寺河矿煤地质产甲烷微生物菌群的保藏和产甲烷性能

Preservation and methane production of methanogenic microbial consortia from coal geological environment in Sihe Mine

【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 ℃ 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 ℃. 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 ℃, 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 ℃, in which microbial activity of methanogens could be sustained at a better status and methanogens had good methane production ability.