微囊藻毒素降解菌的筛选鉴定及其对藻毒素MC-LR的降解
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摘要
针对近年来微囊藻毒素(MCs)的全球性污染问题,本研究以蓝藻爆发较严重和频繁的巢湖为研究对象,对其进行了较为详细的研究。本文以MCs为唯一碳氮源,采用富集驯化培养的办法,从巢湖蓝藻爆发的底泥中分离筛选MCs降解菌,同时对筛选出的MCs降解菌进行鉴定,主要通过形态观察、生理生化实验以及16S rDNA序列分析来确定其分类学地位,为巢湖的藻毒素污染的微生物降解治理提供有效地菌源。本研究从巢湖底泥中分离纯化出一株MCs降解菌株M6,其16S rDNA的长度为1408bp,经鉴定该降解菌株M6为蜡状芽孢杆菌(Bacillus cereus)。
为了进一步优化该降解菌株M6的降解效果,本研究以MCs为唯一的碳源和氮源,分别从不同的外加碳源和氮源,pH值,接种量,MCs的初始浓度等方面,考察这些因素对降解菌株M6降解MC-LR效果的影响,并初步确定该降解反应的最适环境条件。结果表明外加碳源甘油可大幅促进降解菌株M6的生长及其对MC-LR的降解效率,降解率可达到66.2%；外加氮源硝酸铵可以促进降解菌株M6对MC-LR的降解效率；菌株在pH值为7.0-8.0时,菌株的生长状况良好,并且对MC-LR的降解效率都比较高,当培养基中的pH值为8.0时,MC-LR的降解效率达到最大,可达到62.0%；当接种量介于1%-10%时,菌株的生长量及MC-LR的降解效率均随接种量的增大而增大,降解效率最高可达51.3%；当MC-LR初始浓度在1.95-15.6mg/L之间时,MC-LR的降解效率随着MC-LR的初始浓度的增大而逐渐增大,当初始浓度为15.6mg/L时,MC-LR的降解效率最高为59.2%。
本研究还对降解菌株M6降解MC-LR的降解机理进行了初步的探究,对降解菌株M6降解MC-LR进行了定位研究,考察MC-LR诱导前后的降解菌M6进行了SDS-PAGE全细胞电泳,确定经MC-LR诱导前后胞内蛋白质的变化,并且从不同pH值,接种量,MCs的初始浓度等方面,考察这些因素对降解菌株M6胞内粗酶液降解MC-LR的影响。结果表明,降解菌M6降解MC-LR的活性物质位于细胞内,属于胞内酶,SDS-PAGE全细胞电泳发现至少有三种酶参与了MC-LR的降解过程,而且实验表明这三种酶为降解菌本身的组织酶而不是诱导酶。当反应体系中胞内粗酶液浓度达到404.9mgL,MC-LR的初始浓度为10mg/L,pH为8.0,时,MC-LR的降解率达到最高,16h降解率可达98.7%。同时本研究还在不同条件下对降解菌株M6进行培养,提取粗酶液并降解一定量的MC-LR,以初步了解环境因素对产酶量多少的影响,发现当在外加碳源为甘油、氮源为硝酸铵,pH为8,接种量为10%,MC-LR初始浓度为15.6mg/L的诱导条件下,菌株M6的胞内粗提液对MC-LR有较大的降解效率,说明在此条件下降解MC-LR的酶表达量较多。
For the global pollution problem of microcystins (MCs) in recent years, the research choose Chaohu Lake where blue-green algae is more serious and frequent as research object and carry out some detailed studies. In this paper, MCs degradation bacterium is separated and identificated in the way of enrichment training based on MCs as the only carbon source. Taxonomy status of MCs degradation bacterium is determined by the morphology, physiological biochemistry experiment and16S rDNA sequence analysis to provide effective bacterium source for the cyanobacteria toxin microbial degradation of pollution control in the chaohu lake. The result indicats that the MCs degradation bacterium named M6with length of1408bp shows similarity of99%to candle shape Bacillus.
In order to optimize the degradation effect of the MCs degradation bacterium, this study research the efect to degradation effect of some facts such as carbon, nitrogen source, pH value, quantity and the initial concentration of the MCs based on the MCs as the only carbon, nitrogen source. It also determines the optimal environment degradation of the reaction of degradation and the maximum efficiency. The result indicates that plus carbon source (glycerin) can largely promote the growth of the M6degrading bacteria and the degradation of efficiency and the degradation rate of MC-LR, up to66.2%. Plus plus nitrogen source (ammonium) can promote the degradation efficiency of MC-LR. When the pH value is7.0-8.0, the growth of the strains and the degradation of efficiency of MC-LR is higher. When the pH value is8.0, the degradation efficiency reachs the maximum value, up to62.0%. When inoculation amount ranges from1%-10%, the degradation rate of MC-LR grows as the degradation rate increases and the degradation of highest efficiency strains is51.3%. When initial concentration of MC-LR is between1.95to15.6, the degradation rate of MC-LR increses. When the initial concentration of MC-LR is15.6mg/L, MC-LR degradation rate is up to59.2%.
This study also researches degradation mechanism of M6and positions the degradation bacterium M6degradation MC-LR. At the same time, it carry out the SDS-PAGE whole cell electrophoresis on M6before and after being inducted by MC-LR to be sure the change of the cell protein. It also investigates efect of environmental factors such as pH value, quantity and the initial concentration of the MCs on the inside the cell thick liquid enzyme degradation effect. The result shows that degradation bacterium M6degradation MC-LR of the active material is located in cells, belonging to the intracellular enzyme, based on SDS-PAGE whole cell electrophoresis, we find that at three enzymes at least joins in the MC-LR of degradation process, and these three enzymes is degradation bacterium itself, not the organization of the enzyme inducible enzyme. When the cell in thick liquid enzyme concentration reached404.9mg/L, MC-LR of initial concentration of10mg/L and pH values8.0in the system, MC-LR degradation rate reaches the highest, up to98.7%after16h. At the same time the research inducts degradation bacterium M6in different conditions, thick liquid extraction enzyme degradation and a certain amount of MC-LR preliminary understanding the environment factors on the enzyme production quantity of how much influence, we find that when have carbon source glycerin, ammonium nitrate nitrogen source, pH values8, inoculated quantity is10%and initial concentration of MC-LR is15.6mg/L, intracellular coarse extraction liquid of strains M6have larger degradation of efficiency to MC-LR. It indicates that drop solution-LR enzyme expression of MC is more in this condition.
为了进一步优化该降解菌株M6的降解效果,本研究以MCs为唯一的碳源和氮源,分别从不同的外加碳源和氮源,pH值,接种量,MCs的初始浓度等方面,考察这些因素对降解菌株M6降解MC-LR效果的影响,并初步确定该降解反应的最适环境条件。结果表明外加碳源甘油可大幅促进降解菌株M6的生长及其对MC-LR的降解效率,降解率可达到66.2%；外加氮源硝酸铵可以促进降解菌株M6对MC-LR的降解效率；菌株在pH值为7.0-8.0时,菌株的生长状况良好,并且对MC-LR的降解效率都比较高,当培养基中的pH值为8.0时,MC-LR的降解效率达到最大,可达到62.0%；当接种量介于1%-10%时,菌株的生长量及MC-LR的降解效率均随接种量的增大而增大,降解效率最高可达51.3%；当MC-LR初始浓度在1.95-15.6mg/L之间时,MC-LR的降解效率随着MC-LR的初始浓度的增大而逐渐增大,当初始浓度为15.6mg/L时,MC-LR的降解效率最高为59.2%。
本研究还对降解菌株M6降解MC-LR的降解机理进行了初步的探究,对降解菌株M6降解MC-LR进行了定位研究,考察MC-LR诱导前后的降解菌M6进行了SDS-PAGE全细胞电泳,确定经MC-LR诱导前后胞内蛋白质的变化,并且从不同pH值,接种量,MCs的初始浓度等方面,考察这些因素对降解菌株M6胞内粗酶液降解MC-LR的影响。结果表明,降解菌M6降解MC-LR的活性物质位于细胞内,属于胞内酶,SDS-PAGE全细胞电泳发现至少有三种酶参与了MC-LR的降解过程,而且实验表明这三种酶为降解菌本身的组织酶而不是诱导酶。当反应体系中胞内粗酶液浓度达到404.9mgL,MC-LR的初始浓度为10mg/L,pH为8.0,时,MC-LR的降解率达到最高,16h降解率可达98.7%。同时本研究还在不同条件下对降解菌株M6进行培养,提取粗酶液并降解一定量的MC-LR,以初步了解环境因素对产酶量多少的影响,发现当在外加碳源为甘油、氮源为硝酸铵,pH为8,接种量为10%,MC-LR初始浓度为15.6mg/L的诱导条件下,菌株M6的胞内粗提液对MC-LR有较大的降解效率,说明在此条件下降解MC-LR的酶表达量较多。
For the global pollution problem of microcystins (MCs) in recent years, the research choose Chaohu Lake where blue-green algae is more serious and frequent as research object and carry out some detailed studies. In this paper, MCs degradation bacterium is separated and identificated in the way of enrichment training based on MCs as the only carbon source. Taxonomy status of MCs degradation bacterium is determined by the morphology, physiological biochemistry experiment and16S rDNA sequence analysis to provide effective bacterium source for the cyanobacteria toxin microbial degradation of pollution control in the chaohu lake. The result indicats that the MCs degradation bacterium named M6with length of1408bp shows similarity of99%to candle shape Bacillus.
In order to optimize the degradation effect of the MCs degradation bacterium, this study research the efect to degradation effect of some facts such as carbon, nitrogen source, pH value, quantity and the initial concentration of the MCs based on the MCs as the only carbon, nitrogen source. It also determines the optimal environment degradation of the reaction of degradation and the maximum efficiency. The result indicates that plus carbon source (glycerin) can largely promote the growth of the M6degrading bacteria and the degradation of efficiency and the degradation rate of MC-LR, up to66.2%. Plus plus nitrogen source (ammonium) can promote the degradation efficiency of MC-LR. When the pH value is7.0-8.0, the growth of the strains and the degradation of efficiency of MC-LR is higher. When the pH value is8.0, the degradation efficiency reachs the maximum value, up to62.0%. When inoculation amount ranges from1%-10%, the degradation rate of MC-LR grows as the degradation rate increases and the degradation of highest efficiency strains is51.3%. When initial concentration of MC-LR is between1.95to15.6, the degradation rate of MC-LR increses. When the initial concentration of MC-LR is15.6mg/L, MC-LR degradation rate is up to59.2%.
This study also researches degradation mechanism of M6and positions the degradation bacterium M6degradation MC-LR. At the same time, it carry out the SDS-PAGE whole cell electrophoresis on M6before and after being inducted by MC-LR to be sure the change of the cell protein. It also investigates efect of environmental factors such as pH value, quantity and the initial concentration of the MCs on the inside the cell thick liquid enzyme degradation effect. The result shows that degradation bacterium M6degradation MC-LR of the active material is located in cells, belonging to the intracellular enzyme, based on SDS-PAGE whole cell electrophoresis, we find that at three enzymes at least joins in the MC-LR of degradation process, and these three enzymes is degradation bacterium itself, not the organization of the enzyme inducible enzyme. When the cell in thick liquid enzyme concentration reached404.9mg/L, MC-LR of initial concentration of10mg/L and pH values8.0in the system, MC-LR degradation rate reaches the highest, up to98.7%after16h. At the same time the research inducts degradation bacterium M6in different conditions, thick liquid extraction enzyme degradation and a certain amount of MC-LR preliminary understanding the environment factors on the enzyme production quantity of how much influence, we find that when have carbon source glycerin, ammonium nitrate nitrogen source, pH values8, inoculated quantity is10%and initial concentration of MC-LR is15.6mg/L, intracellular coarse extraction liquid of strains M6have larger degradation of efficiency to MC-LR. It indicates that drop solution-LR enzyme expression of MC is more in this condition.
引文
[1]许秋瑾,高光,陈伟民.微囊藻毒素的研究进展,中国公共卫生,2002,06：761-762
[2]屠清瑛,顾丁锡,尹澄清,等.巢湖富营养化的研究.合肥：中国科学技术大学出版社.1990
[3]谢平.微囊藻毒素对人类健康影响相关研究的回顾.湖泊科学(J.LakeSci.),2009,21(5)：603-613.
[4]Carmichael WW, Azevedo SMFO, An JS, Molica RJR, Jochimsen EM, Lau S, Renehart KL, Shaw GR, Eaglesham GK. Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ Health Perspect,2001, 109:663-668.
[5]王歧山.巢湖鱼类区系研究.安徽大学学报(自然科学版),1987,2：70-78
[6]颜京松,张玉书,王美珍.巢湖鱼产潜力估算及其合理开发利用的一些建议.农村生态环境,1987,2：34-41
[7]何家苑,何振荣,俞敏娟等.东湖蓝藻有毒水华种类组成及毒性研究.东湖生态学研究.北京：科学出版社,1990.395-402
[8]Keil C, Forchert A, Fastner J, Szewzyk U, et al. Toxicity and microcystin content of extracts from a Planktothrix bloom and two laboratory strains [J]. WaterRes, 2002,36:2133-2139.
[9]Carmichael,W.W.,C.L.A.Jones,N.A.Mahmood,andW.C.Theiss. Algal toxins and water based diseases. Crit. Rev. Environ. Control.1985.15:275-313.
[10]Hitzfeld B C,Hoger S J, Dietrich D R. Cyanobacterial toxins:Removal during Drinking Perspect, Water Treatment, and HumanRisk Assessment[J]. Environ Health Perspect,2000,108:113-122
[11]刘贞秋,蒙仁宪.巢湖浮游蓝藻的初步研究[J].海洋湖沼通报,1989,2：35-41.
[12]张红,黄勇,姚筠,等.巢湖藻类遥感监测和气象因子分析[J].环境科 学与技术,2009,32(1)：118-121.
[13]谢杰,王心源,张洁等.基于TM/ETM+影响分析巢湖叶绿素a浓度变化趋势[J].中国环境科学,2010,30(5)：677-682.
[14]Carmichael, W. W. Cyanobacteria secondary metabolites—the cyano-toxins. J. Appl. Bacteriol.1992.72:445-459.
[15]张青学,俞敏娟.铜绿微囊藻伽ciorycstisaeurginosa)水华毒性及毒素的研究[J]环境科学学报,1989,9(1)：86-94.
[16]Harada K I. Isolation and characteriaztion of the minor components associated wih microcystins LR and RR in the Cyanobacterium (blue-green).Toxicon, 1990,28:55.
[18]邓义敏.滇池水体中微囊藻毒素的研究.云南环境科学,2000,19(增刊)
[19]Tsuji K T,Watanuki F Stasility of microcystins from cy-anobacteria IV[M]. Effect of chlorination on decomposition.Toxicon,1997,35 (7):1033-1041.
[20]韩志国,武宝轩,郑解生.淡水水体中的蓝藻毒素研究进展[J].暨南大学学报,2001,22(3)：129-135.
[21]Watanabe M F, Tsuji K, Watanabe Y, et al. Release of heptapeptide toxin (microcystin) during the decomposition process of microcystis aeruginosa. Nat.Toxins, 1992(a),1:48-53.
[22]Lukae M and Aegerter R. Influence of trace metals on growth and toxin production of microcystis aeruginosa. Toxicon,1993,31(3):293-305.
[23]Mei ner K,Kittmann E.Borner T.Toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa contain sequences homologous to peptide synthetase genes. FEMS Microbiology Letters.1996,135:295-303.
[24]Haider S, Naithani V, Viswanathan PN, Kakkar P. Cyanobacterial toxins: a growing environmental concern. Chemosphere,2003,52:1-21.
[25]柳丽丽,钟儒刚,曾毅.微囊藻毒素及其毒性研究进展[J].卫生研究,2006,35(2)：247-249.
[26]Rudolf-Bohner,S.,D.F.Mierke,andL.Moroder.1994.Molecularstructure of the cyanobacterial tumour-promoting microcystins. FEBS Lett.1994.349:319-323
[27]Jochimsen EM, Carmichael WW, An J, et al. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil [J]. New Eng J Med,1998, 338:873-878.
[28]Harada K I, Motoji O. Detection and identification of microcystins in the drinking water of Haimen City, China. Nuatral Toxins,1996 (4):277-283.
[29]赵金明,蒋颂辉,朱惠刚.藻毒素对实验性大鼠肝癌的促进作用[J].中国公共卫生,2003,19(6)：694-696.
[30]郑树.大肠癌防治现场研究.中国肿瘤[J],2005,14(6)：352-354.
[31]Falconer IR. An overview of problems caused by toxic blue-green algae (cyanobacteria) in drinking and recreational water. Environmental Toxicol.,1999, 14(1):5-12.
[32]许敏,赵以军.水华和赤潮的毒性及其检测与分析.湖泊科学,2001,13(4)：376-384
[33]Hiripi L, Nagy L. Insect (Locusta migratroria migratorioides) test monitoring the toxicity of cyanbacteria. Neurotoxicology,1998,19(4):605-608.
[34]徐立红,陈加平,黄兆辉等.用蛋白磷酸酶抑制法测定水体中的微囊藻毒素类物质[J].环境科学学报,1999,19(5)：536-539
[35]Baker S A. LC-GC Supplement,Current Trends and Developments in Sample Preparation,1998, S37-S40
[36]朱光灿,吕锡武.藻毒素在传统净水工艺中的去除特性[]J.环境化学,2002,21(6)：585-588.
[37]Himberg K,Kerjola A M,Pyysalo H et al,The Effect of Water Treatment Processes on the Removal of Hepatotoxins From Microcystins and Oscillatoria Cyanobacteria:a Laboratory Study.Wat.Res,1989,23 (8):979-984.
[38]Neumann U, Weckesser J. Elimination of microcystin peptide toxins from water by reverse osmosis [J]. Environ Toxicol and Water Qual,1998,13:156-164.
[39]左金龙,崔福义,刘智晓.饮用水中蓝藻毒素污染研究进展[J].环境污染治理技术与设备[J],2006,7(3)：8-13.
[40]Lam A, Prepas E, Spink D, et al. Control of hepatotoxic phytoplankton blooms implication for humance health [J]. Water Res,1995,29:1845-1854.
[41]余国忠,王吉生.饮用水处理中藻毒素污染及其工艺控制特研究[J].给水排水,2002(3)：26-29.
[42]Welker M. Steinberg c. Indirect photohysis of cyanotoxins:one possible mechanism for their low persistence. Wat. Res.1999,33:1150-1152.
[43]张玮,赵以军,孙茜,等.浮水草本浮萍对微囊藻毒素影响的试验[J].环境科学学报,2006,26(1)：85-90.
[44]Guo NC, Xie P. Development of tolerance against toxic Microcystis aerug inosa in three cladocerans and the ecological imp lications [J]. Environmental Pollution,2006,143:513-518.
[45]吴振斌,陈辉蓉,雷腊梅等.人工湿地系统去除藻毒素研究[]J.长江流域资源与环境,2000,9(2)：242—247.
[46]李家就、钱望新,高营养化湖泊水源生物预处理研究.中国给水排水,1992,Vo1.8,No.6：4-7
[47]Tsuji K, Watanuki T, Kondo F, Watanabe MF, Nakazawa H, Suzuki M, Uchida H, Harada KI. Stability of microcystins from cyanobacteria-IV. effect of chlorination on decomposition. Toxicon,1997,35(7):1033-1041.
[48]Jones G J, Bourne D G, Robert L, et al. Degradation of the cyanobacterial hepatotoxin microcystin by aquatic baeteria[J].Natural Toxins, 1994,2(4); 228-235
[49]Cousins I.T.,Beating D.J., James H.A.,Sutton A.Biodegradation of microcystin-LR by indigenous mixed bacterial populations.Wat.Res.,1996,30(2): 481-485.
[50]Takenaka S, Watanabe MF. Microcystin LR degradation by Pseudomonas aeruginosa alkaline protease. Chemosphere,1997,34(4):749-757.
[51]闫海,邓义敏,邹华,李贤良,叶常明.降解微囊藻毒素菌种的筛选与活性研究.环境科学,2004,25(6)：49-53.
[52]徐春霞,何宏胜,张超,吕乐,闫海.高效降解微囊藻毒素食酸戴尔福特菌USTB-04的培养与活性研究.环境工程学报,2007,1(5)：21-24.
[53]Zhang ML, Pan G, Yan H. Biodegradation of Microcystin-RR by a New Isolated Sphingopyxis sp.USTB-05.,2010,22(2):168-175.
[54]周洁,闫海,何宏胜,钟广荣,张超,林海.食酸戴尔福特菌USTB04生物降解微囊MCs的活性研究.科学技术与工程,2006,6(2)：166-170.
[55]吴涓,钟升,王光云,李玉成.一株降解微囊藻毒素菌种的鉴定及其活性研究.中国环境科学,2011,31(1)：116-122.
[56]钟升,吴涓,王光云.微囊藻毒素降解菌的筛选、鉴定及其降解活性的研究.生物学杂质,2010,27(6)：57-60.
[57]Bourne DG Jones G J,Blakeley R, Jones A.,Negri A.P.,Riddles P.Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.Applied and Environ.Microbiology.1996,62(11):4086-4094.
[58]徐亚同、史家梁、张明著.污染控制微生物工程.北京：化学工业出版社,2001,1200
[59]Kato H, Imanish SY, Tsuji K, Harada KI. Microbial degradation of cyanobacterial cyclic pep tides. Water Research,2007,41(8):1754-1762.
[60]何宏胜,闫海,周洁,葛世友,肖宝清,吕乐.筛选菌种酶催化降解微囊藻毒素的特点.环境科学,2006,7(6)：1171-1175.
[61]苑宝玲,陈彩云,李云琴,刘波,史怀.假单胞菌胞内酶粗提液对藻毒素MC-LR的降解,环境化学,2009,28(6)：854-858
[62]Bourne DG, Riddles P, Jones GJ, Smith W, Blakeley RL. Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR. Environmental Toxicol.,2001,16:523-534.
[63]东秀珠,蔡妙英.常见细菌鉴定手册.北京：科学出版社,2001.
[64]布坎南,吉本斯.伯杰氏细菌鉴定手册.第8版.北京：科学出版社,1984
[65]汪家政,范明.蛋白技术手册.北京：科学出版社,2000：42-47.
[66]Valeria AM, Ricardo EJ, Stephan P. Degradation of Microcystin-RR by Sphingomonas sp. CBA4 Isolated from San Roque Reservoir(Cordobar Argentina). Biodegradation,2006,17(5):447-455
[67]陈彩云,苑宝玲,李艳波,刘波,史怀.假单胞菌降解微囊藻毒素的效能及酶作用机理.水生生物学报,2009,33(5)：951-956.
[2]屠清瑛,顾丁锡,尹澄清,等.巢湖富营养化的研究.合肥：中国科学技术大学出版社.1990
[3]谢平.微囊藻毒素对人类健康影响相关研究的回顾.湖泊科学(J.LakeSci.),2009,21(5)：603-613.
[4]Carmichael WW, Azevedo SMFO, An JS, Molica RJR, Jochimsen EM, Lau S, Renehart KL, Shaw GR, Eaglesham GK. Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ Health Perspect,2001, 109:663-668.
[5]王歧山.巢湖鱼类区系研究.安徽大学学报(自然科学版),1987,2：70-78
[6]颜京松,张玉书,王美珍.巢湖鱼产潜力估算及其合理开发利用的一些建议.农村生态环境,1987,2：34-41
[7]何家苑,何振荣,俞敏娟等.东湖蓝藻有毒水华种类组成及毒性研究.东湖生态学研究.北京：科学出版社,1990.395-402
[8]Keil C, Forchert A, Fastner J, Szewzyk U, et al. Toxicity and microcystin content of extracts from a Planktothrix bloom and two laboratory strains [J]. WaterRes, 2002,36:2133-2139.
[9]Carmichael,W.W.,C.L.A.Jones,N.A.Mahmood,andW.C.Theiss. Algal toxins and water based diseases. Crit. Rev. Environ. Control.1985.15:275-313.
[10]Hitzfeld B C,Hoger S J, Dietrich D R. Cyanobacterial toxins:Removal during Drinking Perspect, Water Treatment, and HumanRisk Assessment[J]. Environ Health Perspect,2000,108:113-122
[11]刘贞秋,蒙仁宪.巢湖浮游蓝藻的初步研究[J].海洋湖沼通报,1989,2：35-41.
[12]张红,黄勇,姚筠,等.巢湖藻类遥感监测和气象因子分析[J].环境科 学与技术,2009,32(1)：118-121.
[13]谢杰,王心源,张洁等.基于TM/ETM+影响分析巢湖叶绿素a浓度变化趋势[J].中国环境科学,2010,30(5)：677-682.
[14]Carmichael, W. W. Cyanobacteria secondary metabolites—the cyano-toxins. J. Appl. Bacteriol.1992.72:445-459.
[15]张青学,俞敏娟.铜绿微囊藻伽ciorycstisaeurginosa)水华毒性及毒素的研究[J]环境科学学报,1989,9(1)：86-94.
[16]Harada K I. Isolation and characteriaztion of the minor components associated wih microcystins LR and RR in the Cyanobacterium (blue-green).Toxicon, 1990,28:55.
[18]邓义敏.滇池水体中微囊藻毒素的研究.云南环境科学,2000,19(增刊)
[19]Tsuji K T,Watanuki F Stasility of microcystins from cy-anobacteria IV[M]. Effect of chlorination on decomposition.Toxicon,1997,35 (7):1033-1041.
[20]韩志国,武宝轩,郑解生.淡水水体中的蓝藻毒素研究进展[J].暨南大学学报,2001,22(3)：129-135.
[21]Watanabe M F, Tsuji K, Watanabe Y, et al. Release of heptapeptide toxin (microcystin) during the decomposition process of microcystis aeruginosa. Nat.Toxins, 1992(a),1:48-53.
[22]Lukae M and Aegerter R. Influence of trace metals on growth and toxin production of microcystis aeruginosa. Toxicon,1993,31(3):293-305.
[23]Mei ner K,Kittmann E.Borner T.Toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa contain sequences homologous to peptide synthetase genes. FEMS Microbiology Letters.1996,135:295-303.
[24]Haider S, Naithani V, Viswanathan PN, Kakkar P. Cyanobacterial toxins: a growing environmental concern. Chemosphere,2003,52:1-21.
[25]柳丽丽,钟儒刚,曾毅.微囊藻毒素及其毒性研究进展[J].卫生研究,2006,35(2)：247-249.
[26]Rudolf-Bohner,S.,D.F.Mierke,andL.Moroder.1994.Molecularstructure of the cyanobacterial tumour-promoting microcystins. FEBS Lett.1994.349:319-323
[27]Jochimsen EM, Carmichael WW, An J, et al. Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil [J]. New Eng J Med,1998, 338:873-878.
[28]Harada K I, Motoji O. Detection and identification of microcystins in the drinking water of Haimen City, China. Nuatral Toxins,1996 (4):277-283.
[29]赵金明,蒋颂辉,朱惠刚.藻毒素对实验性大鼠肝癌的促进作用[J].中国公共卫生,2003,19(6)：694-696.
[30]郑树.大肠癌防治现场研究.中国肿瘤[J],2005,14(6)：352-354.
[31]Falconer IR. An overview of problems caused by toxic blue-green algae (cyanobacteria) in drinking and recreational water. Environmental Toxicol.,1999, 14(1):5-12.
[32]许敏,赵以军.水华和赤潮的毒性及其检测与分析.湖泊科学,2001,13(4)：376-384
[33]Hiripi L, Nagy L. Insect (Locusta migratroria migratorioides) test monitoring the toxicity of cyanbacteria. Neurotoxicology,1998,19(4):605-608.
[34]徐立红,陈加平,黄兆辉等.用蛋白磷酸酶抑制法测定水体中的微囊藻毒素类物质[J].环境科学学报,1999,19(5)：536-539
[35]Baker S A. LC-GC Supplement,Current Trends and Developments in Sample Preparation,1998, S37-S40
[36]朱光灿,吕锡武.藻毒素在传统净水工艺中的去除特性[]J.环境化学,2002,21(6)：585-588.
[37]Himberg K,Kerjola A M,Pyysalo H et al,The Effect of Water Treatment Processes on the Removal of Hepatotoxins From Microcystins and Oscillatoria Cyanobacteria:a Laboratory Study.Wat.Res,1989,23 (8):979-984.
[38]Neumann U, Weckesser J. Elimination of microcystin peptide toxins from water by reverse osmosis [J]. Environ Toxicol and Water Qual,1998,13:156-164.
[39]左金龙,崔福义,刘智晓.饮用水中蓝藻毒素污染研究进展[J].环境污染治理技术与设备[J],2006,7(3)：8-13.
[40]Lam A, Prepas E, Spink D, et al. Control of hepatotoxic phytoplankton blooms implication for humance health [J]. Water Res,1995,29:1845-1854.
[41]余国忠,王吉生.饮用水处理中藻毒素污染及其工艺控制特研究[J].给水排水,2002(3)：26-29.
[42]Welker M. Steinberg c. Indirect photohysis of cyanotoxins:one possible mechanism for their low persistence. Wat. Res.1999,33:1150-1152.
[43]张玮,赵以军,孙茜,等.浮水草本浮萍对微囊藻毒素影响的试验[J].环境科学学报,2006,26(1)：85-90.
[44]Guo NC, Xie P. Development of tolerance against toxic Microcystis aerug inosa in three cladocerans and the ecological imp lications [J]. Environmental Pollution,2006,143:513-518.
[45]吴振斌,陈辉蓉,雷腊梅等.人工湿地系统去除藻毒素研究[]J.长江流域资源与环境,2000,9(2)：242—247.
[46]李家就、钱望新,高营养化湖泊水源生物预处理研究.中国给水排水,1992,Vo1.8,No.6：4-7
[47]Tsuji K, Watanuki T, Kondo F, Watanabe MF, Nakazawa H, Suzuki M, Uchida H, Harada KI. Stability of microcystins from cyanobacteria-IV. effect of chlorination on decomposition. Toxicon,1997,35(7):1033-1041.
[48]Jones G J, Bourne D G, Robert L, et al. Degradation of the cyanobacterial hepatotoxin microcystin by aquatic baeteria[J].Natural Toxins, 1994,2(4); 228-235
[49]Cousins I.T.,Beating D.J., James H.A.,Sutton A.Biodegradation of microcystin-LR by indigenous mixed bacterial populations.Wat.Res.,1996,30(2): 481-485.
[50]Takenaka S, Watanabe MF. Microcystin LR degradation by Pseudomonas aeruginosa alkaline protease. Chemosphere,1997,34(4):749-757.
[51]闫海,邓义敏,邹华,李贤良,叶常明.降解微囊藻毒素菌种的筛选与活性研究.环境科学,2004,25(6)：49-53.
[52]徐春霞,何宏胜,张超,吕乐,闫海.高效降解微囊藻毒素食酸戴尔福特菌USTB-04的培养与活性研究.环境工程学报,2007,1(5)：21-24.
[53]Zhang ML, Pan G, Yan H. Biodegradation of Microcystin-RR by a New Isolated Sphingopyxis sp.USTB-05.,2010,22(2):168-175.
[54]周洁,闫海,何宏胜,钟广荣,张超,林海.食酸戴尔福特菌USTB04生物降解微囊MCs的活性研究.科学技术与工程,2006,6(2)：166-170.
[55]吴涓,钟升,王光云,李玉成.一株降解微囊藻毒素菌种的鉴定及其活性研究.中国环境科学,2011,31(1)：116-122.
[56]钟升,吴涓,王光云.微囊藻毒素降解菌的筛选、鉴定及其降解活性的研究.生物学杂质,2010,27(6)：57-60.
[57]Bourne DG Jones G J,Blakeley R, Jones A.,Negri A.P.,Riddles P.Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.Applied and Environ.Microbiology.1996,62(11):4086-4094.
[58]徐亚同、史家梁、张明著.污染控制微生物工程.北京：化学工业出版社,2001,1200
[59]Kato H, Imanish SY, Tsuji K, Harada KI. Microbial degradation of cyanobacterial cyclic pep tides. Water Research,2007,41(8):1754-1762.
[60]何宏胜,闫海,周洁,葛世友,肖宝清,吕乐.筛选菌种酶催化降解微囊藻毒素的特点.环境科学,2006,7(6)：1171-1175.
[61]苑宝玲,陈彩云,李云琴,刘波,史怀.假单胞菌胞内酶粗提液对藻毒素MC-LR的降解,环境化学,2009,28(6)：854-858
[62]Bourne DG, Riddles P, Jones GJ, Smith W, Blakeley RL. Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR. Environmental Toxicol.,2001,16:523-534.
[63]东秀珠,蔡妙英.常见细菌鉴定手册.北京：科学出版社,2001.
[64]布坎南,吉本斯.伯杰氏细菌鉴定手册.第8版.北京：科学出版社,1984
[65]汪家政,范明.蛋白技术手册.北京：科学出版社,2000：42-47.
[66]Valeria AM, Ricardo EJ, Stephan P. Degradation of Microcystin-RR by Sphingomonas sp. CBA4 Isolated from San Roque Reservoir(Cordobar Argentina). Biodegradation,2006,17(5):447-455
[67]陈彩云,苑宝玲,李艳波,刘波,史怀.假单胞菌降解微囊藻毒素的效能及酶作用机理.水生生物学报,2009,33(5)：951-956.