CHARACTERIZATION OF METHANOTROPHIC ACTIVITIES BY MICROBIAL COMMUNITY IN ZODLETONE SPRING FOR POTENTIAL MITIGATION OF METHANE EMISSION IN ABANDONED GAS AND OIL WELLS

Methane is a key greenhouse gas contributing toward roughly 20% of global warming and projected to be 28-fold more potent than CO₂. Methane flux from anthropogenic sources is of great concern in terms of global warming. Abandoned oil and gas wells (AOG) are often high in salinity, providing additional challenge for microbial methane oxidation or methanotrophy. In the US, more than three million AOGs were estimated to be emitting 0.28 million metric tons of methane into the atmosphere in 2018. We aim to identify and characterize microbial community that can oxidize methane under saline condition typically found in oil wells.

Here we provide evidence of methane oxidation in sediment samples collected from Zodletone spring, a sulfur-rich spring in Oklahoma, USA. Microcosms were setup with sediment samples and mineral salts medium in serum bottles with 1% (v/v) methane in the headspace. Bottles were sealed with rubber septa and aluminum crimp and incubated at 30°C. Methane concentration was measured by gas chromatograph. Bottles were repeatedly fed methane for 3 months to develop enrichment culture. The enrichment culture degrades methane in the presence of NaCl concentration ranging from 0 M to 2.5 M with highest rate at 1 M. Amplicon sequencing of 16S rRNA-gene from DNA extracted from both original sediment and the enrichment at 2.5 M salinity revealed the phylum Balneolota (64%) and Proteobacteria (28%) to be the most abundant in the enrichment, while in the original sediment their abundance was relatively low (18% and 13% respectively). The most abundant methanotroph was Methylohalobrius, a known halophilic methanotroph. We detected the presence of particulate methane monooxygenase (pMMO) gene in both sediment and enrichment. However, the gene for soluble MMO (sMMO) was absent. Our experiments showed enhanced pMMO activity when the enrichment was provided with 2-10 μM CuSO₄ as co-factor, with optimal concentration of 6 μM of Cu⁺. Our current efforts to isolate a pure culture of methanotroph from the enrichment are not successful. We will continue to culture the organisms on various media and growth conditions. Given the lack of mitigation strategy for this significant source of methane and severe knowledge gap on halophilic methanotrophs, isolation and characterization of methanotrophs in saline condition is crucial.