丹江口库区表层浮游细菌群落组成与PICRUSt功能预测分析
|
摘要
浮游细菌是水生生态系统的重要组成部分,是水体中氮素生物地球化学循环的主要驱动力.本研究于2016年5月采集丹江口库区库心和渠首2个生态位点表层水样,采用16S r DNA Miseq高通量测序技术研究其群落组成,发现其主要由变形菌门、放线菌门、拟杆菌门等12门、139属细菌组成,渠首样品浮游细菌群落多样性高于库心样品. PICRUSt功能预测分析表明,浮游细菌涉及氨基酸运输和代谢、转录、能量产生和转换等24个基因功能家族,表现出功能上的丰富性.其中35个参与氮代谢的KO(表示通路)中库心高于渠首为20个,渠首高于库心的为15个.两样品中检测到涉及固氮作用(nif H)、硝化作用(hao)、反硝化作用(nar G、nir K、nor B、nos Z)、氮同化还原及异化还原作用(nas A、nar B、nap A、nir A、nir B、nrf A)参与氮循环的关键基因相对丰度.综合基因功能家族预测基因拷贝数和氮循环相关基因丰度分析,丹江口库区库浮游细菌氮代谢能力整体趋势为库心高于渠首.本研究从细菌群落组成、功能角度初步分析了丹江口库区不同生态位点氮循环的差异,为丹江口水库水环境保护提供了参考依据.
Bacterioplankton are important components of aquatic ecosystems,and play a crucial role in the global biogeochemical cycle of nitrogen. In this study,surface-water samples were collected from Kuxin( the center of Danjiangkou Reservoir) and Qushou( the Middle Route of the South-to-North Water Diversion Project's canal head) in the Danjiangkou Reservoir in May 2016. Total DNA was isolated,and high-throughput sequencing was performed to analyze the community structure and diversity of bacteria. Phylogenetic analyses of 16 S r DNA sequences showed that bacterial communities included species from 12 major phylogenetic groups. The predominant phylogenetic groups included Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, and Verrucomicrobia. The microbial biodiversity of Danjiangkou Reservoir bacterioplankton was greater in water samples from Qushou than in those from Kuxin.PICRUSt( phylogenetic investigation of communities by reconstruction of unobserved states) was used to determine the metabolic and functional abilities of the observed bacterial communities. Our results revealed a wide genetic diversity of organisms involved in various essential processes,such as Amino acid transport and metabolism,Transcription,Energy production and conversion,and Carbohydrate transport and metabolism. Thirty-five metabolic pathways involving nitrogen were detected. Key genes impacting the nitrogen cycle were detected in both the Kuxin and Qushou samples,and these encoded enzymes involved in nitrogen fixation( nif H),nitrification( hao),denitrification( nar G、nir K、nor B、nos Z),and assimilatory and dissimilatory nitrogen reduction to ammonia( nas A、nar B、nap A、nir A、nir B、nrf A). Copy numbers of 16 S rRNA genes of each detected phylotype of Danjiangkou Reservoir bacterioplankton were uniformly higher in water samples from Kuxin than in water samples from Qushou. Our analyses of differences in nitrogen abatement potential between water samples,based on bacterial community composition and function,provide a foundation for setting water environmental protection policies in the Danjiangkou Reservoir.
Bacterioplankton are important components of aquatic ecosystems,and play a crucial role in the global biogeochemical cycle of nitrogen. In this study,surface-water samples were collected from Kuxin( the center of Danjiangkou Reservoir) and Qushou( the Middle Route of the South-to-North Water Diversion Project's canal head) in the Danjiangkou Reservoir in May 2016. Total DNA was isolated,and high-throughput sequencing was performed to analyze the community structure and diversity of bacteria. Phylogenetic analyses of 16 S r DNA sequences showed that bacterial communities included species from 12 major phylogenetic groups. The predominant phylogenetic groups included Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, and Verrucomicrobia. The microbial biodiversity of Danjiangkou Reservoir bacterioplankton was greater in water samples from Qushou than in those from Kuxin.PICRUSt( phylogenetic investigation of communities by reconstruction of unobserved states) was used to determine the metabolic and functional abilities of the observed bacterial communities. Our results revealed a wide genetic diversity of organisms involved in various essential processes,such as Amino acid transport and metabolism,Transcription,Energy production and conversion,and Carbohydrate transport and metabolism. Thirty-five metabolic pathways involving nitrogen were detected. Key genes impacting the nitrogen cycle were detected in both the Kuxin and Qushou samples,and these encoded enzymes involved in nitrogen fixation( nif H),nitrification( hao),denitrification( nar G、nir K、nor B、nos Z),and assimilatory and dissimilatory nitrogen reduction to ammonia( nas A、nar B、nap A、nir A、nir B、nrf A). Copy numbers of 16 S rRNA genes of each detected phylotype of Danjiangkou Reservoir bacterioplankton were uniformly higher in water samples from Kuxin than in water samples from Qushou. Our analyses of differences in nitrogen abatement potential between water samples,based on bacterial community composition and function,provide a foundation for setting water environmental protection policies in the Danjiangkou Reservoir.
引文
[1]Hanson C A,Fuhrman J A,Horner-Devine M C,et al.Beyond biogeographic patterns:processes shaping the microbial landscape[J].Nature Reviews Microbiology,2012,10(7):497-506.
[2]Newton R J,Jones S E,Eiler A,et al.A guide to the natural history of freshwater lake bacteria[J].Microbiology and Molecular Biology Reviews,2011,75(1):14-49.
[3]任丽娟,何聃,邢鹏,等.湖泊水体细菌多样性及其生态功能研究进展[J].生物多样性,2013,21(4):421-432.Ren L J,He D,Xing P,et al.Bacterial diversity and ecological function in lake water bodies[J].Biodiversity Science,2013,21(4):421-432.
[4]Chen P,Li L,Zhang H B.Spatio-temporal variations and source apportionment of water pollution in Danjiangkou Reservoir Basin,central China[J].Water,2015,7(6):2591-2611.
[5]陈兆进,丁传雨,朱静亚,等.丹江口水库枯水期浮游细菌群落组成及影响因素研究[J].中国环境科学,2017,37(1):336-344.Chen Z J,Ding C Y,Zhu J Y,et al.Community structure and influencing factors of bacterioplankton during low water periods in Danjiangkou Reservoir[J].China Environmental Science,2017,37(1):336-344.
[6]Kuypers M M M,Marchant H K,Kartal B.The microbial nitrogen-cycling network[J].Nature Reviews Microbiology,2018,16(5):263-276.
[7]杨雪琴,连英丽,颜庆云,等.滨海湿地生态系统微生物驱动的氮循环研究进展[J].微生物学报,2018,58(4):633-648.Yang X Q,Lian Y L,Yan Q Y,et al.Microbially-driven nitrogen cycling in coastal ecosystems[J].Acta Microbiologica Sinica,2018,58(4):633-648.
[8]van Dijk E L,Auger H,Jaszczyszyn Y,et al.Ten years of nextgeneration sequencing technology[J].Trends in Genetics,2014,30(9):418-426.
[9]Gao W L,Chen Z J,Li Y Y,et al.Bioassessment of a drinking water reservoir using plankton:high throughput sequencing vs.traditional morphological method[J].Water,2018,10(1):82.
[10]陈兆进,陈海燕,李玉英,等.南水北调中线干渠(河南段)浮游细菌群落组成及影响因素[J].中国环境科学,2017,37(4):1505-1513.Chen Z J,Chen H Y,Li Y Y,et al.Community structure and influencing factors of bacterioplankton in the Main cancel of the Mid-line Project of South-to-North Water Division in sections of Henan Province[J].China Environmental Science,2017,37(4):1505-1513.
[11]张明露,刘文君,李翠萍,等.南水北调中线水源水的细菌群落和致病菌分析[J].给水排水,2015,41(6):32-36.
[12]Dang C Y,Liu W,Lin Y X,et al.Dominant role of ammoniaoxidizing bacteria in nitrification due to ammonia accumulation in sediments of Danjiangkou reservoir,China[J].Applied Microbiology and Biotechnology,2018,102(7):3399-3410.
[13]丁传雨,郑远,任学敏,等.能源植物修复土壤镉污染过程中细菌群落分析[J].环境科学学报,2016,36(8):3009-3016.Ding C Y,Zheng Y,Ren X M,et al.Changes in bacterial community composition during the remediation of Cdcontaminated soils of bioenergy crops[J].Acta Scientiae Circumstantiae,2016,36(8):3009-3016.
[14]Li Z,Lu L H,Guo J S,et al.Responses of spatial-temporal dynamics of bacterioplankton community to large-scale reservoir operation:a case study in the Three Gorges Reservoir,China[J].Scientific Reports,2017,7(1):42469.
[15]Peter H,Sommaruga R.Shifts in diversity and function of lake bacterial communities upon glacier retreat[J].The ISMEJournal,2016,10(7):1545-1554.
[16]Chen Z J,Zheng Y,Ding C Y,et al.Integrated metagenomics and molecular ecological network analysis of bacterial community composition during the phytoremediation of cadmiumcontaminated soils by bioenergy crops[J].Ecotoxicology and Environmental Safety,2017,145:111-118.
[17]Chen Z J,Yuan J,Sun F,et al.Planktonic fungal community structures and their relationship to water quality in the Danjiangkou Reservoir,China[J].Scientific Reports,2018,8(1):10596.
[18]Langille M G I,Zaneveld J,Caporaso J G,et al.Predictive functional profiling of microbial communities using 16S rRNAmarker gene sequences[J].Nature Biotechnology,2013,31(9):814-821.
[19]董志颖,洪慢,胡晗静,等.过量氮输入对寡营养海水细菌群落代谢潜力的影响[J].环境科学学报,2018,38(2):457-466.Dong Z Y,Hong M,Hu H J,et al.Effect of excess nitrogen loading on the metabolic potential of the bacterial community in oligotrophic coastal water[J].Acta Scientiae Circumstantiae,2018,38(2):457-466.
[20]Bowman J S,Ducklow H W.Microbial communities can be described by metabolic structure:a general framework and application to a seasonally variable,depth-stratified microbial community from the coastal West Antarctic Peninsula[J].PLo SOne,2015,10(8):e0135868.
[21]Zhang Y,Li J,Cheng X H,et al.Community differentiation of bacterioplankton in the epipelagic layer in the South China Sea[J].Ecology and Evolution,2018,8(10):4932-4948.
[22]Ward C S,Yung C M,Davis K M,et al.Annual community patterns are driven by seasonal switching between closely related marine bacteria[J].The ISME Journal,2017,11(11):2637.
[23]Xiong J B,Xiong S L,Qian P,et al.Thermal discharge-created increasing temperatures alter the bacterioplankton composition and functional redundancy[J].AMB Express,2016,6(1):68.
[24]阴星望,田伟,丁一,等.丹江口库区表层沉积物细菌多样性及功能预测分析[J].湖泊科学,2018,30(4):1052-1063.Yin X W,Tian W,Ding Y,et al.Composition and predictive functional analysis of bacterial communities in surface sediments of the Danjiangkou Reservoir[J].Journal of Lake Sciences,2018,30(4):1052-1063.
[25]孙峰,田伟,张菲,等.丹江口库区库滨带植被土壤细菌群落多样性及PICRUSt功能预测分析[J].环境科学,2019,40(1):421-429.Sun F,Tian W,Zhang F,et al.Composition and predictive functional analysis of rhizosphere bacterial communities in riparian buffer strips in the Danjiangkou Reservoir,China[J].Environmental Science,2019,40(1):421-429.
[26]国家环境保护总局.水和废水监测分析方法[M].(第四版).北京:中国环境科学出版社,2002.
[27]Caporaso J G,Kuczynski J,Stombaugh J,et al.QIIME allows analysis of high-throughput community sequencing data[J].Nature Methods,2010,7(5):335-336.
[28]Quast C,Pruesse E,Yilmaz P,et al.The SILVA ribosomal RNA gene database project:improved data processing and webbased tools[J].Nucleic Acids Research,2013,41(D1):D590-D596.
[29]Schloss P D,Westcott S L,Ryabin T,et al.Introducing mothur:open-source,platform-independent,communitysupported software for describing and comparing microbial communities[J].Applied and Environmental Microbiology,2009,75(23):7537-7541.
[30]Parks D H,Tyson G W,Hugenholtz P,et al.STAMP:statistical analysis of taxonomic and functional profiles[J].Bioinformatics,2014,30(21):3123-3124.
[31]Le Brun E,Kang S.A comparison of computationally predicted functional metagenomes and microarray analysis for microbial Pcycle genes in a unique basalt-soil forest[J].F1000 Research,2018,7:179.
[32]王朱珺,王尚,刘洋荧,等.宏基因组技术在氮循环功能微生物分子检测研究中的应用[J].生物技术通报,2018,34(1):1-14.Wang Z J,Wang S,Liu Y Y,et al.The applications of metagenomics in the detection of environmental microbes involving in nitrogen cycle[J].Biotechnology Bulletin,2018,34(1):1-14.
[33]Thajudeen J,Yousuf J,Veetil V P,et al.Nitrogen fixing bacterial diversity in a tropical estuarine sediments[J].World Journal of Microbiology and Biotechnology,2017,33(2):41.
[34]Watanabe K,Komatsu N,Ishii Y,et al.Effective isolation of bacterioplankton genus Polynucleobacter from freshwater environments grown on photochemically degraded dissolved organic matter[J].FEMS Microbiology Ecology,2009,67(1):57-68.
[35]Horn M A,Ihssen J,Matthies C,et al.Dechloromonas denitrificans sp.nov.,Flavobacterium denitrificans sp.nov.,Paenibacillus anaericanus sp.nov.and Paenibacillus terrae strain MH72,N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa[J].International Journal of Systematic and Evolutionary Microbiology,2005,55:1255-1265.
[36]Jetten M S M.The microbial nitrogen cycle[J].Environmental Microbiology,2008,10(11):2903-2909.
[37]Orsi W D,Smith J M,Liu S T,et al.Diverse,uncultivated bacteria and archaea underlying the cycling of dissolved protein in the ocean[J].The ISME Journal,2016,10(9):2158-2173.
[38]Zeng Y H,Kasalicky V,Simek K,et al.Genome sequences of two freshwater betaproteobacterial isolates,Limnohabitans species strains Rim28 and Rim47,indicate their capabilities as both photoautotrophs and ammonia oxidizers[J].Journal of Bacteriology,2012,194(22):6302-6303.
[2]Newton R J,Jones S E,Eiler A,et al.A guide to the natural history of freshwater lake bacteria[J].Microbiology and Molecular Biology Reviews,2011,75(1):14-49.
[3]任丽娟,何聃,邢鹏,等.湖泊水体细菌多样性及其生态功能研究进展[J].生物多样性,2013,21(4):421-432.Ren L J,He D,Xing P,et al.Bacterial diversity and ecological function in lake water bodies[J].Biodiversity Science,2013,21(4):421-432.
[4]Chen P,Li L,Zhang H B.Spatio-temporal variations and source apportionment of water pollution in Danjiangkou Reservoir Basin,central China[J].Water,2015,7(6):2591-2611.
[5]陈兆进,丁传雨,朱静亚,等.丹江口水库枯水期浮游细菌群落组成及影响因素研究[J].中国环境科学,2017,37(1):336-344.Chen Z J,Ding C Y,Zhu J Y,et al.Community structure and influencing factors of bacterioplankton during low water periods in Danjiangkou Reservoir[J].China Environmental Science,2017,37(1):336-344.
[6]Kuypers M M M,Marchant H K,Kartal B.The microbial nitrogen-cycling network[J].Nature Reviews Microbiology,2018,16(5):263-276.
[7]杨雪琴,连英丽,颜庆云,等.滨海湿地生态系统微生物驱动的氮循环研究进展[J].微生物学报,2018,58(4):633-648.Yang X Q,Lian Y L,Yan Q Y,et al.Microbially-driven nitrogen cycling in coastal ecosystems[J].Acta Microbiologica Sinica,2018,58(4):633-648.
[8]van Dijk E L,Auger H,Jaszczyszyn Y,et al.Ten years of nextgeneration sequencing technology[J].Trends in Genetics,2014,30(9):418-426.
[9]Gao W L,Chen Z J,Li Y Y,et al.Bioassessment of a drinking water reservoir using plankton:high throughput sequencing vs.traditional morphological method[J].Water,2018,10(1):82.
[10]陈兆进,陈海燕,李玉英,等.南水北调中线干渠(河南段)浮游细菌群落组成及影响因素[J].中国环境科学,2017,37(4):1505-1513.Chen Z J,Chen H Y,Li Y Y,et al.Community structure and influencing factors of bacterioplankton in the Main cancel of the Mid-line Project of South-to-North Water Division in sections of Henan Province[J].China Environmental Science,2017,37(4):1505-1513.
[11]张明露,刘文君,李翠萍,等.南水北调中线水源水的细菌群落和致病菌分析[J].给水排水,2015,41(6):32-36.
[12]Dang C Y,Liu W,Lin Y X,et al.Dominant role of ammoniaoxidizing bacteria in nitrification due to ammonia accumulation in sediments of Danjiangkou reservoir,China[J].Applied Microbiology and Biotechnology,2018,102(7):3399-3410.
[13]丁传雨,郑远,任学敏,等.能源植物修复土壤镉污染过程中细菌群落分析[J].环境科学学报,2016,36(8):3009-3016.Ding C Y,Zheng Y,Ren X M,et al.Changes in bacterial community composition during the remediation of Cdcontaminated soils of bioenergy crops[J].Acta Scientiae Circumstantiae,2016,36(8):3009-3016.
[14]Li Z,Lu L H,Guo J S,et al.Responses of spatial-temporal dynamics of bacterioplankton community to large-scale reservoir operation:a case study in the Three Gorges Reservoir,China[J].Scientific Reports,2017,7(1):42469.
[15]Peter H,Sommaruga R.Shifts in diversity and function of lake bacterial communities upon glacier retreat[J].The ISMEJournal,2016,10(7):1545-1554.
[16]Chen Z J,Zheng Y,Ding C Y,et al.Integrated metagenomics and molecular ecological network analysis of bacterial community composition during the phytoremediation of cadmiumcontaminated soils by bioenergy crops[J].Ecotoxicology and Environmental Safety,2017,145:111-118.
[17]Chen Z J,Yuan J,Sun F,et al.Planktonic fungal community structures and their relationship to water quality in the Danjiangkou Reservoir,China[J].Scientific Reports,2018,8(1):10596.
[18]Langille M G I,Zaneveld J,Caporaso J G,et al.Predictive functional profiling of microbial communities using 16S rRNAmarker gene sequences[J].Nature Biotechnology,2013,31(9):814-821.
[19]董志颖,洪慢,胡晗静,等.过量氮输入对寡营养海水细菌群落代谢潜力的影响[J].环境科学学报,2018,38(2):457-466.Dong Z Y,Hong M,Hu H J,et al.Effect of excess nitrogen loading on the metabolic potential of the bacterial community in oligotrophic coastal water[J].Acta Scientiae Circumstantiae,2018,38(2):457-466.
[20]Bowman J S,Ducklow H W.Microbial communities can be described by metabolic structure:a general framework and application to a seasonally variable,depth-stratified microbial community from the coastal West Antarctic Peninsula[J].PLo SOne,2015,10(8):e0135868.
[21]Zhang Y,Li J,Cheng X H,et al.Community differentiation of bacterioplankton in the epipelagic layer in the South China Sea[J].Ecology and Evolution,2018,8(10):4932-4948.
[22]Ward C S,Yung C M,Davis K M,et al.Annual community patterns are driven by seasonal switching between closely related marine bacteria[J].The ISME Journal,2017,11(11):2637.
[23]Xiong J B,Xiong S L,Qian P,et al.Thermal discharge-created increasing temperatures alter the bacterioplankton composition and functional redundancy[J].AMB Express,2016,6(1):68.
[24]阴星望,田伟,丁一,等.丹江口库区表层沉积物细菌多样性及功能预测分析[J].湖泊科学,2018,30(4):1052-1063.Yin X W,Tian W,Ding Y,et al.Composition and predictive functional analysis of bacterial communities in surface sediments of the Danjiangkou Reservoir[J].Journal of Lake Sciences,2018,30(4):1052-1063.
[25]孙峰,田伟,张菲,等.丹江口库区库滨带植被土壤细菌群落多样性及PICRUSt功能预测分析[J].环境科学,2019,40(1):421-429.Sun F,Tian W,Zhang F,et al.Composition and predictive functional analysis of rhizosphere bacterial communities in riparian buffer strips in the Danjiangkou Reservoir,China[J].Environmental Science,2019,40(1):421-429.
[26]国家环境保护总局.水和废水监测分析方法[M].(第四版).北京:中国环境科学出版社,2002.
[27]Caporaso J G,Kuczynski J,Stombaugh J,et al.QIIME allows analysis of high-throughput community sequencing data[J].Nature Methods,2010,7(5):335-336.
[28]Quast C,Pruesse E,Yilmaz P,et al.The SILVA ribosomal RNA gene database project:improved data processing and webbased tools[J].Nucleic Acids Research,2013,41(D1):D590-D596.
[29]Schloss P D,Westcott S L,Ryabin T,et al.Introducing mothur:open-source,platform-independent,communitysupported software for describing and comparing microbial communities[J].Applied and Environmental Microbiology,2009,75(23):7537-7541.
[30]Parks D H,Tyson G W,Hugenholtz P,et al.STAMP:statistical analysis of taxonomic and functional profiles[J].Bioinformatics,2014,30(21):3123-3124.
[31]Le Brun E,Kang S.A comparison of computationally predicted functional metagenomes and microarray analysis for microbial Pcycle genes in a unique basalt-soil forest[J].F1000 Research,2018,7:179.
[32]王朱珺,王尚,刘洋荧,等.宏基因组技术在氮循环功能微生物分子检测研究中的应用[J].生物技术通报,2018,34(1):1-14.Wang Z J,Wang S,Liu Y Y,et al.The applications of metagenomics in the detection of environmental microbes involving in nitrogen cycle[J].Biotechnology Bulletin,2018,34(1):1-14.
[33]Thajudeen J,Yousuf J,Veetil V P,et al.Nitrogen fixing bacterial diversity in a tropical estuarine sediments[J].World Journal of Microbiology and Biotechnology,2017,33(2):41.
[34]Watanabe K,Komatsu N,Ishii Y,et al.Effective isolation of bacterioplankton genus Polynucleobacter from freshwater environments grown on photochemically degraded dissolved organic matter[J].FEMS Microbiology Ecology,2009,67(1):57-68.
[35]Horn M A,Ihssen J,Matthies C,et al.Dechloromonas denitrificans sp.nov.,Flavobacterium denitrificans sp.nov.,Paenibacillus anaericanus sp.nov.and Paenibacillus terrae strain MH72,N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa[J].International Journal of Systematic and Evolutionary Microbiology,2005,55:1255-1265.
[36]Jetten M S M.The microbial nitrogen cycle[J].Environmental Microbiology,2008,10(11):2903-2909.
[37]Orsi W D,Smith J M,Liu S T,et al.Diverse,uncultivated bacteria and archaea underlying the cycling of dissolved protein in the ocean[J].The ISME Journal,2016,10(9):2158-2173.
[38]Zeng Y H,Kasalicky V,Simek K,et al.Genome sequences of two freshwater betaproteobacterial isolates,Limnohabitans species strains Rim28 and Rim47,indicate their capabilities as both photoautotrophs and ammonia oxidizers[J].Journal of Bacteriology,2012,194(22):6302-6303.