Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates

Abstract

Objectives: Methanogenic Archaea are considered as extremely oxygen-sensitive organisms, and their culture is fastidious, requiring specific equipment. We report here conditions allowing the cultivation of Methanobrevibacter smithii in an anaerobic chamber without the addition of hydrogen. Methods: We first enriched the stool sample in an anaerobic liquid medium. To cultivate M. smithii with Bacteroides thetaiotaomicron and other hydrogen-producing bacteria on solid medium in an anaerobic chamber, we divided the agar plates into two compartments and seeded each strain on each compartment. Methane production was assessed by gas chromatography, and the growing colonies were authenticated by MALDI-TOF MS. Results: We successfully cultured M. smithii from a liquid culture medium inoculated with stool collected from a healthy donor in an anaerobic chamber. The isolation in pure culture permitted successful culture on agar medium by our performing a co-culture with B. thetaiotaomicron. We also successfully tested the co-cultivation of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen in different amounts. Agar colonies of methanogens were obtained by co-culture with these bacteria, and methane production was detected. Conclusions: We propose a new approach to isolate and cultivate new strains of M. smithii by using a cocultureebased technique that can facilitate and make available the isolation of new methanogenic Archaea strains in clinical microbiology laboratories.

Syntrophy via Interspecies H 2 Transfer between Christensenella and Methanobrevibacter Underlies Their Global Cooccurrence in the Human Gut

“…Such cross-feeding likely explains why the relative abundances of M. smithii and fermenting bacteria are often positively correlated (21,23,24). Several studies have shown that in the laboratory, M. smithii can grow from the H 2 provided by Bacteroides thetaiotaomicron, a common gut commensal bacterium (25)(26)(27). Given that the cultured representatives of the Christensenellaceae ferment simple sugars (16,28) and that their genomes contain hydrogenases (29), we predicted that members of the Christensenellaceae produce H 2 used by M. smithii as a substrate in methanogenesis.…”

Syntrophy via interspecies H2transfer betweenChristensenellaandMethanobrevibacterunderlies their global co-occurrence in the human gut

“…3g-j) but does not aggregate in mono-culture before 7-10 days of culture (data not shown). In contrast, B. thetaiotaomicron, used here as a positive control based on previous reports that it supports the growth of M. smithii via H 2 production (25, 26), did not flocculate when grown alone, (Fig. 2c) and, when co-cultured with M .…”

“…thetaiotaomicron has previously been shown to support the growth of M . smithii in co-culture (25, 26). B .…”

“…Such cross-feeding likely explains why the relative abundances of M. smithii and fermenting bacteria are often positively correlated (21, 23, 24). Several studies have shown that in the laboratory, M. smithii can grow from the H 2 provided by Bacteroides thetaiotaomicron , a common gut commensal bacterium (25–27). Given that the cultured representatives of the Christensenellaceae ferment simple sugars (16, 28), and that their genomes contains hydrogenases (29), we predicted that members of the Christensenellaceae produce H 2 used by M. smithii as a substrate in methanogenesis.…”

Biogas Production from Oil Palm Empty Fruit Bunches and Palm Oil Decanter Cake using Solid-State Anaerobic co-Digestion

“…Hydrogenotrophic methanogens of Methanospirillum sp., Methanobrevibacter sp., and Methanoculleus sp. [40][41][42] and aceticlastic methanogen of Methanothrix sp. [43] were predominant archaea in 35 • C anaerobic sludge.…”