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TRF-CUT, an ARB-implemented tool, was developed to predict in silico the terminal restriction fragments of aligned small-subunit rRNA gene or functional gene sequences. Application of this new tool to perform directed terminal restriction fragment length polymorphism analysis of pmoA products obtained from a forest soil revealed that novel cluster I methanotrophic bacteria were dominant.

Soil microeukaryotes may trophically benefit from plant biopolymers. However, carbon transfer from cellulose into soil microeukaryotes has not been demonstrated so far. Microeukaryotes assimilating cellulose-derived carbon in oxic and anoxic soil slurries were therefore examined by rRNA-based stable-isotope probing. Bacteriovorous flagellates and ciliates and, likely, mixotrophic algae and saprotrophic fungi incorporated carbon from supplemental [U- 13 C]cellulose under oxic conditions. A previous study using the same soil suggested that cellulolytic Bacteria assimilated 13 C of supplemental cellulose. Thus, it can be assumed that ciliates, cercozoa, and chrysophytes assimilated carbon by grazing upon and utilizing metabolic products of Bacteria that hydrolyzed cellulose in the soil slurries.

Methanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and acetogenesis in anoxic microcosms of soil from the fen Schlöppnerbrunnen. Formate dissimilation also yields CO 2 as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO 2 -utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [ 13 C]formate or 13 CO 2 (i.e., [ 13 C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes, mcrA (encoding the alpha-subunit of methyl-coenzyme M reductase), and fhs (encoding formyltetrahydrofolate synthetase). Methanogens, acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated with Methanocellaceae , Methanobacteriaceae , Acetobacteraceae , and Rhodospirillaceae were 13 C enriched (i.e., labeled) in [ 13 C]formate treatments, whereas genes affiliated with Methanosarcinaceae , Conexibacteraceae , and Solirubrobacteraceae were labeled in 13 CO 2 treatments. [ 13 C]acetate was enriched in [ 13 C]formate treatments, but labeling of known acetogenic taxa was not detected. However, several phylotypes were affiliated with acetogen-containing taxa (e.g., Sporomusa ). Methanosaetaceae -affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives. Crenarchaeota constituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schlöppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate that Methanocellaceae , Methanobacteriaceae , Methanosarcinaceae , and Methanosaetaceae were linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.

Norway spruce ( Picea abies ) forests exhibit lower annual atmospheric methane consumption rates than do European beech ( Fagus sylvatica ) forests. In the current study, pmoA (encoding a subunit of membrane-bound CH 4 monooxygenase) genes from three temperate forest ecosystems with both beech and spruce stands were analyzed to assess the potential effect of tree species on methanotrophic communities. A pmoA sequence difference of 7% at the derived protein level correlated with the species-level distance cutoff value of 3% based on the 16S rRNA gene. Applying this distance cutoff, higher numbers of species-level pmoA genotypes were detected in beech than in spruce soil samples, all affiliating with upland soil cluster α (USCα). Additionally, two deep-branching genotypes (named 6 and 7) were present in various soil samples not affiliating with pmoA or amoA . Abundance of USCα pmoA genes was higher in beech soils and reached up to (1.2 ± 0.2) × 10 8 pmoA genes per g of dry weight. Calculated atmospheric methane oxidation rates per cell yielded the same trend. However, these values were below the theoretical threshold necessary for facilitating cell maintenance, suggesting that USCα species might require alternative carbon or energy sources to thrive in forest soils. These collective results indicate that the methanotrophic diversity and abundance in spruce soils are lower than those of beech soils, suggesting that tree species-related factors might influence the in situ activity of methanotrophs.