Isolation and characterization of a new Methanoculleus bourgensis strain KOR-2 from the rumen of Holstein steers

Objective To isolate and identify new methanogens from the rumen of Holstein steers in Korea. Methods Representative rumen contents were obtained from three ruminally cannulated Holstein steers (793±8 kg). Pre-reduced media were used for the growth and isolation of methanogens. Optimum growth temperature, pH, and sodium chloride (NaCl) concentration as well as substrate utilization and antibiotic tolerance were investigated to determine the physiological characteristics of the isolated strain. Furthermore, the isolate was microscopically studied for its morphology. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification. Results One strain designated as KOR-2 was isolated and found to be a non-motile irregular coccus with a diameter of 0.2 to 0.5 μm. KOR-2 utilized H2+CO2 and formate but was unable to metabolize acetate, methanol, trimethylamine, 2-propanol, and isobutanol for growth and methane production. The optimum temperature and pH for the growth of KOR-2 were 38°C and 6.8 to 7.0, respectively, while the optimum NaCl concentration essential for KOR-2 growth was 1.0% (w/v). KOR-2 tolerated ampicillin, penicillin G, kanamycin, spectromycin, and tetracycline. In contrast, the cell growth was inhibited by chloramphenicol. Phylogenetic analysis of 16S rRNA and mcrA genes revealed the relatedness between KOR-2 and Methanoculleus bourgensis. Conclusion Based on the physiological and phylogenetic characteristics, KOR-2 was thought to be a new strain within the genus Methanoculleus and named Methanoculleus bourgensis KOR-2. Keywords: Methanogen; Rumen; 16S rRNA; mcrA; Methanoculleus bourgensis