一株功能益生菌的简易发酵及其在凡纳滨对虾(Litopenaeus vannamei)生物絮团养殖中的应用
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摘要
对一株有脱氮作用的巨大芽孢杆菌(Bacillus megaterium)进行简易发酵,以对虾饲料及赤砂糖为培养基,通过正交实验及发酵条件优化,在对虾饲料3 g/L、赤砂糖6 g/L、接种量1×10~8 CFU/ml、发酵温度31℃、装液量40%条件下,发酵24 h可获得活菌数为1.16×10~(10) CFU/ml的发酵产物。使用发酵得到的巨大芽孢杆菌进行凡纳滨对虾(Litopenaeus vannamei)生物絮团养殖实验。结果显示,添加芽孢杆菌和赤砂糖的增强絮团组的絮团形成速度较添加赤砂糖的絮团组及传统养殖的对照组明显提升(P<0.05),整体上增强絮团组的亚硝酸氮水平与絮团组和对照组差异显著(P<0.05)。养殖结束时,添加巨大芽孢杆菌组的对虾体长、体重水平均显著高于另2组。本研究建立了一种简单可行的功能益生菌发酵方式,并验证添加功能益生菌可提高生物絮团技术在对虾养殖中的效果。
Shrimp aquaculture, as one of the main components of the aquaculture industry, has produced enormous economic benefits and social benefits. But the sustainable development of the shrimp aquaculture has been affected and restricted by the discharge of the waste and the frequent occurrence of diseases. Bio-floc technology by adding brown sugar, molasses and other carbon source in breed pool to promote heterotrophic microbial growth and reproduction to control the water pollution, shows great prospects for development in shrimp culture. In this study, we established a method of simplified fermentation that used a strain of Bacillus megaterium with the activity of denitrification. And we used prawn feed and brown sugar as the medium. Results showed that the best ratio of medium was prawn feed 3 g/L, brown sugar 6 g/L and inoculation volume 1×10~8 CFU/ml through the orthogonal experiment. And the other conditions were optimized by single experiment. We got the fermentation products with the viable cell number was 1.16×10~(10) CFU/ml that fermented with 31℃, 40% liquid volume and 24 hours. Then we fed the Litopenaeus vannamei through the bio-floc technology by using the fermentation products. The result showed that the volume of bio-floc was formed more quickly in dominated bio-floc group(added bacillus and sugar) than bio-floc group(just added sugar) and control(traditional culture pattern)(P<0.05), and the overall concentration level of nitrite-N in dominated bio-floc group was much lower than the other groups(P<0.05). At the end of the culture, the highest of the average body length and weight in prawns was obtained in dominated bio-floc group, and Bio-floc group and control were significantly lower(P<0.05). This study established a simplified and feasible method for the fermentation of probiotic bacteria. And the results showed the effectiveness of the bio-floc technology in prawn culture is verified by the additiong probiotics.
Shrimp aquaculture, as one of the main components of the aquaculture industry, has produced enormous economic benefits and social benefits. But the sustainable development of the shrimp aquaculture has been affected and restricted by the discharge of the waste and the frequent occurrence of diseases. Bio-floc technology by adding brown sugar, molasses and other carbon source in breed pool to promote heterotrophic microbial growth and reproduction to control the water pollution, shows great prospects for development in shrimp culture. In this study, we established a method of simplified fermentation that used a strain of Bacillus megaterium with the activity of denitrification. And we used prawn feed and brown sugar as the medium. Results showed that the best ratio of medium was prawn feed 3 g/L, brown sugar 6 g/L and inoculation volume 1×10~8 CFU/ml through the orthogonal experiment. And the other conditions were optimized by single experiment. We got the fermentation products with the viable cell number was 1.16×10~(10) CFU/ml that fermented with 31℃, 40% liquid volume and 24 hours. Then we fed the Litopenaeus vannamei through the bio-floc technology by using the fermentation products. The result showed that the volume of bio-floc was formed more quickly in dominated bio-floc group(added bacillus and sugar) than bio-floc group(just added sugar) and control(traditional culture pattern)(P<0.05), and the overall concentration level of nitrite-N in dominated bio-floc group was much lower than the other groups(P<0.05). At the end of the culture, the highest of the average body length and weight in prawns was obtained in dominated bio-floc group, and Bio-floc group and control were significantly lower(P<0.05). This study established a simplified and feasible method for the fermentation of probiotic bacteria. And the results showed the effectiveness of the bio-floc technology in prawn culture is verified by the additiong probiotics.
引文
Avnimelech Y.Carbon/nitrogen ratio as a control element in aquaculture systems.Aquaculture,1999,176(3-4):227-235
Chen K,Li JS,Yang HT,et al.Phosphate solubilizing abilities of Bacillus megaterium strain P1 and its fermentation conditions.Soil and Fertilizer Science in China,2010(4):73-76[陈凯,李纪顺,杨合同,等.巨大芽孢杆菌P1的解磷效果与发酵条件研究.中国土壤与肥料,2010(4):73-76]
Chen LL,Dong HB,Li ZJ,et al.Review of the application and perspective of biofloc technology in shrimp culture.Marine Science,2014,38(8):103-108[陈亮亮,董宏标,李卓佳,等.生物絮团技术在对虾养殖中的应用及展望.海洋科学,2014,38(8):103-108]
Claudia K,Josef BC,Thibault G.Debottlenecking recombinant protein production in Bacillus megaterium under large-scale conditions-targeted precursor feeding designed from metabolomics.Biotechnology&Bioengineering,2012,109(6):1538-1550
Deng YN,Zhao P,Sun YZ,et al.Conditions for bio-floc formation and its effects in closed culture system of Litopenaeus vannamei.Progress in Fishery Sciences,2012,33(2):69-75[邓应能,赵培,孙运忠,等.生物絮团在凡纳滨对虾封闭养殖试验中的形成条件及作用效果.渔业科学进展,2012,33(2):69-75]
Devaraja TN,Yusoff FM,Shariff M.Changes in bacterial populations and shrimp production in ponds treated with commercial microbial products.Aquaculture,2002,206(3-4):245-256
FAO.FAO yearbook.Fishery and Aquaculture Statistics,2012
Ge HX,Li J,Chen P,et al.The immune response of Litopenaeus vannamei and its susceptibility to Vibrio parahaemolyticus under stress caused by ammonia nitrogen at different concentrations.Progress in Fishery Sciences,2014,35(6):76-82[葛红星,李健,陈萍,等.氨氮胁迫下凡纳滨对虾对副溶血弧菌的易感性.渔业科学进展,2014,35(6):76-82]
Ge HX,Li J,Chen P,et al.Effects of the fermentation solution of wheat bran,cane sugar and Bacillus substilis on water quality and growth performance of cultured Penaeus vannamei in indoor industrial facilities.Chinese Fishery Quality and Standards,2013,3(4):55-62[葛红星,李健,陈萍,等.麦麸、蔗糖和芽孢杆菌发酵液对室内工厂化养殖凡纳滨对虾水质和生长的影响.中国渔业质量与标准,2013,3(4):55-62]
Kanjanachumpol P,Kulpreecha S,Tolieng V.Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019.Bioprocess and Biosystems Engineering,2013,36(10):1463-1474
Ray AJ,Dillon KS,Lotz JM.Water quality dynamics and shrimp(Litopenaeus vannamei)production in intensive,mesohaline culture systems with two levels of biofloc management.Aquacultural Engineering,2011,45(3):127-136
Sun JJ,Ding ML.Effect of ammonia-N on anti-disease ability of Penaeus chinensis.Oceanologia et Limnologia Sinica,1999,30(3):267-72[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]
Wang L,Li J,Zhang PY.Laboratory study on treatment of eutrophicated scenery water body by Bacillus megaterium.Ecology and Environmental Sciences,2009,18(1):75-78[王琳,李季,张鹏岩.巨大芽孢杆菌对富营养化景观水体的净化效果.生态环境学报,2009,18(1):75-78]
Yang ZW,Yang K,Zhang Z,et al.Research on the biofloc bacterial community structure during larval rearing of Litopenaeus vannamei using metagenome sequencing.Journal of Fujian Fisheries,2015,37(2):91-97[杨章武,杨铿,张哲,等.基于宏基因组测序技术分析凡纳滨对虾育苗中生物絮团细菌群落结构.福建水产,2015,37(2):91-97]
Zhang HH,Wang XH,Li C,et al.Isolation and identification of a Bacillus sp.and application in bioflocs technology shrimp culture system.Progress in Fishery Sciences,2016,37(2):111-118[张欢欢,王秀华,李晨,等.一株芽孢杆菌的分离鉴定及在生物絮团对虾养殖中的应用.渔业科学进展,2016,37(2):111-118]
Zhang SP,Liu SM,Wang JX,et al.Optimizing fermentation condition for the antagonistic Bacillus subtilis NJ-18 strain.Journal of Nanjing Agricultural University,2010,33(2):58-62[章四平,刘圣明,王建新,等.枯草芽孢杆菌生防菌株NJ-18的发酵条件优化.南京农业大学学报,2010,33(2):58-62]
Zhang YY,Zhao M,Zhang N.Optimization of culture medium and fermentation conditions for compound probiotics of Bacillus.Journal of Northeast Forestry University,2012,40(3):93-97[张媛媛,赵敏,张宁.复合益生菌芽孢杆菌发酵培养基及条件的优化.东北林业大学学报,2012,40(3):93-97]
Zhao P,Huang J,Wang XH.The application of bioflocs technology in high-intensive,zero exchange farming systems of Marsupenaeus japonicus.Aquaculture,2012,s354-355(2):97-106
Zhou XJ,Qiu DQ.Effects of nitrite nitrogen and Vibrio parahaemolyticus on some immunity indicators of Litopenaeus vannamei.Journal of Hydroecology,2009,2(1):49-53[周鲜娇,邱德全.亚硝酸氮和副溶血弧菌对凡纳滨对虾部分免疫指标的影响.水生态学杂志,2009,2(1):49-53]
1)Zhang XG.The application and research of bio-floc technology in factory farming systems of Litopenaeus vannamei.Master′s Thesis of Ocean University of China,2012,34-38[张许光.生物絮团技术在凡纳滨对虾工厂化养殖中的应用与研究.中国海洋大学硕士研究生学位论文,2012,34-38]
2 )Zhao P.The study and application of bio-flocs technology in seawater aquaculture.Doctoral Dissertation of Shanghai Ocean University,2011,32-42[赵培.生物絮团技术在海水养殖中的研究与应用.上海海洋大学博士研究生学位论文,2011,32-42]
1)Zhang XG.The application and research of bio-floc technology in factory farming systems of Litopenaeus vannamei.Master′s Thesis of Ocean University of China,2012,9-21[张许光.生物絮团技术在凡纳滨对虾工厂化养殖中的应用与研究.中国海洋大学硕士研究生学位论文,2012,9-21]
1)Zhao P.The study and application of bio-flocs technology in seawater aquaculture.Doctoral Dissertation of Shanghai Ocean University,2011,32-42[赵培.生物絮团技术在海水养殖中的研究与应用.上海海洋大学博士研究生学位论文,2011,32-42]
Chen K,Li JS,Yang HT,et al.Phosphate solubilizing abilities of Bacillus megaterium strain P1 and its fermentation conditions.Soil and Fertilizer Science in China,2010(4):73-76[陈凯,李纪顺,杨合同,等.巨大芽孢杆菌P1的解磷效果与发酵条件研究.中国土壤与肥料,2010(4):73-76]
Chen LL,Dong HB,Li ZJ,et al.Review of the application and perspective of biofloc technology in shrimp culture.Marine Science,2014,38(8):103-108[陈亮亮,董宏标,李卓佳,等.生物絮团技术在对虾养殖中的应用及展望.海洋科学,2014,38(8):103-108]
Claudia K,Josef BC,Thibault G.Debottlenecking recombinant protein production in Bacillus megaterium under large-scale conditions-targeted precursor feeding designed from metabolomics.Biotechnology&Bioengineering,2012,109(6):1538-1550
Deng YN,Zhao P,Sun YZ,et al.Conditions for bio-floc formation and its effects in closed culture system of Litopenaeus vannamei.Progress in Fishery Sciences,2012,33(2):69-75[邓应能,赵培,孙运忠,等.生物絮团在凡纳滨对虾封闭养殖试验中的形成条件及作用效果.渔业科学进展,2012,33(2):69-75]
Devaraja TN,Yusoff FM,Shariff M.Changes in bacterial populations and shrimp production in ponds treated with commercial microbial products.Aquaculture,2002,206(3-4):245-256
FAO.FAO yearbook.Fishery and Aquaculture Statistics,2012
Ge HX,Li J,Chen P,et al.The immune response of Litopenaeus vannamei and its susceptibility to Vibrio parahaemolyticus under stress caused by ammonia nitrogen at different concentrations.Progress in Fishery Sciences,2014,35(6):76-82[葛红星,李健,陈萍,等.氨氮胁迫下凡纳滨对虾对副溶血弧菌的易感性.渔业科学进展,2014,35(6):76-82]
Ge HX,Li J,Chen P,et al.Effects of the fermentation solution of wheat bran,cane sugar and Bacillus substilis on water quality and growth performance of cultured Penaeus vannamei in indoor industrial facilities.Chinese Fishery Quality and Standards,2013,3(4):55-62[葛红星,李健,陈萍,等.麦麸、蔗糖和芽孢杆菌发酵液对室内工厂化养殖凡纳滨对虾水质和生长的影响.中国渔业质量与标准,2013,3(4):55-62]
Kanjanachumpol P,Kulpreecha S,Tolieng V.Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019.Bioprocess and Biosystems Engineering,2013,36(10):1463-1474
Ray AJ,Dillon KS,Lotz JM.Water quality dynamics and shrimp(Litopenaeus vannamei)production in intensive,mesohaline culture systems with two levels of biofloc management.Aquacultural Engineering,2011,45(3):127-136
Sun JJ,Ding ML.Effect of ammonia-N on anti-disease ability of Penaeus chinensis.Oceanologia et Limnologia Sinica,1999,30(3):267-72[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]
Wang L,Li J,Zhang PY.Laboratory study on treatment of eutrophicated scenery water body by Bacillus megaterium.Ecology and Environmental Sciences,2009,18(1):75-78[王琳,李季,张鹏岩.巨大芽孢杆菌对富营养化景观水体的净化效果.生态环境学报,2009,18(1):75-78]
Yang ZW,Yang K,Zhang Z,et al.Research on the biofloc bacterial community structure during larval rearing of Litopenaeus vannamei using metagenome sequencing.Journal of Fujian Fisheries,2015,37(2):91-97[杨章武,杨铿,张哲,等.基于宏基因组测序技术分析凡纳滨对虾育苗中生物絮团细菌群落结构.福建水产,2015,37(2):91-97]
Zhang HH,Wang XH,Li C,et al.Isolation and identification of a Bacillus sp.and application in bioflocs technology shrimp culture system.Progress in Fishery Sciences,2016,37(2):111-118[张欢欢,王秀华,李晨,等.一株芽孢杆菌的分离鉴定及在生物絮团对虾养殖中的应用.渔业科学进展,2016,37(2):111-118]
Zhang SP,Liu SM,Wang JX,et al.Optimizing fermentation condition for the antagonistic Bacillus subtilis NJ-18 strain.Journal of Nanjing Agricultural University,2010,33(2):58-62[章四平,刘圣明,王建新,等.枯草芽孢杆菌生防菌株NJ-18的发酵条件优化.南京农业大学学报,2010,33(2):58-62]
Zhang YY,Zhao M,Zhang N.Optimization of culture medium and fermentation conditions for compound probiotics of Bacillus.Journal of Northeast Forestry University,2012,40(3):93-97[张媛媛,赵敏,张宁.复合益生菌芽孢杆菌发酵培养基及条件的优化.东北林业大学学报,2012,40(3):93-97]
Zhao P,Huang J,Wang XH.The application of bioflocs technology in high-intensive,zero exchange farming systems of Marsupenaeus japonicus.Aquaculture,2012,s354-355(2):97-106
Zhou XJ,Qiu DQ.Effects of nitrite nitrogen and Vibrio parahaemolyticus on some immunity indicators of Litopenaeus vannamei.Journal of Hydroecology,2009,2(1):49-53[周鲜娇,邱德全.亚硝酸氮和副溶血弧菌对凡纳滨对虾部分免疫指标的影响.水生态学杂志,2009,2(1):49-53]
1)Zhang XG.The application and research of bio-floc technology in factory farming systems of Litopenaeus vannamei.Master′s Thesis of Ocean University of China,2012,34-38[张许光.生物絮团技术在凡纳滨对虾工厂化养殖中的应用与研究.中国海洋大学硕士研究生学位论文,2012,34-38]
2 )Zhao P.The study and application of bio-flocs technology in seawater aquaculture.Doctoral Dissertation of Shanghai Ocean University,2011,32-42[赵培.生物絮团技术在海水养殖中的研究与应用.上海海洋大学博士研究生学位论文,2011,32-42]
1)Zhang XG.The application and research of bio-floc technology in factory farming systems of Litopenaeus vannamei.Master′s Thesis of Ocean University of China,2012,9-21[张许光.生物絮团技术在凡纳滨对虾工厂化养殖中的应用与研究.中国海洋大学硕士研究生学位论文,2012,9-21]
1)Zhao P.The study and application of bio-flocs technology in seawater aquaculture.Doctoral Dissertation of Shanghai Ocean University,2011,32-42[赵培.生物絮团技术在海水养殖中的研究与应用.上海海洋大学博士研究生学位论文,2011,32-42]