Inhibitory effects of ammonia on methanogen mcrA transcripts in anaerobic digester sludge

Methanogens in anaerobic ammonia-rich digesters show differential responses to ammonia stress. The mechanism for this is poorly understood. In the present study, we determined the rates of methane production, the composition of methanogen mcrA (the gene coding for the alpha subunit of methyl-coenzyme M reductase) and their transcripts in response to ammonium addition in the anaerobic sludge retrieved from a full-scale digester treating swine manure. The rate of CH4 production substantially reduced with increased addition of ammonium. The analysis of natural C-13 abundances of CH4 and CO2 indicated that the aceticlastic methanogenesis was more sensitive than hydrogenotrophic methanogenesis. Quantitative PCR analysis revealed that mcrA copy number decreased by one order of magnitude in the treatment with a large amount of ammonium (10g NH4+-NL-1) but did not change much with treatments of smaller amounts (3 and 7gNH(4)(+)-NL-1) compared with the control. T-RFLP analysis of mcrA compositions showed that the structure of the methanogen community remained highly stable, with Methanosaetaceae dominating the methanogen community in all incubations. The composition of mcrA transcripts, however, showed a substantial response to the addition of ammonium. The relative abundance of Methanosaetaceae transcripts declined with increasing amounts of ammonium, whereas the transcript level of Methanobacteriales mcrA was relatively resistant. The differential responses corresponded to the shift of methanogenic pathway inferred from C-13 isotope fractionation. Our study suggests that methanogens in anaerobic sludge have a strong mcrA transcriptional response to ammonia stress without a change in the community structure.