Metagenomic Assembly of Methanotrophic and Methylotrophic Bacterial Genomes from a Salt-Tolerant Zodletone Enrichment Culture

Methane is a potent greenhouse gas with a global warming potential 28 to36 times higher than CO2, significantly contributing to climate change. Conventional mitigation strategies, such as mechanically plugging abandoned oil and gas (AOG) wells, are often costly and ineffective. Microbial methane oxidation offers a promising alternative for reducing emissions from these wells. This study aimed to enrich a salt-tolerant methane-oxidizing microbial community from sediment collected at the sulfur-rich Zodletone Spring, Oklahoma.Enrichment cultures were established in serum bottles containing mineral salts medium supplemented with 2.5 M NaCl and 1% methane in the headspace. Despite multiple isolation attempts using various culturomics techniques, including dilution-to-extinction, pure cultures of methane-oxidizing microorganisms could not be obtained. Serial 10-fold dilutions (10E-1 to 10E-14) were performed, with methane oxidation monitored at each dilution. Oxidation was observed at 10E-9 after 25 weeks of incubation, but no activity was detected at higher dilutions, even with extended incubation.To characterize the community structure and function, total genomic DNA was extracted from the 10E-9 culture using the DNeasy PowerSoil Pro Kit. Shotgun metagenome sequencing was conducted (BMKgene, USA) on the Illumina platform. The genomic DNA was sheared into short fragments, end-repaired, A-tailed, and ligated with Illumina adapters. After PCR amplification, size selection, and purification, the sequencing library was prepared. The final dataset consisted of 72,203,262 reads, totaling 11,004,544,590 bp after cleanup.