A versatile medium for cultivating methanogenic archaea

Abstract

Background: Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis and Methanobrevibacter arboriphilicus have been cultured from human digestive microbiota. Each one of these fastidious methanogenic archaea requires a specific medium for its growth, hampering their routine isolation and the culture.

Methodology/principal findings: A new culture medium here referred as SAB medium was optimized and tested to cultivate methanogens associated with human microbiota, as well as two mesophile methanogens Methanobacterium beijingense and Methanosaeta concilii. It was further tested for the isolation of archaea from 20 human stool specimens including 10 specimens testing positive for PCR detection of M. smithii. After inoculating 10(5) colony-forming-unit archaea/mL or 1 g stool specimen in parallel in SAB medium and reference DSMZ medium in the presence of negative controls, growth of archaea was determined by optical microscopy and the measurement of methane production by gas chromatography. While the negative controls remained sterile, all tested archaea grew significantly more rapidly in SAB medium than in reference medium in 1-3 days (P<0.05, Student test). Among PCR-positive stool specimens, 10/10 grew in the SAB medium, 6/10 in DSMZ 119 medium, 5/10 in DSMZ 322 medium and 3/10 in DSMZ 334 c medium. Four out of ten PCR-negative stool specimens grew after a 3-week incubation in the SAB-medium whereas no growth was detected in any of the reference media. 16S rRNA gene sequencing yielded 99-100% sequence similarity with reference M. smithii except for one specimen that yielded 99-100% sequence similarity with reference Methanobrevibacter millerae.

Conclusions/significance: SAB medium allows for the versatile isolation and growth of methanogenic archaea associated with human gut microbiota including the archaea missed by inoculation of reference media. Implementation of the SAB medium in veterinary and medical microbiology laboratories will ease the routine culture-based detection of methanogenic archaea in clinical and environmental specimens.

Figure 1. Growth time of ten methanogenic archaea strains growing in culture medium SAB-medium or the standard DSMZ media.

(Triplicate experiment).

Figure 2. Growth monitoring of the five human-associated archaea strains by monitoring the CH₄ production and H₂ used by methanogens growing in culture medium SAB-medium or the standard DSMZ media.

A: Methanobrevibacter smithii; B: Methanobrevibacter oralis; C: Methanosphaera stadtmanae; D: Methanomassilicoccus luminyensis; E: Methanobrevibacter arboriphilicus (Triplicate experiment).