典型煤层水微生物产甲烷潜力及其群落结构研究

Methanogenic potential and community structure of coalbed-methane production water microbiome

摘要:

内蒙古自治区二连盆地、海拉尔盆地是我国重要的煤层气产区，其中生物成因煤层气是煤层气的重要来源，但复杂物质转化产甲烷相关微生物群落结构及功能尚不清楚。[目的] 研究煤层水中的微生物代谢挥发性脂肪酸产甲烷的生理特征及群落特征。[方法] 以内蒙古自治区二连盆地和海拉尔盆地的四口煤层气井水作为接种物，分别添加乙酸钠、丙酸钠和丁酸钠厌氧培养；定期监测挥发性脂肪酸降解过程中甲烷和底物的变化趋势，应用高通量测序技术，分析原始煤层气井水及稳定期产甲烷菌液的微生物群落结构。[结果] 除海拉尔盆地H303煤层气井微生物不能代谢丙酸外，其他样品均具备代谢乙酸、丙酸和丁酸产生甲烷的能力，其生理生态参数存在显著差异，产甲烷延滞期依次是乙酸 < 丁酸 < 丙酸；最大比产甲烷速率和底物转化效率依次是丙酸 < 乙酸 < 丁酸。富集培养后，古菌群落结构与煤层气井水的来源显著相关，二连盆地优势古菌为氢营养型产甲烷古菌Methanocalculus（相对丰度13.5%-63.4%）和复合营养型产甲烷古菌Methanosarcina（7.9%-51.3%），海拉尔盆地的优势古菌为氢营养型产甲烷古菌Methanobacterium（24.3%-57.4%）和复合营养型产甲烷古菌Methanosarcina（29.6%-66.5%）；细菌群落则与底物类型显著相关，硫酸盐还原菌Desulfovibrio（12.0%-41.0%）、互营丙酸氧化菌Syntrophobacter（39.6%-75.5%）和互营丁酸菌Syntrophomonas（8.5%-21.9%）分别在乙酸钠、丙酸钠和丁酸钠处理组显著富集。[结论] 煤层气井水微生物可降解挥发性脂肪酸（乙酸、丙酸和丁酸）并具有产甲烷潜力；乙酸可能被古菌直接代谢产甲烷，而丙酸和丁酸通过互营细菌和产甲烷古菌代谢产甲烷。Desulfovibrio、Syntrophobacter和Syntrophomonas分别在乙酸、丙酸和丁酸代谢过程中发挥了重要作用。这些结果为煤层气生物强化开采提供了一定的微生物资源基础。

关键词:生物成因煤层气;挥发性脂肪酸;产甲烷;厌氧培养;高通量测序;互营代谢

Abstract:

Biogenetic coalbed methane is a significant composition in Erlian and Hailar basins. However, little information is available how biogenetic coalbed methane is generated. [Objective] To evaluate the methanogenic potential of volatile fatty acids by the coalbed-methane production water microbiome, and to observe the shift of microbial community structure involved. [Methods] Simulation experiments were performed by inoculating coalbed-methane production water from four coalbed methane wells of Erlian and Hailar basins. Acetate, propionate and butyrate were selected as substrates. Methane production and substrate utilization were measured over time. The microbial communities of coalbed methane production water and enrichment cultures after incubation were analyzed through high-throughput sequencing of 16S rRNA gene. [Results] With the exception of H303 coalbed water without propionate degradation, all coalbed water microbiomes had the ability to degrade acetate, propionate and butyrate under methanogenic degradation. The maximum specific methane production rate, substrate conversion ratio and lag-phase period exhibited great difference in different volatile fatty acid group. The maximum specific methane production rate and substrate conversation ratio was highest in the butyrate group and the lag-phase was the shortest with acetate addition. High-throughput sequencing analysis reveals that the archaeal community structure of enrichment cultures was significantly correlated with the source of coalbed water. The predominant archaeal groups of Erlian basin were hydrogenotrophic Methanocalculus (13.5%-63.4%) and Methanosarcina (7.9%-51.3%). The predominant archaea of Hailar basin were hydrogenotrophic Methanobacterium (24.3%-57.4%) and Methanosarcina (29.6%-66.5%). The enriched bacterial community structure is significantly correlated with substrates, sulfate-reducing Desulfovibrio (12.0%-41.0%), syntrophic propionate-degrading Syntrophobacter (39.63%-75.45%) and syntrophic butyrate-degrading Syntrophomonas (8.5%-21.9%) were enriched in acetate, propionate and butyrate group, respectively. [Conclusion] The coalbed-methane production water microbiome possessed potential of methanogenic volatile fatty acid degradation, among which acetate was probably fermented by methanogens, propionate and butyrate are consumed by syntrophic bacteria Syntrophobacter and Syntrophomonas respectively. This result will contribute to the development research of microbial enhanced methane recovery from coalbed.

Key words:biogenetic coalbed methane;volatile fatty acid;methanogenesis;anaerobic incubation;high-throughput sequencing;syntrophic metabolism