Unraveling methanogenesis processes and pathways for Quaternary shallow biogenic gas in aquifer systems through...

Shallow biogenic gas is crucial in global warming and carbon cycling. Considering the knowledge gap in the understanding of methanogenesis and metabolic mechanisms within shallow groundwater systems, we investigated Quaternary shallow biogenic gas resources from the Hetao Basin in North China, which were previously underexplored. We systematically analyzed the genesis of gas and formation water, microbial communities, methanogenic processes, and pathways using geochemistry, genomics, and transcriptomics. Our findings indicated that active freshwater environments are conducive to microbial activity and the generation of primary microbial gases. A diverse range of microbes with functions, such as hydrolysis (e.g., Caulobacter), acidogenesis, and hydrogen production (e.g., Sediminibacterium), synergistically contributed to the methanogenic process. Methanogens predominantly comprised hydrogenotrophic methanogens (e.g., Methanobacteriales), although H₂-dependent methylotrophic methanogens (e.g., Methanofastidiosa) were also prevalent. The metabolic processes of the different methanogenic pathways were revealed based on functional gene analysis and mapping results. Furthermore, the composition of the community structure, functional predictions, metagenomics, and metatranscriptomics underscored the contribution of the hydrogenotrophic pathway, which ranged from 52.22 % to 79.23 %. The aceticlastic pathway exhibited high gene abundance and was primarily associated with methylotrophs and other potential pathways. The H₂-dependent methylotrophic methanogenesis pathway was constrained by low metabolic activity. By revealing the methane production mechanism of biogenic gas in shallow aquifer systems, this study provides a new perspective and profound comprehension of its ecological and environmental implications worldwide.