晚期垃圾渗滤液MBR亚硝化系统中细菌及功能菌的多样性
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摘要
为了处理晚期垃圾渗滤液,利用膜生物反应器(membrane bioreactor,MBR)实现了稳定亚硝化.分别构建总细菌通用克隆文库和针对亚硝化功能菌氨氧化菌(ammonia oxidizing bacteria,AOB)的功能基因——amo A基因的克隆文库,来研究稳定期亚硝化系统中微生物多样性.从16S r DNA克隆文库中随机挑选82个阳性克隆子进行序列测定,将测序结果与Genbank中已有模式菌株的序列进行比对后发现,亚硝化系统中主要有4个优势菌群,分别是Proteobacteria类群(64.65%)、未培养菌(uncultured bacterium)类群(18.3%)、Bacteroidetes类群(9.76%)、Firmicutes类群(7.32%).构建针对AOB的amo A功能基因的克隆文库,从文库中挑选73个阳性克隆子进行序列测定,经序列比对后发现在系统中仅检测到了亚硝化单胞菌属(Nitrosomonas)和未培养菌,分别占41.1%和58.9%.这表明系统中起到亚硝化作用的微生物种群主要是亚硝化单胞菌属(Nitrosomonas).此外,未培养细菌的大量存在表明,系统中还存在着丰富的微生物资源等待进一步的开发利用.
In order to find the better method to deal with late landfill leachate,the stable partial nitrification system was operated in the MBR( membrane bioreactor). The microbial community structure of the stable partial nitrification phase was investigated by the total bacterial clone library and the cloning library of ammonia oxidizing bacteria( AOB) functional gene amo A. 82 positive clones were selected randomly from 16 S rDNA clone library and their sequence was determined. The results show that there are four dominant bacterium groups in the stable partial nitrification phase: Proteobacteria( 64. 65%),uncultured bacterium( 18. 3%),Bacteroidetes( 9. 76%),and Firmicutes( 7. 32%). Building on the cloning library of AOB's functional gene amo A,73 positive clones were selected from the library and their sequence was also determined. Compared with basic local alignment search tool( BLAST),the results show that only the Nitrosomonas and Uncultured bacterium are detected in the system,and their proportion is 41. 1% and 58. 9%,respectively. This phenomenon show that the number of Nitrosomonas is maximum in this system. At the same time,the existence of many unknown bacterial species showedthat there are a large number of microbial resources in the system and needing further development.
In order to find the better method to deal with late landfill leachate,the stable partial nitrification system was operated in the MBR( membrane bioreactor). The microbial community structure of the stable partial nitrification phase was investigated by the total bacterial clone library and the cloning library of ammonia oxidizing bacteria( AOB) functional gene amo A. 82 positive clones were selected randomly from 16 S rDNA clone library and their sequence was determined. The results show that there are four dominant bacterium groups in the stable partial nitrification phase: Proteobacteria( 64. 65%),uncultured bacterium( 18. 3%),Bacteroidetes( 9. 76%),and Firmicutes( 7. 32%). Building on the cloning library of AOB's functional gene amo A,73 positive clones were selected from the library and their sequence was also determined. Compared with basic local alignment search tool( BLAST),the results show that only the Nitrosomonas and Uncultured bacterium are detected in the system,and their proportion is 41. 1% and 58. 9%,respectively. This phenomenon show that the number of Nitrosomonas is maximum in this system. At the same time,the existence of many unknown bacterial species showedthat there are a large number of microbial resources in the system and needing further development.
引文
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[15]XIA S,LI J,WANG R,et al.Tracking composition and dynamics of nitrification and denitrification microbial community in a biofilm reactor by PCR-DGGE and combining fish with flow cytometry[J].Biochemical Engineering Journal,2010,49(3):370-378.
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[18]刘志培,杨惠芳,周培瑾.食酸丛毛单胞菌AN3菌株降解苯胺代谢途径的研究[J].微生物学报,1999,39(5):448-453.LIU Z P,YANG H F,ZHOU P J.Study on the degradation of aniline by the strain of Comamonas acidovorans AN3[J].Acta Microbiologica Sinica,1999,39(5):448-453.(in Chinese)
[19]SCHLEHECK D,VON NETZER F,FLEISCHMANN T,et al.The missing link in linear alkylbenzenesulfonate surfactant degradation:4-sulfoacetophenone as atransient intermediate in the degradation of 3-(4-Sulfophenyl)utyrate by Comamonastestosteroni KF-1[J].Applied and Environmental Microbiology,2010,76(1):196-202.
[20]ANDERSON I C,POTH M,HOMSTEAD J,et al.A comparison of NO and N2O production by the autotrophic nitrifier Nitorsomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis[J].Applied and Environmental Microbiology,1993,59(11):3525-3533.
[21]王成林,周巧红,王亚芬.一株异养硝化细菌的分离鉴定及其亚硝化作用研究[J].农业环境科学学报,2008,27(3):1146-1150.WANG C L,ZHOU Q H,WANG Y F.Isolation and identification of a heterotrophic nitrification bacteria and its role in the study of partial nitrification[J].Journal of Agro-environment Science,2008,27(3):1146-1150.(in Chinese)
[22]HILL V R,KAHLER A M,JOTHIKUMAR N,et al.Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples[J].Applied and Environmental Microbiology,2007,73(13):4218-4225.
[23]CHOUARI R,LE PASLIER D,DAEGELEN P,et al.Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester[J].Environmental Microbiology,2005,7(8):1104-1115.
[24]曾地刚,雷爱莹,彭敏,等.枯草芽孢杆菌的分离及其净化水质的研究[J].水利渔业,2007,27(6):1003-1278.ZENG D G,LEI A Y,PENG M,et al.Isolation of Bacillus subtilis and study on its role of purification water[J].Reservoir Fisheries,2007,27(6):1003-1278.(in Chinese)
[25]邵晴.氨氮和亚硝态氮降解菌的研究[D].无锡:江南大学,2008:19-41.SHAO Q.Study on the degradation of ammonia nitrogen and nitrite nitrogen[D].Wuxi:Jiangnan University,2008:19-41.(in Chinese)
[26]陈尚智,胡勇有.枯草芽孢杆菌对微污染水体的净化作用[J].环境科学学报,2011,31(8):1594-1601.CHEN S Z,HU Y Y.Purification of micro polluted water by Bacillus subtilis[J].Acta Scientiae Circumstantiae,2011,31(8):1594-1601.(in Chinese)
[27]YANG X P,WANG S M,ZHANG D W,et al.Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium,Bacillus subtilis,A1[J].Bioresource Technology,2011,102(2):854-862.
[28]GIOVANNONI S J,MULLINS T D,FIELD K G.Microbial diversity in oceanic systems:rRNA approaches to the study of unculturable microbes[M].Berlin:Springer,1995:217-248.
[29]邵军,孙海美,孙卫玲,等.垃圾渗滤液处理系统中微生物群落结构变化研究[J].北京大学学报,2010,46(3):435-441.SHAO J,SUN H M,SUN W L,et al.Study on the microbial community structure in landfill leachate treatment system[J].Journal of Peking University,2010,46(3):435-441.(in Chinese)
[30]LIMPIYAKORN T,SHINOHARA Y,KURISU F,et al.Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo[J].Fems Microbiology Ecology,2006,54(2):205-217.
[31]LIMPIYAKORN T,KURISU F,SAKAMOTO Y,et al.Effects of ammonium and nitrite on communities and populations of ammonia-oxidizing bacteria in laboratoryscale continuous-flow reactors[J].Fems Microbiology Ecology,2007,60(3):501-512.
[2]FARAH N A,CHRISTOPHER Q L.Treatment of landfill leachate using membrane bioreactors:a review[J].Desalination,2012,287(8):41-54.
[3]STUCZYNSKI T I,MCCARTY G W,SIEBIELEC G.Response of soil microbiological activities to cadmium,lead,and zinc salt amendments[J].Journal of Environmental Quality,2003,32(4):1346-1355.
[4]KARGI F,KONYA I.COD,para-chlorophenol and toxicity removal from para-chlorophenol containing synthetic wastewater in an activated sludge unit[J].Journal of Hazardous Materials,2006,132(2/3):226-231.
[5]STROUS M,HEIJNEN J J,KUENEN J G,et al.The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms[J].Applied Microbiology and Biotechnology,1998,50(5):589-596.
[6]XUE Y,YANG F,LIU S,et al.The influence of controlling factors on the start-up and operation for partial nitrification in membrane bioreactor[J].Bioresource Technology,2009,100(3):1055-1060.
[7]李芸,熊向阳,李军,等.膜生物反应器处理晚期垃圾渗滤液亚硝化性能及其抑制动力学分析[J].中国环境科学,2016,36(2):419-427.LI Y,XIONG X Y,LI J,et al.Performance and inhibition kinetics of membrane bioreactor in treatment of advanced landfill leachate[J].China Environmental Science,2016,36(2):419-427.(in Chinese)
[8]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):45-59.
[9]SHEN L,YUAN Y,MENG F.Reactor performance and microbial ecology of a nitritation membrane bioreactor[J].Journal of Membrane Science,2014,462(462):139-146.
[10]伍阳.膜反应器脱氮除磷及细菌16S r DNA多样性研究[D].雅安:四川农业大学,2008:18-19.WU Y.Study on the diversity of nitrogen and phosphorus removal bacterium in membrane reactor by 16S r DNA.[D].Yaan:Sichuan Agricultural University,2008:18-19.(in Chinese)
[11]HUANG L N,ZHU S,ZHOU H,et al.Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill[J].FEMS Microbiol Lett,2005,242(2):297-303.
[12]KAPLEY A,PRASAD S,PUROHIT H.Changes in microbial diversity infed-batch reactor operation with wastewater containing nitroaromatic residues[J].Bioresource Technology,2007,98(13):2479-2484.
[13]MUYZER G.DGGE/TGGE a method for identifying genes from natural ecosystems[J].Current Opinion in Microbiology,1999,2(3):317-322.
[14]SNAIDR J,AMANN R,HUBER I,et al.Phylogenetic analysis and in situ identification of bacteria in activated sludge[J].Applied and Environmental Microbiology,1997,63(7):2884-2896.
[15]XIA S,LI J,WANG R,et al.Tracking composition and dynamics of nitrification and denitrification microbial community in a biofilm reactor by PCR-DGGE and combining fish with flow cytometry[J].Biochemical Engineering Journal,2010,49(3):370-378.
[16]BOON N,GORIS J,DE VOS P,et al.Genetic diversity among 3-chloroaniline and aniline-degrading strains of the Comamonadaceae[J].Applied and Environmental Microbiology,2001,67(3):1107-1115.
[17]KHAN S T,HORIBA Y,YAMAMOTO M,et al.Members of the family Comamonadaceae as primary poly(3-hydroxybutyrate-co-3-hydroxyvalerate)degrading deni-trifiers in activated sludge asrevealed by a polyphasic approach[J].Applied and Environmental Microbiology,2002,68(7):3206-3214.
[18]刘志培,杨惠芳,周培瑾.食酸丛毛单胞菌AN3菌株降解苯胺代谢途径的研究[J].微生物学报,1999,39(5):448-453.LIU Z P,YANG H F,ZHOU P J.Study on the degradation of aniline by the strain of Comamonas acidovorans AN3[J].Acta Microbiologica Sinica,1999,39(5):448-453.(in Chinese)
[19]SCHLEHECK D,VON NETZER F,FLEISCHMANN T,et al.The missing link in linear alkylbenzenesulfonate surfactant degradation:4-sulfoacetophenone as atransient intermediate in the degradation of 3-(4-Sulfophenyl)utyrate by Comamonastestosteroni KF-1[J].Applied and Environmental Microbiology,2010,76(1):196-202.
[20]ANDERSON I C,POTH M,HOMSTEAD J,et al.A comparison of NO and N2O production by the autotrophic nitrifier Nitorsomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis[J].Applied and Environmental Microbiology,1993,59(11):3525-3533.
[21]王成林,周巧红,王亚芬.一株异养硝化细菌的分离鉴定及其亚硝化作用研究[J].农业环境科学学报,2008,27(3):1146-1150.WANG C L,ZHOU Q H,WANG Y F.Isolation and identification of a heterotrophic nitrification bacteria and its role in the study of partial nitrification[J].Journal of Agro-environment Science,2008,27(3):1146-1150.(in Chinese)
[22]HILL V R,KAHLER A M,JOTHIKUMAR N,et al.Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples[J].Applied and Environmental Microbiology,2007,73(13):4218-4225.
[23]CHOUARI R,LE PASLIER D,DAEGELEN P,et al.Novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester[J].Environmental Microbiology,2005,7(8):1104-1115.
[24]曾地刚,雷爱莹,彭敏,等.枯草芽孢杆菌的分离及其净化水质的研究[J].水利渔业,2007,27(6):1003-1278.ZENG D G,LEI A Y,PENG M,et al.Isolation of Bacillus subtilis and study on its role of purification water[J].Reservoir Fisheries,2007,27(6):1003-1278.(in Chinese)
[25]邵晴.氨氮和亚硝态氮降解菌的研究[D].无锡:江南大学,2008:19-41.SHAO Q.Study on the degradation of ammonia nitrogen and nitrite nitrogen[D].Wuxi:Jiangnan University,2008:19-41.(in Chinese)
[26]陈尚智,胡勇有.枯草芽孢杆菌对微污染水体的净化作用[J].环境科学学报,2011,31(8):1594-1601.CHEN S Z,HU Y Y.Purification of micro polluted water by Bacillus subtilis[J].Acta Scientiae Circumstantiae,2011,31(8):1594-1601.(in Chinese)
[27]YANG X P,WANG S M,ZHANG D W,et al.Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium,Bacillus subtilis,A1[J].Bioresource Technology,2011,102(2):854-862.
[28]GIOVANNONI S J,MULLINS T D,FIELD K G.Microbial diversity in oceanic systems:rRNA approaches to the study of unculturable microbes[M].Berlin:Springer,1995:217-248.
[29]邵军,孙海美,孙卫玲,等.垃圾渗滤液处理系统中微生物群落结构变化研究[J].北京大学学报,2010,46(3):435-441.SHAO J,SUN H M,SUN W L,et al.Study on the microbial community structure in landfill leachate treatment system[J].Journal of Peking University,2010,46(3):435-441.(in Chinese)
[30]LIMPIYAKORN T,SHINOHARA Y,KURISU F,et al.Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo[J].Fems Microbiology Ecology,2006,54(2):205-217.
[31]LIMPIYAKORN T,KURISU F,SAKAMOTO Y,et al.Effects of ammonium and nitrite on communities and populations of ammonia-oxidizing bacteria in laboratoryscale continuous-flow reactors[J].Fems Microbiology Ecology,2007,60(3):501-512.