Novel molecular markers for the detection of methanogens and phylogenetic analyses of methanogenic communities

Abstract

Methanogenic Archaea produce approximately one billion tons of methane annually, but their biology remains largely unknown. This is partially due to the large phylogenetic and phenotypic diversity of this group of organisms, which inhabit various anoxic environments including peatlands, freshwater sediments, landfills, anaerobic digesters and the intestinal tracts of ruminants. Research is also hampered by the inability to cultivate methanogenic Archaea. Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens. Here, we describe four novel molecular markers that should prove useful in the detailed analysis of methanogenic consortia, with a special focus on methylotrophic methanogens. We have developed and validated sets of degenerate PCR primers for the amplification of genes encoding key enzymes involved in methanogenesis: mcrB and mcrG (encoding β and γ subunits of the methyl-CoM reductase, involved in the conversion of methyl-CoM to methane), mtaB (encoding methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, catalyzing the conversion of methanol to methyl-CoM) and mtbA (encoding methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, involved in the conversion of mono-, di- and trimethylamine into methyl-CoM). The sensitivity of these primers was verified by high-throughput sequencing of PCR products amplified from DNA isolated from microorganisms present in anaerobic digesters. The selectivity of the markers was analyzed using phylogenetic methods. Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.

Anaerobic microbial community response to methanogenic inhibitors 2‐bromoethanesulfonate and propynoic acid

“…Previous studies that compared the methanogen community structures using sequencing of the 16S rRNA gene, mcrA gene, and other functional genes related to methanogenesis have found some quantitative differences depending on the gene sequenced (Dziewit et al. ; Wilkins et al. ), but did not include mock communities for comparison.…”

“…The differences between the predicted and the experimental sequencing results observed for the mock communities can be useful in guiding the analyses of mesocosm samples, as described below. Previous studies that compared the methanogen community structures using sequencing of the 16S rRNA gene, mcrA gene, and other functional genes related to methanogenesis have found some quantitative differences depending on the gene sequenced (Dziewit et al 2015;Wilkins et al 2015), but did not include mock communities for comparison. Given the observations made with the mock communities, we note that our interpretation of sequencing results from unknown mesocosm samples focuses on the comparison of relative abundances between samples.…”

Different response of bacteria, archaea and fungi to process parameters in nine full‐scale anaerobic digesters

“…Acetoclastic and hydrogenotrophic methanogens are well-known contributors in the anaerobic digestion process. Methylotrophic archaea, that utilize methylated compounds (mono-, di-, trimethylamines or dimethylsulfide) as carbon source and hydrogen as energy source, are frequently reported as typical members of microbial biogas plant communities since their description (Chojnacka et al, 2015;Dziewit et al, 2015;Zhou et al, 2017). These methanogenic archaea have typically been found in digestive tracts and are also known to be present in human stool (Gorlas et al, 2012;Vanderhaeghen et al, 2015), human gut (Borrel et al, 2012;Gaci et al, 2014), sheep rumen (Li et al, 2016) and cattle rumen (Seedorf et al, 2015).…”

“…Methanogens are either hydrogenotrophic (reduction of CO 2 with H 2 or formate), acetoclastic (splitting of acetate) or methylotrophic (reduction of methyl groups of methylated compounds e.g. methanol) and gain energy from methane production (Hedderich and Whitman, 2006;Dziewit et al, 2015). Both, hydrogenotrophic and acetoclastic methanogenic archaea have primarily been verified to be responsible for methane production during anaerobic digestion.…”

Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning

“…Using Illumina platform for archaeome analysis, deep sequencing of V6-V8 of 16S rRNA and mcrA gene on Illumina HiSeq was performed (Snelling et al 2014). For deeper characterization of the methanogenic population, also other markers (mcrB, mcrG, mtaB and mtbA) in combination with Illumina MiSeq sequencing have been used (Dziewit et al 2015). In both above mentioned studies, the methodological approach of TruSeq library preparation was used.…”