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Aerobic culture of methanogenic archaea without an external source of hydrogen


Culturing methanogenic archaea is fastidious, expensive, and requires an external source of hydrogen and carbon dioxide. Until now, these microorganisms have only been cultivated under strictly anaerobic conditions. We previously developed a single versatile culture medium containing sugars and anti-oxydants for cultivating all human known methanogens. Performing aerobic cultures in the presence of Bacteroides thetaiotaomicron, which produces hydrogen, allows for cultivation of Methanobrevibacter smithii which itself produces methane. To obtain colonies, we cultivated M. smithii in an agar plate in the upper part of a double chamber flask with a liquid culture of B. thetaiotaomicron in the lower compartment. We subsequently cultured four other methanogenic species for the first time and successfully isolated 13 strains of M. smithii and nine strains of Methanobrevibacter oralis from 100 stools and 45 oral samples. This procedure allows aerobic isolation and antibiotic susceptibility testing. This changes the ability to routinely study methanogens, which have been neglected in clinical microbiology laboratories and may be useful for biogas production.

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Correspondence to D. Raoult.

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This work was funded by Fondation Méditerranée Infection.

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SK, MD, and DR are co-inventors of a patent ref. No.: 1H52437 cas 32fr on the use of the three antioxidants herein reported to cultivate anaerobic bacteria and methanogenic archaea aerobically.

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The reduction of the culture medium by antioxidants; Resazurin was used as a redox indicator (Resazurin has a pink color and becomes transparent in the absence of oxygen). A: culture media at T0. B: culture media after 24-h incubation. 1: negative control without antioxidants. 2: culture medium supplemented by adding 1 g/L of ascorbic acid, 0.1 g/L of uric acid and 0.1 g/L of glutathione. 3: culture medium under anaerobic atmosphere. Culture medium supplemented by the addition of the three antioxidants has become clear (as anaerobic tube) indicating the removal of oxygen after 24 h incubation at 37 °C under aerobic conditions. Culture medium without antioxidants retained its pink color indicating the presence of oxygen. (GIF 106 kb)

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Khelaifia, S., Lagier, JC., Nkamga, V.D. et al. Aerobic culture of methanogenic archaea without an external source of hydrogen. Eur J Clin Microbiol Infect Dis 35, 985–991 (2016).

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  • Biogas Production
  • Methanogenic Archaea
  • Aerobic Culture
  • Clinical Microbiology Laboratory
  • Oral Sample