利用PCR-DGGE分析有机物对厌氧氨氧化菌群的影响
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摘要
为了研究有机物对Anammox菌群的影响,以及微生物与脱氮的关系,为工艺改进提供依据。使用SBR厌氧氨氧化反应器,从反应器不同TOC/NH_4~+-N阶段采集活性污泥样品,利用聚合酶链式反应—变性梯度凝胶电泳(PCR-DGGE)技术,分析了样品中微生物种群结构。结果表明反应器中主要微生物包含变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、厚壁菌门(Firmicutes)和绿菌门(Chlorobi);其中变形菌门(β-变形菌和γ-变形菌)为优势菌群。TOC/NH_4~+-N从0逐渐增加至2.0的过程中,反应器中的反硝化菌(变形菌门)不断增长,Anammox菌群在TOC/NH_4~+-N为0.4阶段得到最大程度的富集,此时反应器内部微生物多样性也最高;随着有机物含量增加,Anammox菌生长受到严重抑制,反应器微生物物种多样性也逐渐下降。荧光定量(qPCR)分析表明Anammox菌含量从1.30×10~(11) copies/mL下降至3.18×10~9 copies/mL,而DB含量从1.57×10~9copies/mL增加至3.74×10~(10) copies/mL。说明随着C/N的增加,反应器脱氮能力逐渐从Anammox过渡到反硝化过程。通过测定反应器内壁附着污泥,还发现其微生物丰度和含量均高于同时期反应器内部活性污泥样品,推测厌氧微生物菌群更适宜在静态基质生长。
The purpose of the research was to study the effect of organics on anammox microbial community and the relationship between microbes and denitrification in order to provide basis for technique improvement. Activated sludge samples were collected from different TOC/NH_4~+-N stages in anammox sequencing batch reactor(SBR) and the microbial population structures were analyzed by polymerase chain reaction-denature gradient gel electrophoresis(PCR-DGGE) technology. The results indicated that the main microorganisms in the reactor included Proteobacteria,Planctomycetes, Firmicutes and Chlorobi. Among them, Proteobacteria(β-proteobacteria and γ-proteobacteria)was the dominant flora. With the increase of TOC/NH_4~+-N from 0 to 2.0, the denitrifying bacteria(Proteobacteria)proliferated continuously while the anammox flora reached its maximum concentration at 0.4 TOC/NH_4~+-N stage in which the reactor had the greatest diversity. With the increase of organic content, the growth of anammox bacteria was severely suppressed and the microbial species diversity of the reactor declined gradually. q PCR analysis showed that the content of anammox bacteria declined from 1.30×10~(11)copies/mL to 3.18×10~9copies/m L while the content of the DB increased from 1.57×10~9copies/m L to 3.74×10~(10)copies/m L. This indicated that the denitrification capacity of the SBR reactor gradually transited from anammox to denitrification with the increase of C/N. By determining the attached sludge in the inner wall of the reactor, it was also found that its microbial abundance and content were higher than those in the reactor-inside activated samples inside the reactor of the same period. It was inferred that the anaerobic microbial flora was more suitable for growth in the static substrate.
The purpose of the research was to study the effect of organics on anammox microbial community and the relationship between microbes and denitrification in order to provide basis for technique improvement. Activated sludge samples were collected from different TOC/NH_4~+-N stages in anammox sequencing batch reactor(SBR) and the microbial population structures were analyzed by polymerase chain reaction-denature gradient gel electrophoresis(PCR-DGGE) technology. The results indicated that the main microorganisms in the reactor included Proteobacteria,Planctomycetes, Firmicutes and Chlorobi. Among them, Proteobacteria(β-proteobacteria and γ-proteobacteria)was the dominant flora. With the increase of TOC/NH_4~+-N from 0 to 2.0, the denitrifying bacteria(Proteobacteria)proliferated continuously while the anammox flora reached its maximum concentration at 0.4 TOC/NH_4~+-N stage in which the reactor had the greatest diversity. With the increase of organic content, the growth of anammox bacteria was severely suppressed and the microbial species diversity of the reactor declined gradually. q PCR analysis showed that the content of anammox bacteria declined from 1.30×10~(11)copies/mL to 3.18×10~9copies/m L while the content of the DB increased from 1.57×10~9copies/m L to 3.74×10~(10)copies/m L. This indicated that the denitrification capacity of the SBR reactor gradually transited from anammox to denitrification with the increase of C/N. By determining the attached sludge in the inner wall of the reactor, it was also found that its microbial abundance and content were higher than those in the reactor-inside activated samples inside the reactor of the same period. It was inferred that the anaerobic microbial flora was more suitable for growth in the static substrate.
引文
Ali M.,Chai L.Y.,Tang C.J.,Zheng P.,Min X.B.,Yang Z.H.,Xiong L.,and Song Y.X.,2013,The increasing interest of anammox research in China:bacteria,process development,and application,Biomed Research International,(6):134914
Bi W.,Li X.,Liu H.W.,Huang Y.,Yuan Y.,and Liu X.,2016,Optimization of PCR-DGGE analysis method for anammox function Microbial,Jiangsu Nongye Kexue(Jiangsu Agricultural Sciences),44(10):110-112(毕玮,李祥,刘恒蔚,黄勇,袁怡,刘忻,2016,厌氧氨氧化功能微生物PCR-DGGE分析方法优化,江苏农业科学,44(10):110-112)
Ding S.,Zheng P.,Lu H.,Chen J.,Mahmood Q.,and Abbas G.,2013,Ecological characteristics of anaerobic ammonia oxidizing bacteria,Applied Microbiology and Biotechnology,97(5):1841-1849
Gao D.W.,Hou G.F.,Tao Y.,and Ren N.Q.,2012,Review of the studies on metabolizing organic matter by Anammox bacteria,Harbin Gongye Daxue Xuebao(Journal of Harbin Institute of Technology),44(2):89-93(高大文,侯国凤,陶彧,任南琪,2012,厌氧氨氧化菌代谢有机物研究,哈尔滨工业大学学报,44(2):89-93)
Kartal B.,Rattray J.,van Niftrik L.A.,Van d.V.J.,Schmid M.C.,Webb R.I.,Webb R.I.,Schouten S.,Fuerst J.A.,DamstéJ.S.,Jetten M.S.M.,and Strous M.,2007,Candidatus“anammoxoglobus propionicus”a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria,Systematic and Applied Microbiology,30(1):39-49
Kowalchuk G.A.,Stephen J.R.,De B.W.,Prosser J.I.,Embley T.M.,and Woldendorp J.W.,1997,Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments,Applied and Environmental Microbiology,63(4):1489-1497
Li X.R.,Du B.,Fu H.X.,Wang R.F.,Shi J.H.,Wang Y.,Jetten M.S.M.,and Quan Z.X.,2009,The bacterial diversity in an anaerobic ammonium-oxidizing(anammox)reactor community,Systematic and Applied Microbiology,32(4):278-289
Liu H.J.,Yang F.L.,Zhang H.M.,Hu S.W.,and Sun C.,2007,Nitrification performance and microbial community analysis in carbon membrane-aerated biofilm reactor,Huanjing Kexue(Environmental Science),28(9):2123-2128(刘惠军,杨凤林,张捍民,胡绍伟,孙翠,2007,炭膜曝气生物膜反应器硝化作用及其微生物群落结构分析,环境科学,28(9):2123-2128)
Lópezgutiérrez J.C.,Henry S.,Hallet S.,Martinlaurent F.,Catroux G.,and Philippot L.,2004,Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR,Journal of Microbiological Methods,57(3):399-407
Ma F.N.,Guo G.,Yin X.M.,Guo X.Q.,Li Y.X.,and Zeng H.C.,2016,Identification of antifungi component of streptomyces ahygroscopicus against Fusarium oxysporum f.sp.cubense Race 4,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(11):3190-3194(马伏宁,郭刚,殷晓敏,郭雪琴,李奕星,曾会才,2016,一株香蕉枯萎病拮抗链霉菌抑菌成分初步分析,分子植物育种,14(11):3190-3194)
Mao N.J.,Ren H.Q.,Geng J.J.,Ding L.L.,and Xu K.,2017,Engineering application of anaerobic ammonium oxidation process in wastewater treatment,World Journal of Microbiology and Biotechnology,33(8):153
Ni B.J.,Hu B.L.,Fang F.,Xie W.M.,Kartal B.,Liu X.W.,Sheng G.P.,Jetten M.,Zheng P.,and Yu H.Q.,2010,Microbial and physicochemical characteristics of compact anaerobic ammonium-oxidizing granules in an upflow anaerobic sludge blanket reactor,Applied and Environmental Microbi ology,76(8):2652-2656
Pathak B.K.,Kazama F.,Saiki Y.,and Sumino T.,2007,Presence and activity of anammox and denitrification process in low ammonium-fed bioreactors,Bioresource Technology,98(11):2201-2206
Wang F.,Liu K.,Lin X.,Zhou Z.,Li X.,and Huang Y.,2017,Effect of different TOC to NH4+-N ratios on nitrogen removal efficiency in the anammox process,Huanjing Kexue(Envi ronmental Science),38(8):3425-3421(王凡,刘凯,林兴,周正,李祥,黄勇,2017,不同TOC/NH4+-N对厌氧氨氧化脱氮效能的影响,环境科学,38(8):3415-3421)
Xie S.M.,Hua L.,Wei Y.X.,Xiang X.,Zhai W.X.,and Xia Z.H.,2017,A rapid laboratory method for isolation and identification of pathogen causing rice bacterial leaf streak,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(5):1927-1932(谢仕猛,花龙,韦永选,向贤,翟文学,夏志辉,2017,水稻细菌性条斑病原菌室内快速分离与鉴定方法,分子植物育种,15(5):1927-1932)
Yang G.F.,Jin R.C.,Yu Y.,and Li L.,2012,Inhibition phenomenon of anammox process,Yinyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),18(3):502-510(阳广凤,金仁村,余妤,李玲,2012,厌氧氨氧化工艺的抑制现象,应用与环境生物学报,18(3):502-510)
Yang Y.,Zuo J.W.,Shen P.,and Gu X.S.,2006,Influence of temperature,pH value and organic substance on activity of anammox sludge,Huanjing Kexue(Environmental Science),27(4):691-695(杨洋,左剑恶,沈平,顾夏声,2006,温度,pH值和有机物对厌氧氨氧化污泥活性的影响,环境科学,27(4):691-695)
Zheng P.,Wu M.S.,and Jin R.C.,2006,Effect of organic matter on performance of anammox reactor,Huanjing Kexue Xuebao(Acta Scientiae Circumstantiae),26(7):1087-1092(郑平,吴明生,金仁村,2006,有机物对ANAMMOX反应器运行性能的影响,环境科学学报,26(7):1087-1092)
Zhu J.P.,Hu Y.Y.,and Yan J.,2006,Main reactions in anaerobic ammonium oxidation reactor under organic carbon condition,Huanjing Kexue(Environmental Science),27(7):1353-1357(朱静平,胡勇有,闫佳,2006,有机碳源条件下厌氧氨氧化ASBR反应器中的主要反应,环境科学,27(7):1353-1357)
Zu B.,Zhang D.J.,Zhang P.,and Bai Y.H.,2007,Factors affecting the integration of methanogenesis with simultaneous anaerobic ammonium oxidation and denitrification,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),13(3):438-442(祖波,张代钧,张萍,白玉华,2007,影响厌氧氨氧化与甲烷化反硝化耦合的因素,应用与环境生物学报,13(3):438-442)
Bi W.,Li X.,Liu H.W.,Huang Y.,Yuan Y.,and Liu X.,2016,Optimization of PCR-DGGE analysis method for anammox function Microbial,Jiangsu Nongye Kexue(Jiangsu Agricultural Sciences),44(10):110-112(毕玮,李祥,刘恒蔚,黄勇,袁怡,刘忻,2016,厌氧氨氧化功能微生物PCR-DGGE分析方法优化,江苏农业科学,44(10):110-112)
Ding S.,Zheng P.,Lu H.,Chen J.,Mahmood Q.,and Abbas G.,2013,Ecological characteristics of anaerobic ammonia oxidizing bacteria,Applied Microbiology and Biotechnology,97(5):1841-1849
Gao D.W.,Hou G.F.,Tao Y.,and Ren N.Q.,2012,Review of the studies on metabolizing organic matter by Anammox bacteria,Harbin Gongye Daxue Xuebao(Journal of Harbin Institute of Technology),44(2):89-93(高大文,侯国凤,陶彧,任南琪,2012,厌氧氨氧化菌代谢有机物研究,哈尔滨工业大学学报,44(2):89-93)
Kartal B.,Rattray J.,van Niftrik L.A.,Van d.V.J.,Schmid M.C.,Webb R.I.,Webb R.I.,Schouten S.,Fuerst J.A.,DamstéJ.S.,Jetten M.S.M.,and Strous M.,2007,Candidatus“anammoxoglobus propionicus”a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria,Systematic and Applied Microbiology,30(1):39-49
Kowalchuk G.A.,Stephen J.R.,De B.W.,Prosser J.I.,Embley T.M.,and Woldendorp J.W.,1997,Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments,Applied and Environmental Microbiology,63(4):1489-1497
Li X.R.,Du B.,Fu H.X.,Wang R.F.,Shi J.H.,Wang Y.,Jetten M.S.M.,and Quan Z.X.,2009,The bacterial diversity in an anaerobic ammonium-oxidizing(anammox)reactor community,Systematic and Applied Microbiology,32(4):278-289
Liu H.J.,Yang F.L.,Zhang H.M.,Hu S.W.,and Sun C.,2007,Nitrification performance and microbial community analysis in carbon membrane-aerated biofilm reactor,Huanjing Kexue(Environmental Science),28(9):2123-2128(刘惠军,杨凤林,张捍民,胡绍伟,孙翠,2007,炭膜曝气生物膜反应器硝化作用及其微生物群落结构分析,环境科学,28(9):2123-2128)
Lópezgutiérrez J.C.,Henry S.,Hallet S.,Martinlaurent F.,Catroux G.,and Philippot L.,2004,Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR,Journal of Microbiological Methods,57(3):399-407
Ma F.N.,Guo G.,Yin X.M.,Guo X.Q.,Li Y.X.,and Zeng H.C.,2016,Identification of antifungi component of streptomyces ahygroscopicus against Fusarium oxysporum f.sp.cubense Race 4,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(11):3190-3194(马伏宁,郭刚,殷晓敏,郭雪琴,李奕星,曾会才,2016,一株香蕉枯萎病拮抗链霉菌抑菌成分初步分析,分子植物育种,14(11):3190-3194)
Mao N.J.,Ren H.Q.,Geng J.J.,Ding L.L.,and Xu K.,2017,Engineering application of anaerobic ammonium oxidation process in wastewater treatment,World Journal of Microbiology and Biotechnology,33(8):153
Ni B.J.,Hu B.L.,Fang F.,Xie W.M.,Kartal B.,Liu X.W.,Sheng G.P.,Jetten M.,Zheng P.,and Yu H.Q.,2010,Microbial and physicochemical characteristics of compact anaerobic ammonium-oxidizing granules in an upflow anaerobic sludge blanket reactor,Applied and Environmental Microbi ology,76(8):2652-2656
Pathak B.K.,Kazama F.,Saiki Y.,and Sumino T.,2007,Presence and activity of anammox and denitrification process in low ammonium-fed bioreactors,Bioresource Technology,98(11):2201-2206
Wang F.,Liu K.,Lin X.,Zhou Z.,Li X.,and Huang Y.,2017,Effect of different TOC to NH4+-N ratios on nitrogen removal efficiency in the anammox process,Huanjing Kexue(Envi ronmental Science),38(8):3425-3421(王凡,刘凯,林兴,周正,李祥,黄勇,2017,不同TOC/NH4+-N对厌氧氨氧化脱氮效能的影响,环境科学,38(8):3415-3421)
Xie S.M.,Hua L.,Wei Y.X.,Xiang X.,Zhai W.X.,and Xia Z.H.,2017,A rapid laboratory method for isolation and identification of pathogen causing rice bacterial leaf streak,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(5):1927-1932(谢仕猛,花龙,韦永选,向贤,翟文学,夏志辉,2017,水稻细菌性条斑病原菌室内快速分离与鉴定方法,分子植物育种,15(5):1927-1932)
Yang G.F.,Jin R.C.,Yu Y.,and Li L.,2012,Inhibition phenomenon of anammox process,Yinyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),18(3):502-510(阳广凤,金仁村,余妤,李玲,2012,厌氧氨氧化工艺的抑制现象,应用与环境生物学报,18(3):502-510)
Yang Y.,Zuo J.W.,Shen P.,and Gu X.S.,2006,Influence of temperature,pH value and organic substance on activity of anammox sludge,Huanjing Kexue(Environmental Science),27(4):691-695(杨洋,左剑恶,沈平,顾夏声,2006,温度,pH值和有机物对厌氧氨氧化污泥活性的影响,环境科学,27(4):691-695)
Zheng P.,Wu M.S.,and Jin R.C.,2006,Effect of organic matter on performance of anammox reactor,Huanjing Kexue Xuebao(Acta Scientiae Circumstantiae),26(7):1087-1092(郑平,吴明生,金仁村,2006,有机物对ANAMMOX反应器运行性能的影响,环境科学学报,26(7):1087-1092)
Zhu J.P.,Hu Y.Y.,and Yan J.,2006,Main reactions in anaerobic ammonium oxidation reactor under organic carbon condition,Huanjing Kexue(Environmental Science),27(7):1353-1357(朱静平,胡勇有,闫佳,2006,有机碳源条件下厌氧氨氧化ASBR反应器中的主要反应,环境科学,27(7):1353-1357)
Zu B.,Zhang D.J.,Zhang P.,and Bai Y.H.,2007,Factors affecting the integration of methanogenesis with simultaneous anaerobic ammonium oxidation and denitrification,Yingyong Yu Huanjing Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),13(3):438-442(祖波,张代钧,张萍,白玉华,2007,影响厌氧氨氧化与甲烷化反硝化耦合的因素,应用与环境生物学报,13(3):438-442)
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