Polyphasic Analyses of Methanogenic Archaeal Communities in Agricultural Biogas Plants

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 NH₃ and NH₄⁺) 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 H₂ or formate as an electron donor and CO₂ 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 methanogens are provided by several physiological groups of bacteria which degrade organic matter, sometimes in close syntrophic interaction with the methanogens (1).Several studies on the microbial diversity present in lab-scale biogas reactors supplied with renewable raw material (7, 57) have been recently published. However, analyses under laboratory conditions do not necessarily reflect conditions in full-scale reactors (35). Therefore, further research on the methanogenic community in full-scale biogas reactors is crucial.Generally, studies regarding the microbial community structure in full-scale biogas reactors have focused on different systems for wastewater treatment or classical biogas plants based on manure digestion (32, 38, 43). In most systems, approximately 70% of the carbon fixed in methane was derived from acetate. Only minor amounts, up to approximately 30%, were deduced from CO₂ (1, 42). Together with the presence of huge assemblages of Methanosarcina sp., it was assumed by some authors that aceticlastic methanogenesis was the predominant pathway for methane formation. Moreover, as shown by other studies, the relative contribution of H₂/CO₂ versus acetate as metabolic precursors for methanogens can be quite different in other anaerobic environments (10, 33, 37). However, the methanogenic microfloras in full-scale biogas reactors supplied with energy crops as a primary or sole substrate have rarely been studied (35, 37, 45).The aim of this study was to gain insight into the diversity of methane-producing Archaea in six full-scale biogas plants supplied with renewable raw material and different types of liquid manure as substrates. Therefore, a polyphasic approach with three different culture-independent techniques (fluorescence in situ hybridization FISH], quantitative PCR Q-PCR], and 16S rRNA gene analysis) to analyze methanogen diversity was carried out to overcome the known limitations of each single approach (15, 46). To analyze potential effects of different process parameters on the methanogenic archaeal community, the reactor performances were correlated with the apparent archaeal diversity.