寺河矿煤地质产甲烷微生物菌群的保藏和产甲烷性能
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摘要
【背景】煤地质产甲烷微生物菌群可以代谢煤基质产生甲烷,对于实现煤层气资源的再利用具有重要意义。【目的】检测产甲烷菌群在保藏过程中群落结构的动态变化以及在产气实验中甲烷气的生成情况,以验证保藏方法的可行性,同时为煤层气的微生物增产奠定基础。【方法】分别于不同温度条件下比较3种菌种保藏方法,即甘油/L-半胱氨酸法、富营养法和煤基-基础盐法。通过产气实验检测不同保藏条件下产甲烷菌群的活力。同时,采用454高通量测序技术测定16S r RNA基因序列,分析25°C条件下煤基-基础盐菌种保藏过程中微生物群落结构的变化。【结果】比较了9组菌种保藏方法,发现菌种最佳保藏条件为25°C的煤基-基础盐保藏。在该条件下保藏的产甲烷菌群活性最高,甲烷生成量最大。以无烟煤为碳源进行产气实验时甲烷生成量为12%-25%,而以褐煤为碳源时甲烷生成量可达24%-73%。在25°C的煤基-基础盐菌种保藏条件下,保藏初期细菌的主要优势菌为假单胞菌属(Pseudomonas),而古菌的主要优势菌为甲烷八叠球菌属(Methanosarcina)。随着保藏时间的增加,细菌的群落结构变化显著,发酵细菌及产氢产乙酸细菌成为优势细菌,古菌的群落结构则相对稳定。【结论】菌种保藏的最佳条件为25°C的煤基-基础盐,保藏的产甲烷菌群能长期维持在较高的活性状态,具有较好的产甲烷能力。
[Background] Methanogenic microbial consortia from coal geological environment can metabolize coal matrix to produce methane, which is of great significance for realizing the reutilization of coalbed methane(CBM) resources. [Objective] In order to prove the feasibility of the methods of culture preservation, community dynamics of methanogens was analyzed and the yeild of methane was tested during the preservation process. Meanwhile, the results would give theoretic basis for microbial enhanced CBM. [Methods] Three culture preservation methods involving glycerol/L-cysteine, eutrophication, and coal-basic salt method were compared at different temperatures. Microbial methanogenic activity in different preservations was tested by gas production. In addition, the compositions of microbial community in coal and basic salt preservation at 25 °C were studied by 454 high-throughput sequencing technology for 16 S r RNA genes of bacteria and archaea. [Results] The preservation methods of 9 groups were compared, and the best culture preservation was the coal and basic salt preservation at 25 °C. Under this condition, the microbial methanogenic activity and the methane production were the highest. The yeild of methane was 12% to 25% and 24% to 73% using anthracite and lignite as carbon sources, respectively. In the coal and basic salt preservation test, the dominant bacteria and archaea in the early period were Pseudomonasand Methanosarcina at 25 °C, respectively. The structure of bacterial community changed dramatically with preservation time. The dominant bacteria changed to fermentative bacteria and acetogenic bacteria. The composition of dominant archaea was relatively stable. [Conclusion] The best preservation was coal and basic salt preservation at 25 °C, in which microbial activity of methanogens could be sustained at a better status and methanogens had good methane production ability.
[Background] Methanogenic microbial consortia from coal geological environment can metabolize coal matrix to produce methane, which is of great significance for realizing the reutilization of coalbed methane(CBM) resources. [Objective] In order to prove the feasibility of the methods of culture preservation, community dynamics of methanogens was analyzed and the yeild of methane was tested during the preservation process. Meanwhile, the results would give theoretic basis for microbial enhanced CBM. [Methods] Three culture preservation methods involving glycerol/L-cysteine, eutrophication, and coal-basic salt method were compared at different temperatures. Microbial methanogenic activity in different preservations was tested by gas production. In addition, the compositions of microbial community in coal and basic salt preservation at 25 °C were studied by 454 high-throughput sequencing technology for 16 S r RNA genes of bacteria and archaea. [Results] The preservation methods of 9 groups were compared, and the best culture preservation was the coal and basic salt preservation at 25 °C. Under this condition, the microbial methanogenic activity and the methane production were the highest. The yeild of methane was 12% to 25% and 24% to 73% using anthracite and lignite as carbon sources, respectively. In the coal and basic salt preservation test, the dominant bacteria and archaea in the early period were Pseudomonasand Methanosarcina at 25 °C, respectively. The structure of bacterial community changed dramatically with preservation time. The dominant bacteria changed to fermentative bacteria and acetogenic bacteria. The composition of dominant archaea was relatively stable. [Conclusion] The best preservation was coal and basic salt preservation at 25 °C, in which microbial activity of methanogens could be sustained at a better status and methanogens had good methane production ability.
引文
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[17]Zhao WJ,Xu SY.A simple and effective method for preserving Xanthomonas canpestris[J].Science and Technology of Food Industry,2005,26(1):131,142(in Chinese)赵文娟,徐升运.一种简单有效的黄原胶菌种保藏方法[J].食品工业科技,2005,26(1):131,142
[18]Satoh K,Tanaka T,Oguro Y,et al.Improvement of preservation method for ammonia-oxidizing bacteria by freeze-drying[J].Soil Science and Plant Nutrition,2004,50(5):777-781
[19]Jiang H,Gou ZX,Han B,et al.Study on preservation methods of mixed methane-oxidizing bacteria[J].Microbiology China,2014,41(7):1463-1469(in Chinese)江皓,缑仲轩,韩冰,等.甲烷氧化混合菌的保藏方法研究[J].微生物学通报,2014,41(7):1463-1469
[20]Klein DA,Flores RM,Venot C,et al.Molecular sequences derived from paleocene fort union formation coals vs.associated produced waters:implications for CBM regeneration[J].International Journal of Coal Geology,2008,76(1/2):3-13
[21]Li DM,Hendry P,Faiz M.A survey of the microbial populations in some Australian coalbed methane reservoirs[J].International Journal of Coal Geology,2008,76(1/2):14-24
[22]Str?po?D,Picardal FW,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
[23]Springer E,Sachs MS,Woese CR,et al.Partial gene sequences for the a subunit of methyl-coenzyme M reductase(mcr I)as a phylogenetic tool for the family Methanosarcinaceae[J].International Journal of Systematic Bacteriology,1995,45(3):554-559
[24]Colosimo F,Thomas R,Lloyd JR,et al.Biogenic methane in shale gas and coal bed methane:a review of current knowledge and gaps[J].International Journal of Coal Geology,2016,165:106-120
[25]Rockne KJ,Chee-Sanford JC,Sanford RA,et al.Anaerobic naphthalene degradation by microbial pure cultures under nitrate-reducing conditions[J].Applied and Environmental Microbiology,2000,66(4):1595-1601
[26]Chen SY,Dong XZ.Proteiniphilum acetatigenes gen.nov.,sp.nov.,from a UASB reactor treating brewery wastewater[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(6):2257-2261
[27]Park SY,Liang YN.Biogenic methane production from coal:a review on recent research and development on microbially enhanced coalbed methane(MECBM)[J].Fuel,2016,166:258-267
[28]Beckmann S,Lueders T,Krüger M,et al.Acetogens and acetoclastic Methanosarcinales govern methane formation in abandoned coal mines[J].Applied and Environmental Microbiology,2011,77(11):3749-3756
[2]Scott AR,Kaiser WR,Ayers WB.Thermogenic and secondary biogenic gases,San Juan Basin,Colorado and New Mexico-implications for coalbed gas producibility[J].AAPG Bulletin,1994,78(8):1186-1209
[3]Faiz M,Hendry P.Significance of microbial activity in Australian coal bed methane reservoirs―a review[J].Bulletin of Canadian Petroleum Geology,2006,54(3):261-272
[4]Rice DD,Claypool GE.Generation,accumulation,and resource potential of biogenic gas[J].AAPG Bulletin,1981,65(1):5-25
[5]Fallgren PH,Zeng CP,Ren ZY,et al.Feasibility of microbial production of new natural gas from non-gas-producing lignite[J].International Journal of Coal Geology,2013,115:79-84
[6]Wang H,Lin H,Dong YB,et al.Experiments on the gas production of brown coal degraded by exogenous methanogens[J].Petroleum Exploration and Development,2012,39(6):764-768(in Chinese)汪涵,林海,董颖博,等.外源产甲烷菌降解褐煤产气实验[J].石油勘探与开发,2012,39(6):764-768
[7]Wang AK,Qin Y,Lan FJ.Processes and possible pathways of biogenic coalbed methane generation from lignites based on parent methanogen[J].Geological Journal of China Universities,2012,18(3):485-489(in Chinese)王爱宽,秦勇,兰凤娟.基于本源菌的褐煤生物气生成过程与可能途径[J].高校地质学报,2012,18(3):485-489
[8]Wang BY,Tai C,Wu L,et al.Methane production from lignite through the combined effects of exogenous aerobic and anaerobic microflora[J].International Journal of Coal Geology,2017,173:84-93
[9]Yang XQ,Wu RW,Han ZY,et al.Analysis of methanogenic community and pathway of coalbed methane fields in the Qinshui Basin based on mcr A gene[J].Microbiology China,2017,44(4):795-806(in Chinese)杨秀清,吴瑞薇,韩作颖,等.基于mcr A基因的沁水盆地煤层气田产甲烷菌群与途径分析[J].微生物学通报,2017,44(4):795-806
[10]Guo HG,Yu ZS,Liu RY,et al.Methylotrophic methanogenesis governs the biogenic coal bed methane formation in Eastern Ordos Basin,China[J].Applied Microbiology and Biotechnology,2012,96(6):1587-1597
[11]Muyzer G,de Waal EC,Uitterlinden AG.Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for16S r RNA[J].Applied and Environmental Microbiology,1993,59(3):695-700
[12]Raskin L,Stromley JM,Rittmann BE,et al.Group-specific 16S r RNA hybridization probes to describe natural communities of methanogens[J].Applied and Environmental Microbiology,1994,60(4):1232-1240
[13]Watanabe T,Asakawa S,Nakamura A,et al.DGGE method for analyzing 16S r DNA of methanogenic archaeal community in paddy field soil[J].FEMS Microbiology Letters,2004,232(2):153-163
[14]Schloss PD,Westcott SL,Ryabin T,et al.Introducing mothur:open-source,platform-independent,community-supported software for describing and comparing microbial communities[J].Applied and Environmental Microbiology,2009,75(23):7537-7541
[15]Midgley DJ,Hendry P,Pinetown KL,et al.Characterisation of a microbial community associated with a deep,coal seam methane reservoir in the Gippsland Basin,Australia[J].International Journal of Coal Geology,2010,82(3/4):232-239
[16]Su LM,Li S.Preservation method for L-arginine-producing strain of Brevibacterium flavum[J].Industrial Microbiology,2009,39(1):60-62(in Chinese)苏令鸣,李爽.产精氨酸的黄色短杆菌菌种保藏方法的研究[J].工业微生物,2009,39(1):60-62
[17]Zhao WJ,Xu SY.A simple and effective method for preserving Xanthomonas canpestris[J].Science and Technology of Food Industry,2005,26(1):131,142(in Chinese)赵文娟,徐升运.一种简单有效的黄原胶菌种保藏方法[J].食品工业科技,2005,26(1):131,142
[18]Satoh K,Tanaka T,Oguro Y,et al.Improvement of preservation method for ammonia-oxidizing bacteria by freeze-drying[J].Soil Science and Plant Nutrition,2004,50(5):777-781
[19]Jiang H,Gou ZX,Han B,et al.Study on preservation methods of mixed methane-oxidizing bacteria[J].Microbiology China,2014,41(7):1463-1469(in Chinese)江皓,缑仲轩,韩冰,等.甲烷氧化混合菌的保藏方法研究[J].微生物学通报,2014,41(7):1463-1469
[20]Klein DA,Flores RM,Venot C,et al.Molecular sequences derived from paleocene fort union formation coals vs.associated produced waters:implications for CBM regeneration[J].International Journal of Coal Geology,2008,76(1/2):3-13
[21]Li DM,Hendry P,Faiz M.A survey of the microbial populations in some Australian coalbed methane reservoirs[J].International Journal of Coal Geology,2008,76(1/2):14-24
[22]Str?po?D,Picardal FW,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
[23]Springer E,Sachs MS,Woese CR,et al.Partial gene sequences for the a subunit of methyl-coenzyme M reductase(mcr I)as a phylogenetic tool for the family Methanosarcinaceae[J].International Journal of Systematic Bacteriology,1995,45(3):554-559
[24]Colosimo F,Thomas R,Lloyd JR,et al.Biogenic methane in shale gas and coal bed methane:a review of current knowledge and gaps[J].International Journal of Coal Geology,2016,165:106-120
[25]Rockne KJ,Chee-Sanford JC,Sanford RA,et al.Anaerobic naphthalene degradation by microbial pure cultures under nitrate-reducing conditions[J].Applied and Environmental Microbiology,2000,66(4):1595-1601
[26]Chen SY,Dong XZ.Proteiniphilum acetatigenes gen.nov.,sp.nov.,from a UASB reactor treating brewery wastewater[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(6):2257-2261
[27]Park SY,Liang YN.Biogenic methane production from coal:a review on recent research and development on microbially enhanced coalbed methane(MECBM)[J].Fuel,2016,166:258-267
[28]Beckmann S,Lueders T,Krüger M,et al.Acetogens and acetoclastic Methanosarcinales govern methane formation in abandoned coal mines[J].Applied and Environmental Microbiology,2011,77(11):3749-3756