A culture-dependent analysis of anaerobic methylotrophs in oil reservoir systems

Obtaining pure cultures and enrichment systems of methylotrophic anaerobic microorganisms capable of utilizing methyl compounds is critical to studying the carbon cycle in subsurface anoxic oil reservoirs. Culture-independent methods have been instrumental in uncovering the rich diversity of microorganisms in oil reservoirs. However, there remains a notable scarcity of methylotrophic microorganisms from oil reservoirs obtained by culture-dependent methods. In this study, we used five different methyl compounds, namely methanol, methylamine, dimethylamine trimethylamine, and methyl sulfide, as the sole substrates to isolate methyl-utilizing microorganisms from oil reservoirs, and H₂ was also added together with each of these methyl compounds in a separate isolation experiment, to facilitate H₂-dependent methylotrophic growth and metabolism. Notably, the highest colony numbers in roll tubes were achieved when using methanol as the substrate. A total of 306 pure strains, representing eight genera were obtained, and these microorganisms have rarely been reported for their ecological roles in oil reservoir systems. Following isolation, each strain was tested for utilization of methanol as the sole carbon and energy sources after a second transfer, including headspace gas assay and microbial cell observations. In addition, enrichment cultures amended with each of the five different methyl compounds with or without the addition of H₂ gas were established from four oil reservoir samples. Further experiments showed that the archaea enriched with methyl substrates from different oil reservoirs were almost all Methanobacteria, but after adding H₂, the H₂-dependent methylotrophic methanogen Ca. Methanomethylica was enriched in most of the enrichment cultures. On the contrary, the addition of H₂ has less impact on the bacterial communities. The isolation of pure cultures has significantly enhanced our understanding of the diversity and ecophysiology of methylotrophic microorganisms in oil reservoirs. This study has provided useful insights into methyl-based methane generation within oil reservoirs, contributing to further understanding of the microbial ecology and carbon cycle in anoxic oil reservoirs.