发酵温度对青海农用沼气池微生物群落的影响
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摘要
为了研究发酵温度对青海农用沼气池微生物群落结构的影响,以沼气池全年6个温度时期的24个泥样为研究对象,采用变性梯度凝胶电泳分析样品中细菌与古菌的群落结构及其变化差异.结果显示:发酵温度的波动变化对微生物群落结构、多样性及沼气池产气量影响显著.细菌在温度最高(九月中旬)和最低(三月中旬)的2个采样时期多样性最高,古菌仅在温度最高时期多样性最高.在细菌类群中,厚壁菌门Firmicutes、拟杆菌门Bacteroidetes和变形菌门Proteobacteria是所有样品中丰度最高的类群(三者总和>72.43%),是发酵系统中重要的功能细菌类群.细菌属分类水平上,理研菌科佩特里单胞菌属Petrimonas、梭菌属Clostridium、泰氏菌属Tissierella和假单胞菌属Pseudomona是所有样品的优势类群,总丰度比例较为恒定(约32.40%).在古菌类群中,甲烷微菌目是最优势类群,丰度为40.62%～64.74%.产甲烷菌属Methanogenium是所有样品中主要的产气功能种群,丰度波动范围为32.62%～55.74%,随全年温度的变化而波动,此与沼气的产气规律相一致.
In order to investigate the effect of fermentation temperature on the microbial community structure of the rural household biogas digesters in Qinghai Province, twenty-four mud samples from six fermentation temperature periods through the year were collected and analyzed for their changes in microbial community structure using denaturing gradient gel electrophoresis(DGGE). Results showed that fermentation temperature had significant effects on the structure and diversity of microbial communities, and biogas production. Bacteria showed the highest species richness in both the highest(mid-September) and the lowest(mid-March) sampling temperature period, whereas archaea showed the highest species diversity only in mid-September. Firmicutes, Bacteroidetes and Proteobacteria were the most abundant bacterial phyla detected in all samples(total abundance > 72.43%), all of which are important functional bacterial groups in the fermentation system for biogas production. The genera of Petrimas, Clostridium, Tissierella and Pseudomona were the most dominant bacterial groups in all samples, whose total abundance was relatively constant(～32.40%). In archaea, the most dominant group among all samples was the order Methanomicrobiales, with relative abundance of 40.62% ～ 64.74%. The genus Methanogenium was the most dominant species detected for biogas production. Its abundance changed regularly with the fluctuation of fermentation temperature within the range of 32.62% ～ 55.74%, and was consistent with the regularity of biogas production.
In order to investigate the effect of fermentation temperature on the microbial community structure of the rural household biogas digesters in Qinghai Province, twenty-four mud samples from six fermentation temperature periods through the year were collected and analyzed for their changes in microbial community structure using denaturing gradient gel electrophoresis(DGGE). Results showed that fermentation temperature had significant effects on the structure and diversity of microbial communities, and biogas production. Bacteria showed the highest species richness in both the highest(mid-September) and the lowest(mid-March) sampling temperature period, whereas archaea showed the highest species diversity only in mid-September. Firmicutes, Bacteroidetes and Proteobacteria were the most abundant bacterial phyla detected in all samples(total abundance > 72.43%), all of which are important functional bacterial groups in the fermentation system for biogas production. The genera of Petrimas, Clostridium, Tissierella and Pseudomona were the most dominant bacterial groups in all samples, whose total abundance was relatively constant(～32.40%). In archaea, the most dominant group among all samples was the order Methanomicrobiales, with relative abundance of 40.62% ～ 64.74%. The genus Methanogenium was the most dominant species detected for biogas production. Its abundance changed regularly with the fluctuation of fermentation temperature within the range of 32.62% ～ 55.74%, and was consistent with the regularity of biogas production.
引文
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[27]Liu Y,Whitman W B.Metabolic,phylogenetic,and ecological diversity of the methanogenic archaea[J].Annals of the New York Academy of Sciences,2008,1125:171-189.
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[30]Franzmann P D,Liu Y,Balkwill D L,et al.Methanogenium frigidum sp.nov.,a psychrophilic,H2-using methanogen from Ace Lake,Antarctica[J].International Journal of Systematic Bacteriology,1997,47(4):1068-1072.
[31]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.
[32]Strapoc D,Picardal F W,Turich C,et al.Methane-producing microbial community in a coal bed of the Illinois Basin[J].Applied and Environmental Microbiology,2008,74(8):2424-2432.
[33]陈金全,王风平.珠江口海岸带沉积物甲烷相关古菌群落结构及多样性研究[J].云南大学学报(自然科学版),2013,35(2):240-245.Chen J Q,Wang F P.The community structure and diversity of methanogen-related organisms in Pearl River Estuary sediment[J].Journal of Yunnan University,2013,35(2):240-245.
[34]佘晨兴,仝川,王维奇.互花米草沼泽湿地产甲烷古菌的多样性及垂向分布[J].环境科学学报,2014,34(1):186-193.She C X,Tong C,Wang W Q.Vertical distribution and diversity of methanogenic archaea in estuarine marsh dominated by Spartina alterniflora[J].Acta Scientiae Circumstantiae,2014,34(1):186-193.
[35]曾志华,杨民和,佘晨兴,等.闽江河口区淡水和半咸水潮汐沼泽湿地土壤产甲烷菌多样性[J].生态学报,2014,34(10):2674-2681.Zeng Z H,Yang M H,She C X,et al.Diversity of methanogen communities in tidal freshwater and brackish marsh soil in the Min River estuary[J].Acta ecologica sinica,2014,34(10):2674-2681.
[36]席婧茹,刘素琴,李琳,等.硫酸盐还原型甲烷厌氧氧化菌群驯化及其群落特征[J].环境科学,2014,35(12):4602-4609.Xi J R,Liu S Q,Li L,et al.Acclimatization and characteristics of microbial community in sulphate-dependent
[37]Weiss A,Jér?me V,Freitag R.Diversity of the resident microbiota in a thermophilic municipal biogas plant[J].Applied Microbiology and Biotechnology,2008,81(1):163-173.
[38]王彦伟,徐凤花,阮志勇,等.用DGGE和Real-Time PCR对低温沼气池中产甲烷古菌群落的研究[J].中国沼气,2012,30(1):8-12.Wang Y W,Xu F H,Ruan Z Y,et al.Study on the methanogenic archaea communities in low temperature biogas digester by Real-Time PCR and DGGE methods[J].China Biogas,2012,30(1):8-12.
[39]贾璇,任连海,席北斗,等.芦苇厌氧联产氢气-甲烷过程中古菌群落特征解析[J].环境污染与防治,2016,38(7):5-10.Jia X,Ren L H,Xi B D,et al.Archaea community structure succession in anaerobic fermentation of hydrogen and methane coproduction system[J].Environmental Pollution&Control,2016,38(7):5-10.
[40]Plumb J J,Haddad C M,Gibson J A E,et a1.Acidianus sulfidivorans sp.Nov.,an extremely acidophilic,thermophilic archaeon isolated from a solfatara on Lihir Island,Papua New Guinea,and emendation of the genus description[J].Intemational joumal of systematic and evolutionary microbiology,2007,57(7):1418-1423.
[2]Lin Q,De Vrieze J,Li J,et al.Temperature affects microbial abundance,activity and interactions in anaerobic digestion[J].Bioresource Technology,2016,209:228-236.
[3]Dong M H,Yan W,Li Q M,et al.Investigation of methanogenic community structures in rural biogas digesters from different climatic regions in Yunnan,southwest China[J].Current Microbiology,2015,70(5):679-684.
[4]王华,杨光,刘小刚,等.温度对沼气菌群产气能力的影响及菌群变化的研究[J].西北农业学报,2008,17(5):294-297.Wang H,Yang G,Liu X G,et al.The effect of temperature on biogas production capability and the change of microbial community[J].Acta Agriculturae Boreali-occidentalis Sinica,2008,17(5):294-297.
[5]Rui H,Yongze Y,Qianwen C,et al.PCR-DGGE Analysis on microbial community structure of rural household biogas digesters in Qinghai Plateau[J].Current Microbiology,2018,75(5):541-549.
[6]Zanardini E,May E,Inkpen R,et al.Diversity of archaeal and bacterial communities on exfoliated sandstone from Portchester Castle(UK)[J].International Biodeterioration&Biodegradation,2016,109(6):78-87.
[7]Friha I,Karray F,Feki F,et al.Treatment of cosmetic industry wastewater by submerged membrane bioreactor with consideration of microbial community dynamics[J].International Biodeterioration&Biodegradation,2014,88(88):125-133.
[8]Pacwa-P?ociniczak M,P?aza G A,Piotrowska-Seget Z.Monitoring the changes in a bacterial community in petroleum-polluted soil bioaugmented with hydrocarbon-degrading strains[J].Applied Soil Ecology,2016,105:76-85.
[9]Abdallah M B,Karray F,Mhiri N,et al.Prokaryotic diversity in a Tunisian hypersaline lake,Chott El Jerid[J].Extremophiles,2016,20(2):125-138.
[10]Eleutheria N,Maria I,Vasiliki T,et al.Energy recovery and treatment of winery wastes by a compact anaerobic digester[J].Waste&Biomass Valorization,2016,7(4):799-805.
[11]韩睿,陈来生,李莉,等.PCR-DGGE研究青海农村户用沼气池微生物群落结构[J].中国环境科学,2015,35(6):1794-1804.Han R,Chen L S,Li L,et al.Microbial community of rural household biogas digesters in Qinghai by PCR-DGGE[J].China Environmental Science,2015,35(6):1794-1804.
[12]Ye N F,LüF,Shao L M,et al.Bacterial community dynamics and product distribution during pH-adjusted fermentation of vegetablewastes[J].Journal of Applied Microbiology,2007,103(4):1055-1065.
[13]Narihiro T,Terada T,Kikuchi K,et al.Comparative analysis of bacterial and archaeal communities in methanogenic sludge granules from upflow anaerobic sludge blanket reactors treating various foodprocessing,high-strength organic wastewaters[J].Microbes and Environments,2008,24(2):88-96.
[14]Roest K,Heilig H G H J,Smidt H,et al.Community analysis of a fullscale anaerobic bioreactor treating paper mill wastewater[J].Systematic and Applied Microbiology,2005,28(2):175-185.
[15]Lai Q,Shao Z.Pseudomonas xiamenensis sp.nov.,a denitrifying bacterium isolated from activated sludge[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(8):1911-1915.
[16]Klocke M,M?hnert P,Mundt K,et al.Microbial community analysis of a biogas-producing completely stirred tank reactor fed continuously with fodder beet silage as mono-substrate[J].Systematic and Applied Microbiology,2007,30(2):139-151.
[17]张蕾,梁军锋,崔文文,等.规模化秸秆沼气发酵反应器中微生物群落特征[J].农业环境科学学报,2014,33(3):584-592.Zhang L,Liang J F,Cui W W,et al.Characteristics of microbial communities in full-scale biogas digesters with straw as substrate[J].Journal of Agro-Environment Science,2014,33(3):584-592.
[18]袁月祥,文昊深,黄显波,等.玉米秸秆产生物燃气及其微生物群落解析[J].化工学报,2014,65(5):1784-1791.Yuan Y X,Wen H S,Huang X B.Biogas production using cornstalks and prokaryotic community composition[J].Ciesc Journal,2014,65(5):1784-1791.
[19]赵光,马放,孙婷,等.基于高通量测序的寒地沼气池微生物群落解析[J].哈尔滨工业大学学报,2014,46(4):36-42.Zhao G,Ma F,Sun T,et al.Analysis of microbial community in a full-scale biogas digester of cold region using high-throughput sequencing technology[J].Journal of harbin institute of technology,2014,46(4):36-42.
[20]Grabowski A,Tindall B J,Bardin V,et al.Petrimonas sulfuriphila gen.nov.sp.nov.a mesophilic fermentative bacterium isolated from a biodegraded oil reservoir[J].International Journal of Systematic&Evolutionary Microbiology,2005,55(3):1113-1121.
[21]Kr?ber M,Bekel T,Diaz N N,et al.Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-r RNAsequences and metagenome sequence data obtained by 454-pyrosequencing[J].Journal of Biotechnology,2009,142(1):38-49.
[22]Krause L,Diaz N N,Edwards R A,et al.Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor[J].Journal of Biotechnology,2008,136(1/2):91-101.
[23]Kong Y,Nielsen J L,Nielsen P H.Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants[J].Applied&Environmental Microbiology,2004,70(9):5483-5490.
[24]Hess M,Sczyrba A,Egan R,et al.Metagenomic discovery of biomass degrading genes and genomes from cow rumen[J].Science,2011,331(6016):463-467.
[25]Hugenholtz P,Tyson G W,Webb R I,et al.Investigation of candidate division TM7,a recently recognized major lineage of the domain Bacteria with no known pure-culture representatives[J].Applied and Environmental Microbiology,2001,67(1):411-419.
[26]Nielsen P H,Mielczarek A T,Kragelund C,et al.A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants[J].Water Research,2010,44(17):5070-5088.
[27]Liu Y,Whitman W B.Metabolic,phylogenetic,and ecological diversity of the methanogenic archaea[J].Annals of the New York Academy of Sciences,2008,1125:171-189.
[28]Bauer C,Korthals M,Gronauer A,et al.Methanogens in biogas production from renewable resources--a novel molecular population analysis approach[J].Water Science&Technology A Journal of the International Association on Water Pollution Research,2008,58(7):1433-1439.
[29]Wirth R,Kovács E,Maróti G,et al.Characterization of a biogasproducing microbial community by short-read next generation DNAsequencing[J].Biotechnology for Biofuels,2012,5(1):41-47.
[30]Franzmann P D,Liu Y,Balkwill D L,et al.Methanogenium frigidum sp.nov.,a psychrophilic,H2-using methanogen from Ace Lake,Antarctica[J].International Journal of Systematic Bacteriology,1997,47(4):1068-1072.
[31]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.
[32]Strapoc D,Picardal F W,Turich C,et al.Methane-producing microbial community in a coal bed of the Illinois Basin[J].Applied and Environmental Microbiology,2008,74(8):2424-2432.
[33]陈金全,王风平.珠江口海岸带沉积物甲烷相关古菌群落结构及多样性研究[J].云南大学学报(自然科学版),2013,35(2):240-245.Chen J Q,Wang F P.The community structure and diversity of methanogen-related organisms in Pearl River Estuary sediment[J].Journal of Yunnan University,2013,35(2):240-245.
[34]佘晨兴,仝川,王维奇.互花米草沼泽湿地产甲烷古菌的多样性及垂向分布[J].环境科学学报,2014,34(1):186-193.She C X,Tong C,Wang W Q.Vertical distribution and diversity of methanogenic archaea in estuarine marsh dominated by Spartina alterniflora[J].Acta Scientiae Circumstantiae,2014,34(1):186-193.
[35]曾志华,杨民和,佘晨兴,等.闽江河口区淡水和半咸水潮汐沼泽湿地土壤产甲烷菌多样性[J].生态学报,2014,34(10):2674-2681.Zeng Z H,Yang M H,She C X,et al.Diversity of methanogen communities in tidal freshwater and brackish marsh soil in the Min River estuary[J].Acta ecologica sinica,2014,34(10):2674-2681.
[36]席婧茹,刘素琴,李琳,等.硫酸盐还原型甲烷厌氧氧化菌群驯化及其群落特征[J].环境科学,2014,35(12):4602-4609.Xi J R,Liu S Q,Li L,et al.Acclimatization and characteristics of microbial community in sulphate-dependent
[37]Weiss A,Jér?me V,Freitag R.Diversity of the resident microbiota in a thermophilic municipal biogas plant[J].Applied Microbiology and Biotechnology,2008,81(1):163-173.
[38]王彦伟,徐凤花,阮志勇,等.用DGGE和Real-Time PCR对低温沼气池中产甲烷古菌群落的研究[J].中国沼气,2012,30(1):8-12.Wang Y W,Xu F H,Ruan Z Y,et al.Study on the methanogenic archaea communities in low temperature biogas digester by Real-Time PCR and DGGE methods[J].China Biogas,2012,30(1):8-12.
[39]贾璇,任连海,席北斗,等.芦苇厌氧联产氢气-甲烷过程中古菌群落特征解析[J].环境污染与防治,2016,38(7):5-10.Jia X,Ren L H,Xi B D,et al.Archaea community structure succession in anaerobic fermentation of hydrogen and methane coproduction system[J].Environmental Pollution&Control,2016,38(7):5-10.
[40]Plumb J J,Haddad C M,Gibson J A E,et a1.Acidianus sulfidivorans sp.Nov.,an extremely acidophilic,thermophilic archaeon isolated from a solfatara on Lihir Island,Papua New Guinea,and emendation of the genus description[J].Intemational joumal of systematic and evolutionary microbiology,2007,57(7):1418-1423.