Methanogens in biogas production from renewable resources – a novel molecular population analysis approach

The population structure of thermo- and mesophilic biogas reactors digesting maize silage as the sole substrate was investigated employing a novel, highly degenerated PCR-primer pair targeting mcrA/mrtA coding for the key enzyme of methanogens. No sequence affiliating with Methanococcales, Methanopyrales, ANME-, rice or fen soil clusters was detected. Direct MeA PCR-cloning results indicated that Methanobacteriales were the most important methanogens in the thermophilic reactors. 57% and 80% of the analysed sequences affiliated with this order, 14% and 20% with Methanosarcinaceae and 0% and 29% with Methanomicrobiales. Methanomicrobiales dominated in the mesophilic reactors at the given conditions, 69% and 84% of the sequences recovered from direct MeA primed cloning affiliated with this order, 31% and 0% with Methanosarcinaceae and 0% and 16% with Methanobacteriales. No sequence affiliating with Methanosaetaceae was found. MeA primed PCR-single-strand conformation polymorphism indicated that population fluctuations occurred. According to sequence analysis of excised bands, Methanosarcinaceae dominated and Methanobacteriales were significantly represented in the thermophilic fermenter. Only 1 Methanosaetaceae sequence was found. Hydrogenotrophs appear to have a much higher and obligate acetoclastic methanogens a much lower importance than previously thought in biogas production from renewable resources.