed 63 % and 74 % lower current density with respect to the 1.5 mm diameter sized electrodes. A chronoamperometric test using design 3 electrodes at 0.5 V, resulted in a detection limit of 10 ng/ml BPA with a linear range between 10 to 1000 ng/ml. The detection limit obtained using design 3 electrodes, is well below the acceptable threshold level of BPA in drinking water. Conclusion: The results indicate that design 3 electrode array provides a convenient tool for BPA testing in water samples, and hence, further studies will continue to build a novel sensing device based on this array. 43

50 O-BGE4 Development of Pichia pastoris host strain for recombinant protein production Mehmet İnan*, Mert Karaoğlan and Semiramis Yılmaz Department of Food Engineering, Akdeniz University, Antalya, Turkey, Keywords: AOX1 promoter, Pichia pastoris, regulatory proteins Introduction: The methylotrophic yeast Pichia pastoris is used as a host system to produce recombinant proteins for a variety of applications. P. pastoris has many advantages over prokaryotic systems, including the ability to perform many eukaryotic post-transcriptional modifications, and the capability of this yeast to produce heterologous proteins at high levels extracellularly. The AOX1 promoter is strongly repressed in cells grown on glucose, ethanol, acetate and glycerol, but is induced over 1000-fold when cells are shifted to a medium containing methanol as a sole carbon source. The presence of sorbitol, mannitol, trehalose and alanine together with methanol does not repress the AOX1 promoter but methanol is required for induction. Although the alcohol oxidase I (AOX1) promoter is one of the most extensively utilized promoters for recombinant expression in eukaryotic systems, methanol regulation mechanism is not understood well. In this study we have isolated and characterized regulatory proteins that regulates the AOX1 promoter activity in different carbon sources. Methods: P. pastoris MIG1 and MIG2 genes were deleted in auxotrophic mutant strain (JC304) with functional PpADE1 and PpHIS4 genes respectively. Deletion of genes was confirmed by Southern blot and PCR analysis. The MXR2 gene was cloned under GAP promoter in pgapza plazmid. The GAP promoter and MXR2 gene was subcloned into ppiczαa-egfp and ppiczαa-xylb plasmids. Alcohol oxidase, xylanase activities and relative EGFP fluorescence were measured in cells were grown in different carbon sources. Results: We have isolated two repressor proteins (PpMIG1 and PpMIG2) and one activator protein (PpMXR2) which regulates AOX activity in P. pastoris. PpMIG1 and PpMIG2 proteins are responsible for repression of the AOX1 promoter activity in glucose and glycerol containing media at the transcriptional level. Deletion of PpMIG1 and PpMIG2 genes did not affect growth of cells in glycerol and glucose minimal media. However double mutant P. pastoris strain had 50% of the AOX activity in glycerol/methanol containing media compared to that of wild type P. pastoris in only methanol containing media. P. pastoris ( mxr2) mutant strain has lost ability to grow in methanol containing media as sole carbon source. Overexpression of MXR2 gene under the constitutive GAP promoter resulted in AOX activity in glucose/methanol containing media. Discussion & Conclusions: Deletion of repressor proteins (Mig1 and Mig2) and overexpression of activator protein (Mxr2) in strain expressing Enhanced green fluorescent protein (EGFP) and Aspergillus niger Xylanase (XylB) improved recombinant protein production in different carbon sources. Acknowledgements: This research was supported by a Marie Curie reintegration grant IRG from the European Commission and the grant 111T685 from the Scientific and Research Council of Turkey (TUBITAK). 44

51 O-BIM1 Ionic polymer metal composites for biomimetic and biomedical applications İbrahim Şen 1, Okan Özdemir 2, B.Oğuz Gürses 3, Emine Akar 4, Özgün Cem Yılmaz 1, Levent Çetin 1, Yoldaş Seki 1, Mehmet Sarıkanat*,3 1 Dokuz Eylül University, Izmir, Turkey 2 Gediz University, Izmir, Turkey 3 Ege University, Izmir, Turkey 4 Onsekiz Mart University, Çanakkale, Turkey Keywords : Electroactive polymers,,ionic polymer metal composites, smart materials Introduction : Ionic polymer metal composites are electro active polymers which generate motion by applied external electric fields. IPMCs consist of a ionic film between two metallic electrodes. When an external electric field is applied between these electrodes, ions move due the applied field and gather on the one side of material. Due to the unbalanced presence of ions in polymer, bending motion occurs. IPMCs are generally made of Nafion which is fluoropolymer - copolymer coated with gold or platinum. They are considered as soft materials compared to other smart materials like piezoelectric and shape memory materials and can be actuated by low voltages like 3 5 V [1]. Because of their ionic characteristics, they show better performance in ionic environments. These characteristics make suitable choices for applications in human body. There are applications of left ventricular assist device, endoscope, in literature. In this study, mechanical and electrical characteristics of IPMCs are analyzed and their possible biomedical and biomimetic applications are discussed. Methods : Gold electrodes of 45 nm are deposited on 5x50mm Nafion films by physical vapor deposition. A computer based experimental setup is designed for performance measurement of Nafion based IPMC under DC input. Results : Experiments are done under Volts. In Figure 1, it is seen that the maximum tip displacement increases from 3V to 5V but when input voltage is 7V tip displacement reduces slightly. This is an unexpected result because the more electrical potential energy is provided to actuator. Fig. 1. Tip displacement under DC input Hence the more mechanical work is expected to be done but the results show the contrary situation that tip displacement reduces or remains approximately same when some input voltage value is exceeded (5V in our experiments). Discussion & Conclusions : IPMCs can be used as a promising actuator in biomedical applications because of their low voltage actuation properties. But their movement dynamics depend on complex physical relations, thus their input output relations are nonlinear. Acknowledgements : Financial support for this study was provided by TUBITAK-The Scientific and Technological Research Council of Turkey, Project Number: 111M643. [1] M. Shahinpoor and K. J. Kim. (2001). Smart materials and structures, vol. 10, no. 4, p. 819,. 45

52 O-BIM2 Fe3O4 Reinforced Polycaprolactone Nanofibrous Scaffolds Osman Mert Duman 1, Şeyma Çoğan 1, M. Can Minaz 2, Cenk Çelik 1, Aylin Şendemir-Ürkmez 1,2* aege University 1 Ege Faculty University of Engineering Departments Department of Bioengineering, of Bioengineering, Biotechnology 2 bege University Institute of Science Bornova, Department Turkey of Biotechnology Author2 Name*,2 (Times New Roman, 12pt, Centered, Presenting author underlined) Keywords: Fe3O4, electrospinning, PC12, tissue engineering scaffold Introduction: Magnetites (Fe 3O 4) are the most discussed of iron-oxides. The presence of magnetite in the brain is already known. Fe 3O 4 penetrates into the cells and transforms to the Fe +2 ions. These ions show increasing effect on dopamine secretion. This study aims to produce magnetite-polymer composites for tissue engineering purposes and evaluate the effects of Fe 3O 4 addition on attachment and proliferation rate of central nervous system (CNS) neurons. Methods: %12 w/w PCL was mixed with 500µg/ml Fe 3O 4 and 50µg/ml Fe 3O ml syringes were filled with prepared solutions and syringes were placed in syringe pump. 1 ml/h flow rate, 15 kv and 15 cm distance between the needle tip and plate was set up. Produced fibers were collected on glass lamellae fixed on the plate. The viability of model CNS cells, PC12 and B35 on PCL and PCL/Fe 3O 4 surfaces were tested by using MTT assay (Sigma). PC12 and B35 cells cultured on PCL and PCL/Fe 3O 4 surfaces were also immunostained with Alexa Fluor 488 Phalloidin (Invitrogen) to image their actin cytoskeleton. Results: PC12 pre-neuronal cells show less proliferation on fiber surfaces compared to polystyrene surfaces, which may indicate they start differentiating to neurons without the need to use neural growth factor (NGF). Addition of magnetite nanoparticles does not effect proliferation rate of PC12 cells at 24 hours, while it increases the proliferation rate of B35 cells. Fig. 1. SEM images of Fe3O4/PCL nanofibrous composites and SEM image and immunofluorescence staining of B35 cells on Fe3O4/PCL scaffolds. Discussion & Conclusions: On PCL surfaces, the neurons show more polygonal morphology whereas on surfaces with nanoparticles they show more bipolar morphology with longer neurites. This indicates more pronounced neuron phenotype on composite scaffolds. Acknowledgements: This work is partially funded by Ege University Scientific Research Coordination Unit (BAP). [1] Andlin-Sobocki, P. (2005). Value in Health 8(6): 210. [2] De Lau. (2006). The Lancet Neurology, 5(6): [3] Kanwar, J. R. (2012). Nanomedicine: Nanotechnology, Biology, and Medicine, 8(4):

53 O-NBA1 Cytotoxic activity of anthocyanin rich black carrot extract on neuro 2A cell line Cansu Pala 1, Canan Sevimli-Gur 2, Emel Oyku Cetin-Uyanikgil 3, Ozlem Yesil-Celiktas 1 1 Department of Bioengineering, Ege University, Izmir, Turkey 2 Department of Biology, Kocaeli University, Kocaeli, Turkey 3 Department of Biopharmaceutics and Pharmacokinetics, Ege University, Izmir, Turkey Keywords: Anthocyanin, black carrot, cancer, encapsulation, Neuro 2A Introduction: Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths around 13% of all deaths. Among them brain cancer constitutes 1.9% of all cancer cases [1]. Anthocyanins, a class of polyphenolic compounds, are responsible for the color of black carrots [2]. Interest in anthocyanins has intensified due to health promoting ability by reducing the risk of atherosclerosis, cancer, diabetes and neurodegenerative disorders [3]. Black carrots are reported to have high anthocyanin content up to 1750 mg/kg fresh weight [4] exhibiting antioxidant and anti-inflammatory activities. Methods: Process optimization was carried out by using two methodologies: ultrasonic and solvent extraction. The parameters were temperature, liquid:solid ratio and duration. The extract possesing the highest anthocyanin content was encapsulated using sol-gel technique where a biosilica and a dendrimer were formulized. The crude extract was applied to Neuro 2A brain tumor and VERO healthy cell lines and cell viability was measured. Results: Solvent extracts proved to be better than the ultrasound counterparts, the lowest cell viabilities were attained at 40 C. Shorter process times such as 2 h required more solvent but longer process times required 1.6-fold less solvent in order to reach 50% cell viability at a concentration of 3.1 µg/ml. Table 1. Cell Viability (6.25 μg/ml concentration of extracts) Extract Temp ( C) Time (hour) Liquid:Solid Cell Viability E : E : E : E : Discussion & Conclusions: Black carrot is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells. Therefore, black carrot extract alone or in combination with the anticancer drugs may offer a good strategy for the treatment of neuroblastoma. Acknowledgements: This project is supported by the Scientific and Technical Research Council of Turkey (TUBITAK) (113M196). [1] Globocan 2008 Database [2] Arscott SA, Tanumihardjo SA., (2010). Compr Rev Food Sci F 9: [3] Wrolstad, R. E., (2004). Journal of Food Science, 69(5), [4] Mazza, G., Miniati, E., (1993). (p. 362). Boca Raton, FL: CRC Press 47

54 O-NBA2 Phytochemical and Bioactivity Studies on the Roots of Nerium oleander Erdal Bedir 1, Canan Sevimli Gür 2, Feyruz Bozoğlan 3, İlknur Işikoğlu 3, Sinem Şimşek 4, * 1 Ege University, İzmir, TURKEY 2 Kocaeli University, Kocaeli, TURKEY 3 Bionorm Natural Products LTD., İzmir, TURKEY 4 Ege University, İzmir, TURKEY & Gebze Institute of Technology, Kocaeli, TURKEY Keywords: 5 -demethylaquillochin, cancer, cleomiscosin A, cytotoxicity, Nerium oleander Introduction: Nerium oleander (Oleander), a member of Apocynaceae family, is an evergreen medium sized shrub to 6 m high with 5-petalled commonly white or pink flowers, very narrowly elliptic green leaves reaching to 20 cm [1,2]. Oleander contains mainly cardiac glycosides like other members of Apocynaceae family [3]. Additionally, flavanoids, triterpenes, pregnanes, cardenolides and phytosterols were reported [2,4,5]. Biological activities of N. oleander preparations were investigated prevalently showing anticancer, antimicrobial, anti-inflammatory and neuroprotective activities [2,4]. Methods: N. oleander L. was collected from Izmir, Turkey, in June 2010 and the air-dried roots (380 g) were extracted with methanol under reflux 2 x 5 L. By using open column chromatography techniques with different absorbents and/or mobile phase concentrations four compounds were isolated. Structure elucidation of the compounds was achieved by 1D-NMR techniques ( 1 H and 13 C), mass spectrometry and optical rotation. By using MTT assay, molecules were tested for in vitro cytotoxic activity against 7 mammalian cancer cell lines, MDA-MB-231, PC-3, SK-BR-3, HT-29, MCF-7, Neuro-2A, HeLa and normal cell line Vero. Results: Structures of the purified compounds were established as Odoroside H (1), β-sitosterol glucoside (2), Cleomiscosin A (3) and 5 -demethyl aquillochin (4) (Fig 1). Cytotoxic activity results were provided in Table 1. Table 1. IC50 values of 1, 3 and 4 IC 50 (µg/ml)±sem MDA-MB (±0.03) 41.2 (±0.36) 20.6 (±0.73) PC (±0.03) 30.9 (±0.82) 61.4 (±0.34) SK-BR-3 < (±0.64) 9.8 (±0.24) HT (±0.04) 22.8 (±0.43) 21.4 (±0.37) MCF (±0.05) 28.8 (±0.23) 21.6 (±0.52) Neuro 2A < (±0.45) 17.2 (±0.33) HeLa < (±0.11) 2.9 (±0.13) Vero > (±0.56) 16.2 (±0.41) Discussion & Conclusions: Compound 1 exhibited potent and selective cytotoxicity toward all cancer cell lines. Interestingly, compounds 3 and 4 inhibited proliferation of HeLa cell line specifically. Coumarinolignans (3 and 4) were encountered for the first time in Nerium genus. Fig 1. Compounds isolated from Nerium oleander [1] Davis P.H., (1978). Edingburgh University Press, 6 th ed.: [2] Zibbu G., Batra A., (2010). J. Chem. Pharm. Res., 2(6): [3]. Kohls S., Scholz-Böttcher B. M., Teske J., Zarkc P., Rullkötter J. (2012). Phytochemistry 75: [4] Sharma P., Choudhary A. S., Parashar P., Sharma M. C., Dobhal M. P., 2010, Chemistry&Biodiversity, 7: [5] Siddiqui B.S., Khatoon N., Begum S., Farooq A.D., Qamar K., Bhatti H. A., Ali S. K., (2012) Phytochemistry, 77:

55 O-NBA3 Two quinone derivatives from the fungus Septofusidium berolinense and their biological activities Güner Ekiz 1, E. Esin Hameş Kocabaş*,1, Ayşe Nalbantsoy, Erdal Bedir * 1 Ege University, Faculty of Engineering, Department of Bioengineering, Bornova, İzmir, Turkey Keywords: bioactivity, filamentous fungi, secondary metabolites, Septofusidium berolinense Introduction: Natural products have an important source of drug discovery studies and approximately half of the commercial drugs today derived from natural sources [1]. Bioactive natural products can be produced by almost all types of living organisms. Among them fungi are remarkable organisms with unique biochemical pathways that produce a wide range of natural products such as penicillin, cyclosporin, statins, aflatoxins, trichothecenes and ergot alkaloids. Although fungi have proven to be rich source of diverse bioactive secondary metabolites, yet a vast majority of fungi remain to be explored [2]. Due to the increasing demand for new bioactive natural products, the use of fungal diversity in search for new molecules is extremely important. Methods: The fungal strain was isolated from the soil sample collected from Ayvacık, Çanakkale, Turkey, in The isolate was identified as Septofusidium berolinense based on morphological and physiological characteristics. S. berolinense was cultured by solid substrate fermentation which was carried out in 1000 ml Erlenmeyer flasks, each containing 40 g zeolite, 4 g soybean flour, 4 g mannitol and 11 ml distilled water and incubated at 25 C for 14 days. After incubation, the whole culture was extracted with EtOAc and filtered. The filtrate was evaporated under reduced pressure to give crude EtOAc extract. Bioactivity-guided fractionation studied was performed on EtOAc extract by using chromatographic methods (thin layer chromatography, open column chromatography). The structures of the compounds were elucidated on the basis of spectroscopic methods (NMR and MS). The antimicrobial, cytotoxic and antioxidant activities of the compounds were evaluated. Results: The studies performed on the crude EtOAc extract (2.215 g) by using chromatographic methods resulted in the isolation of a new (1) and a known (2) compound. The compound 1 and 2 showed broad-spectrum antibacterial activity against clinical strains of meticillin-resistant Staphylococcus aureus (MRSA), Escherichia coli O157:H7, Pseudomonas aeruginosa and Enterococcus faecium. Free radical scavenging activity of the isolated compounds was determined by the DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical-scavenging assay. Compound 1 showed potent antioxidant activity compared to standard ascorbic acid whereas compound 2 expressed lower activity. Cytotoxicity of the isolated compounds was evaluated versus 6 human cancer cell lines, CaCo-2, HeLa, MCF7, A549, U87MG and PC3 together with 1 normal cell line, VERO by using the MTT assay. Compound 1 and 2 exhibited significant cytotoxic activity against the entire cell lines comparable to that of reference standard doxorubicin. Discussion & Conclusion: Septofusidium berolinense was studied for the first time from chemistry perspective, whic resulted in isolation of a new compound. The purified molecules showed significant bioactivities. : [1] Tringali C., (2001). Bioactive Compounds from Natural Sources, Taylor&Francis Inc., New York, 693p. [2] Berdy J., (2005). J. Antibiot. 58(1):

56 O-BPE3 Automated process control for animal cell culture Neslihan Oezden *1, Christian Klinger *,2, Detlef Eisenkraetzer *,2 1 Anhalt University of Applied Sciences, Köthen Germany 2 Roche Diagnostics GmbH, Penzberg Germany *neslihan.oezden / christian.klinger / detlef.eisenkraetzer ) Keywords: CHO Cell culture, dynamic feeding, fermentation, oxygen uptake rate, pco 2-level Introduction: In the past decade, the production of monoclonal antibodies by genetically modified CHO (Chinese Hamster Ovary) Cells became a significant role in medical treatment, especially in the field of therapy of cancers. Due to the high complexity and low concentrations, fermentations using animal cells show often a lower degree of automation compared to microbial processes. The purpose of this study was to investigate a fully automated bioprocess control as well as dynamic feeding for producing therapeutic antibodies by mammalian cells. Methods: We performed this study in 2 L scale down bioreactors using the SCADA-system TruBio (FINESSE LLC) for process data monitoring; process control and algorithms for adaptation of feed rates. Compared to our standard processes in the highly automated processes the following improvements have been made based on the available on line and at line process values such as: start/stop and set point of feeding rates; shifts of process parameters (e.g. temperature); control of pco 2 level [1]; automated adjustment of inoculation parameters Results: Most of the automation efforts resulted in a reduced need of manual work. Additionally our dynamically adapted feed rate, which was calculated from the oxygen uptake rate (OUR) of the cells, resulted in a performance improvement of the culture. In contrast to other feeding strategies (continuous feed rates and bolus feedings), 20 % higher product titer were obtained by altering feed rates on the basis of oxygen uptake rate. Discussion & Conclusions: In conclusion this work showed a successfully automated process based on established portfolio projects both to reproduce performance with less manual efforts and to increase process performance. Acknowledgements: Britta Schmidt, Roche Diagnostics GmbH; Birgitt Zauner, Roche Diagnostics GmbH; Christoph Knauer, Roche Diagnostics GmbH [1] Eisenkraetzer, D., Gaetgens, J., Jockwer, A., Klinger, C., Noll, T., Suess, B. (2009): Method for Producing Recombinant Proteins with a Constant Content of pco 2 in the Medium; International Publication Number WO 2009/ A1 50

57 O-BPE4 Aeration-enhanced bioethanol production Irem Deniz*,1,2, Esra Imamoglu 1, Fazilet Vardar Sukan 1 1 Ege University, Bioengineering Department, Bornova-Izmir, Turkey 2 Celal Bayar University, Bioengineering Department, Muradiye-Manisa, Turkey * Keywords: Agroindustrial waste, bioethanol, Escherichia coli KO11, microaeration, oxygen transfer rate Introduction: As a result of the diminishing of fossil fuel reserves and the increasing environmental and political pressures; researchers are in search of alternative energy sources like bioethanol which need to be renewable, sustainable, cost-effective and safe. In this study, micro-aerated fermentation conditions were investigated in 5 L stirred tank reactor for ethanol production using quince pomace by Escherichia coli KO11. Methods: Five different aeration rates (0, 0.020, 0.035, and vvm) were performed for varying durations (for the first 0, 6, 8 and 10 hours of the fermentation) and compared in terms of ethanol yield, microbial viability, sugar consumption andaerated power consumption. Results: Air feeding of vvm for the first 8 hours corresponding to the oxygen transfer rate (OTR) of 5 mmol/l/h resulted in the highest ethanol concentration which was 26.25% higher than unaerated conditions (Table1). As the aeration rate and duration were increased, the biomass concentration was increased; however the ethanol yield was decreased.the changes in aeration rate also affected the hydrodynamic parameter of the batches. Volumetric aerated power consumption (P g/v L) which is a function of oxygen mass transfer coefficient (k La), was decreased with the aeration rate (Table 2). Discussion & Conclusions: In this study, it was shown that ethanol production was enhanced at the aeration rate of vvm (p<0.05). Okuda et al [1] used microaerated conditions and maximized ethanol production at OTR=4 mmol/l/h via promoting sugar utilization in of waste house wood hydrolysate compared to unaerated Table 1. Comparison of process parameters. Aeration rate (vvm) Aeration duration (hour) Ethanol g/l YP/S (g/g) YX/S (g/g) % Sugar uptake ratio Table 2. Comparison of hydrodynamic parameters. Aeration rate (vvm) Pg/VL (x10-3 W/m 3 ) kla (s -1 ) OTR (mmol/l/h) conditions. From the data summarized, it was concluded that the microaeration at OTR=5 mmol/l/h increased ethanol yield (Y P/S) by promoting the sugar uptake ratio and biomass yield (Y X/S) compared with that at OTR=0 mmol/l/h. However, the aeration at OTR=16 mmol/l/h resulted in the lowest ethanol concentration due to ethanol consumption by the cells, even though the sugar uptake ratio increased compared with that at OTR =0 mmol/l/h. Acknowledgements: This study was a part of Cost action FP0602 and the authors wish to thank The Scientific and Technical Research Council of Turkey (TUBITAK) for the financial support. Reference [1]Okuda N. et al. (2007). J Biosci&Bioeng. 103 (4):

58 Enzyme Activity (U/L) O-BPE5 Production of recombinant thermostable glucose ısomerase by Pichia pastoris Sena Yaman 1, Pınar Çalık*,2 1 Middle East Technical University, Department of Biotechnology, Ankara, Turkey 2 Middle East Technical University, Department of Chemical Engineering, Industrial Biotechnology and Metabolic Engineering Laboratory, Ankara, Turkey Keywords: Intracellular, glucose isomerase, Pichia pastoris, recombinant enzyme, thermostable. Introduction: Glucose isomerase (EC ) is an enzyme which catalyzes reversible isomerization of D- glucose to D-fructose and D-xylose to D-xylulose. As a result of being one of the key enzymes for the production of high fructose corn syrup (HFCS) and bioconversion of hemicelluloses to ethanol, GI has a commercial importance for the industry [1]. Using high temperatures for HFCS production process leads to high isomerization yields and low microbial contamination risk which makes the thermostability of GI crucial [2]. Pichia pastoris is a methylotrophic yeast which is widely used for the production of recombinant proteins [3]. Methods: In this study, recombinant thermostable GI was produced by P. pastoris under the control of strong and tightly regulated alcohol oxidase 1 (AOX1) promoter. For this purpose, ppicz-a expression vector containing xlya gene which was obtained from extremely thermophilic microorganism Thermus thermophilus, integrated into genome of P. pastoris X33 strain with LiCl method [4]. Intracellular GI production capacity of P.pastoris xlya-mut + was investigated in a defined medium in laboratory scale air-filtered shake bioreactors. AOX1 promoter was induced by methanol (1% v/v) in every 24 hours. After production phase, cells were harvested, disrupted and enzyme activity assay was performed consecutively with cysteinecarbazole method [5]. In order to determine the effect of a co-carbon source on intracellular GI production, experiments were carried out with use of 10 g L -1, 30 g L -1 and 50 g L -1 sorbitol in addition to methanol. Results: Maximum GI activity without addition of sorbitol was obtained as 545 U L -1. The highest GI activity was obtained as U L -1 with sorbitol at concentration of 30 g L -1 at t=24h of the process Time (h) 10 gl-1 sorbitol 30 gl-1 sorbitol 50 gl-1 sorbitol Fig. 1. Effect of sorbitol concentration on glucose isomerase production Discussion & Conclusions: 1.45-fold increase in recombinant GI activity was obtained by adding sorbitol at concentration of 30 g L -1 at the beginning of the process. It is concluded that the presence of sorbitol as a co-carbon source has a positive effect on recombinant thermostable GI production. Acknowledgements: METU-RFP, TÜBİTAK (211T065) [1] Srivastava, P. et. al., (2010), Journal of Enzyme Research, 1:01-10, [2] Bhosale, S. H., et. al., (1996), Microbiological Reviews, 60(2): , [3] Çelik, E. and Çalık, P., (2012), Biotechnol Adv 30: , [4] Invitrogen, (2010), Manual part no [5] Dische, Z., et. al. (1951), J. Biol. Chem 192:

59 O-BPE6 Investigation of thermostable recombinant glucose isomerase production by sucrose-utilizing Escherichia coli Burcu Akdağ 1, Pınar Çalık*,2 1 Middle East Technical University, Department of Biotechnology, Ankara, Turkey 2 Middle East Technical University, Department of Chemical Engineering, Industrial Biotechnology and Metabolic Engineering Laboratory, Ankara, Turkey Keywords: Escherichia coli W, exponential feeding, molasses, thermostable glucose isomerase Introduction: Glucose isomerase (GI) (E.C ) is a type of isomerase which catalyzes the isomeration reaction of glucose to fructose reversibly. GI catalyzes the critical step of the production of high fructose corn syrup (HFCS). In addition, this enzyme is used for conversion of xylose to ethanol in association with other enzymes throughout the bio-fermentations [1]. Thermostable enzymes are favorable in the applications of industrial biotechnology due to decreasing of reaction time, having higher reaction rate, reducing contamination risk and being more stable than mesophilic enzymes [2]. Escherichia coli is one of the widely used host microorganism for the production of recombinant enzymes, due to having well-characterized system and being known its whole genome [3]. In this study, thermostable GI production capacity of E. coli W (NRRL B-766) was investigated. Methods: prseta plasmid carrying the thermostable GI encoding gene of Thermus thermophilus (xyl) [4] was transferred to E. coli W. E. coli W-26 carrying prseta::xyla was selected as the best producing strain due to having high enzyme activity. Recombinant GI production experiments were conducted first at laboratory scale shake bioreactors; thereafter at pilot scale bioreactors. In the four sets of pilot scale bioreactors, effects of molasses feeding strategies with/without nitrogen source were investigated on GI production. Results: Molasses consumption, cell growth, GI activity, by-product formation and fermentation characteristics were investigated in semi-batch bioreactor experiments. The highest cell concentration was obtained as 18.4 g L -1 at t=26 h and the highest recombinant GI activity was achieved as U L -1 at t=16 h of bioprocess in the bioreactor operation strategy where two molasses pulse feedings with nitrogen source and antibiotic was introduced to the bioreactor at t=5 h and t=8 h; followed with semi-batch operation starting at t=11 h in which molasses was fed to the bioreactor with a pre-determined specific growth rate, µ=0.05 h -1. Discussion & Conclusions: Two molasses pulse feeding with (NH 4) 2HPO 4 resulted in i) 1.25-fold higher GI activity than that obtained without any nitrogen source addition; ii) 1.85-fold higher GI activity than that obtained with one molasses pulse feeding without any additional nitrogen; iii) 2.23-fold higher GI activity than that obtained with the strategy without any pulse molasses feeding. It was concluded that molasses pulse feeding with an additional (NH 4) 2HPO 4 increased cell concentration and GI activity. Acknowledgements: METU-RFP, TÜBİTAK (211T065) [1] Bhosale, S. H., et. al., (1996). Microbiological Reviews, 60(2): , [2] Sterner, R., Liebl W., Critical Reviews in Biochemistry and Molecular Biology, 36(1): [3] Lee, S.Y., Trends in Biotech, 14: [4] Angardi, V., Çalık, P., Journal of Chemical Technology and Biotechnology, 88 (5):

60 O-NBA4 Comparison of extraction methods of Echinacea purpurea and determination of cytotoxic activity of obtained extracts Ece Yildiz*, Dilan Karabulut, Ayse Nalbantsoy, Ozlem Yesil-Celiktas Department of Bioengineering, Faculty of Engineering, Ege University, Bornova- Izmir, Turkey Keywords (Bold): Echinacea purpurea, extraction methods optimization, cytotoxic activity, total flavonoids Introduction: Echinacea purpurea is a perennial herb native to the Midwestern region of North America [1]. Medicinal use of Echinacea species concentrated on treating various infectious and immune related disorders. Extensive research over the last half century has revealed several important functions of E. purpurea [2], mainly focusing on immunomodulatory effects, anti-inflammatory and antioxidant activities, anti-viral effects particularly in the prevention and treatment of upper respiratory tract infections [3]. Besides all pharmaceutical effects, it has also a potential role in cancer therapy and prevention [4]. The chemistry and pharmacology of Echinacea species are well documented and several groups of bioactive constituents are considered important for its activity [3] and the content of active phytochemicals in this plant can vary due to extraction methods. Methods: Extraction of bioactive compounds by using different extraction methods, namely subcritical water, supercritical CO 2 and soxhlet extraction, determination of optimum extraction conditions revealing the highest yield and investigation of the cytotoxic effects of extracts on various cancer cell lines such as CaCo-2, MCF-7, A549, U87MG, HeLa, and also a healthy cell line, Vero by using MTT assay were carried out. A Box Behnken statistical design was used to evaluate the effect of pressure ( bar), temperature (40 80 o C) and concentration of ethanol as co-solvent (6 20%) by CO 2 flow rate of 15 g/min for 60 min in supercritical CO 2 extraction. Results: The most effective variables were pressure (p<0.005) and co-solvent ratio (p<0.005) in supercritical CO 2 extraction. Optimum extraction conditions were elicited as 300 bar, 80 o C and 13% yielding mg rutin equivalent total flavonoids/g extract, which were higher than the values obtained by subcritical water (0.022 mg/g) and soxhlet (methanol) (0.238 mg/g) extractions demonstrating challenges as a green separation process with improved product properties for industrial applications. Cytotoxicity analyses showed that extracts did not inhibit cell proliferation, on the contrary enhanced cell proliferation at lower concentrations. Discussion & Conclusions: The results demonstrated that supercritical CO 2 extraction provides a green solution for obtaining E. purpurea extract with much lower operating costs compared with that of other processes. In terms of biological activity, it can be concluded that the exposure up to 50 μg/ml of extracts revealed no significant inhibition on the proliferation of cells. Acknowledgements: The authors are thankful to the Novel Fluidic Technologies and Applications Laboratory of Bioengineering Department for access to the facilities. [1] Barrett B. (2003). Phytomedicine, 10: [2] Kumar A., Rinwa P., Kumar C. M. (2011). Journal of Pharmaceutical Education and Research. 2: [3] Kumar K. M. & Ramaiah S. (2011). International Journal of Pharma and Bio Sciences. 2: [4] Miller S. C. (2012). Biomedical Research. 23:

61 O-NTF1 A novel approach for activity and stability enhancement of protease Deniz Senyay-Oncel*, Aslihan Kazan, Ozlem Yesıl-Celıktas Ege University, Department of Bioengineering, Izmir TURKEY Keywords : Enzyme, immobilization, kinetic parameters, protease, supercritical CO2 Introduction: Enzymatic reactions in supercritical CO 2 (SC-CO 2) have received increased attention during the last decade. The aim of this study was to alter the activity and stability of protease from Bacillus sp. under SC-CO 2 conditions and determine the reusage of the enzyme after consecutive enzymatic reactions with immobilized form. Method: The effects of operational parameters such as pressure ( bar), temperature (28 80 C), CO 2 flow (4 10 gmin 1 ) and time ( min) were evaluated regarding activity and stability of protease. In order to determine the reusability of adsorbed enzyme on NaY zeolite, the activity was assayed several times for the hydrolysis of casein and when a lower activity value than the initial activity of the untreated enzyme ( μmolml - 1 min -1 ) was recorded, the immobilized samples were retreated with SC-CO 2. Consecutive reactions were carried out till the activity can not be increased with SC-CO 2 retreatment in comparison to the initial activity of the untreated enzyme. In addition, the kinetic parameters, ph and temperature stability of SC-CO 2 treated and untreated protease as a model enzyme are presented in this study. Results: Optimum conditions were determined as 300 bar, 54 C, 6 gmin 1 CO 2 flow and 120 min of process duration yielding 54.4% ( μmol/ml/min) higher activity than the untreated enzyme. After ph and temperature stabilization optimum conditions were ph 9 and 37 C for the enzymatic reaction, whereas the kinetic parameter values, V max and K m were calculated using Michaelis-Menten model. Fig.1 Supercrital CO2 plant Discussion & Conclusions: As a result, applications of SC-CO 2 medium for enzymecatalyzed processes are going to become important and sustainable with environmentally friendly and economical synthetic protocols. Acknowledgements: We would like to thank the Scientific and Technological Research Council of Turkey (TUBITAK-112M250) for its financial support. 55

62 O-NTF2 Subcritical water extraction of bioactive compounds from black tea processing waste Gizem Özan, Neslihan Kayra, F.Yeşim Ekinci, Özlem Güçlü Üstündağ* Yeditepe University, Biotechnology, * Istanbul, Turkey Keywords : Antimicrobial, antioxidant, black tea waste, phenolics, subcritical water extraction Introduction: Black tea, produced from Camellia sinensis plant, is one of the most popular beverages worldwide. The major phenolic compounds in black tea -catechins, thearubigins and theaflavinshave been shown to have antioxidant and antimicrobial properties. Black tea processing waste is also rich in these bioactive compounds and thus can be used as raw material for their recovery to develop bioactive extracts. Subcritical water (SCW) extraction, which uses water kept in the liquid state at temperatures higher than its boiling point by the use of pressure, offers a green alternative with higher efficiency to traditional methods utilizing organic solvents. SCW extraction has been successfully used for the recovery of bioactive compounds from natural materials including food and agricultural waste. The objective of this study was to investigate the potential of SCW as a solvent for the extraction of bioactive compounds with antioxidant and antimicrobial activities from black tea processing waste. Methods: Black tea waste samples were obtained from the grading step at a black tea factory in the Black Sea region of Turkey. SCW extraction of waste samples (2g, water flow rate =2ml/min) was carried out at three temperatures (90, 120, 150 ºC) for 30 min at 1000 psi using an extractor built inhouse (extraction column: 1/2 o.d 20 cm). 70% methanol extract (ISO 14502:2-2005) was used as control. Extraction yield was determined gravimetrically. The antioxidant activity and total phenolic content of the extracts were determined using DPPH radical scavenging activity assay and Folin-Ciocalteu method, respectively. The antimicrobial activity of the extracts was determined using agar well diffusion assay against four microorganisms including Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778, Shigella flexneri ATCC 12022, Candida albicans (isolated from plant source). Results: Extraction yields of subcritical water extracts were between % (w/w). Antioxidant activity of subcritical water extracts ranged between and µmol Trolox Equivalent (TE)/ml extract. While the activities obtained at 90 and 120 C were similar, a significantly higher activity was observed as temperature increased to 150 C (p < 0.05). Water extracts had significantly higher activity (p < 0.05) than 70% methanol extract (0.74 µmol TE/ml extract). Total phenolic contents of the subcritical water extracts and 70% methanol extract were similar ( mg Gallic Acid Equivalent/ml extract). Based on these results, the increase in antioxidant activity could be mainly attributed to the differences in the composition of the phenolic compounds rather than their total amount. Only S. aureus and S. flexneri was slightly inhibited by subcritical water extracts at the tested concentration, whereas 70% methanol extract showed clear zone of inhibition against C. albicans and S. aureus. Discussion & Conclusions: Black tea waste can be effectively extracted using SCW for the development of antioxidant extracts. Further characterization of the extracts can provide information on the compounds that contribute to antioxidant activity. The use of higher temperatures warrants further research to achieve further improvements in antioxidant activity. Acknowledgements: Financial support of EU FP7 Marie Curie Reintegration Grant (PLPWE_TEA) is gratefully acknowledged. Equal contribution 56

63 O-BEF1 Microalgae based green diesel production Funda Çetin* 1, F. Meliz Tunç 1, Burcu Yüzüak 1, Yasin Ersöz 1, Seda Gören 1, Raife Müge Duru 1, Ayça Arınan 1 1 Tüpraş- Turkish Petroleum Refineries Corporation, R&D Center, Kocaeli, TURKEY * Keywords: Green diesel, microalgae, next generation fuels Introduction: Third generation biofuel production is among the biofuel technology priorities in many countries by means of availability of abundant resources, the use of algae for CO2 capture and algae oil yield advantages. Although biodiesel is a well-known and commercialized additive to diesel fuel, there are some problems regarding its use. For this reason, the studies are oriented to hydroprocessing to produce a petroleum diesel like fuel so called green diesel. Green diesel is a high cetane, low gravity, aromatics and sulfur free diesel fuel and it is commercially produced by major oil&gas companies from vegetable oils. R&D studies focuses on diesel-type fuel from biomass are by transesterification to obtain biodiesel from microalgae in Europe and US on laboratory and pilot scale, yet green diesel production from microalgae studies are not common in literature. This paper provides the experimental results of Tüpraş s R&D project called Green Diesel Production from Microalgae and assess its results for future investment decisions. The paper will enlighten green diesel production technologies, make a detailed comparison with biodiesel and discuss its current and future technical and economic situation. Green diesel s position and definition on legal framework in Europe and Turkey will be discussed as well. Discussion & Conclusions: As a conclusion, since biodiesel has advantages such as the risky impact on engine parts and the relatively high solubility of water, green diesel may be used as a diesel fuel additive in the future. Due to scarcity of vegetable oil resources, algae based green diesel production may be an important option for future fuels. 57

64 O-BEF2 Utilization of olive pomace by a green technology Fazilet Gurer 1, Aslihan Kazan 1, Melih Soner Celiktas 2, Ozlem Yesil-Celiktas 1* 1 Bioengineering Department, Ege University, Izmir, Turkey 2 Solar Energy Institute, Ege University, Izmir, Turkey Keywords: Cost, olive pomace, optimization, particle size, supercritical CO 2 extraction Introduction: Olive pomace is a by-product of olive oil production process and a very important biomass source for the Mediterranean countries. Every year tons of waste olive pomace are released from olive oil production process and subjected to oil extraction operation. Solvent extraction, mostly with hexane, is a common way for oil extraction from olive pomace [1]. However this extraction way has some disadvantages in industry such as using of an organic solvent, inefficient solvent removal and high energy requirements. As an alternative to organic solvents supercritical fluids can be used in extraction processes. Supercritical fluids are known as green solvents and have some advantages like high diffusivity, low viscosity and elimination of mass transfer limitations by low surface tension. Supercritical CO2 is the one of the most commonly used solvent in supercritical extraction. It is cheap and has some advantages like being non-toxic, noncorrosive and non-explosive over organic solvents and it can be used in food industry with high safety. In olive oil production process approximately kg of olive pomace is produced from 100 kg of olive and kg olive pomace oil can be extracted by conventional extraction methods [2,3]. Methods: Supercritical CO 2 extraction was carried out in 100 ml extractor (SFE 100, Thar Instruments, UK, 2006) with about 30 g of olive pomace. The independent variables were temperature (30, 55, 80 0 C), pressure (120, 210, 300 bar) and flow rate (10, 15, 20 g/min). Conventional oil extraction performed with hexane according to International Standarts ISO 659 (Reference method; Determination of oil content) Standard sieve series ( µm) were used to separate olive pomace into different size ranges. Results: For supercritical CO 2 extraction optimum conditions were found as 68 o C, bar, 20 g/min CO 2 flow rate and the extraction yield was determined as 4.83 % at optimum condition. The particle size was found to have significant effect on oil extraction and the decreasing of particle size from > 2000 μm to μm range increases the extraction yield to %. Discussion&Conclusions: As a result, supercritical CO 2 can be used as a more environmentally friendly way for oil extraction from olive pomace. It is also possible to increase the extraction yield by reducing the particle size of olive pomace. Acknowledgements: This project is supported by the Scientific and Technical Research Council of Turkey (TUBITAK) (113M363). [1] Eroğlu,F., (2006), Y.Lisans Tezi, Gazi Üniversitesi Fen Bilimleir Enstitüsü. [2] [3] Kurtuluş, E., (2003). Yeni ve Yenilenebilir Enerji Kaynakları Sempozyumu ve Sergisi ve Bildiriler Kitabı, Doç.Dr.Mustafa İlbaş, Makine Mühendisleri Odası, Editörler: Yrd.Doç.Dr. Şükrü Su, Yayın No: E/2003/330,EKGM2003, Kayseri. 58

65 O-BEF3 Hydrolysis of hazelnut husk by using commercial enzymes for the production of fermentable sugar Kübra Karaosmanoğlu 1, Orkun Pinar 1, Nihat A. Sayar 1, Dilek Kazan 1,* 1 * Department of Bioengineering, Marmara University, Kadikoy, Istanbul, TURKEY * Keywords: Cellulase, fermentable sugar, hazelnut husk, laccase, lignocellulosic biomass Introduction: Nowadays, studies related to agricultural residues in other words lignocellulosic biomass used in the different bioprocesses such as production of biofuels increasing day by day [1]. Hazelnut husk (hazelnut outer shell) is one of the important lignocellulosic wastes in Turkey which holds 75-80% of the world hazelnut production. According to Fiskobirlik (The Union of Agricultural Cooperatives for the sale of hazelnuts), between 450,000 and 600,000 tons of hazelnut are produced annually in Turkey. As known, 1 kg hazelnut have 1.72 kg hazelnut husk. In this context, approximately 774,000 to 1032,000 tons of waste is left in fields after harvest or incinerated. Different methods as high-temperature hydrolysis with dilute acid solution [2], low-temperature hydrolysis with concentrated acid solution, hydrolysis after pre-treatment with steam, enzymatic hydrolysis used for hydrolysis of lignocellulosic waste materials in which cellulose and hemicellulose is converted into fermentable sugar. Although the yield of acid hydrolysis is higher than enzymatic hydrolysis, resulting by-products (furfural, etc.) in the acid hydrolysis adversely affects the use of hydrolysate in fermentation process [3]. Sugar obtained with enzymatic hydrolysis at more moderate conditions can be fermented easily and in the absence of byproducts, hydrolysate can easily be used in fermentation. On the other hand, it is very hard to obtain higher yield of fermentable sugar due to a layer of lignin. To overcome this problem, a pre-treatment step prior to enzymatic process is required. Hydrolysis must be successful, inexpensive, and has low energy requirements with lower fermentation time as well as low by-products concentration. Therefore, in recent years, studies with enzyme preparations including more than one enzyme for the hydrolysis of lignocellulosic waste have been increased rapidly. Laccases are multicopper oxidases that can catalyze the oxidation of non-phenolic and phenolic lignin related compounds and cellulases are hydrolases that hydrolyse the β-1,4-glucosidic bonds of cellulose to produce smaller β-glucose oligomers and β-d-glucose. In this study, it was glucose oligomers and β-d-glucose. In this study, it was aimed to use commercial laccase and cellulase to hydrolyse hazelnut husk in order to obtain fermentable sugar with higher yield for further processes. Methods: Hydrolysis conditions were optimized using commercial (Novozyme) enzymes, laccase (51003) and cellulase (188), and hazelnut husk (as substrate) at different ratios, with different reaction times and temperatures. During hydrolysis, sugar content and total sugar concentration were determined at different times by the phenol sulfuric acid and DNS method. Cellulase and laccase activities were determined using the method proposed by Ghose [4] and Garcia et al. [5] respectively. In these activity methods, one unit (U) of laccase activity was defined as 1 µmol of oxidized ABTS per minute and one unit (U) of cellulase activity was defined as 1 µmol of liberated glucose per minute under the assay conditions. Results: An appropriate method for hydrolysis of hazelnut husk with laccase and cellulase was obtained as a result of study. Discussion & Conclusions: As a conclusion, this study suggests a solution to overcome the hydrolysis problems as mentioned, particularly for hazelnut husk as a lignocellulosic waste. Acknowledgements: This work was supported by the Turkish Scientific and Technical Research Council (113M053). : [1] Alvira P, Tomás-Pejó E, Ballesteros M, Negro M.J. (2010). Bioresource Technology 101: [2] Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S.I, Leea Y.C. (2005). Analytical Biochemistry 339: [3] Palmqvist E, Hahn-Hägerdal B. (2000). Bioresour.Technol. 74: [4] Ghose T.K. (1987). International Unionof Pure and Applied Chemistry 59(2): [5] Garcia T. A, Santiago M. F, Ulhoa C. J. (2006). Biotechnology Letters 28:

66 O-BPE7 Photoheterotrophic adaptation of microalgae and enhanced lipid production under different conditions Duygu Ayyildiz-Tamis 1, Müge Isleten-Hosoglu 1 and Murat Elibol 1 * 1 Ege University, Faculty of Engineering, Bioengineering Department, Bornova, Izmir, Turkey * Keywords : Bioreactor, Ettlia texensis, lipid, optimization, photoheterotrophic Introduction: Microalgae are seen to be the fuel of the future due to their high biomass productivity and ability to accumulate high quantity of lipid and carbohydrate inside their cells for biodiesel and bioethanol production, respectively. To achieve economic feasibility and industrial application of this technology, the selection and growth of highly productive algal strains is gaining importance [1]. Chlorella protothecoides, Nannochloropsis sp., Chlorella vulgaris, and Ettlia oleoabundans (formerly known as Neochloris oleoabundans) have been identified as the most promising strains for biofuels production. In addition to that, different cultivation modes like photoheterotrophic and/or heterotrophic conditions can offer a well controlled process and provide the possibility to maximize biomass and lipid productivities on a large scale [2, 3]. Methods: Biomass and lipid productivities of Nannochloropsis sp., Chlorella sp and Ettlia texensis which were obtained from our local resources in Turkey were investigated under photoautotrophic conditions. Among them the highest biomass productivity (0.05 g/l.d) were achieved with Ettlia texensis. Ettlia cultures is known as a freshwater species capable of storing large amounts of lipids composed of triacylglycerides (TAG) at a level of up to 80% of its total lipid composition and suitable for the production of biofuels. Thus, Ettlia species seem to as a promising algal species for biofuel production. Consequently Ettlia texensis was investigated under photoheterotrophic conditions. Before adapting it to photoheterotrophic conditions Ettlia texensis was made axenic with an improved protocol, and then potential use of various carbon and nitrogen sources for culturing Ettlia texensis in photoheterotrophic batch cultures were studied. In a scale-up attempt, productions were accomplished in a 3 L stirred tank bioreactor. Results: Searching the preferences of the carbon and nitrogen source disclosed that this strain could grow photoheterotrophically well with glucose and yeast extract. In the optimized medium, the highest biomass productivity and total lipid content achieved were 0.97 g/l.d and 26% of dry weight basis, respectively. Moreover, the major fatty acid methyl esters were C16:0; C18:1; C18:2 and C18:3. The final biomass and lipid productivities obtained in a 3 L stirred tank bioreactor with 250 rpm agitation rate were 0.92 g/l.d and 322 mg/l.d, respectively. Discussion & Conclusions: E. texensis could be potent candidate for commercial production in the bioreactor photoheterotrophically. Acknowledgements: This research was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) with 109M227 project number, and EBILTEM (Ege University Science and Technology Center, (2010/BIL/013 project number). The authors wish to thank Prof. Dr. Meltem Conk-Dalay (Ege University, Faculty of Engineering, Bioengineering Department) for supplying algal culture. [1] Lam, M.K., Lee, K.T. (2011). Biotechnol. Adv. 30 (3): [2] Xu, H., Miao, X., Wu, Q. (2006). J. Biotechnol. 126: [3] Isleten-Hosoglu, M., Ayyildiz-Tamis, D., Zengin, G., Elibol, M. (2013). Bioresource Technol. 131:

67 O-BPE8 Human growth hormone production by metabolically engineered Bacillus subtilis: feeding strategy development Burcu Gökbudak 1, Pınar Çalık*,1, Tunçer H. Özdamar 2 1 Department of Chemical Engineering, Industrial Biotechnology and Metabolic Engineering Laboratory, Middle East Technical University, Ankara, Turkey 2 Department of Chemical Engineering, Biochemical Reaction Engineering Laboratory, Ankara University, Ankara, Turkey Keywords: Bacillus subtilis, feeding strategy, recombinant human growth hormone, semi-batch Introduction: Human growth hormone (hgh) is anionic, non-glycosylated, four-helix bundle protein known as somatotropin, having a molar mass of 22 kda, and 191 amino acid residues. It has been used to treat hypopituitary, dwarfism, injuries, bone fractures, bleeding ulcers, burns, etc.. Bacillus subtilis is an attractive host for producing heterologous pharmaceutical proteins, like human growth hormone, extracellularly due to its naturally high secretion capacity [1]. Annotation of genome sequence and subsequent international programmes of systematic gene disruption, functional analysis and regulatory network studies makes B. subtilis the focus of interest for genome scale metabolic engineering studies. Recently, from our research group Özdamar et al. (2009) demonstrated for the first time an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hgh) from B. subtilis [2]. In this work, effect of feeding strategies on human growth hormone (rhgh) production by B. subtilis (scoc - ) carrying pmk4::pre(subc)::hgh was investigated. Methods: Pilot scale bioreactor experiments were conducted using V=3.0 dm 3 bioreactor (BBraun, Germany) consisted of temperature, ph, foam, air inlet and stirring rate controls, at ph C=7.5 controlled-ph operation at the oxygen transfer conditions as air inlet rate of Q O/V R=0.5 vvm, and agitation rate of N=750 min -1. Results: Exponential feeding of glucose and glucose based semi-defined medium at predetermined specific growth rates within µ 0= h -1 were carried out. The cell, substrate consumption,fermentation and oxygen transfer characteristics as well as the formation of rhgh, and by-products were investigated in semi-batch bioreactor experiments. The highest rhgh concentration was achieved as C rhgh=0.40 g L -1 at t=28 h by glucose based semi-defined medium feeding at the pre-determined specific growth rate of µ 0=0.17 h -1, where rhgh production was 2.9-fold higher than that obtained in batch-production operation; furthermore, the highest cell concentration was obtained as C X=4.4 g L -1 at t=30 h. The overall product and cell yield on total substrate were found as g kg -1 and 0.73 g g -1, respectively. Discussion & Conclusions: Semi-batch glucose based semi-defined medium, containing glucose, peptone, (NH 4) 2HPO 4 and trace elements, feeding increased rhgh production. Acknowledgements: This work was supported by the Middle East Technical University Research Fund through the project BAP [1] Nakayama, A., Ando, K., Kawamura, K., Mita, I., Fukazawa, K., Hon, M., Honjo, M., Frutani, Y. (1988). Journal of Biotechnology, 8: [2] Özdamar, T.H., Şentürk, B., Yılmaz, Ö.D., Kocabaş, P., Çalık, G., Çelik, E., Çalık, P. (2009). Biotechnology Progress, 25(1):

68 O-BPE9 Hydrogen production by mutant strains of Chlamydomonas reinhardtii Suphi S. Oncel 1 *, Ayse Kose 1, Fazilet Vardar Sukan 1, Cecilia Faraloni 2, Giuseppe Torzillo 2, Esra Imamoğlu 1, Murat Elibol 1 1 Bioengineering, Ege University, Turkey 2 CNR, Italy Keywords: Biohydrogen, bioenergy, Chlamydomonas reinhardtii, microalgae Presenter Author Introduction: The green microalgae Chlamydomonas reinhardtti is well-known specie in the terms of H 2 production by photo fermentation and has been studying for a long time for sustainable energy development [1]. Although the H 2 production yield is promising; there are some bottlenecks to enhance the yield and efficiency to focus on a welldesigned, sustainable production and also scaling up for further studies [1]. D1 protein of photosystem II (PSII) plays an important role in photosystem damage repair and related to H 2 production. Because Chlamydomonas is the model algae and the genetic basis is well-studied; metabolic engineering tools are intended to use for enhanced production. Mutations are focused on D1 protein coding gene which aims long-lasting hydrogen production by blocking the PSII repair system thus O 2 sensitive hydrogenases catalysis hydrogen production for a longer period of time under anaerobic and sulfur deprived conditions [2]. Considering their photosystems; one of the essentials for hydrogen production is light source and intensity. Methods: In laboratory scale, the effect of 50,70, 80 and 100 μmol photons m -2 s -1 (two sides), and 100, 140, 160 and 200 μmol photons m -2 s -1, (one side) was studied for Chlamydomonas reinhardtii (CC124) and D1 mutants (D240, D239-40, D240-41) during growth (aerobic) and hydrogen production (sulfur starved anaerobic) phase. Biohydrogen is analyzed by Gas Chromotography (GC). Results: The photosynthetic activity measured as effective quantum yield (ΔF/F m), Photograph by two sides, reflected a faster decrease than cultures illuminated by one side. The values were less than within 48 h, maintained for 2 days, whereas ΔF/F m strongly declined after 72 h (<0.100). With the decrease of ΔF/F m, cultures illuminated by two sides started hydrogen production earlier (~ h). CC124 produced ml of H 2 in all conditions, but mutants exhibited in a range of ml for low light intensities and a total amount of H 2 350±50 ml (one side) and 250±20 ml (two sides) higher than CC124 at high intensities. Hydrogen production period was 5± 2 days for CC124 and mutants showed a longer production time for 9±2 days. Discussion & Conclusions: The results showed that the light exposure during the hydrogen production process, play an important role in protein degradation. This fact was underlined by the results obtained with the D1 protein mutant strain, which resulted more sensitive to the different light exposure. H 2 productions for mutant strains have a significant effect in terms of productivity, yield and production time. Acknowledgements: The authors would like to thank TUBITAK, CNR Italy and Ege University Bioengineering Department. [1] Melis, A., Happe, T. (2001). Plant. Physiol. 127: [2] Scoma T., Torzillo G. (2012). Journal of Biotechnology 157:


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71 P-BCA1 Stem cell application and their functional effect on the cells of bone and teeth Feyzan Ozdal Kurt*,1, Mahmud Ozkut 2, Isıl Aydemir 2, H. Seda Vatansever 2, M. Ibrahim Tuglu 2 1 Celal Bayar University, Faculty of Sciences & Letters Department of Biology, Muradiye Campus, Muradiye-Manisa, Turkey 2 Celal Bayar University, Medical Faculty Department of Histology-Embriyology, Manisa, Turkey Keywords: Odontoblast, osteoblast, stem cell, tissue engineering nt Introduction: The usage of stem cells in cell therapies for regenerative medicine progressively increases today and it has a great promising potential for the future. Mesenchymal stem cells can be distinguished by the adhesive properties and they easily obtain, proliferate and differentiate in culture [1]. They have an ability to interact with biomaterials and transplate to in vivo regeneration for treatment with the help of tissue engineering. The bone and teeth have special tissues which cannot be regenerated where stem cells may be very useful [1, 2]. Results from this study were evaluated through histology, morphometry, and immunocytochemistry and scanning electron microscopy. Methods: Mesenchymal stem cells from bone marrow, dental pulp and adipose tissue were cultured and their differentiation to osteoblast and odontoblast were determined by markers. Differentiated cells were placed to biomaterials such as hydroxyapatite, titanium and calcium phosphate scaffold for the implantation of experimental wound place. The healing process was analyzed by staining of Haematoxylen & Eosine, Vonkossa, Alizarin red, osteocalcine, osteonectine, dentin sialoprotein and by scoring of histological appearance during healing. Ultrastructure of the cells were investigated for the behavior of the cells on the biomaterials. Results: Mesenchymal stem cells were attached and proliferate in the culture and were differentiated to osteoblast and odontoblast which were proved by markers. Their behavior was changed by the interaction with biomaterials and after implantation they achieved to stay, proliferate and show function in wound environment. They cause to faster, better and more functional healing either in bone or teeth. Their functional activity was more obvious when they were used for critical size defect of mandibular which were demonstrated by radiology, morphology and histology. The behavior of the stem cells were changed by the effect of biomaterials which were made cells more adhesive and interactive with better spreading and more protrusions, lamellapodia and stoplasmic process which extend inside of the pores. The best source of odontoblastic differentiation was the stem cells of adipose tissue compared to that of dental pulp and bone marrow. Together with all these observations, mesenchymal stem cells showed clear functioning on the wound healing. Discussion & Conclusions: This study showed that the best source of mesenchymal stem cells was the adipose tissue and it was also the best candidate for functional repair. The cells from all sources were effective and significantly improved the healing process which was proved by speed, efficacy, quality and cost criteria. As a result, especially with the help of tissue engineering, mesenchymal stem cells will get more success in the field of health with the possibilities of producing bioengineer-teeth and bioengineer-bone. : [1] Lü, L.X., Zhang, X.F., Wang, Y.Y., Ortiz, L., Mao, X., Jiang, Z.L., Xiao, Z.D., Huang, N.P. (2013). ACS Appl Mater Interfaces 23;5: [2] Estrela, C., Alencar, A.H., Kitten, G.T., Vencio, E.F., Gava, E. (2011). Braz Dent J. 22:

72 P-BEF1 in vitro shoot regeneration from protoplasts and selection of somaclones in African violet (Saintpaulia ionantha H. Wendl.) Irmak Cakin 1, Aynur Gurel*,2 1 Biotechnology Department, Graduate School of Nature and Applied Sciences, Ege University, Izmir, Turkey 2 Bioengineering Department, Faculty of Engineering, Ege University, Izmir, Turkey * Author Keywords: African violet, protoplast, Saintpaulia ionantha H. Wendl., shoot regeneration, somaclonal selection Photograph Introduction: Saintpaulia ionantha H. Wendl. (African violet), belongs to the family Gesneriaceae, is the most commercial and ornamental plant. Saintpaulia species has many cultivars with different flower colors, leaf shapes, vegetative habit, flowering time and flower duration to meet requirements of domestic and international markets. There are about varieties in African violet produced by using conventional breeding techniques [1] based on making hybridization mainly, mutation and selection. Recently, tissue culture techniques including gene transferring systems and somatic hybridization methods have been used in order to develop new varieties and also complete the breeding programs established by conventional breeding techniques in this species. In African violet, some characters such as transport ability, cold tolerance, disease and pest resistance have to be improved too [2,3,4]. African violet present a good model system related with in vitro regeneration researches due to their high regenerative abilities and capacities [1,5,6,7]. Whereas shoot regeneration rate was determined between % from vegetative segments of this species [8], somatic embryo formation rates were found as 32.7±1.7% from leaf explants and 63.3±3.2% from embryogenic calli [7]. Shoot regeneration from protoplasts and fusions established among protoplasts belonging to different genotypes to obtain new combinations of nuclear and organellar genes is very important to increase genetic variability in African violet. Discussion & Conclusions: In vitro shoot regeneration rate from protoplasts ranged between 2-68% in Saintpaulia ionantha H. Wendl [2,9,10,11]. Somaclonal variations lead to genetic and epigenetic changes depending on genotype, age of donor plant, cytogenetic changes, DNA methylation, explant type, plant hormones in the culture medium and culture duration. Somaclonal variations in plants directly regenerated from leaf segments were determined on flowering period, number of flowers per plant and flower size in African violet [12]. Somaclonal variations in plants regenerated by tissue cultures changed between 2-10% in African violet [5]. It is very important that developing efficient protocols on protoplast isolation and fusion to create genetic variability in African violet. Plant breeders will be able to develop new varieties in a very short time by micropropagation of somaclones selected for desirable characters in this ornamental plant species. [1] Daud, N., Taha, R., (2008). Pakistan Journal of Biological Sciences, 11(7): [2] Winkelmann, T., Grunewaldt, J., (1992). Gartenbauwissenschaft, 57(6): [3] Khan, S., Naseeb, S., Ali, K., (2007). Pakistan Journal of Botany, 4: [4] Ghasemi, Y., Nematzadeh, G.A., Hosseini, S., (2012). Biharean Biologist, 6(2): [5] Rout, G., Mohapatra, A., Jain, M.S., (2006). Biotechnology Advances, 24: [6] Taha, R.M., Daud, N., Hasbullah, N.A., (2009). Pakistan Journal of Biological Sciences, 9: [7] Taha, R., Daud, N., Hasbullah, N.A., (2010). Acta Horticulturae, 865: [8] Sunpui, W., Kanchanapoom, K., (2002). The Songklanakarin Journal of Science and Technology, 3: [9] Winkelmann, T., (1993). African Violet, 46(6): [10] Winkelmann, T., Grunewaldt, J., (1994). Plant Cell Reports, 14: [11] Winkelmann, T., Grunewaldt, J., (1995). Plant Breeding, 114(4): [12] Hoshino, Y., Nakano, M., Mii, M., (1995). Plant Cell Reports, 14:

73 P-BEF2 Plant secondary metabolite production via co-culture systems and their bioreactor applications Meltem Bayraktar 1, Aynur Gurel*,2 1 Agricultural Biotechnology Department, Faculty of Agriculture, Ahi Evran University, Kirsehir, Turkey 2 Bioengineering Department, Faculty of Engineering, Ege University, Izmir, Turkey Keywords: Bioreactor, co-culture, in vitro, plant, secondary metabolite Introduction: Co-culture is an in vitro system where two or more cultures are grown in the same culture device and same nutritive medium [1,2]. Intra-species and inter-species co-culture systems are a relatively new area in biotechnological research of higher plants [3] and nowadays, these systems are preferred for overproduction of secondary metabolite [2]. Discussion & Conclusions: The co-culture systems can be established diversely: organ+organ co-cultures [4], microorganism+plant cell/tissues or organs cocultures [5,2], cell suspension culture+hairy root co-cultures [1]. In organ+organ co-cultures, metabolites produced by one organ are released into medium and other organ takes these metabolites for biosynthesis of different compounds [4]. Atropa belladonna transformed roots and Duboisia hybrid shooty teratomas have been successfully cocultured in a 2 x dual bioreactor system for the production of scopolamine [4]. Ammi majus hairy roots and Ruta graveolens shoots were co-cultured for production of furanocoumarins [6]. Co-culture of ginseng (Panax ginseng C.A. Meyer) and echinacea [Echiancea purpurea (L.) Moench.] adventitious roots have been established for the production secondary metabolites. Adventitious roots of ginseng and echinacea were cultured in different proportions in 5-L capacity airlift bioreactors [7]. Genista tinctoria L. hairy roots and shoots were co-cultured in a prototype basketbubble bioreactor for synthesis and biotransformation of large amounts of phytoestrogens [3]. Co-culture with Pseudomonas sp. or Enterobacter sp. increased guanosine, inosine, and trigonelline production compared to the control in in vitro PLB (protocormlike body) cultures of Pinellia ternata. Co-culture with living Pseudomonas sp. cells increased the growth and differentiation rate of P. ternata PLBs [5]. When Taxus chinensis suspension cells were co-cultured with other organisms, such as fungi, the production of taxol enhanced [8]. Co-cultures of Linum album cells with arbuscular mycorrhiza-like fungi, Piriformospora indica and Sebacina vermifera, increased podophyllotoxin and 6- methoxypodophyllotoxin production. These two fungi have phytopromotional effect on plant cell in suspension cultures and enhance plant biomass and production of secondary metabolites. Cocultivation of live plant and fungus cells activate defense system of plant cells and subsequently, they release signaling molecules which are responsible for production of defense compounds like podophyllotoxins. This system also provide a valuable tool to study symbiotic mycorrhizal association in plants at cellular level [9]. : [1] Guillon, S., Trémouillaux-Guiller, J., Pati, K., Rideau, M., Gantet, P., (2006). Trends in Biotechnology, 24(9): [2] Weathers, P.J., Towler, M.J., Xu, J., Applied Microbiology and Biotechnology, 85: [3] Łuczkiewicz, M., Kokotkiewicz, A., (2005). Plant Science, 169: [4] Mahagamasekera, M.G.P., Doran, P.M., (1998). Phytochemistry, 47(1): [5] Liu, Y.H., Lianga, Z.S., Chenc, B., Yanga, D.F., Liuc, J.L., (2010). Enzyme and Microbial Technology, 46: [6] Sidwa-Gorycka, M., Królicka, A., Kozyra, M., Głowniak, K., Bourgaud, F., Łojkowska, E., Plant Science, 165: [7] Wu, C.H., Niranjana-Murthy, H., Hahn, E.J., Paek, K.Y., (2008). Acta Physiologiae Plantarum, 30: [8] Lim, E.K., Bowles, D., (2012). Current Opinion in Biotechnology, 23: [9] Baldi, A., Jain, A., Gupta, N., Srivastava, A.K., Bisaria, V.S., (2008). Biotechnology Letters, 30:

74 P-BEF3 The production of bacterial biofertilizers using carriers Emek Aslan* 1, Fazilet Vardar Sukan 1, Hatice Ozaktan 2 1 Ege University, Faculty of Engineering, Deparment of Bioengineering, Bornova, Izmir, Turkey 2 Ege University Faculty of Agriculture Department. of Plant Protection, Bornova, Izmir, Turkey Keywords: Biofertilizer, carrier, PGPR, SEM, soilless culture Introduction: Plant growth promoting rhizobacteria (PGPR) are becoming increasingly important as biofertilizers in agriculture. A microorganism may function optimally in vitro, but formulation of that organism into an end-user product is a difficult step one [1, 2]. Aim of this study is to produce bacterial bioinoculants of two PGPR strains by using carriers and by encapsulation. Methods: Two isolates of PGPR, Pseudomonas putida str. 18/1K and Bacillus spp. strain 66/3 which were proven to stimulate growth promotion in previous researches were included in this study to produce biofertilizers In lab-scale using soilless culture substrates, perlite, zeolite and cocopeat. Additionally PGPR were formulated using alginate beads [3, 4]. PGPR were screened for their existence on the surface with Scanning Electron Microscobe (SEM). Irrigation simulations in vitro and in vivo were carried out for observing the colonization of the both PGPR. The bifofertilizers were tested for the shelf life viability performance in two different storage conditions. Promising results for viability of bacteria on the substrates were observed. Results : For both PGPR strains, bacterial colonization on the the three substrates were 10 9 cfu/g and viability observed from the alginate beads were 10 7 cfu/g. SEM images proved the exsitance of the two PGPR on the surface of the substrates after filtering. In Irrigation simulations (in vitro and in vivo) the colonization of the both PGPR strains on the Rhizosphere found to be 10 6 cfu/g root which is reported to be enough for growth stimulation in plants. The shelf life viability performance In the 90th day was around cfu/g which is acceptable. Discussion & Conclusions: Using biofertilizers is a rising trend in agricultural production especially in developing countries. Inoculation of local bacterial strains and application of the bioinoculant with carriers are important parameters for adaptation of the microorganism and for the success of the biofertilizer. The results of the study are promising for the future prospects for the agricultural production in Turkey. (National Patent process is ongoing. 2012/00239) [1] Bashan, Y. (1998). Biotechnology Advances, Vol. 16, No. 4, pp [2] Chen et al. (1996). The use of yield increasing bacteria (YIB) as plant growth promoting rhizobacteria in Chinese agriculture. In: Management of soil borne diseases, R.S. Uthkede and W.K., Gupta (Eds), Ludhiana: Kalyani Publishers: [3] Bashan, Y., (1986). Applied and Enviromental Microbiology [4] Daza et al. (2000). Soil Biology & Biochemistry 32:

75 P-BGE1 in vitro screeening of cotton genotypes for drought tolerance Eminur Elci 1 1 Department of Biotechnology, Progen Seed Inc., Antakya, Hatay, Turkey Keywords: Cotton, drought stress, germination, PEG-6000, tolerance Introduction: Cotton (Gossypium hirsutum L.) is an economically important crop, worldwide. The tetraploid species, G. barbadense and G. hirsutum, dominate world cotton production with a large number of improved G. hirsutum is the principal cultivated cotton and accounts for about 90% of the world s cotton production. Drought stress or water deficit is a major abiotic stress that effects plant growth, yield and quality. Selection of drought tolerant genotypes under field conditions is expensive and time required. Osmotic solutions are used to impose water stress reproducibly under in vitro conditions [1]. Polyethyleneglycol (PEG) widely used to induce water stress, is a non-ionic water polymer, which is not expected to penetrate into plant tissue rapidly [2]. This study has been performed to evaluate cotton tolerance to osmotic stress induced by PEG. Methods: In this study, cotton genotypes were evaulated for drought tolerance under PEG-6000 induced water stress at germination and seedling stage. Eleven different cotton cultivars were evaluated. A hundred delinted seeds of each genotype were surface sterilized and then induced on PEG-infused Gamborgs B5-MS media containing different levels (0g/L, 20g/L, 40g/L, 60g/L) of PEG-6000 with three replicates. Germination and important seedling characters like root length and weight; shoot length and weight were recorded. ST506 and Sphinx cultivars were used as drought sensitive and tolerant genotypes, respectively. Results: The variance analysis (ANOVA) showed that, there were significantly difference between eleven cultivars, PEG levels and their interactions. The results of this study reveal that different concentrations of PEG had a significant effect on the all measured traits. The most significance were obtained at seedling stage comparing with germination stage. There was no germination under the highest stress conditions after fourteen days for all cultivars. Discussion & Conclusions: According to germination and growth parameters under stress conditions, cv. BA 525 was found the most drought tolerant cultivar. The results showed that PEG-6000 can be used for pre-selection of drought tolerant cotton genotypes and this cost and time effective screening method can be used for screening large number of germplasm. Acknowledgements: This study was supported by TÜBİTAK-TEYDEB/ Project. [1] Nepomuceno, A.L., Oosterhuis, D.M., Stewart. J.M. (1998). Environmental and. Experimental Botany, 40: [2] Pandey, R., Agarwal, R.M. (1998). Physiological and Molecular Biology of Plants, 4:

76 P-BGE2 Assessment of genetic purity of soybean genotypes by microsatellite markers Eminur Elci 1 1 Department of Biotechnology, Progen Seed Inc, Antakya, Hatay, Turkey Keywords: Genetic purity, SSR, soybean Introduction: Soybean (Glycine max L.) is the most important grain legume as a source of protein and oil, worldwide. The cultivated soybean G. max belongs to family Leguminosae. It is essential to assess genetic purity of parental lines and hybrids for seed suppliers before marketing. Molecular marker technology provides effective, fast, accurate and appropriate tool for crop improvement, determination of genetic diversity, paternity tests and varietal purity analysis. Microsatellites, which are show codominant inheritance, locus-specific with high information content and PCR-detectable, are the most popular molecular markers. In this study, SSRs (Simple sequence repeats) were used for the assessment of genetic purity of highly efficient Bravo soybean cultivar. Methods: Bravo, which is the commercially important soybean cultivar produced by Progen Seed Inc., was examined in this study. Ten seeds of each parental lines were mixed and a hundred seeds of cultivars were randomly selected and used for genetic purity analysis. Seeds were homogenized by TissueLyser (Qiagen). DNAs were extracted according to Doyle and Doyle CTAB method [1]. The quality and quantity of extracted DNAs were measured by NanoDrop (Thermo). After dilutions, PCR was carried out using 20 SSR markers obtained from GeneBank. The final PCR products were visualized under UV light after electrophoresis on ethidium bromide-stained 2 % agarose gels. Results: SSR markers which are used in this study were found highly polimorphic. The average number of alleles of each SSR locus is found three and the number of alleles ranged from two to four. According to three replicates of experiment, none or one off-type was determined in hundred genotypes (Figure 1). According to SSR marker analysis, genetic purity of cv. Bravo was determined 99 %. M Fig. 1. Agarose gel electrophoresis results of genetic seed purity testing of cv. Bravo using SoySatt309 primer. M: 100 bp marker (Fermentas). Arrow indicates off-type. Discussion & Conclusions: The results showed that, the tested cv. Bravo. has the purity between the avarage values, and SSR markers could detect genetic purity of soybean seeds accurately that could not be distinguished by morphological markers. [1] Doyle, J.J., Doyle, J.L. (1990). Focus 12:

77 P-BGE3 Estimation of irradiation dose in the flesh of irradiated fish by RAPD PCR Ergun Sakalar 1*, Suhendan Mol 2 1 Department of Bioengineering, Faculty of Engineering, Canakkale Onsekiz Mart University, Canakkale, Turkey 2 Department of Seafood Processing Technology, Faculty of Fisheries, Istanbul University, Istanbul, Turkey Keywords: DNA damage, fish, food, irradiation, RAPD-PCR Introduction: Public concerns about whether irradiated foods are healthy and against. The negative perception of nuclear technology presence makes a controversial issue in the availability of food irradiation technology. In the forefront these days of the elimination of factors that adverse effects on human health, in the future to make it perfect for the production of safe food, the development of alternative technologies have been aimed for traceability of irradiated foods and measurement of technologies of radiation dose applied to foods[1]. Methods: In this study, RAPD PCR were used for the development of metodologies that can able to estimate irradiation dose. Distinguishing between irradiated and non irradiated fish was identified. Fish (Oncorhynchus mykiss) samples were exposed to radiation doses of 0.250, 0.500, 1, 3, 5, 7 and 9 kgy in gamma cell. DNAs were extracted from irradiated samples before and after storage. ERP primers were designed to produce randomly amplifications on the DNA of the irradiated samples, and RAPD PCR was applied and obtained the agarose gel profiles of amplicons. In addition, DNA fragmentation occurring in each dose was determined by DNA Comet test (Table 1) for the purpose of verification of RAPD PCR. In addiation, irradiated products were storaged for 1and 3 monts. The effect of storage on irradiated fish meats was examined and detectable limit of irradiation dose was determined by RAPD PCR. Results: A molecular methodology allowing to easily qualitative analysis of irradiated fish and estimation of dose were developed for the first time. These methodology allowes the analysis of the irradiated fish stored for a period of three months up to the dose limit of around 0.5 kgy (Fig.not shown) Fig. 1. RAPD profiles of fish irradiated with different dose Table 1. The changes that occur in the tail and head of comets depending on the irradiation doses Dose (kgy) Comet tail (µm) Comet head (µm) Discussion & Conclusions: This study can be used as basis for the identification of the dose of irradiation by looking at the loss of specific bands for specific doses of irradiation (Fig.1). Acknowledgements: The authors would like to thank the Office of Scientific Research project (Grant No ), and the Biology Department of Fatih University that has provided necessary facilities. [1] Stefanova, R., Vasilev, V.N., Spassov, S.L., (2010), Food Anatlitic Methods 3:

78 P-BGE4 Estimation of radiation dose by DNA Comet Assay and Electrophoresis of irradiated fish DNA Ergun Sakalar Department of Bioengineering, Faculty of Engineering, Canakkale Onsekiz Mart University, Canakkale, Turkey (*Corresponding Author) Keywords: Comet assay, elektrophoresis, fish, genomic DNA, irradiation Introduction: The radiation treatment of foods like sea food is gaining importance throughout the world. Irradiated food should be adequately labelled according to international and national guidelines. Therefore, there is a need for methods that allow detection of irradiated food [1], as well as for methods that provide a reliable dose estimate. In recent years, the comet assay is a rapid technique for analyzing DNA damage in individual cells [2], but further this tecnique is required to extend the full potential with use of new verification techniques. Methods: Fish samples were exposed to radiation doses of 0, 250, 0.500, 1, 2, 4 and 8 kgy in gamma cell. For each sample, about 1.0 g of very thin slices of fish meat were cut with ascalpel from the fish meat, transferred to a small beaker with ice-cold PBS and stirred. The suspensions were left to settle on ice for about 5min. The supernatant was used as a cell suspension and was mixed with 1 ml of low melting agarose. A 100µl of this mixture was spread on precoated slides. The coated slides were immersed in lysis buffer for 5-7 min. Using the same buffer but devoid of SDS, electrophoresis was performed at 2 V/cm for 2 min. Ethidium bromide staining was employed to visualize DNA. Slides were examined using a microscope. The DNA comets were evaluated by measuring the tail length, tail moment and tail DNA % of comets. All measurement data were analyzed using BS 200 ProP with software image analysis. In addition to Comet Assay, DNAs were extracted from all samples after irradiation. The genomic DNA extracts (5 μl) from fish meats were mixed with 2 μl of gel loading solution, and transferred into 1% agarose gel containing 0.1 mg/ml ethidium bromide in 0.5 X TBE buffer, and electrophoresed for 25 min at 110 V. Genomic DNA was imaged by a UV transilluminator for estimation of genomic DNA bp-fragment size after IR for the purpose of verification of Comet assay. Results: The non-irradiated and irradiated fish samples were clearly distinguishable just by naked eye inspection at the slide under the microscope. The lenght of comet tails increased whereas the amount in the comet head decreases with increasing radiation dose. Based on size of the comets for different doses of radiation, a rough dose estimation may be possible. Gel image of DNA derived from irradiated fish validated that a notable reduction in molecular weight of DNA occured with increase of ionising radiation (IR) dose. In addition, the total intensity of fluorescence decreased with increase of IR dose, and this indicated a decrease in double stranded DNA. Agarose gel image of DNA obtained from non-irradiated fish exhibited a significant amount of 1000-bp DNA and above Discussion & Conclusions: The DNA isolated from irradiated fish can be used in electrophoresis to endorse and control comet eassay results. [1] Stefanova et al (2010), Food Anl. Meth, 3, [2] Erel et al (2009). Radi. Phys. Chem.78:

79 P-BGE5 Potential use of molecular markers in cotton cultivar development Filiz Altan *,1, Abdullah Kahraman 2 1 Department of Molecular Biology and Genetics, Faculty of Sciences, Muğla Sıtkı Koçman University, Muğla -Turkey 2 Department of Field Crops, Faculty of Agriculture, Harran University, S. Urfa- Turkey Keywords: Cotton, fiber quality, molecular markers Introduction: Cotton is a high value crop that is produced as a raw material for the textile industry. Advances in molecular biology and biotechnology have resulted in the development new techniques. The new techniques provide greater flexibility in selection of cotton fiber quality especially strength and length for processing. Molecular markers offer efficient tools for cotton to meet the rapid advances in the textile industry to obtain high productivity [1, 2]. Application of molecular marker assisted selection (MAS) for cotton cultivar development could be easily applied without screening through biotic and abiotic stresses by detection of the presence of any coupling markers [3]. Conclusions: This review lists potential use molecular markers for selection of lines that are resistant to various biotic and abiotic stress factors. [1] Preetha, S., Raveendren, T.S., (2008). Biotechnology and Molecular Biology Review, Vol. 3 (2), [2] Russel, J.K., John, Y., Park, Y., Lazo, G., (2001). Euphytica, 121, [3] Collard, B.C.Y., Jahufer, M.Z.Z., Brouwer, J.B., Pang, E.C.K., (2005). Euphytica, 142,

80 P-BGE6 Linkage mapping of NBS profiling markers in lentil genome Hulya Yilmaz Temel*,1, Abdullah Kahriman 2, Bahattin Tanyolac 1 1 Ege University Deparment of Bioengineering, Izmir, Turkey 2 Harran University, Department of Field Crops, Sanlıurfa, Turkey Keywords: Lentil, mapping, RIL, NBS Introduction: Plants defend themselves against pathogen attacks with resistance (R) genes. The protein products of R-genes encode receptors that specificially recognize pathogen avirulance (Avr) proteins or guard critical components of the plant s physiological processes. Resistance genes which belong to NBS-LRR containing R-gene family have important role in pathogen detection and plant defense. NBS profiling is a PCR based approach that produces many markers in resistance genes and resistance gene analogs (RGAs). Lentil is a diploid (2n = 2x = 14 chromosomes) and self pollinating crop with 4,063 Mbp haploid genome. Lentil has the highest source of protein after soybeans and hemp. In additional to nutritional importance, it also takes role in nitrogen fixation from the atmosphere and forming nitrogen lumps in the soil. Methods: In this study, 101 individuals developed from a Precoz X WA cross, with singleseed descent until the F 7 generation are genotyped by using 38 enzyme-primer combinations. NBS markers were visualized on LICOR platform and 602 markers were scored. Linkage map of lentil is constructed by using JoinMap4 software. The maximum number of markers in one linkage group was 54, the minimum was just 5. Discussion & Conclusions: With this research, we aimed to map the NBS profiling markers in lentil genome. This is the first linkage map constructed using NBS markers. This map will allow to isolate resistance genes to biotic and abiotic stress conditions. Acknowledgements: We acknowledge the Scientific and Technological Research Council of Turkey (TUBITAK) for providing funds for the project (TUBITAK-110O361). [1] Van der Linden, G., Doret C., Wouters V. M., Elena Z. K., Marinus J. M., Smulders B. V. (2004). Theoretical and Applied Genetics 109: [2] Calenge, F., Van der Linden C. G., Van de Weg E., Schouten H. J., Van Arkel G., Denance C., Durel C. E. (2005). Theoretical and Applied Genetics 110: Results: Three hundred and ninety three out of 602 (%65) were mapped on 12 major and 2 minor linkage groups. The 393 markers across cm provided an average marker density of 1 marker per 2.6 cm. Linkage group sizes were ranging from cm to 8.5 cm with a mean length of 73 cm and 28 markers per linkage group. 74

81 P-BGE7 Screening of signal sequences for extracellular production of Aspergillus niger xylanase in Pichia pastoris Hilal Yıldız, Mert Karaoğlan and Mehmet İnan* Department of Food Engineering, Akdeniz University, Antalya, Turkey Keywords: AOX1 promoter, leader sequences, Pichia pastoris, xylanase Introduction: The methylotrophic yeast Pichia pastoris is a widely used for recombinant protein production such as pharmaceuticals and industrial enzymes. Main advantages of P. pastoris are the ability to reach very high cell densities during fermentation in a defined minimal medium and high level secretion of foreign proteins. Purpose of this study was to screen for optimum secretion signal for recombinant xylanase production. Methods: The codon-optimized xylb gene coding for endo-β-1,4-xylanase of Aspergillus niger was subcloned into ppicz B vector with EcoRI and XbaI restriction sites. Resulting plasmid was named ppiczb-xylb. Four different leader sequences were evaluated for extracellular production. These were namely: Saccharomyces cerevisiae -mating factor, HSA (Human Serum Albumin), P. pastoris PHO1 (Acid Phosphatase) and PpPIR1 (Protein with Internal Repeats) secretion signals. Leader sequences were cloned into ppiczb-xylb plasmid with AsuII and EcoRI restriction sites. P. pastoris KM71H (aox1 - ) strain was used as production strain. A single copy clones were selected by Southern blot analysis. Recombinant enzyme production was carried out under the regulation of the AOX1 promoter in methanol containing media at ph 6 and 28 o C in a 250 ml shake flask containing 50 ml of BMGY for growth and BMMY for induction. Results: After 48 h of induction, enzyme activities for clones carrying -mating factor, HSA, PHO and PIR1 as leader sequence were 4247, 4252, 4147 and 5366 U/mL, respectively. Optimum ph and temperature for the enzyme activity was determined as ph 4-5 and 50 C, respectively. Discussion & Conclusions: Pichia pastoris PIR1 leader sequence resulted highest xylanase enzyme activity at shake-flask scale among the tested leader sequences. Acknowledgements: This research was supported by Akdeniz University grant

82 P-BGE8 Functional characterization of ADH genes from Pichia pastoris Mert Karaoğlan, Fidan Erden, Mehmet İnan* Department of Food Engineering, Akdeniz University, Antalya Turkey Keywords: ADH1, ADH3, alcohol dehydrogenase, ethanol metabolism, Pichia pastoris Introduction: Pichia pastoris has ability to grow very high cell densities in a simple defined medium at large scale. It can reach very high cell densities due to its Crabtree negative nature. Therefore it is not expected to produce ethanol in aerobic conditions and high glucose concentrations. However, our previous studies showed that P. pastoris produces ethanol as a by-product in aerobic fermentation conditions which may cause repression of the AOX1 promoter and results in reduced productivity [1]. Alcohol dehydrogenase gene(s) of P. pastoris has not been characterized yet. Only two ADH genes (PpADH1 and PpADH3) has been annotated from the genomes of P. pastoris GS115, DSMZ and CBS 7435 strains based on the sequence homology to ADH genes of Saccharomyces cerevisiae. However, molecular level studies and characterization of the genes has not been done yet. Results: The ADH3 defective strain has lost ability to grow on minimal ethanol media, but able to produce ethanol in minimal glucose media. The results showed that P. pastoris ADH3 gene was the only gene responsible for ethanol catabolism. However, the ADH1 gene did not have any role in ethanol metabolism at conditions tested. Discussion & Conclusions: P. pastoris ADH3 gene was the only gene responsible for catabolism of ethanol. The ADH gene(s) responsible for ethanol production are under investigation. Acknowledgements: This project was supported by the grant 111T905 from the Scientific and Research Council of Turkey (TUBITAK). [1] Inan, M. and Meagher, M. M. (2001). J. Biosci. Bioeng., 92: Methods: In this study, the functional characterization of P. pastoris PpADH1 and PpADH3 were studied. The ADH1 and ADH3 genes were disrupted with functional zeocin resistance gene (Sh ble) and PpHIS4 gene, respectively, in GS115 strain. Deletions of both genes were confirmed by Southern blot analysis and PCR methods. The growth and ethanol production characteristics of wild type and mutant strains were tested in minimal media supplemented glucose and ethanol as carbon sources. 76

83 P-BGE9 Development of Alcohol Oxidase (AOX) defective strains of Pichia pastoris for recombinant protein production Mert Karaoğlan and Mehmet İnan* Department of Food Engineering, Akdeniz University, Antalya Turkey * Keywords: AOX-defective strain, EGFP, Pichia pastoris, xylanase Introduction: Pichia pastoris is an expression system for the production of recombinant proteins. The genome of P. pastoris contains two copies of the alcohol oxidase gene namely AOX1 and AOX2. The AOX1 promoter which is responsible for 85% of total AOX activity is used in recombinant protein production. P. pastoris host strains have three different phenotypes depending on methanol utilization ability. Methanol utilization plus (Mut + ) strains have both genes intact and can utilize methanol similar to wild-type strain. Methanol utilization slow (Mut s ) strain has defective AOX1 gene and growth on methanol relies on the AOX2 gene only with reduced rate. Methanol utilization minus (Mut - ) in which both AOX1 and AOX2 genes are defective and cannot grow on methanol as sole carbon source. Although three phenotypes of P. pastoris strains are available, mut - strains are not utilized as much as mut + and mut s strains for recombinant protein production due to genetic instability of mut - strains and unavailable shakeflask screening protocols for mut - strains. Therefore, we have generated stable mut - strain and tested for recombinant protein production. Methods: The AOX1 and AOX2 genes were inactivated by inserting the functional PpADE1 and PpHIS4 genes in P. pastoris JC304 (his4, ade1) strain, respectively. AOX-defective strains were named MK321 (aox1 - ), MK431 (aox2 - ) and MK500 (aox1 - aox2 - ). The recombinant protein production in these strains was studied in shake-flask conditions. Enhanced Green Fluorescence Protein (EGFP) and Aspergillus niger Xylanase (XylB) genes were subcloned into ppiczαa with XhoI and XbaI restriction sites and transformed into new strains for extracellular expression. Commercially available strains of P. pastoris X33 (wt) and KM71H (aox1 - ) which are mostly-used in recombinant protein production were also used as control strains. The clones with a single-copy vector were selected by Southern blot analysis and protein productions were tested at shake-flask level in methanol containing media supplemented with sorbitol. Extracellular production of proteins has been confirmed using SDS-PAGE and Western Blot analysis. Results: P. pastoris MK500 is capable of producing higher specific yield of EGFP (FU/OD) and Xylanase (U/OD) compared to P. pastoris X33 which is the most commonly used in recombinant protein production. EGFP fluorescence unites for X33 and MK500 were 3406 and FU/OD, respectively. Xylanase activities were 79.8 and U/OD for X33 and MK500, respectively. Discussion & Conclusions: The results showed that newly developed P. pastoris (mut - ) is capable of producing higher specific yield of proteins compared to P. pastoris X33 which is the most commonly used in recombinant protein production. The new strain which cannot utilize methanol as a sole carbon source requires methanol only as an inducer; in turn reduces total methanol utilized especially at large scale processes. Acknowledgements: This project was supported by Akdeniz University The Scientific Research Projects Coordination Unit with project number

84 P-BGE10 Whole genome sequencıng of an EPS producer Brevibacillus thermoruber Songul Yasar Yildiz 1,*, Margarita Kambourova 2, Kazim Yalcin Arga 1, Ebru Toksoy Oner 1 1 Department of Bioengineering, Marmara University, Istanbul, Turkey, 2 Department of Extremophilic Bacteria, Institute of Microbiology, BAS, Sofia, Bulgaria Keywords: Brevibacillus thermoruber, exopolysaccharide, whole genome sequencing Introduction: Extremophilic microorganisms evolved many adaptation strategies to extreme conditions one of which involves the production and excretion of high molecular weight exopolysaccharides (EPSs) having unique physicochemical and rheological properties with novel functionality. Due to their advantages like high EPS production rates due to their fast metabolic activities and lower viscosity of the fermentation medium, thermophiles can be considered as good cell factories for industrially important biopolymers [1]. However, there is very limited literature on the microbial EPS production by thermophiles. Consequently, within the scope of this study, an EPS producer Brevibacillus thermoruber strain was selected as a model organism to investigate EPS production mechanisms. Methods: As a first, whole genome sequencing was performed in duplicate via high-throughput Illumina HiSeq 2000 next generation sequencing technology. Short reads were assembled into genome sequence via SOAPde novo. Assembly result is local assembled and optimized according to paired-end and overlap relationship via mapping reads to contig. The annotation of the draft genome was done via the RAST annotation server [2]. Results: The draft genome of B. thermoruber was structured with 25 contigs/15 scaffolds with a genome size of 4.43 Mbp. The resultant draft genome had a mean GC content of 58.46%, 4,446 coding sequences and 112 RNA genes. Of the coding sequences, 42% could be assigned to subsystems. Putative functions could be assigned to 3020 protein-coding genes, whereas 1426 hypothetical proteins had no match to any known proteins. Table 1. Assembly Results Content Scaffold Contig Total Number (#) Total Length (bp) 4,433,503 4,433,037 N50 (bp) 596, ,785 N90 (bp) 332, ,246 Max Length (bp) 783, ,797 Min Length (bp) Sequence GC (%) 58,46 58,46 Discussion & Conclusions: In systems biology research, the microbial genome sequence is the starting point for detailed analysis of identifying gene-protein associations and metabolic reconstruction. Therefore, whole genome sequencing of Brevibacillus thermoruber will provide additional information to enhance understanding of the genetic and metabolic network of thermophilic bacteria. Acknowledgements: This study is financially supported by TUBITAK through projects TBAG- 111T016 and MAG-110M613, and MONM TK02-46/2009 [1] Nicolaus B., Kambourova M., Toksoy Oner E. (2010). Enviromental Tech., 31 (10), [2] Aziz R.K., Bartels D., Best A.A., DeJongh M., Disz T., Edwards R.A., Formsma K., Gerdes S. et al., (2008). BMC Genomics, 9,

85 P-BGE11 Using ipbs-retrotransposons-based phylogeny analysis in common bean Tala Kianoosh 1, Seda Nemli 1,M. Bahattin Tanyolac 1* 1 Department of Bioengineering, Ege University, Izmir/TURKEY Keywords:, Common bean, genetic diversity, retrotransposons, structure Introduction: ipbs (inter-primer binding site), which is based on highly or universally conserved retrotransposon primer binding site sequences, is based on presence of a trna complement as a reverse transcriptase PBS in LTR retrotransposons. And also, this method as such has proved to be a powerful DNA fingerprinting technology without the need for prior sequence information. Methods: A total of 67 common bean genotypes collected from different countries (17 of which had been introduced from different countries, while the remaining 50 were from Turkey). In this study a total 83 ipbs primers was attempted[1]. Selected 45 ipbs primers were useful in the production of polymorphic bands for PCR analysis. Results: A total number of fragments generated were 174, of which all were polymorphic fragments. Genetic similarity estimates were performed using Numerical Taxonomy and Multivariate Analysis System (NTSYS)-pc version 2.1 software[2]. STRUCTURE software was used to discriminate the 67 common bean genotypes into 4 major groups (K=4) that correlated with genotypes location. Discussion & Conclusions: This study also demonstrates the utility and role of ipbsretrotransposons can be used to study genetic diversity and these markers seems to be universal and transferable across many organisms in which retrotransposons have ipbs elements. [1]Kalendar (2010). Theor Appl Genet. 121: [2]Rohlf (2002). Exeter Publishing, Setauket, NY, US. 79

86 P-BGE12 Comparative analysis of psoriasis transcriptome across different platforms Tuba Sevimoğlu*,1, Kazım Yalçın Arğa 2 1 Marmara University,Department of BioEngineering, Istanbul, Turkey 2 Marmara University, Department of BioEngineering, Istanbul, Turkey Keywords: Gene expression, microarray, platform comparison, psoriasis Introduction: Psoriasis is an autoimmune disease that affects approximately 2% of the world s population [1]. Genetic and environmental factors play a significant role. It is a debilitating disease that can greatly impact on quality of life. Though there are several published gene expression studies, comparison between different microarray platforms for psoriasis has not been done before. Methods: A comparison of expression data from three independent studies, GSE14905, GSE41745 and GSE40033, in which raw data was deposited in Gene Expression Omnibus has been performed. A total of 81 patients were involved in this study. These datasets are from three different microarray platforms: Affymetrix, Illumina and Agilent. Each dataset was statistically analyzed in Bioconductor ( in order to identify differentially expressed genes (DEGs). DEGs were selected according to computed p-values (p<0.05), and up/down-regulation were identified according to fold changes. The DEGs with fold change of more than two were accepted as up-regulated and the DEGs with fold change of less than 0.5 were accepted as down-regulated. The pathway, disease and gene ontology enrichment analyses were carried out by the DAVID bioinformatics tool [2]. The visualization of the protein-protein interaction network was performed by Cytoscape [3]. Hub proteins were found by Cytoscape plugin Cytohubba [4]. Results: Twelve DEGs have been found to be common between the datasets. Three of the genes were down-regulated and the rest were upregulated. Five of the common genes have been linked to psoriasis in other studies. Ten of the twelve DEGs have been mapped in a protein interactions network. Two of the common DEGs have no known interactions. Comparison of enrichment analysis shows that all three datasets have a common pathway: Vascular smooth muscle contraction. Discussion & Conclusions: Despite differences in microarray platforms, sample sizes and sampling sites common DEGs have been found. This result ensures that different platforms can be compared. This study provides additional insight into the molecular mechanism of psoriasis. [1] Piruzian, (2010). BMC Systems Biology, 4:41 [2] Huang et al, (2007). Nucleic Acids Res. July; 35,W169 W175. [3] Shannon et. al. (2003). Genome Res. Nov;13(11): [4] Lin et. al. (2008). Nucleic Acids Res. 36. W438- W

87 P-BIM1 in vitro characterization of cell encapsulating poly(ethylene glycol) scaffolds cultured under mineralizing conditions Cigdem Demirkaya 1, Yoke Chin Chai 2,3 Abhijith Kundadka Kudva 3,4, Aylin Sendemir Urkmez 1, Jennifer Patterson 3,4,* 1 Bioengineering Department, Ege University, Izmir, Turhey 2 Skeletal Biology and Engineering Research Center 3 Research Division Prometheus 4 Department of Metallurgu and Materials Engineering, KU Leuven, Belgium Keywords : Hydrogel, mineralization, poly(ethylene glycol), 3D scaffold Introduction: Many researchers are investigating methods through which biomaterials, from ceramics to polymers, can be better integrated with natural bone. Some approaches focus on the creation of functional polymer scaffolds that mimic the bone extracellular matrix (ECM), direct biomineralization, and stimulate cell adhesion, proliferation, migration, and differentiation. Hydrogels are polymer-based materials with a high water content and thus suitable to use as scaffolds for tissue regeneration. Hydrogels made from poly(ethylene glycol) (PEG) are one of the most known and studied hydrogel systems because they offer good biocompatibility, non-immunogenicity, and resistance to protein adsorption. Endfunctionalized multi-arm PEG precursors can be crosslinked into an enzymatically degradable hydrogel network using di-thiol containing protease substrates in a Michael-type addition reaction [1]. Using the same chemistry, various bioactive peptides can be incorporated into the PEG hydrogels to enhance the adhesion of cells and promote a biomimetic environment for cells encapsulated in the hydrogel. The most commonly researched adhesive peptide, Arg-Gly-Asp (RGD), is found in cell-binding domains of several ECM proteins. Methods: Human periosteum-derived cells (hpdcs) were encapsulated in degradable PEG hydrogels that were modified with an RGDcontaining peptide. The PEG scaffolds were incubated in different culture conditions consisting of growth medium supplemented with calcium (Ca2+) and phosphate (Pi). The Presto Blue metabolic activity assay for cell proliferation, staining/microscopy for hpdc attachment, Live/Dead assay for cell viability, and alkaline phosphatase activity assay for cell differentiation were performed at 0, 7, 14, 21, 28, 35, and 42 days. NanoCT was used to characterize the distribution and volume of mineral deposited within the scaffold. Results: According to the Presto Blue assay, after 21 days, cells encapsulated in PEG and cultured in growth medium with dexamethasone were continuing to proliferate. Metabolic activity of encapsulated cells cultured in growth medium as well as growth medium with Ca2+ and Pi peaked at 14 days and then decreased. From images of phalloidin and DAPI stained hydrogels, hpdcs in PEG gels cultured in medium containing Ca2+ and Pi exhibited a more spread morphology than the other conditions. NanoCT analysis showed that incubation of cell-free hydrogels in medium containing Ca2+ and Pi led to mineral deposition primarily on the surface of the hydrogels whereas cell encapsulation in the hydrogels led to mineral being deposited also inside the scaffolds. Discussion & Conclusions: In conclusion, we have identified culture conditions that support the growth of hpdcs in a 3D PEG scaffold while promoting construct mineralization. : [1] Patterson, J., Hubbell, J.A., (2010). Biomaterials. 31:

88 P-BIM2 The Utilization of Bacterial Cellulose and Hydroxyapatite- Bacterial Cellulose Composite as a Tissue Engineering Scaffold Ece Bayır 1, Eyüp Bilgi 1, E. Esin Hameş-Kocabaş 2, Aylin Şendemir- Ürkmez 1,2, * 1 Department of Biomedical Technologies, Graduate School of Natural and Applied Sciences, Ege University, Izmir, Turkey 2 Department of Bioengineering, Faculty of Engineering, Ege University, Izmir, Turkey Keywords : Bacterial cellulose, Hydroxyapatite, EPC, SaOS-2, scaffold Introduction: The advances in natural polymers have utilized their use in biomedical field due to their sustainability, biodegradability and higher biocompatibility compared to synthetic polymers [1]. Bacterial cellulose (BC) is a biopolymer produced by certain bacteria and has same chemical properties with plant cellulose [2]. The unique properties such as high surface area per unit mass, nano-micro porous structure, hydophilicity, biocompatibility, purity makes it an attractive candidate for a wide range of usage in biomedical and biotechnological applications [3]. Hydroxyapatite (HAp) can be found in bones within the human body. In vitro studies have revealed that biopolymer-hap composites have a great potential for application in tissue engineering and bone regeneration [4]. The aim of the study is to investigate the usage of BC and BC-HAp composites as a scaffold for human endothelial progenitor (EPC) and human osteosarcoma (SaOS 2) cell lines. Methods: BC is obtained from static cultures of Gram-negative bacteria, Gluconacetobacter xylinus ATCC in Hestrin & Schramm (H&S) medium. BC-HAp composites were produced by adding HAp powder in culture medium. EPC and SaOS 2 cell lines were cultured on both BC and BC- HAp composites at 37 C in an atmosphere of 5% CO 2 using a humidified incubator. Results: BC-HAp composites were characterized with FTIR, SEM and XRD. Viability of EPC and SaOS 2 cell lines on BC and BC-HAp composites were assessed by MTT assay and cell lines were monitored with fluorescence microscope. Fig 1. Bacterial cellulose scaffolds form a strong hydrogel when hydrated Discussion & Conclusions: We suggest that BC can be used as tissue engineering scaffold for angiogenesis and vascularization studies and BC- HAp composites can be used for repairing of bone defects. [1] Petersen, N., and Gatenholm, P., (2011). Appl Microbiol Biotechnol 91: [2] Vitta, S., and Thiruvengadam, V., (2012). Current Science 102: [3] Fu, L., et al., (2013). Carbohydrate Polymers 92: [4] Saska, S., et al., (2011). International Journal of Biomaterials 2011:

89 P-BIM3 A preliminary study for the assessment of I 125 absorption by kaolin, pumice and clinoptilolite Zeynep Yılmaz 1, İmran Göker* 1, Can Duman 2, Sevgi Özyeğin 3 1 Department of Biomedical Engineering, İstanbul Arel University, İstanbul, Turkey 2 Department of Medicinal Biology, Çanakkale 18 Mart University, Çanakkale, Turkey 3 Keywords : Absorption, biomaterial, clinoptilolite, kaolin, radioactivity Introduction: Aluminosilicate minerals are high absorptive materials with mesoporous structure and they are used in bioabsorption systems [3]. Their structures enable them to absorb radioactive elements such as uranium, cesium, iodine and radon [2,4]. I 125 is used in thyroid therapy. After the therapy, patients emit radioactivity via skin, faeces and urine for a given period depending on the administrated dosage. This emission can cause serious contamination in wastewater [5]. Therefore, contamination control and storage applications are expensive. Methods: In our preliminary study, we used calibrated kaoline, clinoptilolite and pumice. Samples are crushed in agate mortar and sieved through 30 mesh sieve. Subsieve material was used. I 125 -bound anti-shgb (370kBq ( 10 mikrocurie) in 22 ml) was studied. Measurements were done by SR 300 AutoRIA (SL 300, Stratec Biomedical AG, Germany) in triplicate. Radioactivity was recorded by means of gamma radiation per minute. Statistical analyses were made by F-test. Results: In our study, we have found out that standardized kaolin, clinoptilolite and pumice have high I 125 sorption capacity. The results demonstrated that absorption rate of clinoptilolite and pumice is significantly higher than kaoline (p<0.001, p<0.01 respectively). But difference between clinoptilolite and pumice is less significant than those expressed (p<0.05). Table 1. Radioactivity absorption of kaolin, clinoptilolite and pumice Sorption rate (%) (mean ± SD) mg mg mg Clinopti lolite (n=7) Pumice (n=7) Kaolin (n=7) 6.8 9± ± ± ± ± ± ± ± ±0.27 Discussion & Conclusions: Our samples were capable of binding I 125 [1]. There are similar studies showing that kaoline and clinoptilolite are used orally for absorption of radiation [3]. So, clinoptilolite could be used to bind iodine when it is taken orally. Further studies are required to reveal the effectiveness of clinoptilolite for the absorption of radioactivity. Since kaolin and pumice has good binding properties for I 125 in faeces, it could be used for elimination of patients faecal wastes. Because of the high mineral potential of Turkey, these results could lead new applications of these minerals in many areas. [1]Osmanlioglu, A. E. (2007). Applied Radiation and Isotopes 65, [2]Osmanlioglu, A. E. (2002). Waste Management, 22, [3]Pavelic, K and Mirko Hadzija M. (2003). Marcel Dekker, Inc., New York. [4]Mokobia, C.E. (2011). Scientia Africana, 10, [5]Khalil, M. M. (2011). Springer Verlag, Berlin Heidelberg 83

90 P-BPE1 Carotenoid accumulation in 2-methyl imidasole and amitrole treated Dunaliella salina Arzu Yıldırım*,1, İsmail Hakki Akgun 1, Meltem Conk Dalay 1 1 Ege University, Faculty of Engineering, Bioengineering Department, Izmir, Turkey Keywords: Carotenoids, chemical inhibitors, Dunaliella salina Introduction: Imidazoles are group of chemical inhibitors shown to inhibit the enzymes responsible from cyclisation of crotenoid molecules, therefore; they have been used to regulate carotenoid biosynthesis in different organisms. 2-methyl imidasole (2MI) was successfully applied for lycopene production from Blakeslea trispora, a flamentous fungus known as microbial producer of β-carotene [1]. Similarly, Amitrole treatment of maise was resulted in the accumulation of lycopene in the leaves with a small amounts of β-carotene and xantophylls as well [2]. Dunaliella salina is a commercially valuable species of green microalgae with the ability to produce high amounts of natural β-carotene in its unicell. Methods: Amitrole and 2-methylimidazole were added to the cultures of D. salina in different concentrations and the effects on carotenoid composition were investigated on the cultures incubated at 28 C followed by 35 C on a rotary shaker (100 rpm) with 12:12 h illumination of 50 µmol m -2 s -1. Results: 2-methylimidazole at 3 mm concentration was highly toxic to the cells, so that the culture was completely bleached after the 10th day of the experiment. 2MI at 1 mm concentration showed the highest level of lutein content (1,5 fold) respect to control cells. The increase in the temperature resulted an enhancement in the accumulation of both clorophylls and carotenoids in the amitrol treated D. salina cells as expected, Presenter Author Photograph however; lycopene was not observed in both 2MI and amitrole treated cultures in U-HPLC analysis. In order to enhance lycopene accumulation in the cell, D. salina was transformed with phytoene synthase gene, which is responsible to synthase the first carotenoid in the pathway. Discussion & Conclusions: Phytoene enriched D. salina is expected to show lycopene accumulation after the treatment of cyclisation inhibitors as suggested [3]. The results of this study may give the opportunity to use D. salina as a host for the production of valuable carotenoids other than β- carotene. Acknowledgements: This work has been supported by the projects from Turkish General Directorate of Agricultural Research and Policy (TAGEM) [1] Pegklidou, K., Mantzouridou, F. and Tsimidou, M.Z., (2008). J. Agric. Food Chem. 56: [2] Dalla Vecchia, F., Barbato, R., La Rocca, N., Moro, I., Rascio, N. (2001). J. Exp. Bot. 52(357): [3] Shaish, A., Avron, M. and Ben-Amotz, A., (1990). Plant Cell Physiol. 31(5):

91 Ln (Reducing Sugar) (mg/ml) P-BPE2 Modeling reducing sugar release from ammonia treated wheat straw Sonnur Yazıcı, Aslı Isci* Ankara University, Food Engineering Department, Ankara, Turkey P Keywords: Aqueous ammonia soaking, enzymatic hydrolysis, lignocellulosic biomass, pretreatment, wheat straw Introduction: Biorenewable resources and the fuels produced from them are poised to reduce our dependence on petroleum, create new domestic job opportunities and improve environmental quality. Bioethanol is one of the most widely promoted and promising alternative fuels. Cellulosic ethanol has the advantage of abundant and diverse feedstock. It also promotes the utilization of agricultural residues [1]. The objectives of this study were to determine the effect of various operating variables on enzymatic digestibility of wheat straw. It was also aimed to determine whether an empirical model can be developed that relates total reducing sugars released during enzymatic hydrolysis to pretreatment variables. Methods: Milled wheat straw samples were pretreated with 10% and 30% of aqueous ammonia in screw-capped glass bottles at 30, 50 and 70 C for 15, 30, and 45 hours. Solid to liquid ratio of 1:6 was applied. Enzymatic hydrolysis experiments were at 5% (w/w) solid loading, 50 C and 150 rpm for 96 hours, using cellulase enzyme (Accelerase 1500). Samples were analyzed for glucose and xylose content using HPLC. Total reducing sugars in the samples were determined using (DNS) method [2]. Results: Maximum enzymatic hydrolysis yields (76% for glucose and 81% for xylose) were achieved when the samples were pretreated with 30% ammonia at 50 C for 45h. As the severity of the pretreatment was increased, higher reducing sugar release was observed. It was also found that hydrolysis of polymeric carbohydrates to reducing sugars vary exponentially with the reciprocal of the pretreatment temperature (Fig. 1). For 10% ammonia loading, reducing sugar concentration can be predicted using the following equation, RS e PT PT T while at 30% ammonia loading reducing sugar concentration (mg/ml) is correlated to pretreatment time (h) and temperature (K) as follows RS ( PT 12490) e PT T /Pretreatment Temperature (1/K) 15h-10% 15h-30% 30h-10% 30h-30% 45h-10% 45h-30% Fig. 1. Change of natural logarithm of total reducing sugar concentrations with reciprocal of pretreatment temperatures Discussion & Conclusions: Ammonia soaking is an effective pretreatment method for wheat straw. An empirical model was developed that relates reducing sugar concentrations to pretreatment time and temperature. The correlation between ammonia soaking conditions and reducing sugars could be an area of further studies. [1] S. Kim, B. E. Dale. (2004). Biomass and Bioenergy 26: [2] G. L. Miller. (1959). Analytical Chemistry 31:

92 P-BPE3 Biological properties of microalgal enzymatic hydrolysates from Chlorella vulgaris SAG b Ayse Kose 1, Suphi Oncel 1 *, Murat Elibol 1 1 Ege University, Faculty of Engineering, Bioengineering Department, Bornova, Izmir, Turkey Keywords: Bioengineering, bioreactor, enzymatic hydrolysis, microalgae Introduction: Immunonutrition is a new concept and highlight worthy issue for daily life of human beings in terms of balanced nutrition and immune defense systems. Daily life stress, inadequate nutrition, acute or chronic diseases require to fulfill the gap in the immune enhancing dietary products. To avoid protein malnutrition; single cell proteins like bacteria, yeast and microalgae are used as dietary supplements [1]. Among these organisms microalgae have unique features like generally regarded as safe organisms (GRAS), easy cultivation for mass cultures, low cost and land requirement, ability to use broad range of nutrients compared to field crops. Also microalgae have some valuable biocompounds for pharmaceutical and neutriceutical uses [2, 3]. Among microalgae Chlorella vulgaris is protein - rich green unicellular freshwater microalgae with fast growing, antitumor, immune enhancing features. But although they have high amounts of proteins it is not able for non-ruminants to digest the whole cells because of thick cellulosic cell walls [4, 5]. Methods: Cells grown in sucrose, fructose, glucose as carbon sources; proteose peptone, yeast extract and sodium acetate as nitrogen sources are selected. BG11 medium is used as control media for algae culturing. 1 g/l and 5 g/l of carbon and nitrogen sources are chosen as minimum and maximum levels. The cells are hydrolysed with pancreatin enzyme (SIGMA) in order to break down the cell walls. Dry weight, total protein, total sugar and fatty acid analyses are done to determine the biochemical composition. Results: The effect of carbon and nitrogen sources to cell growth and hydrolysis is searched. The cultures grown in 5 g/l glucose showed a highest dry weight of 2.31±0.16 g/l among all samples and for nitrogen experiments 5 g/l proteose peptone has 1.13±0.08 g/l dry weight compared to control BG11 cultures which have 0.83±0.06 g/l dry weight. The total protein contents of the cells showed that addition of nitrogen sources into cultures increases the protein content which has highest yield of total protein in proteose peptone experiments (76.46±3.6 mg/l). Also sugar content showed similar results. The fatty acid content for samples was 20-25% of dry weight % of the proteins are hydrolysed with pancreatin enzyme. Discussion & Conclusions: The results are promising for further utility of hydrolysed algae cell proteins for pharmaceutical and neutriceutical industries. The disruption of intact cells results in the ability to use the amino acids and other components like fatty acids, pigments with higher digestibility. Acknowledgements: The authors would like to thank Ege University BAP Committee for financial support. [1] Becker, E.W. (2007). Biotechnology Advances 25: [2] Borowitzka, M.A. (1995). Journal of Applied Phycology 7:3-15. [3]Janczyk P. (2007). Animal Feed Science and Technology 132: [4] Morris, H.J. (2008). Bioresource Technology 99: [5] Morris, H.J. (2011) Journal of Medicinal Foods

93 P-BPE4 Lactic acid production profile of some Lactobacilli Tugba Aksoy, Bilge Hilal Cadırcı* Bioengineering Department, Faculty of Natural Sciences and Engineering, Gaziosmanpasa University, Tokat Keywords: Biomaterial, fermentation, Lactobacilli, poly lactic acid, purification Introduction: Poly lactic acid (PLA) is aliphatic polyester which is polymerized by pure L-LA or D- LA, or mixtures of both components. PLA is very attractive biomaterial for textiles and food packaging industry and biomedical applications like sutures, clips and drug delivery systems [6]. Lactic acid especially L-Lactic acid is produced as a by-product of fermentation procedure by microorganisms. Lactic acid bacteria are a group of gram (+), non-spore forming, cocci or rod shaped microorganisms which produce one or two moles of lactate per one glucose molecule via hetero- or homo-fermentation, respectively [1] Although global lactic acid production reached 450,000 tonnes with an average annual growth of 10%, it doesn t have the deserved attraction in Turkey. In this study we aimed to isolate homofermentative lactic acid bacteria and make them to produce lactic acid from by products like corn and to purify and characterize lactic acid as a component of PLA. Methods: Lactic acid bacteria were isolated from decimal dilution of yoghurt by pour-plate methods on de Mann Rogosa Sharp (MRS) Agar. The cultures were incubated microaerobically at 37 C for 3 days. Colonies with different colony shape were selected and stored in skimmed milk with 20 % glycerol at -80 C [4]. Fermentation profiles of the isolated were examined individually by Benedict reaction. Briefly, lactic acid bacteria were incubated in yeast-glucose medium (Na 2HPO 4,30 mm; glucose, 10 mm; yeast extract, 1.8 gl; NH 4Cl, 6 mm. The glucose was made up in a l00x stock solution, filter sterilized and added after autoclaving of the medium, [2] media till glucose in the media is consumed which is detected by Benedict Reaction [3]. Fermentation state was determined by titration of lactic acid with NaOH which is monitored by phenol phthalein. Lactic acid was purified from fermentation broth by esterification and hydrolysis method [5]. Results&Conclusion: Thirteen lactic acid bacteria were isolated from yogurt and the fermentation patterns were detected. Lactic acid purification is performed. Some modification strategies aim at tailoring the properties of PLA-based materials for certain applications will be done in our department for the following studies. [1] Beasley, S. (2004). Yliopistopaino, Helsinki, ISBN [2] Gisi, D. (1996) Microbial Diversity Course, MBL, 1-6 [3] Patel, M. A., Ou, M. S., Harbrucker, R., Aldrich, H. C., Buszko, N. L., Ingram, L. O. and Shanmugam, K. T., (2006). Applied and Environmental Microbiology 72/5: [4] Patil, M. M., Pal, A., Anand, T. and Ramana, K. V. (2010). Indian Journal of Biotechnology 9: [5] Sun X., Wang Q., Zhao W., Ma H. and Sakata K. (2006). Separation and Purification Technology 49/1: [6] Xiao, L., Wang, B., Yang, G., and Gauthier, M. (2012). Biomedical Science, Engineering and Technology Dhanjoo N. Ghista (Ed.) ISBN:

94 P-BPE5 Comprehensive procedure to obtain axenic cultures of local microalgae species for photoheterotrophic and heterotrophic microalgal growth and lipid production Duygu Ayyildiz-Tamis 1, Müge Isleten-Hosoglu 1 and Murat Elibol 1 1 Ege University, Faculty of Engineering, Bioengineering Department, Bornova, Izmir, Turkey Keywords: Axenic culture, biodiesel, eicosapentaenoic acid, photoheterotrophic cultivation Introduction: In recent years, much attention has been attracted to effective mass cultivation of microalgae for lipid production. The selection of suitable microalgae species with a high biomass and lipid productivity has been gaining importance [1-3]. Photoheterotrophic and/or heterotrophic cultivations in which microalgae can grow on organic substrates can offer a well-controlled process. Also, they provide the possibility to maximize microalgal growth and lipid production on a large scale compared to autotrophic cultivation [1-5]. The first important step before adaptation of microalgae to photoheterotrophic and/or heterotrophic conditions is to obtain their axenic cultures. Methods: A comprehensive procedure to obtain axenic cultures of selected microalgae species Cylindrotheca closterium, Ettlia Texensis, Chlorella sp., Dunaliella sp and Nannochloropsis sp. from our local isolates were established. Axenic isolation of these species was achieved by different procedures involving filtration, Percoll density gradient centrifuge, treatment with antibiotic mixture (penicillin, streptomycin sulfate, gentamycin sulfate), growth on agar medium or in liquid medium with serial dilutions. Axenic status of cultures were confirmed after sub-culturing three times in their sterile medium with glucose (1g/L) and on nutrient agar and also with 4',6-diamidino- 2-phenylindole (DAPI) staining. After that, those microalgae species were grown under autotrophic, photoheterotrophic and/or heterotrophic conditions. Results: Final biomass and lipid productivities and the fatty acid compositions of Cylindrotheca closterium, Ettlia Texensis, Chlorella sp., Dunaliella sp and Nannochloropsis sp under autotrophic conditions was shown in Table 1. Table 1: Biomass productivity and fatty acid composition of the selected microalgae species under photoautotrophic conditions. In photoheterotropic condition, Ettlia texensis provided the highest biomass productivity and total lipid content of 0.97 g/l.d and 26% of dry weight basis, respectively. Chlorella sp provided the highest biomass productivity and total lipid content of 0.36 g/l.d and 25% of dry weight basis, respectively. In addition, Dunaliella sp and Nannochloropsis sp. gave roughly 2-fold biomass productivity under photoheterotropic condition. Discussion & Conclusions: The eicosapentaenoic acid (EPA) content of total fatty acids makes C. closterium as a potential source for the production of ω-3 polyunsaturated fatty acid. Acknowledgements: This work is supported through research grants from Ege University Science-Technology and Research Center (EBILTEM-2010/BIL/013) and also The Scientific and Technological Research Council of Turkey (TUBITAK) with a project number of 109M227. [1] O. Perez-Garcia, et al. (2011). Water Res. 45: [2] R. Vazhappilly, F. Chen, J. Am. (1998). Oil Chem. Soc. 75: [3] H. Xu, et al. (2006). Biotechnol. 126: [4] Isleten-Hosoglu, M., Ayyıldız-Tamis, D., Zengin, G., Elibol, M. (2013). Bioresource Technol. 131: [5] Isleten-Hosoglu, M., Gultepe, I., Elibol, M. (2012). Biochem Eng J. 61:

95 P-BPE6 Optimization of human lysozyme production by Kluyveromyces lactis K7 in biofilm reactors Duygu Ercan, Ali Demirci * Department of Agricultural and Biological Engineering The Pennsylvania State University, University Park, PA, USA * Keywords: Biofilm, growth parameters, human lysozyme, Kluyveromyces lactis K7, plastic composite Introduction: Lysozyme is an antimicrobial compound without any acute toxicity. Therefore, lysozyme has been used in pharmaceutical and food industries. Nowadays, egg is the commercial source of lysozyme. However, human lysozyme is better and safer than egg-white lysozyme. Therefore, the production of human lysozyme is needed to meet the demand. Kluyveromyces lactis K7 is genetically modified microorganism, which can produce human lysozyme. A novel bioreactor, which is biofilm reactor with plastic composite support (PCS), can be used to enhance the fermentation process because it provides high cell concentrations with low nutrition requirements for the fermentation medium. Methods: Three factors Box Behnken design was used to find optimum growth parameters combination and fermentation medium composition. Fig 1. Biofilm reactor design with PCS. Results: Both lysozyme and biomass production decreased when the temperature decreased (Fig. 2). Moreover, ph and aeration decrease caused increase in lysozyme production. In contrast to lysozyme production conditions, biomass production increased when the aeration level and ph increased. Although the effect of lactose on production was higher than the effects of casamino acid and yeast nitrogen base, both casamino acid and yeast nitrogen base concentration decrease caused decrease in lysozyme production. Fig 2. Effects of parameters on lysozyme production. Discussion & Conclusions: Based on the results of optimization of growth parameters, different ph and aeration shift strategies were tested and the ph decrease without any control provided the highest lysozyme production level (141 U/ml) without aeration at 25 C and no aeration. Then, by the optimization of medium composition, the production level was increased to U/ml with the medium containing the lactose, yeast nitrogen base, and casamino acid concentrations at 16.3, 0.8, and 1.2%, respectively. In conclusion, higher production level and production rate were observed in biofilm reactor ( U/ml and 4 U/ml/h) than suspended cell reactor (110 U/ml and 2 U/ml/h). Acknowledgements: This work was supported in part by Turkish Ministry of Education by providing scholarship to Duygu Ercan, the Pennsylvania Experiment Station, and Agricultural Sciences Graduate Student Competitive Grants Program. 89

96 P-BPE7 Long term monitoring of an anaerobic digester treating chicken manure with a high ammonia content Duygu Karaalp 1, Gülizar Çalışkan 2, Nuri Azbar*,2 1 Ege University, Biotechnology Department, Institute of Science, Izmir, Turkey 2 Ege University, Bioengineering Department, Engineering Faculty, Izmir, Turkey (*Corresponding Author) Keywords : Anaerobic fermentation, biogas, BMP, chicken manure Introduction: In parallel to the increase in the production of chicken and other side products, tremendous amount of waste material has also been generated throughout Turkey. The current disposal method for chicken manure in Turkey is generally direct release to receiving media including land and aquatic environment. In this study, we have demonstrated the use of chicken manure alone using a fed-batch anaerobic process configuration with varying dry matter content Methods : A continuously stirred tank reactor (2 liter size fermentor with a fully automatic control via PLC) was used as a mesophilic (37 o C) anaerobic reactor to examine the effect of high loadings of dry matter and ammonia on the methane yield of the system. The retention time was kept at 30 days and the loading rate was maintained in the range kg VS m -3 d -1. Results: Fig.1-2 depicts some of the operational parameters that have been monitored for over 550 days. It was seen that high initial dry matter content negatively affects the process performance. On the other hand, dilution of feed down to 3-5% DM provides quite stable performance with high gas production. Q (L/grVS) 1,2 1,0 0,8 0,6 0,4 0,2 0,0 Q(L/grVS) Time (day) Fig.2. The time course of TN, DM%, Free NH3 Discussion & Conclusions: A few studies, including our previous study, have demonstrated that acclimation of methanogenic consortia to high ammonia levels was effective to raise ammonia tolerance for methane production. Use of this technology could possibly deliver substantial dry methane fermentation of chicken manure by maintaining the level of ammonia below the inhibition level of methane production. Acknowledgements: The authors would like to express appreciation for the support of the TÜBİTAK [Project Number = 111Y019]. : [1] Bujoczek, G., Oleszkiewicz, J., Sparling, R., Cenkowski, S. (2000). J. agric. Engng Res. 76; [2] Callaghan, F.J., Wase, D.A.J., Thayanithy, K., Forster, C.F. (2002). Biomass and Bioenergy 27;

97 P-BPE8 Cultivation of Botryococcus braunii for production of hydrocarbons and effects of cryopreservation method on cell viability Ece Yildiz 1, *, Aslihan Kazan 1, Ozlem Yesil-Celiktas 1 Department of Bioengineering, Faculty of Engineering, Ege University, Bornova- Izmir, Turkey Keywords: Botryococcus braunii, cell viability;,cryopreservation, cultivation, microalgae Introduction: Botryococcus braunii is a green colonial fresh water microalgae that have the ability to synthesise and accumulate variety of lipids including numerous hydrocarbons [1]. B. braunii produces three types of hydrocarbons, associated with three chemical races A (alkadienes, trienes), B (triterpenes) and L (tetraterpene) [2]. Although the levels and distribution of hydrocarbons vary with algal origin, 95 % of the produced hydrocarbons are localized outside the cell wall [3]. It s extremely energy-consuming process to synthesize highenergy hydrocarbon in cells, which means the growth rate of B. braunii is low. The doubling generation time of B. braunii is usually 6-7 days in natural condition and about 3-5 days in shaking condition. However, B. braunii could grow fast under optimal conditions [4]. In the changing energy scenario for renewable energy sources, hydrocarbons and exopolysaccharides of B. braunii are potential sources for the production of renewable polymers and biodegradable products. In light of this, the biotechnological exploitation of this potential microalga for hydrocarbons needs to be explored [5]. Methods: In this study cultivation of B. braunii and investigation of the freezing procedures and their effects on cell viability were investigated. For this purpose, firstly B.braunii was cultivated using BG- 11 medium at 25 o C under aeration and 60 µem -2.s -1 light intensity and the production volume was scaled up from 250 ml to 10 L. After cultivation, microscobic screening of microalgae was carried out and biomass from production medium was separated by centrifugation. Subsequently, algae biomass was lyophilized and recultivated in plates in order to examine cell viability. medium was separated by centrifugation. Subsequently, algae biomass was lyophilized and recultivated in plates in order to examine cell viability. Results: The results along with microscopic investigation indicated that the freezing step had an adverse effect on cell viability. Therefore DMSO and glucose solutions with different concentrations were used as cryoprotectants to sustain higher cell viability after freezing. Initial results revealed the significance of both the concentrations of cryoprotectants and the biomass. Discussion & Conclusions: The experiments to improve the cell viability after cryopreservation are still in progress. Acknowledgement: Financial support provided by EU FP7 SPLASH Project (311956) is highly appreciated. [1] Rao A. R., Ravishankar G. A., Sarada R. (2012). Bioresource Technology. 123: [2] Metzger P., Largeau C. (2005). Applied Microbiology Biotechnology. 66: [3] Mendes R. L., Helena L. Fernandes, Coelho J. A. P., Cabral J. M. S., Palavra A. M. F. & Novais J. M. (1994). Journal of Applied Phycology. 6: [4] Zhang H., Wang W., Li Y., Yang W., Shen G. (2011). Biomass and Bioenergy. 35: [5] Dayananda C., Sarada R., Bhattacharya S., Ravishankar G. A. (2005). Process Biochemistry. 40:

98 P-BPE9 Invastigating effects of different parameters on biomass and extract yields of red seaweed, Jania rubens, to optimize its biotechnological production for industrial applications Gamze Turan 1*, Cigdem Demırkaya 2, Zeliha Demirel 2, Meltem Conk Dalay 2 1 Ege University, Fisheries Faculty, Department of Aquaculture, Bornova, Izmir, Turkey 2 Ege University, Faculty of Engineering, Department of Bioengineering, Bornova, Izmir, Turkey Keywords: Algal biotechnology, cultivation, Jania rubens, 0ptimization, red seaweed Introduction: The objective of this study, which is supported by an EU Project titled MAREX (Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications) was to investigate the growth and bioactive molecule yields of native red seaweed species, Jania rubens, which distributed along the coast of Turkey and has high potential for industrial applications. Methods: Jania rubens samples were collected from the coast of Urla (38 21'52 N, 26 46'12 E), Izmir, Turkey. Some seaweed material was taken and used for determining crude extract of the samples at the beginning of the study. Collected Jania individuals were placed in the growing vessels where a total of 12 treatment groups were applied. Growth rates of Jania were measured daily and presented as percentage. Experimental treatment groups were included: Group 1 (0% nitrogen), Group 2 (100% nitrogen), Group 3 (200% nitrogen), Group 4 (with % salinity), Group 5 (with % 0 25 salinity), Group 6 (with % salinity), Group 7 (no illumination), Group 8 (high illumination), Group 9 (high Jania density), Group 10 (low Jania density), Group 11 (with aeration) and Group 12 (with high temperature). The lyophilised Jania samples were extracted with 80% ethanol using Soxhlet apparatus and the extract yields were measured volumetrically and presented as percent dry weight. All data with three replicates were analyzed using MEANS Procedure and represented as mean ± sd. Biomass and extraction yields differencies between the treatment groups were analyzed using Duncan s Multiple Comparison Test in SPSS statistic program and the values are 0.05 were accepted as signaficant. Results: Overall, biomass and extract yields of Jania were experiment treatment group- depended. Jania growth was found to be quite manageable for production. The highest biomass yield was recorded as 3.92± 0.49 1 and the highest extract yield was found to be 7.45 ± 1.20 % in Group 10 among the experimental treatment groups. Discussion & Conclusions: From the result of the study, it was concluded that the red seaweed Jania rubens can be produced via biotechnological processes or optimized culture conditions in a sustainable program with the aim of developing Jania-based agents to the stage where they are available for various industrial applications [1], such as nutraceuticals, cosmetics, agrochemicals, biosensors and other speciality chemicals. Acknowledgements: This study is a part of MAREX EU Project and the authors thank to EU-FP7 for supporting MAREX project. : [1] McHugh, D. J. (2003). A guide to the Seaweed Industry. FAO Food and Agriculture Organization of the United Nations, Roma, Italy, 103 p. 92

99 P-BPE10 Exploring marine resources for bioactive compounds: from discovery to sustainable production and industrial applications (MAREX) Meltem Conk Dalay 1, Gamze Turan 2, *, Esra Imamoglu 1, Zeliha Demirel 1 1 Ege University, Faculty of Engineering, Department of Bioengineering, Izmir, Turkey 2 Ege University, Fisheries Faculty, Department of Aquaculture, Izmir, Turkey Keywords: Bioactive compounds, marine resources Introduction: The MAREX project is a joint effort of 19 academic, research institute, and industrial partners from 13 countries for exploring and researching of marine bioactive compounds. MAREX is funded by the European Union Seventh Framework Programme grant no. FP7-KBBE MAREX plans to go through the whole discovery path from sourcing of marine organisms from the sea to cultivation, compound isolation, characterization, bioactivity screening, synthetic optimization, and finally to the evaluation of industrial feasibility. Biodiversity in the seas is only partly explored, although marine organisms are excellent sources for many industrial products. Through close cooperation between industrial and academic partners, the MAREX project have collected, isolated and classified marine organisms, such as micro- and macroalgae, cyanobacteria, sea anemones, tunicates and fish from the Atlantic, Pacific and Indian Oceans as well as from the Mediterranean, Baltic and Arabian Seas. Extracts and purified compounds of these organisms have been studied for several therapeutically and industrially significant biological activities, including anticancer, antiinflammatory, antiviral and anticoagulant activities by applying a wide variety of screening tools, as well as for ion channel/receptor modulation and plant growth regulation. Chromatographic isolation of bioactive compounds has been followed by structural determination. Sustainable cultivation methods for promising organisms and biotechnological processes for selected compounds have been developed, as well as biosensors for monitoring the target compounds. The work will entail sustainable organic synthesis of selected active compounds and new derivatives, and development of selected hits to lead compounds [1]. Fig. 1. Marex brochure Discussion & Conclusions: MAREX will expand marine compound libraries. MAREX innovations will be targeted for industrial product development in order to improve the growth and productivity of European marine biotechnology. Acknowledgements: This project is a part of MAREX (Novel marine bioactive compounds for European industries) project and the authors wish to thank EU- FP7 for the financial support. [1] 93

100 P-BPE11 Development of functional food products with free from food concept Gokcen Komen 1, Sebnem Harsa*,1 1 Izmir Institute of Technology, Izmir, Turkey Keywords: Free-from, functional food, lactic acid bacteria, sourdough Introduction: Chronic diseases, food allergies and intolerances become more of an issue in today s populations and their awareness is increasing dramatically. Individuals on a special diet are searching for food products free from gluten, wheat, milk, egg, yeast, soy, salt, sugar, fat, nut etc. Since it s possible for a person to have sensitivity to more than one food component, excluding one component from a food product is not enough to fulfill the need. Hence, there is a vital necessity to consider the whole picture thoroughly and generate solutions in this respect. From that standpoint, we come across a novel and promising concept named as free from food. Free from foods do not include any allergens and unhealthful food compounds as far as possible. By being nutritious and exerting several health benefits, they can be considered as functional foods. Although products by excluding only one or two compounds have been studied to a great extend, studies about the development of food products free from multiple compounds is really at the beginning. Development of those kinds of products is a challenging task and needs special attention. The requirement for a multidisciplinary approach for the free from product development process results in the consolidation of food chemistry, food biotechnology and food technology disciplines. In addition, the efforts exerted during the product design stage are directly fall into the product development engineering field. Discussion & Conclusions: In this review, development techniques and approaches for the free from food products will be presented with the model based on gluten-free bakery product. There are several negative outcomes of a free from product. Products for special diet needs generally have poor quality (texture, aroma, sensory properties, shelf-life ect.). Since the absence of gluten and exclusion of other technologically important ingredients from the formulations because of their allergenic potential, the products should be fortified with the ingredients those have technological functions and/or some processing techniques should be utilized for the quality of final product. The ingredients for that purpose should be naturally gluten-free and free from other allergens. For bakery products, sourdough fermentation is the primary candidate for process development. The metabolites of lactic acid bacteria such as exopolysaccharides and bacteriocins result a product with improved texture and longer shelf-life. On the other hand, using starter cultures, selected according to the required improvement in final product (choosing exopolysaccharide, bacteriocin and/or specific aroma producers), may be beneficial to enhance sourdough process. By combining the suitable approaches with the appropriate raw materials and processing techniques, it may be possible to solve problems related with this health related diet issue and produce healthier and highquality novel free-from food products. 94

101 P-BPE12 Production of phytase by microbial submerged fermentation and optimization of process conditions Hasan Bugra Coban, Ali Demirci * Department of Agricultural and Biological Engineering The Pennsylvania State University, University Park, PA, USA. * Keywords: Aspergillus ficuum, fermentation, growth parameters, optimization, phytase. Introduction: Phytase is an important industrial enzyme, which breaks down phytate, the major phosphorus source in the nature. Phytate causes malnutrition in animals and humans by binding several divalent minor nutrients in the bodies. Phytase is also used to decrease phosphorus excretion in monogastric animals' manure to reduce the negative effects on the environment. Therefore, phytase has been used in diets as additive for years. However, the current production by solid state fermentation cannot meet the demand and limiting the use due to high cost. This project is undertaken to increase phytase production in submerged fermentation, which can make the phtase production commercially more feasable. Methods: In this study, phytase activity was increased by the selection of the best producer microbial specie and the optimization of growth and medium parameters in submerged fermentations by Box-Behnken designs. Phytase activity was determined with spectrophotometer as explained by Kim et al. [1]. Results: Aspergillus ficuum was selected as the best producer for the study. Growth parameters were optimized in 2-L bioreactors for the selected producer by surface response methodology as 33 o C, 4.5 ph, and 0.9 vvm. Moreover, optimum glucose, phytate, and CaSO 4 concentration in the fermentation medium were determined as 126 g/l, 14 g/l, and 1.1 g/l, respectively. Under these optimized conditions, phytase activity was enhanced to 3.45 U/ml from 1.02 U/ml in shakeflask fermentation. Fig. 1. Optimum growth and medium parameters for phytase production. Discussion & Conclusions: Phytase activity was increased more than 3 fold after optimization of growth and medium parameters comparing to shakeflask fermentations. Overall, this study demonstrated that phytase can be produced by submerged fermentation successfully. Acknowledgements: This work was supported in part by Turkish Ministry of Education by providing scholarship to Hasan Bugra Coban and the Pennsylvania Experiment Station. [1] Kim, Y. O., H. K. Kim, K. S. Bae, J. H. Yu, and T. K. Oh. (1998). Enz. Microb. Technol. 22:

102 P-BPE13 Production of Trichoderma harzianum EGE-K38 micropropagules by solid state fermentation using two different bioreactor configurations Işık Coban 1, Sayit Sargin *,1 1 Ege University Engineering Faculty Department of Bioengineering, Izmir, Turkey Keywords: Trichoderma harzianum, solid state fermentation, horizontal drum bioreactor, tray bioreactor, biocontrol agent Introduction: The use of biological agents to control various plant diseases has been considered as an environmentally friendly alternative to widespread application to synthetic pesticides[1,3]. Trichoderma type fungi can be used to control a wide spectrum of plant pathogen fungi as a biocontrol agent. Therefore effective production of Trichoderma micropropagules is very important since they are commercially preferred due to their plant growth stimulation characteristic and high activity on soil bioremediation[2]. In this study, production of Trichoderma harzianum EGE-K38 micropropagules were performed in solid state fermentation conditions by using horizontal drum bioreactor and tray bioreactor. Methods: In two different bioreactor systems, the effect of fermentation temperature, air flow rate, substrate bed thickness, initial moisture content of the substrate, inoculation volume, inoculum spore concentration and the effect of using spores or mycelia containing agar pieces as inoculum were determined individually and the results were compared in respect to each bioreactor system. Results: Under optimum conditions cfu/g substrate of micropropagule count was achieved in horizontal drum bioreactor, while it was cfu/ g substrate for tray bioreactor system. Fig. 1. Tray Bioreactor(a) and Horizontal Drum Bioreactor(b) Acknowledgements: The authors wish to thank Ege University Research Fund of EBİLTEM for financial support of this study under the grant number 2010/BİL/022. [1] Butt, T.M., Jackson, C.W., Magan, N. (eds). (2001). CABI International, Wallingford, Oxon, U.K. [2] Elad, Y., Chet, I. and Kantan, J. (1980b),, Phytopathology, [3] Vinale, F., Sivasithamparam, K., Ghisalberti, E.L., Marra, R., Woo, S.L., Lorito, M. (2008). Soil Biology and Biochemistry 40:

103 P-BPE14 Fruit pomace waste for bioethanol production Irem Deniz*,1,2, Esra Imamoglu 1, Fazilet Vardar Sukan 1 1 Ege University, Bioengineering Department, Bornova-Izmir, Turkey 2 Celal Bayar University, Bioengineering Department, Muradiye-Manisa, Turkey * Keywords: Agroindustrial waste, bioethanol production, Escherichia coli KO11, quicepomace. Introduction: Bioethanol is considered as the best alternative energy resource; however an inexpensive and easily pre-treatable carbon source has still not been fixed and established for ethanol production. The aim of this study was to determine the most appropriate agro-industrial waste as a carbon source for bioethanol production from E. coli KO11. Methods: The cells were incubated on an orbital shaker containing 150 ml of media at the temperature of 30 C during 60 hours of the fermentation period. The experimental design was achieved by two different agro-industrial wastes including quince and apple pomace individually supported with Luria-Bertani (LB) medium in 250- ml-shaken-flasks at different shaking frequencies (200 to 500 rpm) and the productivities of ethanol fermentations were compared with the synthetic LB media containing glucose, sucrose or fructose as carbon sources.the sugar contents of the agroindustrial wastes are listed in Table 1. Results: The highest ethanol concentration of agroindustrial wastes (15.74 g/l) was obtained with quince pomace at 300 rpm and this value was similar to the ethanol concentrations of and g/l obtained with synthetic LB medium containing fructose and glucose, respectively (Table2). Discussion & Conclusions: Ethanol yields were at the highest and lowest value when glucose and sucrose was used as carbon sources, respectively (see Table 2). This may be caused the fact that at glycolysis, 1.73, 1.33 and 0.75 mol NADPH were produced per glucose, fructose and sucrose, Table 1. Sugar contents of studied materials [1]. Agroindustrial waste Quince pomace Glucose (%) 4.72 Fructose (%) 9.13 Sucrose (%) 1.72 Apple pomace Table 2. Comparison of process parameters. Ethanol (g/l) Sugar uptake ratio (%) μ (1/h) YP/S (g/g) YX/S (g/g) Glucose Fructose Sucrose Quince Apple respectively. Kiefer et al. [2] demonstrated the lower product and biomass yield when sucrose was used as the main substrate. In this study, it was shown that the utilization of fruit pomace without delignification and enzyme pretreatment steps in bioethanol production could lead to higher ethanol yields. Fruit pomace could be taken into the consideration as a cost-effective alternative source to the conventional agroindustrial wastes for further bioethanol production researches. Acknowledgements: This study was a part of Cost action FP0602 and the authors wish to thank The Scientific and Technical Research Council of Turkey (TUBITAK) for the financial support. [1] Cemeroglu B., Karadeniz F. (2001). Gıda Teknolojisi Dernegi Yayınları pp [2] Kiefer P. et al. (2002). Journal of Industrial Microbiology & Biotechnology, 28:

104 P-BPE15 Effect of different nitrogen sources on mannanase production from carob pod extract using recombinant Aspergillus sojae Ercan Yatmaz 1, Mustafa Germec 1, Ercan Karahalil 1, Irfan Turhan*,1 1 Akdeniz University Faculty of Engineering Department of Food Engineering, Antalya, Turkey * Keywords: β-mannanase, Aspergillus sojae transformant 1: AsT1, carob pod Introduction : Microbial mannanase have become biotechnologically importance since they target the hydrolysis of complex polysaccharides of plant tissues into simple molecules like mannooligosaccharides and mannoses. These enzymes can occur spontaneously in plants and animals, in addition can be produced from microorganisms. Microbial mannanases that are obtained by fermentation method are used widely in the food and feed, detergent industry. β-mannanases are greatly influenced by nutritional and physicochemical factors, such as ph, nitrogen and carbon sources, initial sugar content, inorganic salts, and agitation. Carob has been widely grown in Mediterranean region for a long time. It was not able to be utilized as more than a tree and has been neglected to be grown in our country. When the fruits are ripe enough to be harvested, it has 91-92% total dry matter and 62-67% total soluble solids which consist of 34-42% sucrose, 10-12% fructose and 7-10% glucose. With their suitable content, carob extract or fruit are known to be utilized by biotechnological methods, especially fermentation. It is well known that, fermentation media must satisfy all the nutritional requirements of microorganisms. Most industrial molds can utilize both inorganic and organic nitrogen sources. They are generally supplied in byproducts of other industries, such as corn steep liquor, yeast extract, peptone, and soy meal. Methods : In this study, different quantities (%0.5, %0.75, %1) of yeast extract (YE), beef extract (BE) and ammonium nitrate (NH 4NO 3) (AN) were used to be nitrogen sources for fermentation media. The aim of this research will be designed not only to evaluate the potential of carob extract for mannanase production but also to determine the effect of different nitrogen sources on mannanase production. Recombinant Aspergillus sojae (ATCC 11906) was used for production of mannanase in carob extract. Fermentation samples were analyzed for ph, residual sugar, biomass, and betamannanase activity. Results : Fermentations were performed with various amounts of different nitrogen sources. Results showed that the maximum mannanase activity was obtained from 0.5% of AN to be U/ml while the lowest mannanase activity was U/ml with 1% of AN. There was also negative correlation between mannanase activity and amount of nitrogen sources. It means that higher amount of nitrogen source in fermentation media resulted in lower mannanase activity. YE(0,5%) :423.6 U/ml, YE(0,75%) :304.8 U/ml, YE(1%) : U/ml BE(0,5%) : 472 U/ml, BE(0,75%) : U/ml, BE(1%) : U/ml AN(0,5%) : U/ml, AN(0,75%) : U/ml, AN(1%) : U/ml Discussion & Conclusions : In our previous studies, mannanase production from carob pod extract was performed and the highest mannanase activity was found to be U/ml. It can be easily seen that different nitrogen sources in fermentation media can be an alternative method to enhance mannanase activity. Acknowledgements : This study was supported by the TUBITAK foundation (Project number: 112 O 167). 98

105 P-BPE16 Enhanced mannanase production by using microparticles in recombinant Aspergillus sojae fermentation Ercan Yatmaz 1, Mustafa Germec 1, Ercan Karahalil 1, Irfan Turhan*,1 1 Akdeniz University Faculty of Engineering Department of Food Engineering, Antalya, Turkey * Keywords: Aluminium oxide, mannanase, microparticle, recombinant Aspergillus sojae Introduction: The role of mannanases in the paper and pulp industry is well established and recently they have found application in the food and feed technology, fruit juice clarification, fruit maseration, oil drilling and detergent industry. Mannanases are mostly extracellular enzymes hydrolysing the 1,4-β-D-mannosidic linkages in mannans, galactomannans, glucomannans and galactoglucomannans. Mannanase are enzymes produced mainly from microorganisms but mannanases produced from plants has also been reported. However, microbes are the most potent producers of mannanase and represent the preferred source of enzymes in view of their rapid growth, limited spaces required for cultivation, and ready accessibility to genetic manipulation. Microbial mannanase have become biotechnologically importance since they target the hydrolysis of complex polysaccharides of plant tissues into simple molecules like manno-oligosaccharides and mannoses. Filamentous fungi such as recombinant Aspergillus sojae is important biocatalyst for industrial production of mannanase enzymes. But, there are some problems during the fermentation process such as limited oxygen, substrate transfer, high broth medium viscosity, excessive biomass. Because microparticles caused a change of growth morphology from pellets (thickness of biomass layer) to single hyphae. Therefore, mannanase enzyme productivity could be considerable high. In the literature, mannanase production from different carbon source such as glucose, sucrose, molasses, and carob pod extract were performed by recombinant Aspergillus sojae without any separation and purification process and mannanase activities were found to be U/ml, U/ml, 482 U/ml, and U/ml, respectively. Besides, different quantities (0.5%, 0.75%, 1%) of nitrogen sources (yeast extract, beef extract, ammonium nitrate) were used to produce of mannanase by recombinant Aspergillus sojae in carob pod extract. And, the maximum mannanase activity was determined to be U/ml with 0.5% ammonium nitrate. Methods: In the present study, different concentrations of microparticles (1, 5, 10 g/l of aluminum oxide) in fermentation medium were used not only to enhance mannanase activity but also to control of morphology in recombinant Aspergillus sojae. This study was performed with fed-batch fermentation and conditions were 4% of initial sugar concentration, rpm agitation rate (speed was gradually increased), 1% of inoculation rate, 1vvm aeration rate, ph 5 and 30ºC. Results : The results showed that the maximum mannanase activity was determined to be U/ml with using 5 g/l of aluminum oxide. Discussion & Conclusions: Overall, this study demonstrated that the microparticles can be successfully implemented to enhance enzyme activity. Acknowledgements: This study was supported by the TUBITAK foundation (Project number: 112 O 167). 99

106 P-BPE17 Design and optimization diameter of poly lactic acid electrospun nanofibers for controlled release of naltrexone Maniya Habibi 1*, Mohammad Irani 2, Ismaeil Haririan 1, Iman Yousefi 1 Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Sciences,Tehran, Iran 2 Department of Chemical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran * Keywords: Box Behnken design, electroespining nanofiber, naltreoxane, PLA Introduction: Recently, there has been a growing attention in the development of novel drug delivery systems to improve the effectiveness of drugs. A wide range of polymeric materials either biodegradable or non-biodegradable but biocompatible; can be used as drug carriers. Among them, fibrous scaffolds, such as the system made-up by electrospinning [1,2], have been used in several biomedical applications [3]. In this study, sustained release of naltreoxane loaded electrospun poly lactic acid nanofiberous scaffolds prepared by electrospinnig process was investigated. Methods: Naltreoxane- loaded nanofiberous scaffolds were prepared and characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The effect of fabrication parameters such as target - collector distance (TCD), polymer solution concentration, flow rate and applied voltage, on the morphology and diameter of electrospun nanofibers has been studied. A response surface methodology (RSM) based on Box-Behnken design was used to evaluate the effect of key parameters on the fiber diameter. Finally, the release behavior of naltreoxane from the nanofibrous scaffolds was investigated. Results: The SEM micrograph of the distribution of the electrospun naltreoxane loaded electrospun poly lactic acid nanofiberous scaffolds is shown in Figure 1. It seems the samples were uniform. The surface were smooth and no drug crystals were identified, indicating that the drug was finely incorporated into the electrospun fibers. tables. Insert figures and tables after they are cited in the text. was smooth and no drug crystals was identified, indicating that the drug was finely incorporated into the electrospun fibers. Fig.1 SEM image of naltreoxane loaded electrospun poly lactic acid nanofiber Discussion & Conclusions: Based on results, the optimal values of concentration, flow rate, distance and voltage were estimated to be 10%, 0.5 ml/h, 14 cm and 20 kv, respectively, which in turn yielded the minimum fiber diameter of 230 nm. For electrospinning parameters, the experimental nanofiber diameter (235 nm) was 2.5%further than the value predicted by the model (230 nm) which is ignorable. The release behavior of naltreoxane loaded nanofibrous scaffolds indicated that the release rate of naltreoxane from the fibers was dependent on the initial naltreoxane loading. During the whole release time, the rate of naltreoxane release decreased as the nultreoxane content in the fibers increased. The sustained released could last for more than 30 days. [1] M. Qi, X. Li, Y. Yang, S. Zhou. 2008, Europ. J. Pharm. Biopharma. 70, [2] J. Xie, Chi-Hwa Wang.2006, Pharm. Res, 23, [3] U.S.Sajeev, K.A.Anand, D.Menon, S.Nasir, 2008, Bull. Mater. Sci, 31,

107 P-BPE18 Evaluation of culture conditions of Dunaliella salina Esra Imamoglu 1, Zeliha Demirel 1, Meltem Conk Dalay*,1 Ege University, Faculty of Engineering, Department of Bioengineering, Izmir, Turkey Keywords: Agitation rate, Dunaliella salina, light intensity, temperature Introduction: Recently, an increasing interest in microalgal biotechnology has emerged due to the variety of biotechnologically interesting compounds that microalgae can synthesize. Dunaliella salina, unicellular green microalgae, halophylic with free wall, is scientifically interesting for its capacity of producing β-carotene and glycerol. D. salina can accumulate a very high concentration of β-carotene (up to 14 %) in cell dry weight under the conditions of high light, high salinity and nutrient deprivation and it is recognized as the best biological source of this carotenoid [1]. The aim of this study was to optimize physical parameters such as light intensity, temperature and agitation rate which might affect the growth of D. salina. Methods: The cells were cultured in 250 ml Erlenmeyer flasks containing 150 ml of F/2 medium at different light intensities, temperatures and agitation rates. The experimental optimization design was performed using Response Surface Methodology (RSM) with Central composite design (CCD). The experimental design was a 2 3 fullfactorial central composite experimental plan with three physical parameters. 5 different temperatures (20, 22, 25, 28 and 30 C), 5 different agitation rates (100, 120, 150, 180 and 200 rpm) and 5 different light intensities (30, 40, 55, 70 and 80 μmol photons m -2 s -1 ) were tested. A total of 18 runs were used to optimize the range and levels of the chosen variables. Each run had been completed in 12 days. Chlorophyll-a (mg/l) and protein amounts (µg/100 µl) were taken as responses of the systems. Results: The optimal growth conditions were found at C under the light intensity of μmol photons m -2 s -1 at the agitation rate of rpm with the Chlorophyll-a concentration of 6.34 mg/l, whereas the protein amount was µg/100 µl at C under the light intensity of μmol photons m -2 s -1 with the agitation rate of rpm for D.salina. The maximum specific growth rate of 0.22 day -1, which corresponded to the doubling time of 3.15 day was obtained for D.salina under determined conditions. Fig. 1. 3D response surface plot showing the mutual effects of light intensity, temperature and agitation rate of D.salina. Discussion & Conclusions: The growth rate obtained under these optimization conditions was 25% higher than the other D.salina production studies. Culturing the microalgae under determined optimization conditions could provide to reach the maximum growth rates. Acknowledgements: This project is a part of MAREX (Novel marine bioactive compounds for European industries) project and the authors wish to thank EU- FP7 for the financial support. [1] Jesus, S.S. and Filho, R.M. (2010). American J. of Biochem. and Biotech., 6 (4):

108 P-BPE19 Comparison of different light colors for the biomass production of Amphora capitellata Zeliha Demirel, Esra Imamoglu, Meltem Conk Dalay* Ege University, Faculty of Engineering, Department of Bioengineering, Izmir, Turkey Keywords: Amphora capitellata, biomass, light colors Introduction: Diatoma species, which are widely distribute in marine coastal waters, were collected for a European 7th Framework project titled; Exploring Marine Resources For Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications (MAREX). Amphora capitellata, isolated from Aegean Sea in Urla-Izmir, Turkey, was found more effective on various pharmaceutical activities for industrial applications and therefore this strain was selected for biomass production. The quality and quantity of light may affect the biomass production of microalgae. Diatoms are much more adaptable than many plants to different colors and types of light [1]. The aim of this study was to determine the effect of different light colors (red, blue and white) on the biomass production of Amphora sp. in the air-lift photobioreactor. Methods: The cells were grown in F/2 medium under 20±2 ºC at the light intensities of 23 µmol photons m -2 s -1 in the air-lift photobioreactor for 12 days. The determination of the biomass were carried out by the measuring the absorbance values (556 and 635 nm) using a UV-VIS spectrometer (Ultrospec 1100 pro, Amersham Biosciences) and by counting the cells with hemocytometer using an Olympus phase-contrast microscope. The specific growth rate (µ) of the cells was calculated from the initial logarithmic phase of growth for at least 48 h, as µ = ln X 2 - lnx 1/dt, where X 2 is the final cell concentration, X 1 is the initial cell concentration and dt is the time required for the increase in concentration from X 1 to X 2. Doubling time (DT) was also calculated as DT = ln 2/µ. Results: Chlorophyll-a concentration reached the maximum level of 4.76 mg/l at the red light intensity of 23 µmol photons m -2 s -1, which was about 1.12 times higher than the blue light intensity at the end of the cultivation period. Furthermore, the maximum cell concentration (5.8 x10 5 cells/ml) was obtained at the red light intensity of 23 μmol photons m -2 s -1 for A. capitellata. Fig. 1. Effect of light color on growth of Amphora capitellata. Discussion & Conclusions: Pena et al. [1] reported that the low photosynthetic photon flux density (PPFD) did not limit the growth of Amphora sp., and this was demonstrated by an erratic growth rate when Amphora sp. was subjected to higher PPFD. On the other hand, this study is a novel approach about the effect of the light colors on the growth of Amphora sp. Acknowledgements: This project is a part of MAREX (Novel marine bioactive compounds for European industries) project and the authors wish to thank EU- FP7 for the financial support. : [1] Pena, M.R. (2007). Journal of Applied Phycology 19:


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