基于好氧甲烷氧化菌的反硝化效能及微生物群落研究
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摘要
采用气体循环序批式生物膜反应器(gcSBBR),构建反硝化型甲烷好氧氧化(AME-D)系统.考察了进水氮负荷的影响,发现氮负荷为0.075kg/(m3·d)时,硝酸盐氮去除率达到98.93%,其反硝化速率为74.25mg/(L·d),系统的甲烷日平均消耗量为35.91%(初期为50%);扫描电子显微镜(SEM)分析结果显示,系统中的微生物主要以短杆菌(12～18μm)为主,并存在少量的丝状菌(长150～200μm);16SrRNA高通量测序结果显示,该系统中的甲烷氧化菌为Methylocaldum、Methylomonas、Methylococcus和Methylococcaceae_unclassified,反硝化菌为Denitratisoma、Hydrogenophaga、Azoarcus、Thiobacillus和Rhodobacter,其中主要的功能微生物为Methylocaldum、Denitratisoma和Hydrogenophaga,系统对氮的去除是由好氧甲烷氧化菌与反硝化菌协同实现.此外,系统中存在大量以甲醇和甲基胺类物质为生长基质的Methylophilaceae_uncultured(30.4%).
An aerobic methane oxidation coupled to denitrification(AME-D) was constructed using a gas circulation sequencing batch biofilm reactor(gcSBBR), and nitrogen loading in influent was observed. It was found that the removal rate of nitrate nitrogen reached 98.93% and the denitrification rate 74.25 mg/(L·d) when the nitrogen load was 0.075 kg/(m~3·d), and the average daily consumption of methane was 35.91%(at the initial stage: 50%). According to scanning electron microscopy(SEM) results, microorganisms in the system were mainly Bacillus brevis(12～18μm), dotted with a few filamentous bacteria(150～200μm). The high-throughput sequencing of 16SrRNA indicated that methanotroph were Methylocaldum, Methylomonas, Methylococcus and Methylococcaceae_unclassified, the denitrifying bacteria Denittrasoma, Hydrogenophaga, Azoarcus, Thiobacillus and Rhodobacter, and the main functional bacteria Methylocaldum, Denittrasoma and Hydrogenophaga in this system. Nitrogen was removed by the synergism of aerobic methanotroph and denitrifying bacteria. In addition, a large volume of Methylophilaceae_uncultured(30.4%), which utilized methanol and methylamine as growth substrates, was found.
An aerobic methane oxidation coupled to denitrification(AME-D) was constructed using a gas circulation sequencing batch biofilm reactor(gcSBBR), and nitrogen loading in influent was observed. It was found that the removal rate of nitrate nitrogen reached 98.93% and the denitrification rate 74.25 mg/(L·d) when the nitrogen load was 0.075 kg/(m~3·d), and the average daily consumption of methane was 35.91%(at the initial stage: 50%). According to scanning electron microscopy(SEM) results, microorganisms in the system were mainly Bacillus brevis(12～18μm), dotted with a few filamentous bacteria(150～200μm). The high-throughput sequencing of 16SrRNA indicated that methanotroph were Methylocaldum, Methylomonas, Methylococcus and Methylococcaceae_unclassified, the denitrifying bacteria Denittrasoma, Hydrogenophaga, Azoarcus, Thiobacillus and Rhodobacter, and the main functional bacteria Methylocaldum, Denittrasoma and Hydrogenophaga in this system. Nitrogen was removed by the synergism of aerobic methanotroph and denitrifying bacteria. In addition, a large volume of Methylophilaceae_uncultured(30.4%), which utilized methanol and methylamine as growth substrates, was found.
引文
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[32]Kalyuhznaya M G,Martens-Habbena W,Wang T,et al.Methylophilaceae link methanol oxidation to denitrification in freshwater lake sediment as suggested by stable isotope probing and pure culture analysis[J].Environmental Microbiology Reports,2009,1(5):385-392.
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[34]Newton R J,Kent A D,Triplett E W,et al.Microbial community dynamics in a humic lake:differential persistence of common freshwater phylotypes[J].Environmental Microbiology,2006,8(6):956-970.
[35]李振灵,丁彦礼,白少元,等.潜流人工湿地基质结构与微生物群落特征的相关性[J].环境科学,2017,(9):3713-3720.Li Z L,Ding Y L,Bai S Y,et al.Correlations between substrate structure and microbial community in subsurface flow constructed wetlands[J].Environmental Science,2017,(9):3713-3720.
[36]Fahrbach M.Denitratisoma oestradiolicum gen.nov.,sp.nov.,a 17beta-oestradiol-degrading,denitrifying betaproteobacterium[J].International Journal of Systematic and Evolutionary Microbiology,2006,56(7):1547-1552.
[37]J?rgensen K S,Pauli A S.Polyphosphate accumulation among denitrifying bacteria in activated sludge[J].Anaerobe,1995,1(3):161-168.
[38]Moreno B,Gómez M A,González-López J,et al.Inoculation of a submerged filter for biological denitrification of nitrate polluted groundwater:a comparative study[J].Journal of Hazardous Materials,2005,117(2/3):141-147.
[39]Mansell B O,Schroeder E D.Hydrogenotrophic denitrification in a microporous membrane bioreactor[J].Water Research,2002,36(19):4683-4690.
[2]Raghoebarsing A A,Smolders A J P,Schmid M C,et al.Methanotrophic symbionts provide carbon for photosynthesis in peat bogs[J].Nature,2005,436(7054):1153-1156.
[3]Han B,Chen Y,Abell G,et al.Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine[J].FEMSMicrobiology Ecology,2009,70(2):40-51.
[4]Ho A,Vlaeminck S E,Ettwig K F,et al.Revisiting methanotrophic communities in sewage treatment plants[J].Applied and Environmental Microbiology,2013,79(8):2841-2846.
[5]Belkhelfa S,Labadie K,Cruaud C,et al.Complete genome sequence of the facultative methylotroph methylobacterium extorquens TK0001isolated from soil in poland[J].Genome Announcements,2018,6(8).
[6]刘洋,陈永娟,王晓燕,等.水库与河流沉积物中好氧甲烷氧化菌群落差异性研究[J].中国环境科学,2018,(5):1844-1854.Liu Y,Chen Y J,Wang X Y,et al.Microbial communities differences between aerobic methanotrophs in Miyun Reservoir and North Canal[J].China Environmental Science,2018,(5):1844-1854.
[7]Ruff S E,Felden J,Gruber-Vodicka H R,et al.In situ development of a methanotrophic microbiome in deep-sea sediments[J].The ISMEJournal,2019,13(1):197-213.
[8]Zhu J,Wang Q,Yuan M,et al.Microbiology and potential applications of aerobic methane oxidation coupled to denitrification(AME-D)process:A review[J].Water Research,2016,90:203-215.
[9]Haroon M F,Hu S,Shi Y,et al.Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage[J].Nature,2013,501(7468).
[10]薛松,张梦竹,李琳,等.甲烷厌氧氧化协同硝酸盐还原菌群驯化及其群落特征[J].环境科学,2018,39(3):1357-1364.Xu S,Zhang M Z,Li L,et al.Acclimatization and community structure analysis of the microbial consortium in nitrate-dependent anaerobic methane oxidation[J].Environmental Science,2018,39(3):1357-1364.
[11]Modin O,Fukushi K,Nakajima F,et al.Nitrate removal and biofilm characteristics in methanotrophic membrane biofilm reactors with various gas supply regimes[J].Water Research,2010,44(1):85-96.
[12]Stein L Y,Klotz M G.Nitrifying and denitrifying pathways of methanotrophic bacteria[J].Biochemical Society Transactions,2011,39(6):1826-1831.
[13]江皓.甲烷氧化混合菌群的富集培养及其治理瓦斯工艺研究[D].北京:清华大学,2010.Jiang H.Enrichment culture of mixed Methanotrophic community and its application in coal mine gas control[D].Beijing:Tsinghua University,2010.
[14]国家环境保护总局.水和废水监测分析方法[M].第4版.中国环境科学出版社,2002.State Environmental Protection Administration.Water and wastewater monitoring and analysis methods[M].4th Ed.China Environmental Science Press,2002.
[15]Sheets J P,Ge X,Li Y.Effect of limited air exposure and comparative performance between thermophilic and mesophilic solid-state anaerobic digestion of switchgrass[J].Bioresource Technology,2015,180:296-303.
[16]Pfluger A R,Wu W,Pieja A J,et al.Selection of Type I and Type IImethanotrophic proteobacteria in a fluidized bed reactor under non-sterile conditions[J].Bioresource Technology,2011,102(21):9919-9926.
[17]Sun F,Dong W,Shao M,et al.Aerobic methane oxidation coupled to denitrification in a membrane biofilm reactor:Treatment performance and the effect of oxygen ventilation[J].Bioresource Technology,2013,145:2-9.
[18]Kampman C,Temmink H,Hendrickx T L G,et al.Enrichment of denitrifying methanotrophic bacteria from municipal wastewater sludge in a membrane bioreactor at 20°C[J].Journal of Hazardous Materials,2014,274:428-435.
[19]周祥玉,赵云飞,李东,等.微氧条件下以甲烷为碳源的反硝化实验研究[J].环境科学学报,2017,(5):1704-1710.Zhou X Y,Zhao Y F,Li D,et al.Experimental study on denitrification using methane as carbon source under microaerobic condition[J].Acta Scientiae Circumstantiae,2017,(5):1704-1710.
[20]卢培利,唐荧霜,丁阿强,等.亚硝酸盐型厌氧甲烷氧化过程强化新视角:排泥及其微生物机制研究[J].环境科学学报,2019:1-9.Lu P L,Tang Y S,Ding A Q,et al.New insight into the enhancement of nitrite-dependent anaerobic methane oxidation:withdrawing sludge and its microbial mechanism[J].Acta Scientiae Circumstantiae,2019:1-9.
[21]Semrau J D,Dispirito A A,Yoon S.Methanotrophs and copper[J].FEMS Microbiology Reviews,2010,34(4):496-531.
[22]Stoecker K,Bendinger B,Schoning B,et al.Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase[J].Proceedings of The National Academy of Sciences of the United States of America,2006,103(7):2363-2367.
[23]Sheng G,Yu H,Li X.Extracellular polymeric substances(EPS)of microbial aggregates in biological wastewater treatment systems:Areview[J].Biotechnology Advances,2010,28(6):882-894.
[24]Manz W,Wagner M,Amann R,et al.In-situ characterization of the microbial consortia active in 2 waste-water treatment plants[J].Water Research,1994,28(8):1715-1723.
[25]Bowman J P.The Family Methylococcaceae[J].The Prokaryotes:Gammaproteobacteria,2014:411-440.
[26]Kalyuzhnaya M G,Puri A W,Lidstrom M E.Metabolic engineering in methanotrophic bacteria[J].Metabolic Engineering,2015,29:142-152.
[27]Bowman J.The Methanotrophs-The Families Methylococcaceae and Methylocystaceae[M]//The Prokaryotes:Volume 5:Proteobacteria:Alpha and Beta Subclasses,New York,NY:Springer New York,2007:266-289.
[28]Doronina N,Kaparullina E,Trotsenko Y.The family methylophilaceae[J].The Prokaryotes:Alphaproteobacteria and Betaproteobacteria,2014:869-880.
[29]Beck D A C,Mctaggart T L,Setboonsarng U,et al.The expanded diversity of methylophilaceae from Lake Washington through cultivation and genomic sequencing of novel ecotypes[J].PLOS ONE,2014,9(e1024587).
[30]Xia F,Zou B,Shen C,et al.Complete genome sequence of Methylophilus sp TWE2 isolated from methane oxidation enrichment culture of tap-water[J].Journal of Biotechnology,2015,211:121-122.
[31]Beck D A C,Kalyuzhnaya M G,Malfatti S,et al.A metagenomic insight into freshwater methane-utilizing communities and evidence for cooperation between the Methylococcaceae and the Methylophilaceae[J].PEERJ,2013,1(e23).
[32]Kalyuhznaya M G,Martens-Habbena W,Wang T,et al.Methylophilaceae link methanol oxidation to denitrification in freshwater lake sediment as suggested by stable isotope probing and pure culture analysis[J].Environmental Microbiology Reports,2009,1(5):385-392.
[33]Yang Y,Li S,Gao Y,et al.Environment-driven geographical distribution of bacterial communities and identification of indicator taxa in Songhua River[J].Ecological Indicators,2019,101:62-70.
[34]Newton R J,Kent A D,Triplett E W,et al.Microbial community dynamics in a humic lake:differential persistence of common freshwater phylotypes[J].Environmental Microbiology,2006,8(6):956-970.
[35]李振灵,丁彦礼,白少元,等.潜流人工湿地基质结构与微生物群落特征的相关性[J].环境科学,2017,(9):3713-3720.Li Z L,Ding Y L,Bai S Y,et al.Correlations between substrate structure and microbial community in subsurface flow constructed wetlands[J].Environmental Science,2017,(9):3713-3720.
[36]Fahrbach M.Denitratisoma oestradiolicum gen.nov.,sp.nov.,a 17beta-oestradiol-degrading,denitrifying betaproteobacterium[J].International Journal of Systematic and Evolutionary Microbiology,2006,56(7):1547-1552.
[37]J?rgensen K S,Pauli A S.Polyphosphate accumulation among denitrifying bacteria in activated sludge[J].Anaerobe,1995,1(3):161-168.
[38]Moreno B,Gómez M A,González-López J,et al.Inoculation of a submerged filter for biological denitrification of nitrate polluted groundwater:a comparative study[J].Journal of Hazardous Materials,2005,117(2/3):141-147.
[39]Mansell B O,Schroeder E D.Hydrogenotrophic denitrification in a microporous membrane bioreactor[J].Water Research,2002,36(19):4683-4690.
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