摘要
伴随着对虾产业的高速发展，对虾养殖产业现代化、工厂化和集约化程度的大幅度提高，对虾养殖业给周边环境带来的污染问题日益引起人们的关注，减轻对虾养殖业对周边环境的污染是对虾养殖业面临的亟待解决的问题之一。在水产养殖过程中，对养殖环境做出合理调控，减少养殖用水的使用量，建立零换水系统成为减少对虾养殖业对周边环境污染的有效途径之一。目前，很多研究集中在对养殖环境水质的处理上，对通过改良对虾养殖环境底质，从而使整个养殖环境得到优化的研究较少。有益菌（如芽孢杆菌、光合细菌等）在调控养殖环境水质方面效果明显，但是人们对其调控水质的作用机理研究较少。近些年来，由于固定化微生物技术所具有的优点，人们已经开始使用固定化微生物技术对养殖环境和养殖废水进行处理。但是，由于养殖环境的特殊性，固定化微生物技术在水产养殖中的应用需要更高要求。因此，针对以上几点，本文设计如下实验内容。
本论文的主要研究内容如下：1.利用两种农业废弃物（甘蔗渣和牡蛎壳粉）作为对虾养殖环境中的底质，研究底质改良对零换水条件下对虾养殖环境的影响，所测指标包括：水质因子（氨态氮、亚硝态氮、叶绿素a）、底质和水体中的细菌密度（总菌密度和氮循环菌密度）、浮游动物密度和对虾生长指标等；2.研究了一株溶藻弧菌（Z5）作为有益菌在不同C:N条件下对不同种类氮源的利用；3溶藻弧菌(Z5)固定化小球对养殖环境中的氨态氮和亚硝态氮去除效果。作者期望通过以上研究能够对零换水条件下对虾系统中的人工底质的构建、溶藻弧菌作为有益菌对水质的调控作用以及调控的作用机理和固定化微生物技术对养殖环境因子（氨态氮和亚硝态氮）的改良等研究提供参考，为减少养殖废水排放，减轻对虾养殖业对周边环境的污染做出贡献。本论文实验设计及主要研究结果如下
1.底质改良对零换水条件下凡纳滨对虾养殖环境的影响
牡蛎壳粉（OP），甘蔗渣(BA)，牡蛎壳粉与甘蔗渣混合（OS），以及底泥（FS）作为底质，比较它们在零换水条件下对凡纳滨对虾高密度养殖环境的影响。测定的指标包括水质指标，底质及水体中的总菌量及氮循环菌量，浮游动物密度和对虾生长指标等。研究结果表明：在养殖后期，BA组中的TAN浓度低于0.36mg/L，显著低于其他各组中的TAN浓度（P <0.05）。OS组中的NO_2-N浓度低于0.50mg/L，显著低于其它各组中的NO_2-N浓度（P <0.05）。以上结果可能与其相对应组中高密度的亚硝化细菌和硝化细菌有关。新的底质促进了养殖环境中细菌的生长，OP、BA和OS组底质中的细菌密度比FS组中底质中的细菌密度多出一个数量级。甘蔗渣的添加对水体中浮游动物的密度有显著影响，甘蔗渣添加组（BA组和OS组）中浮游动物密度显著高于没有甘蔗渣添加组（FS组和OP组）中的浮游动物密度（P <0.05）。综合指标显示，BA和OS这两种材料的加入有效的改善了养殖环境中的水质，提高了对虾的产量并且降低了对虾的饵料系数。新的底质，尤其是甘蔗渣（BA）和甘蔗渣与壳粉混合（OS）能够有效的改善零换水条件下凡纳滨对虾高密度的养殖环境。
2.甘蔗渣不同添加量对对虾养殖环境的影响
向凡纳滨对虾（Litopenaeus vannamei）养殖环境中添加不同量的甘蔗渣（0.5kg/m~2、1kg/m~2、1.5kg/m~2和2kg/m~2，BA)与牡蛎壳粉（5kg/m~2，OP）作为底质，通过对养殖水体中的水质指标、水体及底质中的总菌、氮循环菌、浮游动物（>160μm）数量变动以及对虾生长指标的检测，评估甘蔗渣不同添加量对养殖环境和养殖动物的影响，处理组分别为BA0.5、BA1、BA1.5和BA2。研究发现，甘蔗渣不同添加量对养殖环境中水质及细菌密度的有一定的影响，投放甘蔗渣促进了浮游动物数量的增长，但投放甘蔗渣的处理组之间浮游动物数量差异不显著。水体中悬浮的甘蔗颗粒在提高养殖环境中的营养元素利用率上有着重大的贡献。但是，过量投放甘蔗渣会对养殖环境造成压力，甘蔗渣的适宜添加量为0.5-1kg/m~2。
3.不同C:N条件下一株溶藻弧菌对不同种类氮源的吸收特性
在不同C:N（5、10、15、20）条件下研究了一株溶藻弧菌（Z5）作为有益菌对单一氮源（有机氮源、NH_4-N、NO_2-N）及混合氮源（有机氮源与NH_4-N、有机氮源与NO_2-N、NH_4-N与NO_2-N）的吸收利用特性及蛋白酶、谷氨酰胺合成酶(GS)和亚硝酸还原酶（NiR）等3种氮代谢相关酶的活力响应。研究结果表明：溶藻弧菌（Z5）对有机氮与无机氮的吸收均受到环境中碳含量的影响，在C:N20时，NH_4-N和NO_2-N的吸收率分别是C:N5时吸收率的3.39倍和2.25倍。在C:N15和C:N20的水平下，溶藻弧菌（Z5）对NH_4-N和NO_2-N的吸收没有显著性差异（P>0.05）。溶藻弧菌（Z5）吸收利用NH_4-N和NO_2-N途径分别为NH_4-N→细菌菌体蛋白和NO_2-N→NH_4-N→细菌菌体蛋白。在高C:N条件下，溶藻弧菌（Z5）对无机氮源（NH_4-N和NO_2-N）的吸收增强，细菌菌体蛋白中来自NH_4-N和NO_2-N的量分别由C:N5时的25.0%和19.4%上升到C:N20时的41.3%和43.0%。与NO_2-N相比，溶藻弧菌（Z5）优先吸收利用NH_4-N。GS酶活力受NH_4-N调控明显，相比之下，蛋白酶和NiR酶受氮源种类调控不明显。
4.溶藻弧菌（Z5）固定化小球对养殖环境中NH_4-N和NO_2-N的去除效果
应用固定化微生物技术，以海藻酸钠为包埋材料，包埋甘蔗渣和溶藻弧菌（Z5）后形成固定化小球（SA-小球），研究了SA-小球对包埋细菌的影响、对NO_2-N的降解能力和额外碳源添加对SA-小球降解能力的影响以及SA-小球对对虾养殖环境中TAN和NO_2-N的去除效果。研究结果表明：SA-小球的最适的初始包埋菌量在10~4cfu/球-105cfu/球，最大载菌量可达到1.06×10~8cfu/球。平均每个SA-小球对NO_2-N的降解总量可达到0.06mg。额外碳源的添加能够增强SA-小球对NH_4-N和NO_2-N的降解。通过50天的对虾养殖实验发现，SA-小球能够有效的去除养殖水体中TAN和NO_2-N，利用SA-小球降解水体中的TAN和NO_2-N的效果和使用换水的方法达到的效果相同。此外，使用SA-小球处理养殖水体，减少了养殖过程中的换水量，并且对虾的产量也可以达到换水养殖对虾产量的70%。在养殖后期，尽管SA-小球部分分解，但是其内包含的甘蔗渣附着有大量的细菌，能够继续起到调控水质的作用。
The important research contents in this experiment as follows:1. Oyster shellpowder (OP) and sugarcane bagasse (BA) were used as bottom substrates to researchtheir effects on Litopenaeus vannamei intensive culture under zero-water exchangecondition. Parameters including water quality (TAN concentration, NO_2-Nconcentration, Chl.a concentration), bacterial density (total bacterial and nitrogencycle bacterial densities) in the bottom and water, zooplankton densities, and shrimpgrowth performance were investigated；2. Different nitrogen species assimilated by aVibrio alginolyticus (Z5) strain under different C/N ratios；3. Ammonia nitrogen andnitrite nitrogen removal in shrimp culture by Vibrio alginolyticus (Z5) strainimmobilized in SA-beads. The pupose of this experiment was provided as referencefor the research of optimization of bottom substrate on shrimp culture, the regulativemechanism of probiotic on water quality, and application of immobilizedmicroorganism technology on shrimp culture. In addition, contribute to reduce thedischarge of wastewater to environment in aquaculture. The significant results werelisted below:
1. Effect of optimization of bottom substrate on shrimp culture under zero-waterexchange condition
Oyster shell powder (OP), sugarcane bagasse (BA), OP and BA mixture (OS),and fresh soil (FS) were used as bottom substrates to compare their effects onLitopenaeus vannamei intensive culture under zero-water exchange condition.Parameters including water quality, total bacterial and nitrogen cycle bacterialdensities in the bottom and water, zooplankton densities, and shrimp growth performance were investigated. Total ammonia-nitrogen (TAN) in BA was below0.36mg/L which was significantly lower than those in other treatments at the end of theculture (P <0.05). NO_2-N concentrations in OS were below0.50mg/L which wassignificantly lower than those in other treatments (P <0.05). This result wasconsistent with the relatively higher ammonia-oxidizing and nitrite-oxidizing bacterialdensities, respectively. The Chl.a concentrations in OP, BA, and OS exhibited a slowincreasing trend during the entire period, whereas these in FS kept a low level and didnot increase significantly. The bacterial densities in the bottom substrates of OP, BA,and OS were one order of magnitude greater than those in FS. The zooplanktondensities in treatments containing the substrate of BA (BA and OS) were significantlyhigher than those without BA (FS and OP)(P <0.05). These increased bacterial andzooplankton generated synergistic effects along with improved water quality usingBA and OS on shrimp growth performance, which was demonstrated in highersurvival rates and yields, and lower feed conversion rates. We first demonstrate thebeneficial effects of using appropriate bottom substrates, indicating that BA and OSare effective for improving the performance of L. vannamei intensive culture withoutwater exchange.
2. Effect of sugarcane bagasse as bottom substrates at different amounts onLitopenaeus vannamei intensive culture
Different amounts of bagasse (0.5kg/m~2,1kg/m~2,1.5kg/m~2and2kg/m~2,bagasseshort for BA) mixing with oyster shell powder（5kg/m~2, oyster shell powder short forOP） were added as bottom substrates into Litopenaeus vannamei intensive culture toestimatethe effect on the aquaculture environment and shrimp by investigating waterquality, total bacterial and nitrogen cycle bacterial densities in the bottom and water,zooplankton (>160μm) densities and shrimp growth performance. The groups wereBA0.5, BA1, BA1.5and BA2, respectively. Result showed that there were somesignificance differences in water quality and bacterial density in water and bottomsubstrates with different amounts of bagasse. BA could promote the growth ofzooplankton strongly; nevertheless, there were no significance difference with addition different amounts of BA. In addition, BA suspended in water couldcontribute to improve the utilization of nutrient substance in aquacultureenvironment.However, addition excessive BA would cause the environment pressure,so it was good the amount of BA added between0.5-1kg/m~2.
3. Different nitrogen species assimilated by a Vibrio alginolyticus (Z5) strain underdifferent C/N ratios
Nitrogen assimilation characteristics of a Vibrio alginolyticus (Z5) wereinvestigated under single nitrogen source (organic nitrogen, NH_4-N, or NO_2-N) andmixed nitrogen source (organic nitrogen and NH_4-N, organic nitrogen and NH_4-N, orNH_4-N and NO_2-N) at different C/N ratios. The activities of protease, glutaminesynthetase and nitrite reductase involved in nitrogen metabolism were also determined.The results showed that the assimilation rates of both organic nitrogen and inorganicnitrogen of Vibrio alginolyticus (Z5) were affected by C/N ratio. The assimilationrates of NH_4-N and NO_2-N under C/N ratio of20were3.39times and2.25timeshigher than those under C/N ratio of5. There were no significant differences in theassimilation of NH_4-N and NO_2-N between C/N of15and C/N of20(P>0.05). Theassimilation processes of NH_4-N and NO_2-N were NH_4-N→bacterial protein andNO_2-N→NH_4-N→bacterial protein, respectively. The assimilation rates of inorganicnitrogen (NH_4-N and NO_2-N) increased with higher C/N; and the protein nitrogencontent of the bacteria enriched from NH_4-N and NO_2-N increased from25.0%and19.4%under C/N of5to41.3%and43.0%under C/N of20, respectively. Moreover,Vibrio alginolyticus (Z5) preferred to assimilate NH_4-N than NO_2-N. Additionally, theactivity of glutamine synthetase was regulated significantly by NH_4-N while theactivities of protease and NiR were not regulated significantly by the nitrogen species.
4. Ammonia nitrogen and nitrite nitrogen removal in shrimp culture by Vibrioalginolyticus (Z5) immobilized in SA-beads
In this study, SA-beads which formed of the Vibrio alginolyticus (Z5), sodiumalginate and sugarcane bagasse were studied for the ammonia nitrogen (NH_4-N) and nitrite nitrogen (NO_2-N) removal in shrimp cultrue. In addition, the variation ofbacterial density embedded in SA-beads was evaluated. The results indicatedSA-beads might have a maximum capacity of1.06×10~8cfu/bead. However, optimuminitial bacteria density embedded in SA-beads should be10~4cfu/bead-105cfu/bead.The maximum NO_2-N degradation rate of SA-beads could reach to8.44mg/L·day,and the average NO_2-N degradation quantity of each bead was0.06mg. The additionof carbon source could accelerate the degradation of NH_4-N and NO_2-N for-beads. Inorder to evaluate activity of SA-beads in shimp culture,50days cultivationexperiment was carry out in aquariums. The NH_4-N and NO_2-N concentrations whichwater treatment with SA-beads were below1.55mg/L and1.62mg/L at later period,repectively, which were lower than those in control treatment (P <0.05). There wasno significant difference in NH_4-N and NO_2-N concentrations which water treatmentwith SA-beads and exchanging water (P>0.05), and the yield which water treatmentwith SA-beads could reach about70%of those which water treatment withexchanging water. Moreover, POC and DOC concentrations in water were enhancedby SA-beads added. In late period, even though some SA-brads broken, the sugarcanebaggse contained in SA-beads might be suspend in water to continue to keep goodwater quality.
引文
Allen, A.E., Howard-Jones, M.H., Booth, M.G., et al. Importance of heterotrophic bacterialassimilation of ammonium and nitrate in the Barents Sea during summer. Journal of MarineSystems,2002,38:93-108
Anderson, R.K., Parker, P.L., Lawrence, A. A13C/12C tracer study of the utilization of presentedfeed by a commercially important shrimp Penaeus vannamei in a pond grow-out system.Journal of the World Aquaculture Society,1987,18(3):148-155
Arnold, S.J., Coman, F.E., Jackson, C.J., et al. High-intensity, zero water-exchange production ofjuvenile tiger shrimp, Penaeus monodon: An evaluation of artificial substrates and stockingdensity. Aquaculture,2009,293:42-48
Aduzzaman, M., Wahab, M.A., Verdegem, M.C.J., et al. C/N ratio control and substrate additionfor periphyton development jointly enhance freshwater prawn Macrobrachium rosenbergiiproduction in ponds. Aquaculture,2008,280:117-123
Austin, B.L.F., Stuckey, P.A.W., Robertson, I., et al. A probiotic strain of Vibrio alginolyticuseffective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum andVibrio ordalii. Journal of Fish Disease,1995,18:93-96
Avnimelech, Y. C/N ratio as a control element in aquaculture systems. Aquaculture,1999,176:227-235
Avnimelech, Y. Feeding with microbial flocs by tilapia in minimal discharge bio-flocs technologyponds. Aquaculture,2007,264:140-147
Avnimelech, Y., Kochva, M., Diab, S. Development of controlled intensive aquaculture systemswith a limited water exchange and adjusted C to N ratio. Israeli Journal ofAquaculture-Bamidgeh,1994,46:119-131
Avnimelech, Y., Ritvo, G. Shrimp and fish pond soils: processes and management. Aquaculture,2003,220:549-567
Avnimelech, Y., Verdegem, M.C.J., Kurup, M., et al. Sustainable land-based aquaculture: rationalutilization of water, land and feed resources. Mediterranean Aquaculture Journal,2008,1(1):45-55
Bergheim, A., sg rd, T. Waste production from aquaculture. In: Baird, D.J., et al., eds.Aquaculture and Water Resource Management. Oxford, UK: Blackwell,1996.50-80
Beveridge, M.C.M., Begum, M., Frerichs, G.N., et al. The ingestion of bacteria in suspension bythe tilapia Oreochromis niloticus. Aquaculture,1989,81:373-378
Bothe, H, Jost, G, Schloter, M, et al. Molecular analysis of ammonia oxidation and denitrificationin natural environments. FEMS Microbiol. Rev.2000,24:673-690.
Boyd, C.E. Water quality in ponds for aquaculture. Shrimp Mart (Thai), Songkhla, Thailand,1996,pp:482
Boyd, C.E. Water quality management and aeration in shrimp farming. In Fisheries and AlliedAquaculture Departmental Series, vol.2. Alabama Agricultural Experiment Station, AuburnUniversity, Alabama,1989
Bradford, M.M. A rapid and sensitive method f or quantitation of microgram quantities of proteinutilizing the principle of protein dye binding. Analytical Biochemistry,1976,72:248-254.
Bradley, P.B., Lomas, M.W., Bronk, D. A. Inorganic and organic nitrogen use by phytoplanktonalong chepeake bay, measured using a flow cytometric sorting approach. Estuaries andCoasts,2010,33:971-984.
Briggs, M.R.P., Funge-Smith, S.J. A nutrient budget of some intensive marine shrimp ponds inThailand. Aquaculture and Fisheries Management,1994,25:789-811.
Browdy, C.L., Bratvold, D., Stokes, A.D., et al. In: Browdy, C.L., Jory, D.E, eds. Perspectives onthe application of closed shrimp culture systems: The New Wave, Proceedings of the SpecialSession on Sustainable Shrimp Culture, Aquaculture2001. Baton Rouge, U: The WorldAquaculture Society,2001.20-34
Burford, M.A, Thompson, P.J., Mcintosh, R.P., et al. The contribution of flocculated material toshrimp (Litopenaeus vannamei) nutrition in a high-intensity, zero-exchange system.Aquaculture,2004,232:525-537
Cao, G.M., Zhao, Q.X., Sun, X.B., et al. Characterization of nitrifying and denitrifying bacteriacoimmobilized in PVA and kinetics model of biological nitrogen removal by coimmobilizedcells. Enzyme Microb Tech,2002,30:49-55
Cheng, S.Y., Chen, J.C. Accumulation of nitrite in the tissues of Penaeusm monodon exposed toelevated ambient nitrite after different time periods. Arch. Environ. Contam. Toxicol.,2000，39:183-192
Chien, Y.H. Water quality requirements and management for marine shrimp culture. In: Wyban, J,eds. Proceedings of the Special Session on Shrimp Farming. Baton Rouge, LA, U: WorldAquaculture Society,1992.144-156.
Chien, Y.H. The Management of Sediment in Prawn Ponds. Proceedings of the Third BrazilianShrimp Farming Congress. Joao Pessoa, PB, Brazil.1989.
Colt, J.E., Armstrong, D.A. Nitrogen toxicity to crustaceans, fish and mollusk. In Allen, L.J.,Kinney, E.C. Proceedings of the Bio-Engineering Symposium for Fish Culture. Bethesda,MD: Fish Culture Section of the American Fisheries Society,1981.34-47
Crab, R., Avnimelech, Y., Defoirdt, T., et al. Nitrogen removal techniques in aquaculture for asustainable production. Aquaculture,2007,270:1-14
Crab, R., Chielens, B., Wille, M., et al. The effect of different carbon sources on the nutritionalvalue of bioflocs, a feed for Macrobrachium rosenbergii postlarvae. Aquac. Res.,2010,41:559-567
Crab, R., Kochva, M., Verstraete, W., et al. Bio-flocs technology application in over-wintering oftilapia. Aquacultural Engineering,2009,40:105-112.
Davis, T.A., Volesky, B., Mucci, A. A review of the biochemistry of heavy metal biosorption bybrown algae. Water Research,2003,37:4311-4330
De Schryver, P., Crab, R., Defoirdt, T., et al. The basics of bio-flocs technology: The added valuefor aquaculture. Aquaculture,2008,277:125-137
Decamp, O., Cody, J., Conquest, L., et al. Effect of linity on natural community and production ofLitopenaeus vannanmei (Boone), within experimental zero-water exchange culture systems.Aquaculture Research,2003,34:345-355
Dierberg, F.E., Kiattisimkul, W. Issues, impacts, and implications of aquaculture in Thailand ofshrimp. Environmental Management,1996,20:649-666
Ebeling, J.M., Timmons, M.B., Bisogni, J.J. Engineering analysis of the stoichiometry ofphotoautotrophic, autotrophic and heterotrophic removal of ammonia-nitrogen in aquaculturesystems. Aquaculture,2006,257:346-358
Eiler, A., Langenheder, S., Bertilsson, S., et al. Heterotrophic bacterial growth efficiency andcommunity structure at different natural organic carbon concentrations. Applied andEnvironmental Microbiology,2003,69(7):3701-3709
Eisenberg, D., Gill, H.S., fluegl, G.M., et al. Structure-function relationships of glutaminesynthetases. Biochim. Biophys. Acta,2000,1477：122-145
Emerenciano, M., Ballester, E.L.C., Cavalli, R.O., et al. Effect of Biofloc technology (BFT) on theearly postlarval stage of pink shrimp Farfantepenaeus paulensis: growth performance, floccomposition and linity stress tolerance. Aquacult. Int.,2011,19:891-901
Fisher,S.H. Regulation of nitrogen metabolism in Bacillus subtilis: vive la difference! MolecularMicrobiology,1999,(2):223-232
Gao, L., Shan, H.W., Zhang, T.W., et al. Effects of carbohydrate addition on Litopenaeusvannamei intensive culture in a zero-water exchange system. Aquaculture,2012,342-342:89-96
Garriques, D., Arevalo, G. An evaluation of the production and use of a live bacterial isolate tomanipulate the microbial flora in commercial production of Penaeus vannamei postlarvae inEcuador. In: Browdy C L, Hopkins J S ed. Swimming through Troubled Water. Proceedingsof the Special Session on Prawn Farming, Aquaculture'95. World Aquaculture Society,1995.53-59
Gatesoupe, F.J. Siderophore production and probiotic effect of Vibrio sp. associated with turbotlarvae, Scophthalmus maximus. Aquat. Living Resour.,1997,10:239-246.
Ginige, M.P, Hugenholtz, P., Daims, H., et al. Use of stable-isotope probing, full-cycle rRNAanalysis, and fluorescence in situ hybridization-microautoradiography to study amethanol-fed denitrifying microbial community. Appl. Environ. Microbiol.2004,70(1):588-596.
Goldman, J.C. Growth of marine bacteria in batch and continuous culture under carbon andnitrogen limitation. Limmol. Oceanogr.,2000,45(4):789-800
Gombotz, W.R., Wee, S.F. Protein release from alginate matrices. Adv Drug Deliver Rev,2012,64:194-205
Granéli, E., Carlsson, P., Legrand, C. The role of C, N and P in dissolved and particulate organicmatter as a nutrient source for phytoplankton growth, including toxic species. AquaticEcology,1999,33:17-27.
Gullian, M., Thompson, F., Rodriguez, J. Selection of probiotic bacteria and study of theirimmunostimulatory effect in Penaeus vannamei. Aquaculture,2004,233:1-14
Hargreaves, J.A. Photosynthetic suspended-growth systems in aquaculture. Aquacult. Eng.,2006,34:344-363.
Hill, C.B., Khan, E. A Comparative Study of Immobilized Nitrifying and Co-ImmobilizedNitrifying and Denitrifying Bacteria for Ammonia Removal from Sludge DigesterSupernatant. Water Air Soil Poll,2008,195:23-33
Hobbie, J.E., Daley, R.J., Jasper, S. Use of nuclepore filters for counting bacteria by fluorescencemicroscopy. Appl. Environ. Microbiol.,1977,33:1225-1228.
Hopkins, J.S., Sandifer, P.A., Browdy, C.L. Sludge management in intensive pond culture ofshrimp: Effect of management regime on water quality, sludge characteristics, nitrogenextinction, and shrimp production. Aquacultural Engineering,1994,13:11-30
Hsieh, Y.L., Tseng, S.K., Chang, Y.J. Nitrification using polyvinyl alcohol-immobilized nitrifyingbiofilm on an O2-enriching membrane. Biotechnol Lett,2002,24:315-319
Huang, X.H., Li, C.L., Zheng, L., et al. The toxicity of NO2-N on Litopenaeus vannamei andeffects of NO2-N on factors relating to the anti-disease ability. Acta Hydrobiol. Sin.,2006,30(4):466-471
Ivanov, V., Stabnikova, O., Sihanonth, P., et al. Aggregation of ammonia-oxidizing bacteria inmicrobial biofilm on oyster shell surface. World Journal of Microbiology and Biotechnology,2006,22:807-812.
Jackson, C., Preston, N., Thompson, P. J., et al. Nitrogen budget and effluent nitrogen componentsat an intensive shrimp farm. Aquaculture,2003,218:397-411
Jensen, F.B. Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem.Physiol. A Mol. Integr. Physiol.,2003,135:9-24
Jorgensen, N.O.G., Kroer, N., Coffin, R.B. Utilization of dissolved nitrogen by heterotrophicbacterioplankton: effect of substrate C/N ratio. Applied and Environmental Microbiology,1994,60(11):4124-4133
Jorgensen, N.O.G., Kroer, N., Coffin, R.B., et al. Relations between bacterial nitrogen metabolismand growth efficiency in an estuarine and an open-water ecosystem. Aquatic microbialecology,1999,18:247-261
Keymi, M.A., Saad, C.R., Sijam, K., et al. Effect of Bacillus subtilis on growth development andsurvival of postlarvae Macrobrachium rosenbergii (de Man). Aquacult Nutr,2007,13:131-136
Khin, T., Annachhatre, A.P. Novel microbial nitrogen removal processe. Biotechnology Advances,2004,22:519-532
Kim, J.K, Park, K.J., Cho, K.S., et al. Aerobic nitrification–denitrification by heterotrophicBacillus strains. Bioresource Technology,2005,96(17):1897-1906
Kim, S.K., Kong, I., Lee, B.H., et al. Removal of ammonium-N from a recirculation aquaculturalsystem using an immobilized nitrifier. Aquacult Eng,2000,21:139-150
Kindaichi, T., Ito, T., Okabe, S. Ecophysiological interaction between nitrifying bacteria andheterotrophic bacteria in autotrophic nitrifying biofilms as determined bymicroautoradiography-fluorescence in situ hybridization. Appl. Environ. Microbiol,2004.70(3):1641-1650
Kirchman, D.L., Keil, R.G. Carbon limitation of ammonium uptake by heterotrophic bacteria insubarctic Pacific. Limnol. Oceanogr.,1990,36(6):1258-1266
Koza, M. Toyocerin (Bacillus toyoi) as growth promoter for animal feeding. Microbiol alimentnutr,1986,4:121-135
Kroer, N., Jorgensen, N.O.G., Coffin, R.B. Utilization of dissolved nitrogen by heterotrophicbacterioplankton: a comparison of three ecosystems. Applied and EnvironmentalMicrobiology,1994,60(11):4116-4123
Lee, J., Cho, M.H. Removal of nitrogen in wastewater by polyvinyl alcohol(PVA)-immobilizationof effective microorganisms. Korean J Chem Eng.,2010,27(1):193-197
Li, X., Hu, H. Y., Gan, K., et al. Growth and nutrient removal properties of a freshwater microalgaScenedesmus sp. LX1under different kinds of nitrogen sources. Ecological Engineering,2010,36:379-381.
Liang, Y., Leonard, J.J., Feddes, J.J.R., et al. Influence of carbon and buffer amendment onammonia volatilization in composting. Bioresource Technology,2006,97:748-761.
Lighmer, D.V.A. Handbook of Pathology and Diagnostic Procedures for Diseases of PenaeidShrimp．LA：Wodd Aquaculture Society，1996.
Lin, Y.F., Jing, S.R., Lee, D.Y, et al. Performance of a constructed wetland treating intensiveshrimp aquaculture wastewater under high hydraulic loading rate. Environmental Pollution,2005,134:411-421
Lin, Y.F., Jing, S.R., Lee, D.Y., et al. Nutrient removal from aquaculture wastewater using aconstructed wetlands system culture. Aquaculture,2002,209:169-184
Liu, K.F, Chiu, C.H., Shiu,Y.L., et al. Effects of the probiotic, Bacillus subtilis E20, on thesurvival, development, stress tolerance, and immune status of white shrimp, Litopenaeusvannamei larvae. Fish Shellfish Immunol,2010,28:837-844
Liu, C.H., Chen, J.C. Effect of ammonia on the immune response of white shrimp Litopenaeusvannamei and its suscep tibility to Vibrio alginolyticus. Fish&Shellfish Imm unology,2004,16:321-333
Liu, Y.X., Yang, T.O., Yuan, D.X., et al. Study of municipal wastewater treatment with oyster shellas biological aerated filter medium. Delination,2010,254:149-153.
Lozinsky, V.I., Plieva, F.M. Polyvinyl alcohol cryogels employed as matrices for cellimmobilization.3. Overview of recent research and developments. Enzyme Microb Tech,1998,23:227-242
Mahious, A.S., Gatesoupe, F.J., Hervi, M., et al. Effect of dietary inulin and oligoccharides asprebiotics for weaning turbot, Psetta maxima (Linnaeus, C.1758). Aquacult Int,2006,14:219-229
Manju, N.J., Deepesh, V., Achuthan, C., et al. Immobilization of nitrifying bacterial consortia onwood particles for bioaugmenting nitrification in shrimp culture systems. Aquaaculture,2009,294(1-2):65-75
Martínez-Córdova, L.R., Pe a-Messina, E. Biotic communities and feeding habits of Litopenaeusvannamei (Boone1931) and Litopenaeus stylirostris (Stimpson1974) in monoculture andpolyculture semi-intensive ponds. Aquacutlture Research,2005,36:1075-1084.
Martinez-Cordova, L.R., Villarreal-Colmenares, H., Porchas-Cornejo, M.A., Naranjo-Paramo, J.,and Aragon-Noriega, A. Effect of aeration rate on growth, survival and yield of white shrimpPenaeus vannamei in low water exchange ponds. Aquacultural Engineering,1997,16:85-90.
Martínez-Espino, R.M., Marhuenda-Egea, F.C, Bonete, M.J. Purification and characterition of apossible assimilatory nitrite reductase from the halophile archaeon Haloferax mediterranei.FEMS Microbiology Letters,2001,196:113-118.
Menasveta, P., Panritdam, T., Sihanonth, P., et al. Design and function of a closed, recirculatingseawater system with denitrification for the culture of black tiger shrimp broodstock.Aquacultural. Engineering,2001,25:35-39
Moriarty, D.J.W. The role of microorganisms in aquaculture ponds. Aquaculture,1997,151:333-349.
Moriarty, D.J.W. Control of luminous Vibrio species in penaeid aquaculture ponds.Aquaculture,1998,164:351-358
Moss, K.P.K., Moss, S. Effects of Artificial Substrate and Stocking Density on the NurseryProduction of Pacific White Shrimp Litopenaeus vannamei. Journal of the World AquacultureSociety,2004,35(4):536-542
Naidu, K.S.B, Devi, K., Adam, K.J. Evaluation of different matrices for production of alkalineprotease from Bacillus subtilis-K30by entrapment technique. Afr J Biochem Res.,2011,5(7):220-225
Namasivayam, C., koda, A., Suzuki, M. Technical note removal of phosphate by adsorption ontooyster shell powder-kinetic studies. J. Chem. TechnolBiot,2005,80:356-358
Naylor, R.L., Goldburg, R.J., Mooney, H., et al. Nature’s subsidies to shrimp and lmon farming.Science,1998,282:883-884.
Naylor, R.L., Goldburg, R.J., Primavera, J.H., et al. Effect of aquaculture on world fish supplies.Nature,2000,405:1017-1024.
Páez-Osuna, F., Guerrero-Galván, S.R., Ruiz-Fernández, A.C. The environmental impact ofshrimp aquaculture and the coastal pollution in Mexico. Mar Pollut Bull,1998,36:65-75
Pandey, A., Soccol, C.R., Nigam, P.Q., et al. Biotechnological potential of agro-industrial residues.I: sugarcane bagasse. Bioresource Technology,2000,74:69-80.
Park, W.H., Polprasert, C. Roles of oyster shells in an integrated constructed wetland systemdesigned for P removal. Ecological Engineering,2008,34:50-56
Parker, R.B. Probiotics, the other half of the antibiotics story. Anim nutr health,1974,29:4-8
Pernthaler, J., GI ckner, F., Sch nhuber, W., et al. Fluorescence in situ hybridization (FISH) withrRNA-targeted oligonucleotide probes. Meth.Microbiol.2001,30:207-226
Pernthaler, A., Pernthaler, J., Amann, R. Fluorescence in situ hybridization and catalyzed reporterdeposition for the identification of marine bacteria. Applied and Environmental Microbiology,2002,68:3094-3101.
Radhakrishnan, M.V, Sugumaran, E. Fluctuations in Zooplankton Density on Sugarcane BagasseSubstrate Used for Fish Culure. American-Eurasian Journal of Scientific Research,2010,5(2):153-155.
Rahmatullah, S.M., Beveridge, M.C.M. Ingestion of bacteria in suspension by Indian major carps(Catla catla, Labeo rohita) and Chinese carps (Hypophthalmichthys molotrix, Aristichthysnobilis). Hydrobiologia,1993,264:79-84
Ramesh, M.R, Shankar, K.M, Mohan, C.V, et al. Comparison of three plant substrates forenhancing carp growth through bacterial biofilm. Aquacult. Eng.1999,19:119-131.
Rao, P.S.S., Karunagar, I., Otta, S.K., et al. Incidence of bacteria involved in nitrogen and sulphurcycles in tropical shrimp culture ponds. Aquaculture International,2000,8:463-472
Reid, B., Arnold, C.R. The intensive culture of the Penaeid shrimp Penaeus vannamei Boone in arecirculating raceway system. Journal of the World Aquaculture Society,1992,23:146-153.
Reitzer, L.J. Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate,asparagine, L-alanine, and D-alanine. In: Neidhardt, F. C., Curtiss, III R., Ingraham. J. L, et al.Escherichia coli and lmonella: Cellular and Molecular Biology. Washington, DC: AmericanSociety for Microbiology Press,1996.391–407
Ross, R.M., Quetin, L. B., Martinson, D.G., et al. Palmer LTER: Patterns of distribution of fivedominant zooplankton species in the epipelagic zone west of the Antarctic Peninsula,1993–2004. Deep-Sea Research. II,2008,55:2086-2105.
Salman, F.M., El-Kadi, R.I., Abdel–Rahman, H., et al. Biologically treated sugar beet pulp as asupplement in goat rations. Int. J. Agric. Biol.,2008,10:412-416
Samocha, T.M., Patnaik, S., Speed, M., et al. Use of molasses as carbon source in limiteddischarge nursery and grow-out systems for Litopenaeus vannamei. Aquacult. Eng.,2007,36:184-191
Schirawski, J., Unden, G. Anaerobic respiration of Bacillus macerans with fumarate,TMAO,nitrate and nitrite and regulation of the pathways by oxygen and nitrate. Arch Microbiol，1995,163:148-154.
Schramm, A, Larsen, L.H., Revsbech, N.P., Ramsing, et al. Structure and function of a nitrifyingbiofilm as determined by in situhybridization and the use of microelectrodes. Appl. Environ.Microbiol.1996,62(12):4641-4647
Schroeder, G.L. Carbon and Nitrogen budget in manured fishponds on Israel’s coastal plain.Aquaculture,1987,62:259-279
Schryver, P.D, Crab, R., Defoirdt, T., et al. The basics of bio-flocs technology: The added value foraquaculture. Aquaculture,2008,277:125-137
Seo, J.K., Jung, I.H., Kim, M.R., et al. Nitrification performance of nitrifiers immobilized in PVA(polyvinyl alcohol) for a marine recirculating aquarium system. Aquacult Eng,2001,24:181-194
Shan, H., Obbard, J.P. Ammonia removal from prawn aquaculture water using immobilizednitrifying bacteria. Appl Microbiol Biotechnol,2001,57:791-798
Song, Z.F., An, J., Fu, G.H., et al. Isolation and characterization of an aerobic denitrifying Bacillussp. YX-6from shrimp culture ponds. Aquaculture,2011,319:188-193
Tango., M.S., Gagnon, G.A. Impact of ozonation on water quality in marine recirculation systems.Aquaculture engineering.2003,29(3-4):125-137
Thompson, F.L., Abreu, P.C., Wasielesky, W. Importance of biofilm for water quality andnourishment in intensive shrimp culture. Aquaculture,2002,203:263-278
Tones, A.B., Dennison, W.C, Preston, N.P. Integrated treatment of shrimp effluent bysedimentation, oyster filtration and macroalgal absorption: A laboratory scalestudy．Aquaculture,2001,193:155-178
Tseng, I.T., Chen, J.C. The immine response of white shrimp Litopenaeus vannamei and itssusceptibility to Vibrio alginolyticus under nitrite stress. Fish Shellfish Immunol.2004,17(4):325-333
Tseng, K.F., Su, H.M., Su, M.S. Culture of Penaeus monodon in a recirculating system.Aquacultural Engineering,1998,17:138-147
Urata, K., Satoh, T. Enzyme localization and orientation of the active site of dissimilatory nitritereductase from Bacillus firmus. Arch Microbiol,1991,156:24-27
Wacker, I., Ludwig, H., Reif, I., et al. The regulatory link between carbon and nitrogenmetabolism in Bacillus subtilis: regulation of the gltAB operon by the catabolite controlprotein CcpA. Microbiology,2003,149(10):3001-3009.
Wang, Y., He, Z. Effect of probiotics on alkaline phosphatase activity and nutrient level insediment of shrimp, Penaeus vannamei, ponds. Aquaculture,2009,287:94-97
Wang, Y.B. Effect of probiotics on growth performance and digestive enzyme activity of theshrimp Penaeus vannamei. Aquaculture,2007,269:259-264
Wasielesky, W., Atwood, H., Stokes, A., et al. Effect of natural production in a zero exchangesuspended microbial floc based super-intensive culture system for white shrimp Litopenaeusvannamei. Aquaculture,2006,258:396-403.
Wu, Z.H., Liu, C.B., Liu, C.R., et al. Histopathological research on chronic poisoning of Penaeuschinensis by nitrite and ammonia. Central China Normal Univ. Natur.,1999,33(1):119-122
Xiao, J.J., Zhu, C.X., Sun, D.Y., et al. Removal of ammonium-N from ammonium-rich sewageusing an immobilized Bacillus subtilis AYC bioreactor system. Journal of EnvironmentalSciences,2011,23(8):1279-1285
Xing, D.L., Huo, T.B., Wu, H.M., et al. Simultaneous digestion for determination of totalphosphorus and total nitrogen in sea water. Journal of Dalian Fisheries University,2006,21(3):219-225
Yang, L. Investigation of nitrification by co-immobilized nitrifying bacteria and zeolite in abatchwise fluidized bed. Water Sci Technol,1997,35:169-175
Zang,W.L., Xu, X.C., Dai, X.L., et al. Toxic effects of Zn2+, Cu2+, Cd2+and NH3on Chineseprawn. Chin. J. Oceanol. Limol.,1993,11(3):254-259
Ziaei-Nejad, S., Rezaei, M.H., Takami, G.A., et al. The effect of Bacillus spp. Bacteria used asprobiotics on digestive enzyme activity, survival and growth in the Indian white shrimpFenneropenaeus indicus. Aquaculture,2005,252:516-524
安建，宋增福，杨先乐，等.好氧反硝化芽孢杆菌筛选及其反硝化特性.环境科学研究，2010，23（1）：100-105
曹煜成，李卓佳，贾小平，等.对虾工厂化养殖的系统结构.南方水产,2006,2(3):72-76
曹煜成，李卓佳，林黑着，等.地衣芽孢杆菌De在优质草鱼养殖中的应用研究.南方水产,2008,4(3)：15-19
陈春云，庄源益，方圣琼.小球藻对养殖废水中N、P的去除研究.海洋环境科学,2009,28(1):9-11.
陈金春，陈国强．微生物学实验指导．北京：清华大学出版社，2005：36-38
崔毅,陈碧鹃,陈聚法.黄渤海海水养殖自身污染的评估.应用生态学报,2005,16(1):180-185
催和，肖乐.2011-2012我国对虾产业发展及展望.中国水产，2012，（7）：25-26
丁天喜，李明云.对虾综合养殖的模式与原理.浙江水产学院学报,1996,15(2):134-139
丁贤，李卓佳，陈永青，等.芽孢杆菌对凡纳对虾生长和消化酶活性的影响.中国水产科学，2004，11（6）：580-584
丁彦文,艾红.微生物在水产养殖中的应用.湛江海洋大学学报,2000,20（1）:68-73.
范成新，杨龙元，张路.太湖底泥及其间隙水中氮磷垂直分布及相互关系分析.湖泊科学，2000,12(4):359-366
付天玺，魏开建，许国煥.芽孢杆菌在水产养殖中的研究和应用概况.水利渔业，2007,27(3):102-104
高磊，包卫洋，张天文，等.水体碳氮比对芽孢杆菌、乳酸菌与弧菌生长、拮抗作用及菌体碳氮比的影响.中国海洋大学学报（自然科学版），2013，43（1）：28-34
顾传辉．人工湿地处理系统概述．中山大学研究生学刊(自然科学版),2001,22(2)：34-40
郭静，Gadafii, L.,郭安南，等.溶藻弧菌相关分离株的分子及VITEK鉴定.水产科学，2012，36（3）：383-390.
韩士群，范成新，严少华.固定化微生物对养殖水体浮游生生的影响及生物除氮的研究.应用与环境生物学报,2006,12(2)：252-254
胡毅，谭北平，麦康森，等.饲料中益生茵对凡纳滨对虾生长、肠道茵群及部分免疫指标的影响.中国水产科学，2008，15（2）：244-250
黄丽彬，陈有容，齐凤兰.蜡状芽孢杆菌在饲料中的应用.粮食与饲料工作，2001，9：32-33.
黄玉玲.我国虾类淡水养殖概述.水利渔业,2003,1:1-3
黄正，李谷,2002.固定化硝化细菌去除养殖废水中氨氮的研究.华中科技大学学报（医学版），31（1）：18-20
姜令绪,潘鲁青,肖国强.氨氮对凡纳滨对虾免疫指标的影响.中国水产科学,2004,11(6):537-541
李纯厚，黄洪辉，林钦,等.海水对虾池塘养殖污染物环境负荷量的研究.农业环境科学学报，2004，23(3):545～550
李辉，徐新阳，李培军，等.人工湿地中氨化细菌去除有机氮的效果.环境工程学报，2008，2（8）：1044-1047
李健，刘得月，刘淇，等.北方地区对虾工厂化养殖技术研究．渔业现代化,2003，1：11-13
李晓燕.利用微生物固定化技术进行养殖水体脱氮的研究.山西大学学报（自然科学版），2008,31（2）：262-264
李卓佳，郭志勋，冯娟，等.应用芽孢杆菌调控虾池微生态的初步研究.海洋科学,2006,30(11):28-31
林金忠，林星.我国对虾养殖模式现状及其发展趋势.现代渔业信息,1999,14（6）:18-21
林亮,李卓佳,郭志勋,等.施用芽孢杆菌对虾池底泥细菌群落的影响.生态学杂志,2005,24(1):26-29
刘川顺，李平，李键.谷氨酰胺合成酶产酶菌株的筛选及酶学性质.农产品加工·学刊，2010，7：4-7.
刘文斌，尹君，方星星，等．3种益生素配伍对异育银鲫(Carassius auratus gibelio)生长、消化及肠道茵群组成的影响．海洋与湖沼，2007，38（1）：29-35
刘忠颖，刘洋，鲍相渤，等.水产养殖有益菌的研究进展.水产科学，2010，29(8)：500-504
罗琳，舒廷飞，温琐茂.水产养殖对近海生态环境的影响.水产科学，2002,21(3):28-30
聂艳丽,刘永国,李娅,等.甘蔗渣资源利用现状及开发前景.林业经济,2007,5:61-63.
潘鲁青，王克行.中华绒螯蟹幼体消化酶活力与氨基酸组成的研究.中国水产科学,1997,4(2):13-20.
潘忠诚，王芳宇，何建国，等．凡纳滨对虾在不同实验条件下感染白斑综合症的研究.衡阳师范学院学报，2008，29(6)：63-67
孙艳，刘飞，宋晓玲，等.饲料中添加益生菌对凡纳滨对虾(Litopenaeus vannamei)非特异免疫基因表达量和抗病力的影响。海洋与湖沼，2012，43（4）：845-851
乔顺风,刘恒义,靳秀云.养殖水体氨氮积累危害与生物利用.河北渔业,2006,1:20-22
沈锦玉，沈智华，尹文林，等.饲喂枯草芽孢杆菌对银鲫等水生动物肠道菌群及消化酶活性的影响。水产科学，2004,28(增刊):146-150
孙国铭，汤建华，仲霞铭.氨氮和亚硝酸氮对南美白对虾的毒性研究.水产养殖，2002，1：22-24.
孙舰军,丁美丽.氨氮对中国对虾抗病力的影响.海洋与湖沼,1999,30(3):267-272
王克行.虾蟹类增养殖学.北京：中国农业出版社，1997.79-81
王兰，廖丽华.光合细菌固定化及对养殖水净化的研究.微生物学杂志，2005,25（3）:50-53
王彦波，许梓荣，邓岳松.水产养殖中氨态和亚硝酸盐氮的危害及治理.饲料工业，2002，23（12）：46-48
邢殿楼，霍堂斌，吴会民，等.总磷、总氮联合消化的测定方法.大连水产学院学报，2006，21（3）：220-225
徐亚同.废水反硝化除氮.上海环境科学,1994,13(10):8-12.
杨希，刘德立，邓灵福，等.蜡状芽孢杆菌好氧反硝化特性研究.环境科学研究，2008，21（3）：155-159.
于明超，李卓佳，文国樑.芽孢杆菌在水产养殖应用中的研究进展.广东农业科学，2007，11：78-51.
俞开康，战文斌，周丽，等.我国沿海养殖对虾的疾病及研究现状.中山大学学报(自然科学版),2000,39(增刊)：2-5
张文强，杜若谦，常传刚，等.南方高位池养殖模式.齐鲁渔业,2003,20(5):17-18
张振奎，张勤，刘振亮,等.南美白对虾淡水养殖发展对策.河北渔业.2005,2:19-20
中华人民共和国国家标准,海洋监测规范, GB17378.4-2007a
中华人民共和国国家标准,海洋调查规范, GB12763.6-2007b
周光正.氨和亚硝酸盐对对虾幼体的毒性.海洋湖沼通报,1991,(2):95-98
周小壮，林小涛，林继辉，等.不同模式对虾养殖的自身污染及其环境效应.生态科学,2004,23(1):68-72
朱学芝,郑石轩,潘庆军,等.芽孢杆菌对凡纳滨对虾免疫和生化指标的影响.饲料研究,2007,4:56-59