利用PCR-DGGE研究膜生物反应器中微生物的群落结构
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摘要
微生物对膜生物反应器等污水处理工艺系统中的主体污染物的降解起着决定性的作用,只有充分认识系统内的微生物群落结构及其功能,才能为改进或优化处理工艺、运行参数和提高污水处理效率提供依据。但是目前污水系统中微生物群落的研究仍以培养分离等传统技术为主,而它们已经无法满足对微生物进行快速、全面、准确的分析和鉴定的要求。本课题利用PCR-DGGE分子生物学技术和系统发育分析等方法,对膜生物反应器中的微生物的群落结构和功能进行研究,并对反应器内的微生物进行分析鉴定。
利用PCR-DGGE分子生物学技术研究MBR反应器培养驯化整个过程中微生物的群落结构及演替规律。在新的MBR环境中,接种的传统活性污泥中的微生物群落在几天内发生了较大的变化,在进污水驯化时,微生物群落遭受了冲击,最后经过培养驯化逐渐趋于稳定,一些菌种逐渐成长为顶级优势微生物,在反应器内占据主导地位。最终该反应器形成了自己独有的微生物群落生态系统。另外,对该反应器的部分优势菌种进行了克隆测序和系统发育分析,通过鉴定获得10条总细菌的16S rDNA序列,它们分别与气单胞菌属(Aeromonas)、假单胞菌(Pseudomonas)、亚硝酸菌属(Nitrosomas)、丛毛单胞菌属(Comamonas)和杆菌(Bacillus)的同源性在97%以上。这些优势微生物在MBR反应器去除有机物的过程中起到了关键的作用。
另外研究了不同的膜生物反应器的微生物群落结构的异同。不同的环境条件、进水水质及运行参数导致了不同膜生物反应器微生物群落的差异。通过巢式PCR-DGGE技术并利用特殊的引物,研究发现MBR反应器中存在比较丰富的硝酸菌和反硝化菌,对这些微生物的群落结构情况进行了分析。通过克隆测序鉴定了部分硝化菌,鉴定出MBR反应器内的5种硝酸菌和3种反硝化菌,并发现反应器内存在好氧反硝化菌,说明MBR反应器内存在好氧反硝化现象。这些硝化菌在MBR反应器的脱氮过程中起着关键的作用。
Microorganism plays a decisive role in the process of degradation of the main pollutants in the membrane bioreactor and other biological wastewater treatment systems. In order to improve or optimize the treatment system, operating parameter, and improve the effect of wastewater treatment, understanding the microbial community structure and function are very important. However, technologies for the research of wastewater treatment’s microbial community mainly are traditional techniques based on culture and separation, and it could not satisfy nowadays the research of microbial community which requires analysis and identification more rapid, comprehensive and exactly. This issue using PCR-DGGE molecular biology technique and the method of phylogenetic analysis to research the microbial community structure and function of membrane bioreactor, and identify the microorganism of the reactor.
Using molecular biology technique of PCR-DGGE to research microbial community structure and succession in the cultivation and domestication process of MBR. The microbial community of traditional activated sludge which is inoculated had a large change in a few days in the new MBR’s environment, and it also caused a impact to microbial community when using wastewater to domesticate. Finally it tend to stable after domestication, some bacterias gradually growed to be dominant species, and are dominated in the reactor. Ultimately the reactor formed its own unique microbial community ecosystem. Moreover, part of reactor’s dominant bacterias are cloning, sequencing and phylogenetic analysising, finally obtain ten bacterial 16S rDNA sequences through identificating, the homology of these bacterias campared with Aeromonas, Pseudomonas, Nitrosomas,Comamonas and Bacillus are greater than 97%. These dominant microorganisms played key roles in the process of removaling organic pollutants in MBR.
In addition, the similarity and difference of microbial community structure between different MBRs are studied. Different environmental condition, water quality and operating parameter resulted in difference of microbial community between different MBRs. Through Nested PCR-DGGE and using special primers it is found that there are rich nitrobacterias and denitrification bacterias in MBRs, the microbial community structure of these bacterias were analyzed. Through cloning and sequencing, five kinds of nitrobacterias and three kinds of denitrification bacterias are identified, and it is found that the existence of aerobic denitrification bacteria in MBRs, it is indicated the existence of the phenomenon of aerobic denitrification in MBRs. These nitrifying bacterias play key roles in denitrifying process in MBRs.
利用PCR-DGGE分子生物学技术研究MBR反应器培养驯化整个过程中微生物的群落结构及演替规律。在新的MBR环境中,接种的传统活性污泥中的微生物群落在几天内发生了较大的变化,在进污水驯化时,微生物群落遭受了冲击,最后经过培养驯化逐渐趋于稳定,一些菌种逐渐成长为顶级优势微生物,在反应器内占据主导地位。最终该反应器形成了自己独有的微生物群落生态系统。另外,对该反应器的部分优势菌种进行了克隆测序和系统发育分析,通过鉴定获得10条总细菌的16S rDNA序列,它们分别与气单胞菌属(Aeromonas)、假单胞菌(Pseudomonas)、亚硝酸菌属(Nitrosomas)、丛毛单胞菌属(Comamonas)和杆菌(Bacillus)的同源性在97%以上。这些优势微生物在MBR反应器去除有机物的过程中起到了关键的作用。
另外研究了不同的膜生物反应器的微生物群落结构的异同。不同的环境条件、进水水质及运行参数导致了不同膜生物反应器微生物群落的差异。通过巢式PCR-DGGE技术并利用特殊的引物,研究发现MBR反应器中存在比较丰富的硝酸菌和反硝化菌,对这些微生物的群落结构情况进行了分析。通过克隆测序鉴定了部分硝化菌,鉴定出MBR反应器内的5种硝酸菌和3种反硝化菌,并发现反应器内存在好氧反硝化菌,说明MBR反应器内存在好氧反硝化现象。这些硝化菌在MBR反应器的脱氮过程中起着关键的作用。
Microorganism plays a decisive role in the process of degradation of the main pollutants in the membrane bioreactor and other biological wastewater treatment systems. In order to improve or optimize the treatment system, operating parameter, and improve the effect of wastewater treatment, understanding the microbial community structure and function are very important. However, technologies for the research of wastewater treatment’s microbial community mainly are traditional techniques based on culture and separation, and it could not satisfy nowadays the research of microbial community which requires analysis and identification more rapid, comprehensive and exactly. This issue using PCR-DGGE molecular biology technique and the method of phylogenetic analysis to research the microbial community structure and function of membrane bioreactor, and identify the microorganism of the reactor.
Using molecular biology technique of PCR-DGGE to research microbial community structure and succession in the cultivation and domestication process of MBR. The microbial community of traditional activated sludge which is inoculated had a large change in a few days in the new MBR’s environment, and it also caused a impact to microbial community when using wastewater to domesticate. Finally it tend to stable after domestication, some bacterias gradually growed to be dominant species, and are dominated in the reactor. Ultimately the reactor formed its own unique microbial community ecosystem. Moreover, part of reactor’s dominant bacterias are cloning, sequencing and phylogenetic analysising, finally obtain ten bacterial 16S rDNA sequences through identificating, the homology of these bacterias campared with Aeromonas, Pseudomonas, Nitrosomas,Comamonas and Bacillus are greater than 97%. These dominant microorganisms played key roles in the process of removaling organic pollutants in MBR.
In addition, the similarity and difference of microbial community structure between different MBRs are studied. Different environmental condition, water quality and operating parameter resulted in difference of microbial community between different MBRs. Through Nested PCR-DGGE and using special primers it is found that there are rich nitrobacterias and denitrification bacterias in MBRs, the microbial community structure of these bacterias were analyzed. Through cloning and sequencing, five kinds of nitrobacterias and three kinds of denitrification bacterias are identified, and it is found that the existence of aerobic denitrification bacteria in MBRs, it is indicated the existence of the phenomenon of aerobic denitrification in MBRs. These nitrifying bacterias play key roles in denitrifying process in MBRs.
引文
[1]迟娟,张国照,李敏哲,等.膜生物反应器处理生活污水的实验研究和工程设计[J].水处理技术,2005,31(10):76-78.
[2] Hugh T,John M.Fouling characteristics of membrane filtration in membrane bioreactors[J].Membrane Technology,2000,122(6):10-13.
[3] Van D L,Roncken G C G.Membrane bioreactor for wastewater treatment:the state of the art and new developments[J].Water Science and Technology,1997,35(10):35-41.
[4]张全忠,韩春梅.膜生物反应器的应用现状及存在的问题[J].环境科学与管理,2005,30(4):42-46.
[5]张传义.膜生物反应器-膜污染控制方法研究[D].北京:中国矿业大学,2003.
[6] Meng F G,Zhang H M,Yang F L,et al.Effect of filamentous bacteria on membrane fouling in submerged membrane bioreactor[J].Journal of Membrane Science,2006,272(3):161-168.
[7] Rosenberger S,Laabs C,Lesjean B,et al.Impact of colloidal and soluble organic material on membrane performance in membrane bioreactors for municipal wastewater treatment[J].Water Research,2006,40(4):710-720.
[8]周健,龙腾锐,苗利利.胞外聚合物EPS对活性污泥沉降性能的影响研究[J].环境科学学报,2004,24(4):613-618.
[9]崔雨新,王小明.分子生物学技术在环境生物学中的应用[J].自然杂志,2001,21(5):295-301.
[10]Arlene K R,Jason R S,David F,et al.Composition and diversity of ammonia-oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater[J].FEMS Micorbiology Ecology,2003,43(2):195-206.
[11]Amann R I,Ludwig W,Schleifer K H.Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J].Microbiological Reviews,1995,59(1):143-169.
[12]高平平,晃群芳,张学礼,等.TGGE分析焦化废水处理系统活性污泥细菌种群动态变化及多样性[J].生态学报,2003,23(10):1963-1969.
[13]谢冰,姜京顺.铜离子冲击下活性污泥微生物多样性的分子生态学分析[J].环境科学学报,2002,22(6):721-725.
[14]Ingela D.Molecular community analysis of microbial diversity[J].Current Opinion in Biotechnology,2002,13(3):213-217.
[15]Widada J,Nojiri H,Omori T.Recent developments in molecular techniques for identification and monitoring of xenobiotic-degrading bacteria and their catabolic genes in bioremediation[J].Applied Microbiology and Biotechnology,2002,60(1/2):45-59.
[16]Wilderer P A,Bungartz H J,Lemmer H,et al.Modern scientific methods and their potential in wastewater science and technology[J].Water Research,2002,36(2):370-393.
[17]Muyzer G . DGGE/TGGE a method for identifying genes from natural ecosystems[J].Current Opinion in Microbiology,1999,2(3):317-322.
[18]Muyzer G,Smalla K.Application of denaturing gradient gel(DGGE) and temperature gradient gel elecrtophoresis(TGGE) in microbial ecology[J].Antonie Van Leeuwenhoek,1998,73(1):127-141.
[19]Muyzer G,Waal E C,Uiterlinden A G.Profiling of complex microbial populations by denaturing gradient gel elecrtophoersis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J] . Applied and Environmental Microbiology,1993,59(3):695-700.
[20]Dabert P,Sialve B,Delgenes J P,et al.Characterisation of the microbial 16S rDNA diversity of an aerobic phosphorus-removal ecosystem and monitoring of its transition to nitrate respiration[J].Applied Microbiology and Biotechnology,2001,55(4):500-509.
[21]Liu W T,Marsh T L,Hans C,et al.Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA[J].Applied and Environmental Microbiology,1997,63(11):4516-4522.
[22]Marsh T L.Terminal restriction fragment length polymorphism(T-RFLP):an emerging method for characterizing diversity among homologous populations of amplification products[J].Current Opinion in Microbiol,1999,2(3):323-327.
[23]Marsh T L,Saxman P,Cole J,et al.Terminal restriction fragment length polymorphism analysis program,a web-based research tool for microbial community analysis[J].Applied and Environmental Microbiology,2000,66(8):3616-3620.
[24]Wikstrom P,Andersson A C,Forsman M.Biomonitoring complex microbial communities using random amplified polymorphic DNA and principal component analysis[J].FEMS Microbiology Ecology,1999,28(2):131-139.
[25]Giovannoni S J,Britschgi T B,Moyer C L,et al.Genetic diversity in sargasso sea bacterioplankton[J].Nature,1990,345(6270):60-63.
[26]Kemp P F,Aller J Y.Bacterial diversity in aquatic and other environments:what 16S rDNA libraries can tell us[J].FEMS Microbiology Ecology,2004,47(2):161-177.
[27]Hugenholtz P,Goebel B M,Pace N R.Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity[J].Journal of Bacteriology,1998,180(18):4765-4774.
[28]Eckburg P B,Bik E M,Bernstein C N,et al.Diversity of the human intestinal microbial flora[J].Science,2005,308(5728):1635-1638.
[29]Woese C R.Bacterial evolution[J].Microbiological Reciews,1987,51(2):221-271.
[30]Hugenholtz P,Goebel B M,Pace N R.Minireview:Impact of culture-independent studies on the emerging phytogenetic view of bacterial diversity[J].Journal of Bacteriology,1998,180(18):4765-4774.
[31]Brenner D J,Krieg N R,Staley J T,et al.Bergey’s manual of systematic bacteriology(Vol 2)[M].Baltimore:Williams and Wilkins,2004.
[32]Utaker J B,Nes I F.A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of ammonia oxidizing bacteria[J]. Systematic and Applied Microbiology,1998,21(1):72-88.
[33]David R B,Richard W C.Bergey’s manual of systematic bacteriology(Vol 1)[M].Baltimore:Williams and Wilkins,2001.
[34]Purkhold U,Roser A P,Juretschko S,et al.Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis : implications for molecular diversity surveys[J] . Applied and Environmental Microbiology,2000,66(12):5368-5382.
[35]Woese C R,Weisburg W G,Hahn C M,et al.The phylogeny of the purple bacteria:the gamma subdivision[J].Systematic and Applied Microbiology,1985,6(1):25-33.
[36]Ward B B,Gregory D O.Worldwide distribution of nitrosococcus oceani,a marine ammonia-oxidizingγ-proteobacterium,detected by PCR and sequencing of 16S rRNA and amoA genes[J].Applied and Environmental Microbiology,2002,68(8):4153-4157.
[37]Prosser J I.Autotrophic nitrification in bacteria[J].Advances in Microbial Physiology,1989,30(2):125-181.
[38]Kuenen J G , Robertson L A . Combined nitrification-denitrification processes[J].FEMS Microbiology Reviews,1994,15(2/3):109-117.
[39]Teske A,Alm E,Regan J M,et al.Evolutionary relationships among ammonia and nitrite-oxidizing bacteria[J].The Journal of Bacteriology,1994,176(21):6623-6630.
[40]Ehrich S,Behrens D,Lebedeva E,et al.A new obligately chemolithoautotrophic,nitrite-oxidizing bacterium,nitrospira moscoviensis sp. nov. and its phylogenetic relationship[J].Archives of Microbiology,1995,164(1):16-23.
[41]Stephen J R,Kowalchuk G A,Bruns M A,et al.Analysis ofβ-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing[J].Applied and Environmental Microbiology,1998,64(8):2958-2965.
[42]Princic A,Mahne I,Megusar F,et al.Effects of pH and oxygen and ammonium concentrations on the community structure of nitrifying bacteria from wastewater [J].Applied and Environmental Microbiology,1998,64(10):3584-3590.
[43]Mccaig A E,Phillips C J,Stephen J R,et al.Nitrogen cycling and community structure of proteobacterialβ-subgroup ammonia-oxidizing bacteria within polluted marine fish farm sediments[J] . Applied and Environmental Microbiology,1999,65(1):213-220.
[44]Donner G,Schwarz K,Hoppe H G,et al.Profiling the succession of bacterial populations in pelagic chemoclines[J].Arch Hydrobiol Spec Issues Advanc Limnol,1996,48(5):7-14.
[45]Santegoeds C M,Muyzer G,Beer D.Successional processes in a bacterial biofilm determined with microsensors and molecular techniques,in:proceedings part I of the‘international symposium environmental biotechnology’ostend[M].Belgium:Ghent University Publishers,1997.
[46]Teske A,Sigalevich P,Cohen Y,et al.Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures[J].Applied and Environmental Microbiology,1996,62(11):4210-4215.
[47]Rowan A K,Snape J R,Fearnside D,et al.Composition and diversity of ammonia-oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater[J].FEMS Micorbiology Ecology,2003,43(2):195-206.
[48]Feng Wang,Siqing Xia,Yigang Fu,et al.Biodiversity analysis of microbial community in the chem-bioflocculation treatment process[J].Biotechnology and Bioengineering,2005,89(6):656-659.
[49]Yan Shi,Siqing Xia,Yigang Fu,et al.DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical-biological flocculation and chemical coagulation systems[J].Applied and Environmental Microbiology,2005,69(1):99-105.
[50]苏俊峰,马放,王弘宇,等.利用PCR-DGGE技术分析生物陶粒硝化反应器中微生物群落动态[J].环境科学学报,2007,27(3):386-390.
[51]张斌,孙宝盛,吴卿,等.应用PCR-DGGE技术解析MBR中微生物群落多样性[J].天津大学学报(自然科学版),2008,41(3):356-361.
[52]陈岭,明镇寰.硝化池中氨氧化细菌amoA基因的检测及其多样性研究[J].浙江大学学报(理学版),2004,31(5):565-569.
[53]孙寓姣,左剑恶,杨洋,等.好氧亚硝化颗粒污泥中硝化细菌群落结构分析[J].环境科学,2006,27(9):1858-1861.
[54]罗鹏,胡超群,谢珍玉,等.凡纳滨对虾咸淡水养殖系统内细菌群落组成的PCR-DGGE分析[J].热带海洋学报,2006,25(2):49-53.
[55]张丹,刘耀平,徐慧,等.OLAND生物脱氮系统中硝化菌群16S rDNA的DGGE分析[J].生物技术,2003,13(5):1-3.
[56]张光亚,方柏山,闵航,等.好氧同时硝化-反硝化菌的分离鉴定及系统发育分析[J].应用与环境生物学报,2005,11(2):226-228.
[57]黄正,陈晓春,刘红艳,等.下流式固定床反应器中反硝化细菌类群及其脱氮效率分析[J].卫生研究,2007,36(1):23-25.
[58]Purohit H J,Kapley A,Moharikar A A,et al.A novel approach for extraction of PCR-compatible DNA from activated sludges amples collected from different biological effluent treatment plants[J].Journal of Microbiological Methods,2003,52(3):315-323.
[59]高平平,赵立平.可用于微生物群落分子生态学研究的活性污泥总DNA提取方法研究[J].生态学报,2002,22(11):2015-2019.
[60]Gilliane K,Verbrugge D,Chasseur M L,et al.PCR typing of tetracycline resistance determinants(Tet A-E) in salmonella enterica serotype hadar and in the microbial community of activated sludges from hospital and urban wastewater treatment facilities in Belgium[J].FEMS Micorbiology Ecology,2000,32(1):77-85.
[61]Asim K B,Meena H M,Joseph L D,et al.Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples[J].Applied and Environmental Microbiology,1991,57(12):3529-3534.
[62]Bourrain M,Achouak W,Urbain V,et al.DNA extraction from activated sludge[J].Current Microbiology,1999,38(6):315-319.
[63]萨姆布鲁克,拉塞尔.分子克隆实验指南(第二版)[M].北京:科学技术出版社,1996.
[64]沃克,拉普勒.分子生物学与生物技术[M].北京:化学工业出版,2003.
[65]张斌,孙宝盛,刘慧娜,等.处理不同废水MBR系统中微生物群落结构的比较[J].环境科学,2008,29(10):2044-2049.
[66]黄正,赵芳,刘红艳,等.活性污泥中硝化细菌16S rDNA鉴定方法研究[J].华中科技大学学报(医学版),2004,33(3):269-272.
[67]Tadashi S,Tadashi N,Motoharu O,et a1.Microbial community of biological phosphorus removal process fed with municipal wastewater under different electron acceptor conditions[J].Applied and Environmental Microbiology,1991,23(5):1211-1224.
[68]Marcus A H,Harold L D,Andreas S,et a1.Nitrous oxide reductase genes(nosZ) of denitrifying microbial populations in soil and the earthworm gut are phylogenetically similar[J].Applied and Environmental Microbiology,2006,72(2):1019-1026.
[69]张于光,李迪强,王慧敏,等.青藏高原三江源地区高寒草甸土反硝化细菌多样性的初步研究[J].科学通报,2006,51(6):715-723.
[70]Heuer H,Martin K,Paul B,et al.Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel electrophoretic separation in denaturing gradients[J].Applied and Environmental Microbiology,1997,63(8),3233-3241.
[71]Henckel T,Friedrich M,Conrad R,et al.Molecular analyses of the methane oxidizing microbial community in rice fields oil by targeting the genes of the16S rRNA,particulate methane monooxygenase,and methanol dehydrogenase[J]. Applied and Environmental Microbiology,1999,65(5):1980-1990.
[72]张维铭.现代分子生物学实验手册(第二版)[M].北京:科学出版社,2003.
[73]黄留玉.PCR最新技术原理、方法及应用[M].北京:化工工业出版社,2005.
[74]Fischer S G,Lerman L S.DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels:correspondence with melting theory[J].Proceedings of the National Academy of Science of USA,1983,80(6):1579-1583.
[75]Myers R M,Fischer S G,Lerman L S,et a1.Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis[J].Nucleic Acids Research,1985,13(9):313l-3145.
[76]Sheffield V C,Cox D R,Lerman L S,et a1.Attachment of a 40-base-pair G+C- rich sequence(GC clamp) to genomic DNA fragments by polymerase chain reaction results in improved detection of single-base changes[J].Proceedings of the National Academy of Sciences of USA,1989,86(1):232-236.
[77]刘新春,吴成强,张昱,等.PCR-DGGE法用于活性污泥系统中微生物群落结构变化的解析[J].生态学报,2005,25(4):842-847.
[78]马克平.试论生物多样性的概念[J].生物多样性,1993,1(1):20-22.
[79]Woese C R,Fox G E.Phylogenetic structure of the prokaryotic domain:the prim ary kingdoms[J].Proceedings of the National Academy of Sciences of USA,1977,74(11):5088-5090.
[80]Higgins D G,Bleasby A J.CLUSTAL V:improved software for multiple sequence alignment[J].Computer Applications in the Biosciences,1991,8(2):189-191.
[81]国家环境保护总局,水和废水监测分析方法编委会.水和废水监测分析方法(第4版)[M].北京:中国环境出版社,2002.
[82]George A K,John R S,Wietse D B,et al.Analysis of ammonia-oxidizing bacteria of theβ-subdivision of the class proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments[J].Applied and Environmental Microbiology,1997,63(4):1489-1497.
[83]刘志恒.现代微生物学[M].北京:科学出版社,2002.
[84]Ye R W,Thomas S M.Microbial nitrogen cycles:physiology,genomics and applications[J].Current Opinion in Biotechnology,2001,4(3):307-312.
[85]Atkinson B W,Mudaly D D,Bux F.Contribution of Pseudomonas spp to phosphorus uptake in the anoxic zone of an anaerobic-anoxic-aerobic continuous activated sludge system[J].Water Science and Technology,2001,43(1):139-146.
[86]臧倩,孙宝盛,魏青.膜生物反应器用于医院废水处理[J].水处理技术,2006,32(9):85-87.
[87]张光亚,陈培钦.好氧反硝化菌的分离鉴定及特性研究[J].微生物学杂志,2005,25(6):23-26.
[88]Robertson L A , Kuenen J G . Aerobic denitrification : a controversy revived[J].Archives of Microbiology,1984,139(4):351-354.
[89]张联璧.产碱杆菌的鉴定[J].临床检验杂志,1999,17(2):123-125.
[2] Hugh T,John M.Fouling characteristics of membrane filtration in membrane bioreactors[J].Membrane Technology,2000,122(6):10-13.
[3] Van D L,Roncken G C G.Membrane bioreactor for wastewater treatment:the state of the art and new developments[J].Water Science and Technology,1997,35(10):35-41.
[4]张全忠,韩春梅.膜生物反应器的应用现状及存在的问题[J].环境科学与管理,2005,30(4):42-46.
[5]张传义.膜生物反应器-膜污染控制方法研究[D].北京:中国矿业大学,2003.
[6] Meng F G,Zhang H M,Yang F L,et al.Effect of filamentous bacteria on membrane fouling in submerged membrane bioreactor[J].Journal of Membrane Science,2006,272(3):161-168.
[7] Rosenberger S,Laabs C,Lesjean B,et al.Impact of colloidal and soluble organic material on membrane performance in membrane bioreactors for municipal wastewater treatment[J].Water Research,2006,40(4):710-720.
[8]周健,龙腾锐,苗利利.胞外聚合物EPS对活性污泥沉降性能的影响研究[J].环境科学学报,2004,24(4):613-618.
[9]崔雨新,王小明.分子生物学技术在环境生物学中的应用[J].自然杂志,2001,21(5):295-301.
[10]Arlene K R,Jason R S,David F,et al.Composition and diversity of ammonia-oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater[J].FEMS Micorbiology Ecology,2003,43(2):195-206.
[11]Amann R I,Ludwig W,Schleifer K H.Phylogenetic identification and in situ detection of individual microbial cells without cultivation[J].Microbiological Reviews,1995,59(1):143-169.
[12]高平平,晃群芳,张学礼,等.TGGE分析焦化废水处理系统活性污泥细菌种群动态变化及多样性[J].生态学报,2003,23(10):1963-1969.
[13]谢冰,姜京顺.铜离子冲击下活性污泥微生物多样性的分子生态学分析[J].环境科学学报,2002,22(6):721-725.
[14]Ingela D.Molecular community analysis of microbial diversity[J].Current Opinion in Biotechnology,2002,13(3):213-217.
[15]Widada J,Nojiri H,Omori T.Recent developments in molecular techniques for identification and monitoring of xenobiotic-degrading bacteria and their catabolic genes in bioremediation[J].Applied Microbiology and Biotechnology,2002,60(1/2):45-59.
[16]Wilderer P A,Bungartz H J,Lemmer H,et al.Modern scientific methods and their potential in wastewater science and technology[J].Water Research,2002,36(2):370-393.
[17]Muyzer G . DGGE/TGGE a method for identifying genes from natural ecosystems[J].Current Opinion in Microbiology,1999,2(3):317-322.
[18]Muyzer G,Smalla K.Application of denaturing gradient gel(DGGE) and temperature gradient gel elecrtophoresis(TGGE) in microbial ecology[J].Antonie Van Leeuwenhoek,1998,73(1):127-141.
[19]Muyzer G,Waal E C,Uiterlinden A G.Profiling of complex microbial populations by denaturing gradient gel elecrtophoersis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J] . Applied and Environmental Microbiology,1993,59(3):695-700.
[20]Dabert P,Sialve B,Delgenes J P,et al.Characterisation of the microbial 16S rDNA diversity of an aerobic phosphorus-removal ecosystem and monitoring of its transition to nitrate respiration[J].Applied Microbiology and Biotechnology,2001,55(4):500-509.
[21]Liu W T,Marsh T L,Hans C,et al.Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA[J].Applied and Environmental Microbiology,1997,63(11):4516-4522.
[22]Marsh T L.Terminal restriction fragment length polymorphism(T-RFLP):an emerging method for characterizing diversity among homologous populations of amplification products[J].Current Opinion in Microbiol,1999,2(3):323-327.
[23]Marsh T L,Saxman P,Cole J,et al.Terminal restriction fragment length polymorphism analysis program,a web-based research tool for microbial community analysis[J].Applied and Environmental Microbiology,2000,66(8):3616-3620.
[24]Wikstrom P,Andersson A C,Forsman M.Biomonitoring complex microbial communities using random amplified polymorphic DNA and principal component analysis[J].FEMS Microbiology Ecology,1999,28(2):131-139.
[25]Giovannoni S J,Britschgi T B,Moyer C L,et al.Genetic diversity in sargasso sea bacterioplankton[J].Nature,1990,345(6270):60-63.
[26]Kemp P F,Aller J Y.Bacterial diversity in aquatic and other environments:what 16S rDNA libraries can tell us[J].FEMS Microbiology Ecology,2004,47(2):161-177.
[27]Hugenholtz P,Goebel B M,Pace N R.Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity[J].Journal of Bacteriology,1998,180(18):4765-4774.
[28]Eckburg P B,Bik E M,Bernstein C N,et al.Diversity of the human intestinal microbial flora[J].Science,2005,308(5728):1635-1638.
[29]Woese C R.Bacterial evolution[J].Microbiological Reciews,1987,51(2):221-271.
[30]Hugenholtz P,Goebel B M,Pace N R.Minireview:Impact of culture-independent studies on the emerging phytogenetic view of bacterial diversity[J].Journal of Bacteriology,1998,180(18):4765-4774.
[31]Brenner D J,Krieg N R,Staley J T,et al.Bergey’s manual of systematic bacteriology(Vol 2)[M].Baltimore:Williams and Wilkins,2004.
[32]Utaker J B,Nes I F.A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of ammonia oxidizing bacteria[J]. Systematic and Applied Microbiology,1998,21(1):72-88.
[33]David R B,Richard W C.Bergey’s manual of systematic bacteriology(Vol 1)[M].Baltimore:Williams and Wilkins,2001.
[34]Purkhold U,Roser A P,Juretschko S,et al.Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis : implications for molecular diversity surveys[J] . Applied and Environmental Microbiology,2000,66(12):5368-5382.
[35]Woese C R,Weisburg W G,Hahn C M,et al.The phylogeny of the purple bacteria:the gamma subdivision[J].Systematic and Applied Microbiology,1985,6(1):25-33.
[36]Ward B B,Gregory D O.Worldwide distribution of nitrosococcus oceani,a marine ammonia-oxidizingγ-proteobacterium,detected by PCR and sequencing of 16S rRNA and amoA genes[J].Applied and Environmental Microbiology,2002,68(8):4153-4157.
[37]Prosser J I.Autotrophic nitrification in bacteria[J].Advances in Microbial Physiology,1989,30(2):125-181.
[38]Kuenen J G , Robertson L A . Combined nitrification-denitrification processes[J].FEMS Microbiology Reviews,1994,15(2/3):109-117.
[39]Teske A,Alm E,Regan J M,et al.Evolutionary relationships among ammonia and nitrite-oxidizing bacteria[J].The Journal of Bacteriology,1994,176(21):6623-6630.
[40]Ehrich S,Behrens D,Lebedeva E,et al.A new obligately chemolithoautotrophic,nitrite-oxidizing bacterium,nitrospira moscoviensis sp. nov. and its phylogenetic relationship[J].Archives of Microbiology,1995,164(1):16-23.
[41]Stephen J R,Kowalchuk G A,Bruns M A,et al.Analysis ofβ-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing[J].Applied and Environmental Microbiology,1998,64(8):2958-2965.
[42]Princic A,Mahne I,Megusar F,et al.Effects of pH and oxygen and ammonium concentrations on the community structure of nitrifying bacteria from wastewater [J].Applied and Environmental Microbiology,1998,64(10):3584-3590.
[43]Mccaig A E,Phillips C J,Stephen J R,et al.Nitrogen cycling and community structure of proteobacterialβ-subgroup ammonia-oxidizing bacteria within polluted marine fish farm sediments[J] . Applied and Environmental Microbiology,1999,65(1):213-220.
[44]Donner G,Schwarz K,Hoppe H G,et al.Profiling the succession of bacterial populations in pelagic chemoclines[J].Arch Hydrobiol Spec Issues Advanc Limnol,1996,48(5):7-14.
[45]Santegoeds C M,Muyzer G,Beer D.Successional processes in a bacterial biofilm determined with microsensors and molecular techniques,in:proceedings part I of the‘international symposium environmental biotechnology’ostend[M].Belgium:Ghent University Publishers,1997.
[46]Teske A,Sigalevich P,Cohen Y,et al.Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures[J].Applied and Environmental Microbiology,1996,62(11):4210-4215.
[47]Rowan A K,Snape J R,Fearnside D,et al.Composition and diversity of ammonia-oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater[J].FEMS Micorbiology Ecology,2003,43(2):195-206.
[48]Feng Wang,Siqing Xia,Yigang Fu,et al.Biodiversity analysis of microbial community in the chem-bioflocculation treatment process[J].Biotechnology and Bioengineering,2005,89(6):656-659.
[49]Yan Shi,Siqing Xia,Yigang Fu,et al.DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical-biological flocculation and chemical coagulation systems[J].Applied and Environmental Microbiology,2005,69(1):99-105.
[50]苏俊峰,马放,王弘宇,等.利用PCR-DGGE技术分析生物陶粒硝化反应器中微生物群落动态[J].环境科学学报,2007,27(3):386-390.
[51]张斌,孙宝盛,吴卿,等.应用PCR-DGGE技术解析MBR中微生物群落多样性[J].天津大学学报(自然科学版),2008,41(3):356-361.
[52]陈岭,明镇寰.硝化池中氨氧化细菌amoA基因的检测及其多样性研究[J].浙江大学学报(理学版),2004,31(5):565-569.
[53]孙寓姣,左剑恶,杨洋,等.好氧亚硝化颗粒污泥中硝化细菌群落结构分析[J].环境科学,2006,27(9):1858-1861.
[54]罗鹏,胡超群,谢珍玉,等.凡纳滨对虾咸淡水养殖系统内细菌群落组成的PCR-DGGE分析[J].热带海洋学报,2006,25(2):49-53.
[55]张丹,刘耀平,徐慧,等.OLAND生物脱氮系统中硝化菌群16S rDNA的DGGE分析[J].生物技术,2003,13(5):1-3.
[56]张光亚,方柏山,闵航,等.好氧同时硝化-反硝化菌的分离鉴定及系统发育分析[J].应用与环境生物学报,2005,11(2):226-228.
[57]黄正,陈晓春,刘红艳,等.下流式固定床反应器中反硝化细菌类群及其脱氮效率分析[J].卫生研究,2007,36(1):23-25.
[58]Purohit H J,Kapley A,Moharikar A A,et al.A novel approach for extraction of PCR-compatible DNA from activated sludges amples collected from different biological effluent treatment plants[J].Journal of Microbiological Methods,2003,52(3):315-323.
[59]高平平,赵立平.可用于微生物群落分子生态学研究的活性污泥总DNA提取方法研究[J].生态学报,2002,22(11):2015-2019.
[60]Gilliane K,Verbrugge D,Chasseur M L,et al.PCR typing of tetracycline resistance determinants(Tet A-E) in salmonella enterica serotype hadar and in the microbial community of activated sludges from hospital and urban wastewater treatment facilities in Belgium[J].FEMS Micorbiology Ecology,2000,32(1):77-85.
[61]Asim K B,Meena H M,Joseph L D,et al.Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples[J].Applied and Environmental Microbiology,1991,57(12):3529-3534.
[62]Bourrain M,Achouak W,Urbain V,et al.DNA extraction from activated sludge[J].Current Microbiology,1999,38(6):315-319.
[63]萨姆布鲁克,拉塞尔.分子克隆实验指南(第二版)[M].北京:科学技术出版社,1996.
[64]沃克,拉普勒.分子生物学与生物技术[M].北京:化学工业出版,2003.
[65]张斌,孙宝盛,刘慧娜,等.处理不同废水MBR系统中微生物群落结构的比较[J].环境科学,2008,29(10):2044-2049.
[66]黄正,赵芳,刘红艳,等.活性污泥中硝化细菌16S rDNA鉴定方法研究[J].华中科技大学学报(医学版),2004,33(3):269-272.
[67]Tadashi S,Tadashi N,Motoharu O,et a1.Microbial community of biological phosphorus removal process fed with municipal wastewater under different electron acceptor conditions[J].Applied and Environmental Microbiology,1991,23(5):1211-1224.
[68]Marcus A H,Harold L D,Andreas S,et a1.Nitrous oxide reductase genes(nosZ) of denitrifying microbial populations in soil and the earthworm gut are phylogenetically similar[J].Applied and Environmental Microbiology,2006,72(2):1019-1026.
[69]张于光,李迪强,王慧敏,等.青藏高原三江源地区高寒草甸土反硝化细菌多样性的初步研究[J].科学通报,2006,51(6):715-723.
[70]Heuer H,Martin K,Paul B,et al.Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel electrophoretic separation in denaturing gradients[J].Applied and Environmental Microbiology,1997,63(8),3233-3241.
[71]Henckel T,Friedrich M,Conrad R,et al.Molecular analyses of the methane oxidizing microbial community in rice fields oil by targeting the genes of the16S rRNA,particulate methane monooxygenase,and methanol dehydrogenase[J]. Applied and Environmental Microbiology,1999,65(5):1980-1990.
[72]张维铭.现代分子生物学实验手册(第二版)[M].北京:科学出版社,2003.
[73]黄留玉.PCR最新技术原理、方法及应用[M].北京:化工工业出版社,2005.
[74]Fischer S G,Lerman L S.DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels:correspondence with melting theory[J].Proceedings of the National Academy of Science of USA,1983,80(6):1579-1583.
[75]Myers R M,Fischer S G,Lerman L S,et a1.Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis[J].Nucleic Acids Research,1985,13(9):313l-3145.
[76]Sheffield V C,Cox D R,Lerman L S,et a1.Attachment of a 40-base-pair G+C- rich sequence(GC clamp) to genomic DNA fragments by polymerase chain reaction results in improved detection of single-base changes[J].Proceedings of the National Academy of Sciences of USA,1989,86(1):232-236.
[77]刘新春,吴成强,张昱,等.PCR-DGGE法用于活性污泥系统中微生物群落结构变化的解析[J].生态学报,2005,25(4):842-847.
[78]马克平.试论生物多样性的概念[J].生物多样性,1993,1(1):20-22.
[79]Woese C R,Fox G E.Phylogenetic structure of the prokaryotic domain:the prim ary kingdoms[J].Proceedings of the National Academy of Sciences of USA,1977,74(11):5088-5090.
[80]Higgins D G,Bleasby A J.CLUSTAL V:improved software for multiple sequence alignment[J].Computer Applications in the Biosciences,1991,8(2):189-191.
[81]国家环境保护总局,水和废水监测分析方法编委会.水和废水监测分析方法(第4版)[M].北京:中国环境出版社,2002.
[82]George A K,John R S,Wietse D B,et al.Analysis of ammonia-oxidizing bacteria of theβ-subdivision of the class proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments[J].Applied and Environmental Microbiology,1997,63(4):1489-1497.
[83]刘志恒.现代微生物学[M].北京:科学出版社,2002.
[84]Ye R W,Thomas S M.Microbial nitrogen cycles:physiology,genomics and applications[J].Current Opinion in Biotechnology,2001,4(3):307-312.
[85]Atkinson B W,Mudaly D D,Bux F.Contribution of Pseudomonas spp to phosphorus uptake in the anoxic zone of an anaerobic-anoxic-aerobic continuous activated sludge system[J].Water Science and Technology,2001,43(1):139-146.
[86]臧倩,孙宝盛,魏青.膜生物反应器用于医院废水处理[J].水处理技术,2006,32(9):85-87.
[87]张光亚,陈培钦.好氧反硝化菌的分离鉴定及特性研究[J].微生物学杂志,2005,25(6):23-26.
[88]Robertson L A , Kuenen J G . Aerobic denitrification : a controversy revived[J].Archives of Microbiology,1984,139(4):351-354.
[89]张联璧.产碱杆菌的鉴定[J].临床检验杂志,1999,17(2):123-125.