Syntrophomonadaceae-affiliated species as active butyrate-utilizing syntrophs in paddy field soil

Abstract

DNA-based stable-isotope probing was applied to identify the active microorganisms involved in syntrophic butyrate oxidation in paddy field soil. After 14 and 21 days of incubation with [U-(13)C]butyrate, the bacterial Syntrophomonadaceae and the archaeal Methanosarcinaceae and Methanocellales incorporated substantial amounts of (13)C label into their nucleic acids. Unexpectedly, members of the Planctomycetes and Chloroflexi were also labeled with (13)C by yet-unclear mechanisms.

Fig. 1.

Dynamics of short-chain fatty acids, headspace gas abundance, and ¹³C ratios during the incubation. (A) Butyrate and acetate concentrations in the soil solution after the addition of sodium butyrate. (B) CH₄ and CO₂ concentrations in the headspace during the whole incubation period. Bt, with addition of sodium butyrate; CK, control check, without butyrate addition. (C) Dynamics of the ¹³CH₄ and ¹³CO₂ atom ratio (percentages) in the headspace after addition of a ¹³C-labeled substrate. Arrows in the figure indicate the time points of butyrate addition. Data are means ± standard errors (n = 3 for panels A and B; n = 2 for panel C).

Fig. 2.

T-RFLP fingerprints of density-resolved bacterial communities generated from selected 16S rRNA gene gradient fractions. (A) Nonlabeled DNA sample of day 21; (B) ¹³C-labeled DNA sample of day 21. Cesium trifluoroacetate BDs (g ml⁻¹) of gradient fractions are given in brackets. The specific fragment lengths (in base pairs) of important T-RFs (as mentioned in the text) are given, and T-RFs with obvious changes are marked with short arrows.

Fig. 3.

Phylogenetic relationship of representative sequences related to Syntrophomonas spp. Clone libraries were generated from density-resolved 16S rRNA genes of anoxic rice soil organisms after incubation with [¹³C]butyrate for 21 days. d21h or d21l, day 21 heavy or light DNA fractions, respectively. Numbers in parentheses (base pairs) indicate in silico T-RF sizes. The scale bar represents 10% sequence divergence. GenBank accession numbers of reference sequences are given.