Using Bacillus amyloliquefaciens for remediation of aquaculture water

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1 Xie et l. SpringerPlus 13, 2:119 SpringerOpen Journl RESEARCH Open Access Using Bcillus myloliquefciens for remedition of quculture wter Fengxing Xie 2*, Ticheng Zhu 3, Fengfeng Zhng 2, Ke Zhou 2, Yujie Zho 2 nd Zhenghu Li 1* Astrct Remedition of quculture wter using microorgnisms like Bcillus species is urgeoning trend for the sustinle development of quculture industries. In this work, Bcillus myloliquefciens strin (nmely B. myloliquefciens HN), isolted from ctivted sludge of polluted river, ws evluted for its potentil in wter remedition using simulted quculture wter. B. myloliquefciens HN exhiited high tolernce towrds 8 mg l -1 of nitrite-n nd mmoni-n. It could effectively remove mg l -1 of nitrite-n, ut ws inefficient in eliminting mmoni-n when the mmoni-n concentrtion ws elow mg l -1. Further studies showed tht the mmoni-n removl y B. myloliquefciens HN ws more efficient t 3 C nd 35 C thn 25 C, nd tht mximum nitrite-n removl rte ws chieved t ph 8. Keywords: Bcillus myloliquefciens, Aquculture wter remedition, Nitrogenous compound, Nitrite removl Introduction The rpid expnsion of intensive quculture industries, re often compnied y rotted uneten feed, sedimenttion of feces nd orgnic residues. The wter qulity rpidly deteriortes s result. In prticulr, nitrogenous compounds such s mmoni nd nitrite quickly uild up, which re oth hrmful to fish nd shrimp even t low concentrtions (Cr et l. 7; Mohptr et l. 12). Wter exchnge cn e pplied to mintin good wter qulity. However, frequent wter exchnge is not only lorious nd costly, ut lso my incur disese cusing gents nd pollute nery rivers nd costl res (Mohptr et l. 12). Therefore, there is n urgent demnd for cost-effective nd environment-friendly pproches for remedition of quculture wter. In recent yers, the use of microorgnisms to improve wter qulity ecomes urgeoning trend (Ninwe nd Selvin 9; Verschuere et l. ). Bcillus species re widely used for wter remedition ecuse they re stle for long period due to spore formtion, esily prepred y * Correspondence: [email protected]; [email protected] 2 Tinjin Reserch Center of Agriculturl Biotechnology, Tinjin 3192, People s Repulic of Chin 1 Tinjin Institute of Industril Biotechnology, Chinese Acdemy of Sciences, 32XiQiDo, Tinjin Airport Economic Prk, Tinjin 338, People s Repulic of Chin Full list of uthor informtion is ville t the end of the rticle fermenttion nd possess ntgonistic effects on pthogens (Hong et l. 5). Strins elonging to severl Bcillus species, such s Bcillus sutilis, Bcillus cereus, Bcillus licheniformis, Bcillus pumilus were isolted nd evluted for their potentil s iologicl gents for wter qulity enhncement. Severl strins with good nitrogen removl properties were thus found. To dte, screening strins with good remedition chrcteristics still remins fundmentl step towrds developing commercil microil gents. Previously, we isolted Bcillus myloliquefciens strin, nmed s B. myloliquefciens HN from the ctivted sludge of polluted river. This strin ws shown to effectively remove nitrogenous compounds nd grow in rod temperture, ph, nd slts concentrtion in preliminry studies. Moreover, no previous studied hs chrcterized the nitrogen removl ility of B. myloliquefciens. Therefore, the im of this study is to evlute B. myloliquefciens HN for its remedition properties using simulted quculture wter. Mterils nd methods Strins nd culture medi B. myloliquefciens HN ws isolted from polluted pool in Xiqing District, Tinjin. The strin ws identified y Agriculturl Culture Collection of Chin, nd deposited t Chin Generl Microiologicl Culture Collection Center (CGMCC No. 3261). 13 Xie et l.; licensee Springer. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Xie et l. SpringerPlus 13, 2:119 Pge 2 of Cell density ( 8 cfu/ml) Cell density ( 8 cfu/ml) ck ck 8 16 Nitrite-N concentrtion (mg/l) Ammoni-N concentrtion (mg/l) Figure 1 Nitrite nd mmoni-n tolernce tests for B. myloliquefciens HN. ) Nitrite tolernce tests; ) Ammoni-N tolernce tests. The culture medium (lso referred s sl medium) for B. myloliquefciens HN contined (per liter): peptone 8 g, eef extrct 3 g, solule strch 5 g, NCl 5 g (plus gr 15 g for solid medium). The simulted quculture wter ws prepred referring to Chen (5) nd LuzE. De- Bshnet (2), which contined (per liter): eef extrct.5 g, sucrose.5 g, NCl.25 g, KH 2 PO 4.75 g. Nitrite nd mmoni-n tolernce tests Sodium nitrite nd mmonium sulfte were dded to the culture medium to mke the finl concentrtions of nitrite-n 1, 5,,,, 8, nd mg l -1, respectively, nd those of mmoni-n,,, 8, nd 16 mg l -1, respectively. All the experiments were repeted thrice with the sl medium s control. Three milliliters of strin culture were inoculted into 25 ml shke flsk with ml culture medi nd cultured t 37 C. Removl tests of nitrite-n nd mmoni-n The simulted polluted wter ws prepred y mking the finl concentrtions of nitrite-n,,, nd 8 mg l -1, respectively, nd the concentrtions of mmoni-n 5,,, nd mg l -1, respectively. Three milliliters of strin culture were inoculted into 25 ml shke flsk with ml culture medi nd cultured t 37 C. Effects of different conditions on the removl of nitrite-n nd mmoni-n The model polluted wter ws prepred to mke the initil concentrtion of nitrite-n ws mg l -1, nd mmoni-n ws mg l -1. The inoculum volume ws 3%, nd cultured t different temperture (25 C, 3 C, nd 35 C), nd different initil ph (ph5., 6., 7., 8., nd 9., djusted y NOH nd HCl). Nitrite-N concentrtion (mg/l) h 24 h 48 h 72 h Ammoni-N concentrtion (mg/l) h 24 h 48 h 72 h Time (h) Time (h) Figure 2 Nitrite nd mmoni-n removl tests for B. myloliquefciens HN. ) Nitrite removl tests; ) Ammoni-N removl tests.

3 Xie et l. SpringerPlus 13, 2:119 Pge 3 of 5 Nitrite-N cocentrtion (mg/l) nitrite-n concentrtion removl rte 25 C 3 C 35 C Temperture 1% % 3 % % Removl rte Ammoni-N concentrtion (mg/l) mmoni-n concentrtion removl rte 25 C 3 C 35 C Temperture 5 45% % 35% 3 25% % 15% % 5% Figure 3 Effects of different temperture on the removl rtes of nitrite-n nd mmoni-n y B. myloliquefciens HN. ) Effects of temperture on nitrite-n removl rte; ) Effects of temperture on the mmoni-n removl rte. Removl rte Anlysis methods The Nesslr method ws used for mmoni determintion. The 1,2-ethnedimine, N-1-nphthlenyl-,dihydrochloride spectrophotometric method ws used for the nitrite mesurement (APHA, 5). The nitrte concentrtion ws mesured using the slicylic (2-hydroxyenzoic) cid method (Ctldo et l. 1975). Cell concentrtion ws determined y plte counting. Results Nitrite nd mmoni-n tolernce tests for of B. myloliquefciens HN For Nitrite-N tolernce test (Figure 1), cell concentrtions of B. myloliquefciens HN with ll tested nitrite- N concentrtions were higher tht of control, illustrting tht the strin could tolerte wide rnge of nitrite-n. The results lso showed tht the presence of nitrite-n could promote the growth of B. myloliquefciens, especilly t low concentrtions (< mg/l). The highest cell concentrtion ws chieved with the nitrite-n concentrtion of mg l -1, which is 8.1% higher thn tht of control. For mmoni-n tolernce test (Figure 1), ll cell concentrtions with tested mmoni-n concentrtions were higher thn tht of control, except the one with 16 mg l -1 mmoni-n. The mmoni-n concentrtion of mg l -1 resulted in the highest cell growth, 79.9% higher thn tht of control. The results indicted tht B. myloliquefciens HN cn tolerte mmoni-n rnging from mg l -1 to 8 mg l -1. An mmoni-n concentrtion of 16 mg l -1 ws shown to inhiit the growth of the strin. Nitrite-N nd mmoni-n removl tests for B. myloliquefciens HN Figure 2 showed tht the nitrite-n could not e detected within 24 h with n initil nitrite-n concentrtion of mg l of the nitrite-n ws removed in 24 h for mg l -1, nd lmost ws removed in 48 h mg l -1 nd.1 mg l -1 of nitrite-n (representing 53.8% Nitrite-N concentrtion (mg/l) nitrite-n concentrtion removl rte ph 8 6 % % Removl rte Ammin-N concentrtion (mg/l) mmin-n concentrtion removl rte ph Figure 4 Effects of ph on the removl rtes of nitrite-n nd mmoni-n. ) Effects of ph on nitrite-n removl rte; ) Effects of ph on the mmoni-n removl rte. 5 % 3 % % Removl rte

4 Xie et l. SpringerPlus 13, 2:119 Pge 4 of 5 nd 21.6% of the totl nitrite-n) ws eliminted for n initil nitrite-n concentrtion of mg l -1 nd 8 mg l -1,respectively. The removl rte, however, did not significntly elevted with the time (the residue nitrite-n even incresed littlefor8mgl -1 ), suggesting tht the mximum removl concentrtion of nitrite-n might e round mg l -1. With high initil mmoni-n concentrtions ( mg l -1 nd mg l -1 ),the removl rtes of mmoni-n were significnt, nd mximum removl rtes of 59.8% nd 79.8% were reched t 48 h (Figure 2). As for low initil mmoni-n concentrtions ( mg l -1 nd mg l -1 ), the removl efficiencies of mmoni-n were not efficient. During the time course, the residue mmoni-n levels fluctuted round mg l -1, suggesting tht B. myloliquefciens HN could not only utilize mmoni-n ut lso produce it. Effects of temperture on the nitrite-n nd mmoni-n removl ilities of B. myloliquefciens HN The nitrite-n removl rtes of B. myloliquefciens HN t 25 C, 3 C nd 35 C ll reched 99% (Figure 3), suggesting temperture (over the experimentl rnge) did not ffect the removl of nitrite-n. The mmoni-n removl rtes t 25 C, 3 C nd 35 C were 29%, 37.5% nd 39.4% respectively (Figure 3), suggesting higher temperture might e preferle for effective removl of mmoni-n y B. myloliquefciens HN. Effects of ph on the nitrite-n nd mmoni-n removl ilities of B. myloliquefciens HN Figure 4 showed tht ph remrkly influenced the removl of nitrite-n y B. myloliquefciens HN. Nitrite-N removl rtes incresed significntly with incresed ph vlue. A mximum removl rte of 96% could e reched t ph 8.. Further increse of ph reduced the removl rte. Ammoni-N removl rtes from ph 5. ~ 9. were etween 32 ~ 38%, suggesting ph hd no significnt effects on the removl of mmoni-n (Figure 4). Discussion In this work, we investigted the potentil of using B. myloliquefciens in the remedition of quculture wter. Prticulrly, its remedition chrcteristics regrding nitrite nd mmoni removl were evluted. The ccumultion of nitrite nd mmoni is highly toxic to qutic fun..1 ~ mg l -1 of nitrite cn cuse 5 mortlity (LC 5) of numer of fish nd shrimp (Philips et l. 2). Ammoni-N cn e toxic to commercilly cultured fish t concentrtions ove 1.5 mg l -1 (Cr et l. 7). Bcillus species re importnt cndidtes for developing commercil iologicl gents for nitrogen removl nd wter qulity enhncement (Hong et l. 5). Previously, few studies reported tht some strins of B. sutilis (Chen nd Hu 11; Meng et l. 9), B. lichenformis (Meng et l. 9) nd B. cereus (Llloo et l. 7) exhiited strong nitrite removl ility. Physiologicl studies on Bcillus spp. lso showed tht Bcillus spp. could utilize nitrte nd nitrite s lterntive electron cceptors nd nitrogen sources (Nkno et l. 1998; Hoffmnn et l. 1998). This study showed tht n B. myloliquefciens strin, isolted from the ctivted sludge, ws lso very efficient nitrite-n clener, which ws le to completely remove mg l -1 of nitrite-n present in the simulted quculture wter within 24 h. In the pst, B. myloliquefciens were pplied in enzyme production (Wei et l. 11), plnt disese control (Alvindi nd Ntsuki 9; Arreol et l. ) nd food preservtion (Wng et l. 6). This is the first study on its potentil ppliction in improving wter qulity. It suggested tht B. myloliquefciens might e n importnt lterntive Bcillus species for nitrite removl. The mmoni-n removl ility of B. myloliquefciens ws not stisfying, ecuse when the mmoni-n concentrtion ws elow mg l -1,theremovlrtewstest 39.4% nd the mmoni-n concentrtion cnnot e reduced elow mg l -1. Pst literture seems to suggest tht Bcillus species re not very efficient in mmoni removl, nd no mmoni removl efficiency of single Bcillus strin hs een reported to exceed 9. Therefore, to crete microl gent which cn simultneously eliminte nitrite nd mmoni, B. myloliquefciens NH cn e formulted with efficient mmoni reducing cteri, such s nitrifying cteri (Meng et l. 9). Competing interests The uthors declre tht they hve no competing interests. Authors contriutions Conceived nd designed the experiments: FX, ZL. Performed the experiments: FX,FZ,KZ,YZ. Anlyzed the dt: FX, TZ, ZL. Wrote the pper: FX, ZT, ZL. All uthors red nd pproved the finl mnuscript. Acknowledgement This work is supported y Key Projects in Science nd Technology Support Progrm of Tinjin, Chin (ZCZDSY6), Mjor Projects in Science nd Technology Support Progrm of Tinjin, Chin (11ZCKFNC5) nd the Knowledge Innovtion Progrm of the Chinese Acdemy of Sciences (KSCX2-EW-G-15-3). Author detils 1 Tinjin Institute of Industril Biotechnology, Chinese Acdemy of Sciences, 32XiQiDo, Tinjin Airport Economic Prk, Tinjin 338, People s Repulic of Chin. 2 Tinjin Reserch Center of Agriculturl Biotechnology, Tinjin 3192, People s Repulic of Chin. 3 Institute of Microiology, Chinese Acdemy of Sciences, Beijing 1, People s Repulic of Chin. Received: 29 Novemer 12 Accepted: 27 Ferury 13 Pulished: Mrch 13 References Alvindi DG, Ntsuki KT (9) Biocontrol ctivities of Bcillus myloliquefciens DGA14 isolted from nn fruit surfce ginst nn crown rot-cusing pthogens. Crop Prot 28(3): Aph A WPCF (5) Stndrd methods for the exmintion of wter nd wstewter. Pulic Helth Assocition, Wshington, DC

5 Xie et l. SpringerPlus 13, 2:119 Pge 5 of 5 Arreol E, Jcos R, Korsten L () Iturin A is the principl inhiitor in the iocontrol ctivity of Bcillus myloliquefciens PPCB4 ginst posthrvest fungl pthogens. J Appl Microiol 8(2): Ctldo D, Hroon M, Schrder L, Youngs V (1975) Rpid colorimetric determintion of nitrte in plnt-tissue y nitrtion of slicylic-cid. 1(6):71 8 Chen GK, Chen G (5) Effect of synthetic wstewter on some physicochemicl properties of soil in Avicenni mrin simulted wetlnd system. Mr Environ Sci 24(2):26 28 Chen S, Hu Y (11) Use of Bcillus sutilis in purifiction of slightly-polluted wter. Act Scienctie Circumstntie 31(8): Cr R, Avnimelech Y, Defoirdt T, Bossier P, Verstrete W (7) Nitrogen removl techniques in quculture for sustinle production. Aquculture 27(1 4):1 14 De-Bshn LE, Moreno M, Hernndez JP (2) Removl of mmonium nd phosphorus ions from synthetic wstewter y the microlge Chlorell vulgris coimmoilizedin lginte eds with the microlge growth promoting cterium Azospirilum rsilense. Wter Res 36(12): Hoffmnn T, Frnkenerg N, Mrino M, Jhn D (1998) Ammonifiction in Bcillus sutilis utilizing dissimiltory nitrite reductse is dependent on resde. J Bcteriol 18(1): Hong HA, Duc H, Cutting SM (5) The use of cteril spore formers s proiotics. FEMS Microiol Rev 29(4): Llloo R, Rmchurn S, Rmduth D, Görgens J, Grdiner N (7) Isoltion nd selection of Bcillus spp. s potentil iologicl gents for enhncement of wter qulity in culture of ornmentl fish. J Appl Microiol 3(5): Meng R, He LS, Xi BD, Hu X, Li YY (9) Experimentl study on purifying quculture wstewter etween Bcillus nd nitrifying cteri. Environ Sci Technol 32(11):28 31 Mohptr S, Chkrorty T, Kumr V, Deoeck G, Mohnt KN (12) Aquculture nd stress mngement: review of proiotic intervention. J Anim Physiol Anim Nutr:1 26. doi:.1111/j x Nkno MM, Hoffmnn T, Zhu Y, Jhn D (1998) Nitrogen nd oxygen regultion of Bcillus sutilis nsdef encoding NADH-dependent nitrite reductse y TnrA nd ResDE. J Bcteriol 18(): Ninwe AS, Selvin J (9) Proiotics in shrimp quculture: venues nd chllenges. Crit Rev Microiol 35(1):43 66 Philips S, Lnroek HJ, Verstrete W (2) Origin, cuses nd effects of incresed nitrite concentrtions in qutic environments. 1(2): Verschuere L, Romut G, Sorgeloos P, Verstrete W () Proiotic cteri s iologicl control gents in quculture. Microiol Mol Biol Rev 64(4): Wng J, Qun C, Xu H, Fn S (6) Antifungl chrcteriztion of Bcillus myloliquefciens Q-12. Food Ferment Ind 32(6):47 5 Wei X, Luo M, Xu L, Zhng Y, Lin X, Kong P, Liu H (11) Production of firinolytic enzyme from Bcillus myloliquefciens y fermenttion of chickpes, with the evlution of the nticogulnt nd ntioxidnt properties of chickpes. J Agric Food Chem 59(8): doi:.1186/ Cite this rticle s: Xie et l.: Using Bcillus myloliquefciens for remedition of quculture wter. SpringerPlus 13 2:119. Sumit your mnuscript to journl nd enefit from: 7 Convenient online sumission 7 Rigorous peer review 7 Immedite puliction on cceptnce 7 Open ccess: rticles freely ville online 7 High visiility within the field 7 Retining the copyright to your rticle Sumit your next mnuscript t 7 springeropen.com