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Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescence in situ hybridization (FISH) was carried out to quantify the methanogenic Archaea in the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophic Methanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order, Methanoculleus was found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic family Methanosaetaceae was determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence of Methanosaetaceae in the biogas reactors and high concentrations of total ammonia (sum of NH 3 and NH 4 ؉ ) was observed.During the last decade the production of biogas from organic materials and residues has increased continuously in order to reduce the greenhouse gas emission resulting from the use of fossil energy sources. The energy-bearing substance of biogas is methane, which is produced as an end product of microbial anaerobic degradation of organic substrates, such as energy crops like maize, grains, grasses, or beets. Research for optimization of biogas production from renewable materials was initially focused on the evaluation of substrate eligibility and on the development and optimization of technical systems. However, biogas formation primarily depends on the structure and activity of the microbial community (28).The key microorganisms in the biogas formation process are the methane-generating microorganisms (methanogens). The capacity for methanogenesis is limited to members of the domain Archaea and, within this domain, on the phylum Euryarchaeota. With respect to the main metabolic precursors used, methanogens are usually divided into two groups: the aceticlastic methanogens that strictly metabolize acetate and the hydrogenotrophic methanogens that use H 2 or formate as an electron donor and CO 2 as a carbon source for their metabolism. Besides these major groups, certain methanogens are also able to convert methyl groups, methylamines, or methanol to methane (23,40). The substrates for the me...

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Aims:  The Archaea diversity was evaluated in an agricultural biogas plant supplied with cattle liquid manure and maize silage under mesophilic conditions. Methods and Results:  Two different genes (16S rRNA; methyl‐coenzyme‐M‐reductase, MCR) targeted by three different PCR primer sets were selected and used for the construction of three clone libraries comprising between 104 and 118 clones. The clone libraries were analysed by restriction fragment polymorphism (RFLP). Between 11 and 31 operational taxonomic units (OTUs) were detected and assigned to orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. Over 70% of all Archaea OTUs belong to the order Methanomicrobiales which mostly include hydrogenotrophic methanogens. Acetotrophic methanogens were detected in minor rates. Similar relative values were obtained by a quantitative real‐time PCR analysis. Conclusions:  The results implied that in this biogas plant the most of the methane formation resulted from the conversion of H2 and CO2. Significance and Impact of the Study:  This study reports, for the first time, a molecular analysis of the archaeal community in this type of agricultural biogas plants. Therein the hydrogenotrophic methanogenesis seems to be the major pathway of methane formation. These results are in contrast with the common thesis that in biogas fermentations the primary substrate for methanogenesis is acetate.

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