Methyl fluoride affects methanogenesis rather than community composition of methanogenic archaea in a rice field soil

Abstract

The metabolic pathways of methane formation vary with environmental conditions, but whether this can also be linked to changes in the active archaeal community structure remains uncertain. Here, we show that the suppression of aceticlastic methanogenesis by methyl fluoride (CH(3)F) caused surprisingly little differences in community composition of active methanogenic archaea from a rice field soil. By measuring the natural abundances of carbon isotopes we found that the effective dose for a 90% inhibition of aceticlastic methanogenesis in anoxic paddy soil incubations was <0.75% CH(3)F (v/v). The construction of clone libraries as well as t-RFLP analysis revealed that the active community, as indicated by mcrA transcripts (encoding the α subunit of methyl-coenzyme M reductase, a key enzyme for methanogenesis), remained stable over a wide range of CH(3)F concentrations and represented only a subset of the methanogenic community. More precisely, Methanocellaceae were of minor importance, but Methanosarcinaceae dominated the active population, even when CH(3)F inhibition only allowed for aceticlastic methanogenesis. In addition, we detected mcrA gene fragments of a so far unrecognised phylogenetic cluster. Transcription of this phylotype at methyl fluoride concentrations suppressing aceticlastic methanogenesis suggests that the respective organisms perform hydrogenotrophic methanogenesis. Hence, the application of CH(3)F combined with transcript analysis is not only a useful tool to measure and assign in situ acetate usage, but also to explore substrate usage by as yet uncultivated methanogens.

Figure 1. Residuals, the difference between real and estimated size, of a FAM-labeled size standard used as ‘sample’ in t-RFLP analysis.

Data from three replicate runs are shown. Fit: fifth order polynomial, red line; 95% prediction intervals: black lines.

Figure 2. Accumulation of acetate and methane (A), and the respective δ¹³C signatures in ‰ VPDB (B) depending on initial concentrations of methyl fluoride; δ¹³C_acetate is the combined signature for both C-atoms.

Data are endpoint measurements and not corrected for initial concentrations. The fitted dose-response curves follow a log-logistic model with the parameters ED₅₀ (effective dose for 50% inhibition), upper limit, and slope, while the lower limit was fixed to the respective averages for 0% CH₃F. ED₅₀, ED₉₀, and ED₉₅ are marked by red lines. (C) Box-plot summarizing accumulation of methane and acetate in control (n = 3) and in samples with CH₃F≥0.75%, n = 6) after 14 days of anoxic incubation.

Figure 3. Multivariate analysis of relative abundances of terminal restriction fragments (tRF).

(A) Biplot of a constrained correspondence analysis (CCA). Two constraints were applied: CH₃F concentration and the type of nucleic acid, i.e. DNA or mRNA. The CCA explains about 71% of overall variation, with CCA1 being the most important axis. The arrows indicate the direction in which constraints correlate with the ordination axes. Confidence ellipses (95%) surround the centers of DNA- and mRNA-derived communities, respectively. Closed circles represent the samples, and black triangles the different tRFs. The triangle surrounded by a red outline corresponds to tRF 133, the numerically dominant fragment. (B) Multivariate regression tree (MRT) based on squared Euclidean distances. The vertical spacing of the branches is proportional to the error in the fit; the first split reduces the error by 75%. The tree is pruned, i.e. the least important splits have been removed. Barplots at the leaves show the relative abundance of different tRFs; from left: 126, 133, 503, 648, 652, 663, 683, 743, and 752 bp. As in panel A, tRF 133 is marked by a red outline.

Figure 4. Neighbor-joining tree based on 147 deduced amino acid positions from 949 mcrA sequences.

Phylogenetic nodes verified by a maximum likelihood tree are marked with closed circles. The outer branches of distinct clusters are collapsed, and those containing OTUs defined in this study are marked in blue. Only representative sequences for the OTUs have been incorporated into the tree and are depicted as ‘OTU name (accession number, number of sequences representing the OTU)’. Environmental clusters were labeled with two reference sequences showing maximum phylogenetic distance within the respective cluster, given as ‘name 1 (accession number 1), name 2 (accession number 2). The corresponding tRFs were calculated in silico using the TRiFLe package and are given to the right. Scale bar: 0.09 changes per amino acid position. The outgroup is Methanopyrus kandleri.