Coalbed Microbial Methane Production And DNA Marker Preparation And Application Of Their Species

Coalbed methane?CBM?is an important unconventional natural gas,and its development and utilization not only can improve our country's energy structure,but also reduce environmental pollution.According to the different causes,CBM can be divided into thermogenic gas and biogenic gas.Among them,biogenic gas is produced by degrading coal under the action of mixed microbial consortia.Based on the mechanism of biogenic gas production,additional biogas is generated by promoting the growth and metabolism of methanogenic microbial consortia in coalbed,which can increase the content of biogas in coalbed,thereby realizing the regeneration and enhancement of the CBM.However,the species of microbes involved in the biogasification process are complex,and the different microbes have different functions.For example,some bacteria need to degrade macromolecules in coal into small molecules before methanogenic archaea converted those small molecules to methane.The analysis of the diversity of microbial communities related to biogenic gas helps to understand the types of functional microbes,and the results would give theoretic basis for realizing the regulation of biogenic gas generation and revealing the mechanism of biogenic gas generation.In this study,biogas production simulation experiments were conducted by using CBM anaerobic enrichment culture sample as inoculum and lignite as carbon source,and the effect of adding NH₄Cl on the production of methane was preliminary studied.Meanwhile the dynamic changes of microbial community and their related functional genes during gas production were analyzed by metagenomic sequencing.Experimental results showed that more methane was generated from the experiment supplemented NH₄Cl compared to the experiment not supplemented with NH₄Cl;In the biogas production experiment with NH₄Cl supplement,with increasing culture time,the proportion of methanogenic archaea increased,mainly Methanosarcina and Methanobacterium.This finding indicated that the pathway of methane formation was mainly a mixed pathway of acetoclastic and hydrogenotrophic;In addition,based on the KEGG metabolic pathway classification,we found that the number of annotated genes related to metabolisms was the most,accounting for 69.30%of all annotated genes in KEGG database.Among them,the genes related to xenobiotics biodegradation and metabolism were mainly involved in the biodegradation process of coal;For nitrogen metabolism analysis,most of the annotated genes were involved in the dissimilatory nitrate reduction,denitrification and nitrogen fixation,indicated that some microbial consortia in the gas production process could use nitrate as an electron acceptor to breathe.However,there was not find functional genes that use ammonia.Moreover,in order to quickly detect the dominant microbes in the process of methane formation,we studied the total genomic DNA of microbes in the formation water from CBM field in Sihe mine and its enrichment culture samples.The V3 and V3-V4 region of 16S rRNA gene from bacteria and archaea were respectively obtained by PCR-DGGE,and connected to pEASY^?-T3 vector for the preparation of species DNA Marker for PCR-DGGE detection of microbial community in coal geological environment.Experimental results showed that the prepared bacteria and archaeal species DNA Marker all had clear,bright and species-specific bands;When the microbial community composition in the enriched culture samples was monitored using the species DNA Marker,it was found that the dominant species detected by DGGE were consistent with high-throughput sequencing results,indicated that the species DNA Marker prepared in this study has high reliability in the rapid detection of microbial diversity by DGGE,and can be applied as molecular standard to study microbial diversity in coal geological environment.The preparation of this series of DNA Marker help to skip the steps of cloning and sequencing in the PCR-DGGE species diversity analysis,and accelerate the process of the experiment.