Hydrogenotrophic methanogens dominate in biogas reactors fed with defined substrates

Methanogenic communities in 200 L biogas reactors containing liquid manure were investigated for 33 d. The reactors were consecutively fed with casein, starch and cream. Real-time PCR with primers targeting the gene for methyl coenzyme-M reductase (mcrA) resulted in copy numbers of up to 2.1 × 109 g dry mass−1. Single strand conformation polymorphism (SSCP) analysis revealed a stable community consisting of few hydrogenotrophic methanogens. One of the two most abundant species was closely related to Methanospirillum hungatei, whereas the other one was only distantly related to other methanogens, with Methanopyrus kandleri being the closest cultivated relative. Most probable number (MPN) cultivations were accomplished with a sample from a 600 m3 reactor from which all manures used in the experiments originated, and equal cell counts of ca. 109 g dry mass−1 were found for cultivations with acetate, H2 and methanol. SSCP analysis of these samples and sequencing of the DNA bands identified different hydrogenotrophic methanogens in all samples, and acetoclastic methanogens closely related to Methanosarcina mazei in the samples cultivated with acetate and methanol. As the acetoclastic species were not found in any other SSCP sample, it was supposed that the ammonia values in the manure of the laboratory biogas reactor, which ranged from 2.48 to 3.61 g NH4-N L−1, inhibited the growth of the acetoclastic methanogens.