Phylogenetic Comparison of the Methanogenic Communities from an Acidic, Oligotrophic Fen and an Anaerobic Digester Treating Municipal Wastewater Sludge

Abstract

Methanogens play a critical role in the decomposition of organics under anaerobic conditions. The methanogenic consortia in saturated wetland soils are often subjected to large temperature fluctuations and acidic conditions, imposing a selective pressure for psychro-and acidotolerant community members; however, methanogenic communities in engineered digesters are frequently maintained within a narrow range of mesophilic and circumneutral conditions to retain system stability. To investigate the hypothesis that these two disparate environments have distinct methanogenic communities, the methanogens in an oligotrophic acidic fen and a mesophilic anaerobic digester treating municipal wastewater sludge were characterized by creating clone libraries for the 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes. A quantitative framework was developed to assess the differences between these two communities by calculating the average sequence similarity for 16S rRNA genes and mcrA within a genus and family using sequences of isolated and characterized methanogens within the approved methanogen taxonomy. The average sequence similarities for 16S rRNA genes within a genus and family were 96.0 and 93.5%, respectively, and the average sequence similarities for mcrA within a genus and family were 88.9 and 79%, respectively. The clone libraries of the bog and digester environments showed no overlap at the species level and almost no overlap at the family level. Both libraries were dominated by clones related to uncultured methanogen groups within the Methanomicrobiales, although members of the Methanosarcinales and Methanobacteriales were also found in both libraries. Diversity indices for the 16S rRNA gene library of the bog and both mcrA libraries were similar, but these indices indicated much lower diversity in the 16S digester library than in the other three libraries.

Methanogenic community composition in an organic waste mixture in an anaerobic bioreactor

“…The PCR reaction was performed in accordance to Steinberg and Regan (Steinberg and Regan, 2008), with the following temperature cycle: initial denaturation step at 95°C for 3 min, followed by five cycles of denaturation at 95°C for 30 s, annealing at 48°C for 45 s and extension at 72°C for 30 s, with a ramp rate of 0.1°C s -1 from the annealing to the extension temperature. These initial five cycles were followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 30 s, followed by a final extension step at 72°C for 10 min.…”

“…This enzyme is unique to methanogens, where the highly conserved gene mcrA is found only in this group of microorganisms, whereas other enzymes involved in methanogenesis, such as methylene tetrahydromethanopterin dehydrogenase and methenyl tetrahydromethanopterin cyclohydrolase appear in another group of C1-utilising microorganisms. Therefore, identifying methanogens that occur in environmental samples by comparative mcrA (as a functional marker) sequence analysis could be performed for the following methanogen groups: Methanosarcinaceae, Methanosaetaceae, Methanobacteriales, Methanococcales and Methanomicrobiales (Steinberg and Regan, 2008).…”

Nitrogen and methanogen community composition within and among three Sphagnum dominated peatlands in Scandinavia

“…The mcrA DNA and cDNA partial sequences were amplified using the primer set mlas and mcrA-rev (Steinberg and Regan, 2008). Five µl of template (10-fold diluted DNA and non-diluted cDNA, respectively) were added to a 20 µl reaction mixture, consisting of 1X SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA) and 300 nM of each primer, 0.3 M of betaine and 250 µg ml -1 of BSA.…”

“…The mcrA region was amplified using the primers mlas and mcrA-rev (Steinberg and Regan, 2008); the forward primer (mlas) was 5'-labelled with 6-carboxyfluorescein (FAM). A 25 µl reaction mix contained one Illustra PuReTaq Ready-ToGo PCR Bead (GE Healthcare, Uppsala, Sweden), 300 nM of each primer, 5 µl of template DNA and RNA-free water.…”

Erratum: Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome

“…The differences observed between our current and previous estimates are likely due to differences in the technologies used (Janssen and Kirs, 2008;Steinberg and Regan, 2008). For instance, biases in the primers used for ribosomal RNA amplification in archaea can under-represent certain groups and because of differences in the primers used, the comparison of abundances of archaea and bacteria is nontrivial (Tymensen and McAllister, 2012).…”