A 'rare biosphere' microorganism contributes to sulfate reduction in a peatland

Abstract

Methane emission from peatlands contributes substantially to global warming but is significantly reduced by sulfate reduction, which is fuelled by globally increasing aerial sulfur pollution. However, the biology behind sulfate reduction in terrestrial ecosystems is not well understood and the key players for this process as well as their abundance remained unidentified. Comparative 16S rRNA gene stable isotope probing (SIP) in the presence and absence of sulfate indicated that a Desulfosporosinus species, which constitutes only 0.006% of the total microbial community 16S rRNA genes, is an important sulfate reducer in a long-term experimental peatland field site. Parallel SIP using dsrAB (encoding subunit A and B of the dissimilatory (bi)sulfite reductase) identified no additional sulfate reducers under the conditions tested. For the identified Desulfosporosinus species a high cell-specific sulfate reduction rate of up to 341 fmol SO₄²⁻ cell⁻¹ day⁻¹ was estimated. Thus, the small Desulfosporosinus population has the potential to reduce sulfate in situ at a rate of 4.0-36.8 nmol (g soil w. wt.)⁻¹ day⁻¹, sufficient to account for a considerable part of sulfate reduction in the peat soil. Modeling of sulfate diffusion to such highly active cells identified no limitation in sulfate supply even at bulk concentrations as low as 10 μM. Collectively, these data show that the identified Desulfosporosinus species, despite being a member of the 'rare biosphere', contributes to an important biogeochemical process that diverts the carbon flow in peatlands from methane to CO₂ and, thus, alters their contribution to global warming.

Fig. 1

Substrate and sulfate measurements during pre-incubation and ¹²C-substrate turnover determinations. (A) Monitoring of indigenous substrate and sulfate concentrations during 4 weeks of pre-incubation of anoxic non-amended peat soil slurries. Averages ± SD are shown (n=6). (B) Time course of ¹²C-substrate turnover in anoxic peat soil slurries in the presence and absence of sulfate. Arrows indicate the time points of substrate additions; sulfate was added only once at the beginning of the experiment. Data points represent average values of three independent soil slurries; standard deviation bars were omitted for better visibility.

Fig. 2

T-RFLP fingerprinting of density-resolved bacterial 16S rRNA genes after two months of SIP incubations in the presence and absence of sulfate. CsCl buoyant densities are given for each fraction. Major bacterial populations are indicated with their respective T-RFs, which were assigned using 16S rRNA gene clone libraries generated from fractions with buoyant densities 1.722 g ml⁻¹ (incubation with sulfate) and 1.719 g ml⁻¹ (incubation without sulfate), respectively (see also Table S1, Fig. S4). T-RFs, which had no assignment according to their respective clone library, are indicated by their length only. The range of ‘heavy’ fractions that yielded no PCR product is indicated above the T-RFLP profiles.

Fig. 3

Phylogenetic consensus tree of 16S rRNA gene clones affiliated to the genus Desulfosporosinus (marked in bold). Clones were grouped according to ≥99% sequence identity; representing T-RFs and number of clones per group are indicated. With one exception, all Desulfosporosinus clones have a 16S rRNA sequence identity of >97% to each other. Parsimony bootstrap values for branches are indicated by solid circles (>90%) and open circles (75 to 90%). GenBank accession numbers of published 16S rRNA gene sequences are indicated behind the name of the respective sequences. The bar represents 1% estimated sequence divergence as inferred from distance matrix analysis.

Fig. 4

Quantification of Desulfosporosinus 16S rRNA genes relative to total 16S rRNA genes of Bacteria and Archaea by quantitative real-time PCR. The relative abundance ± SD of Desulfosporosinus sp. was determined for pristine peat soil samples over the years 2004, 2006, and 2007 (10–20 cm depth; biological replicates, n=3) in comparison to SIP incubations with and without sulfate (technical replicates, n=3). Peat soil of the 10–20-cm depth horizon was also used for the SIP incubations.

Fig. 5

Quantification of Desulfosporosinus 16S rRNA gene numbers by quantitative PCR over a peatland depth profile of 0–30 cm and quantification of potential Desulfosporosinus sulfate reduction rates (SRR). 16S rRNA gene numbers were determined in triplicate cores over the years 2004, 2006, and 2007, with the exception of the 20–30-cm depth, where samples were only available for the year 2007. The distribution of gene numbers is represented in boxplots showing the interquartile range and the median. Whiskers (maximum 1.5-fold interquartile range) represent the data distribution outside the interquartile range; outliers are depicted as black circles. Potential SRR of the Desulfosporosinus population were determined using the estimated cell-specific SRR of the identified peatland Desulfosporosinus sp., the interquartile range of Desulfosporosinus 16S rRNA genes per depth, and an average of 4.4 16S rRNA gene copies per cell (for details see text).

Fig. 6

Phylogenetic consensus tree of deduced DsrAB amino acid sequences longer than 500 amino acids, showing the affiliation of OTUs retrieved from the ‘heavy’ SIP fractions (indicated by a triangle) in comparison to known sulfate reducers and peat soil OTUs retrieved in a previous study from the same and a neighboring peatland (Loy et al., 2004. An OTU comprises all sequences having ≥90% amino acid sequence identity. Deduced DsrAB sequences shorter than 500 amino acids (indicated by dashed branches) were individually added to the distance matrix tree without changing the overall tree topology by using the ARB Parsimony_interactive tool. Parsimony bootstrap values for branches are indicated by solid circles (>90%) and open circles (75 to 90%). The Desulfosporosinus-related dsrA clone (shown in red) was retrieved from the 2-month incubation with sulfate using Desulfosporosinus/Desulfitobacterium-selective primers. GenBank accession numbers of published DsrAB sequences are indicated behind the name of the respective sequences. The bar represents 10% estimated sequence divergence as inferred from distance matrix analysis.