Methanobrevibacter spp. are often the most prominent group of archaea identified in mammalian gut microbial communities and employ a “hydrogenotrophic” pathway for methanogenesis and growth using CO2 and/or formate. Recent studies with ruminant-derived isolates demonstrate that some species can use alternative hydrogen sources, including formate and short-chain alcohols, to facilitate CO2 reduction to methane. Native Australian herbivores are often described as low methane emitting animals, but many species retain a measurable population of methanogenic archaea. As part of a broader characterisation of the methanogenic archaea from these iconic animal species we have isolated a Methanobrevibacter sp. LTK from the faecal sample of a Lumholtz’s Tree Kangaroo (Dendrolagus lumholtzi) which appears to have a diauxic growth profile when CO2, hydrogen and ethanol are provided as substrates.
Faecal samples from a Lumholtz’s Tree Kangaroo maintained at Currumbin Wildlife Sanctuary were used to inoculate a methanogen enrichment medium with added antibiotics and containing methanol and trimethylamine. The headspaces of the enrichment cultures were pressurised with either H2 or H2:CO2 (80:20). Serial transfers of these enrichment cultures followed by a dilution to extinction series with antibiotics produced a culture confirmed by microscopy and PCR to possess cells of a singular morphology and PCR positive only with archaea-domain rrs gene-specific primers. Subsequent sequencing of this amplicon confirmed the recovery of a member of the genus Methanobrevibacter that is most closely affiliated with the lineage containing Methanobrevibacter sp. AbM4. Preliminary growth studies of strain LTK show that when both H2 and ethanol are provided in combination with CO2, the growth pattern appears to be diauxic, with a distinct lag phase occurring between the first (OD600 ~0.3) and second (OD600 ~0.9) growth phases; and culture integrity was confirmed by rrs gene PCR and sequencing as described above. Although the use of short chain alcohols for methanogenesis in the absence of hydrogen has been observed in a limited number of Methanobrevibacter lineages, the diauxic pattern of growth observed here raises the possibility of substrate preferences affecting methanogen growth and thereby, provides novel targets and strategies for controlling their growth and persistence in the mammalian gut.