Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 degrees C) and all other methanongens (84-86 degrees C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.