Detection of active butyrate-degrading microorganisms in methanogenic sludges by RNA-based stable isotope probing

Abstract

Butyrate-degrading bacteria in four methanogenic sludges were studied by RNA-based stable isotope probing. Bacterial populations in the (13)C-labeled rRNA fractions were distinct from unlabeled fractions, and Syntrophaceae species, Tepidanaerobacter sp., and Clostridium spp. dominated. These results suggest that diverse microbes were active in butyrate degradation under methanogenic conditions.

FIG. 1.

Bacterial T-RFLP fingerprinting of density-resolved RNA from representative ¹³C-labeled and unlabeled fractions of sludges MP (A), JET (B), TP (C), and MBF (D). Electropherograms were generated from gradient fractions of RNA from the first [¹³C₄]butyrate incubation as a template. Dashed boxes show T-RFLP fingerprints of density-resolved unlabeled control RNA. Cesium trifluoroacetate BDs (g·ml⁻¹) of gradient fractions are given in brackets. The numbers at the T-RF peaks indicate the T-RF lengths. Representing phylotypes of T-RFs identified by clone analysis are indicated in parentheses.

FIG. 2.

Phylogenetic placement of representative bacterial 16S rRNA phylotypes from ¹³C-labeled RNA fractions. The phylogenetic tree was constructed by the neighbor-joining method. The measured T-RF lengths of phylotypes digested with MspI are shown in brackets. The scale bar represents the number of changes of nucleotides per sequence position. The symbols at each branch point show the bootstrap values obtained with 1,000-resampling analysis. Stars indicate putative butyrate degraders inferred from our results.