Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge

Abstract

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with (14)C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with (13)C(6)-glucose and (13)C(3)-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with (13)C-glucose and (13)C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with (14)C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K(m) for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5-10 mM). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.

Figure 1

MAR-FISH image of acetate-utilizing bacterial and archaeal cells in the anaerobic digester sludge. (a) FISH image and (b) MAR image. In situ hybridization was performed with fluorescein isothiocyanate (FITC)-labeled EUB338-mixed probe and tetramethylrhodamine 5-isothiocyanate (TRITC)-labeled ARC915 probe. After incubating with 2.5 m glucose for 1 h, 740 kBq [2-¹⁴C]acetate was injected into the sample (the radiolabeled acetate concentration was 18% of total acetate concentration), and the samples were incubated for 2 h. Dotted circles indicate MAR-positive bacterial cells. Filamentous archaeal cells are also MAR-positive. Bar represents 10 μm.

Figure 2

A phylogenetic tree showing the affiliation of 16S rRNA clones retrieved from ‘heavy' fraction of the RNA, which was extracted from the anaerobic digester sludge incubated with 2.5 m [¹³C₆]glucose for 48 h (batch incubation). The numbers near branching points indicates bootstrap values. The scale bar represents 5% sequence divergence.

Figure 3

A phylogenetic tree showing the affiliation of 16S rRNA clones retrieved from ‘heavy' fraction of the RNA, which was extracted from the anaerobic digester sludge incubated at 0.5 m [¹³C₃]propionate for 58 h (continuous-flow incubation). The numbers near branching points indicates bootstrap values. The scale bar represents 5% sequence divergence.

Figure 4

MAR-FISH images of acetate-utilizing archaeal and bacterial cells present in the anaerobic digester sludge incubated for 5 h at 0.5 m acetate containing 19% [2-¹⁴C]acetate. In these images, MAR-positive cells are identified with genus-specific probes, which are yellowish owing to the cross-hybridization with domain probes. (a) FITC-labeled MX825 probe-stained filamentous MAR-positive cells. The sample was simultaneously hybridized with TRITC-labeled ARC915 probe. (b) FITC-labeled Syner195 probe-stained MAR-positive cells. The sample was simultaneously hybridized with TRITC-labeled EUB338-mixed probe. Bars represent 10 μm.

Figure 5

Relative abundance of [¹⁴C]acetate-utilizing Methanosaeta and Synergistes group 4 populations at 0.5 m, 1.0 m, 2.5 m, 5 m and 10 m acetate determined by MAR-FISH using probes of MSX825 and Syner195, respectively. The samples were incubated for 5 h at each acetate concentration containing 19% [2-¹⁴C]acetate. The genus-specific probes were always combined with domain-specific probes, and the sample was counter-stained with 4′, 6-diamidino-2-phenylindole (DAPI). Error bars represent the standard errors of duplicated measurements.

Figure 6

Acetate degradation rates of the anaerobic digester sludge incubated at 0.5 m, 1.0 m, 2.5 m, 5 m, 10 m and 20 m acetate containing 185 kBq [2-¹⁴C]acetate for 10 h, which was determined by liquid scintillation counting. Error bars represent the standard errors of duplicated measurements.

Figure 7

Acetate degradation rates of Methanosaeta and Synergistes group 4 in the anaerobic digester sludge at 0.5 m, 1.0 m, 2.5 m, 5 m and 10 m acetate concentrations. The rates were calculated from the values of Figures 5 and 6.