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In this study, methanogenic Archaea were isolated from five full-scale agricultural biogas plants (BGPs) located in Rhineland-Palatinate and Saarland, Germany, digesting maize silage and cattle manure. According to partial 16S rRNA gene sequences, the strains isolated from enrichment cultures were related to Methanoculleus bourgensis, Methanosarcina mazei, Methanosaeta concilii, and Methanobacterium formicicum. The 16S rRNA gene libraries of two representative BGPs screened with the direct amplified rDNA restriction analysis approach also revealed these Archaea to be present. Comparative phylogenetic analyses of reference strains and the isolates of genus Methanobacterium based on 16S and 23S rRNA gene sequences suggest two major groups of isolates, with both of them closely associated with Methanobacterium formicicum strain MF(T). The affiliation of Methanobacterium isolates is further supported by denaturating gradient gel electrophoresis of 16S rRNA gene amplificates, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and specifically amplified polymorphic DNA-PCR (SAPD-PCR), a novel fingerprint approach applied to methanogenic Archaea for the first time. Signature sequence 03Mbf derived from the application of SAPD-PCR was subsequently used to develop a PCR-based primer system for the detection of Methanobacterium formicicum-related isolates and the reference strain in BGP samples. Amplification of 03Mbf fragments down to a minimal titer of 10(3) cells of Methanobacterium formicicum-related isolate Mb9 was possible under BGP fermenter-comparable conditions.

A synthetic DNA fragment containing primer binding sites for the quantification of ten different microbial groups was constructed and evaluated as a reliable enumeration standard for quantitative real-time PCR (qPCR) analyses. This approach has been exemplary verified for the quantification of several methanogenic orders and families in a series of samples drawn from a mesophilic biogas plant. Furthermore, the total amounts of bacteria as well as the number of sulfate-reducing and propionic acid bacteria as potential methanogenic interaction partners were successfully determined. The obtained results indicated a highly dynamic microbial community structure which was distinctly affected by the organic loading rate, the substrate selection, and the amount of free volatile fatty acids in the fermenter. Methanosarcinales was the most predominant methanogenic order during the 3 months of observation despite fluctuating process conditions. During all trials, the modified quantification standard indicated a maximum of reproducibility and efficiency, enabling this method to open up a wide range of novel application options.

The final step of the biogas production process, the methanogenesis, is frequently dominated by members of the genus Methanoculleus. In particular, the species Methanoculleus bourgensis was identified to play a role in different biogas reactor systems. The genome of the type strain M. bourgensis MS2(T), originally isolated from a sewage sludge digestor, was completely sequenced to analyze putative adaptive genome features conferring competitiveness within biogas reactor environments to the strain. Sequencing and assembly of the M. bourgensis MS2(T) genome yielded a chromosome with a size of 2,789,773 bp. Comparative analysis of M. bourgensis MS2(T) and Methanoculleus marisnigri JR1 revealed significant similarities. The absence of genes for a putative ammonium uptake system may indicate that M. bourgensis MS2(T) is adapted to environments rich in ammonium/ammonia. Specific genes featuring predicted functions in the context of osmolyte production were detected in the genome of M. bourgensis MS2(T). Mapping of metagenome sequences derived from a production-scale biogas plant revealed that M. bourgensis MS2(T) almost completely comprises the genetic information of dominant methanogens present in the biogas reactor analyzed. Hence, availability of the M. bourgensis MS2(T) genome sequence may be valuable regarding further research addressing the performance of Methanoculleus species in agricultural biogas plants. (C) 2014 Elsevier B.V. All rights reserved.

The neotype strain Methanobacterium formicicum MFT (DSM1535), a hydrogenotrophic methanogenic Archaeon, was isolated from a domestic sewage sludge digestor in Urbana (IL, USA). Here, the complete genome sequence of the methanogen is reported. The genome is 2,478,074 bp in size, featuring a GC content of 41.23%. M. formicicum MFT encodes several genes predicted to be involved in adaptation to abiotic stress such as high osmolarity. The strain MFT is of biotechnological importance since M. formicicum strains are often found in production-scale biogas plants and it is suggested as a starter culture for the anaerobic biomethanation process. (C) 2014 Elsevier B.V. All rights reserved.

Methanobacterium sp. Mb1, a hydrogenotrophic methanogenic Archaeon, was isolated from a rural biogas plant producing methane-rich biogas from maize silage and cattle manure in Germany. Here we report the complete genome sequence of the novel methanogenic isolate Methanobacterium sp. Mb1 harboring a 2,029,766 bp circular chromosome featuring a GC content of 39.74%. The genome encodes two rRNA operons, 41 tRNA genes and 2021 coding sequences and represents the smallest genome currently known within the genus Methanobacterium. (C) 2013 Elsevier B.V. All rights reserved.

Methanoculleus bourgensis , of the order Methanomicrobiales , is a dominant methanogenic archaeon in many biogas-producing reactor systems fed with renewable primary products. It is capable of synthesizing methane via the hydrogenotrophic pathway utilizing hydrogen and carbon dioxide or formate as the substrates. Here we report the complete and finished genome sequence of M. bourgensis strain MS2 T , isolated from a sewage sludge digester.