Ito T, Yoshiguchi K, Ariesyady HD, Okabe S.. Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge. ISME J 5: 1844-1856

Department of Civil and Environmental Engineering, Graduate School of Engineering, Gunma University, Kiryu, Japan.
The ISME Journal (Impact Factor: 9.3). 05/2011; 5(12):1844-56. DOI: 10.1038/ismej.2011.59
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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.

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Available from: Satoshi Okabe, Jul 15, 2014
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    • "Synergistetes are widely distributed throughout anaerobic environments, especially those associated with the digestive system of animals (Godon et al. 2005). Several bacteria belonging to this phylum are known as acetate-utilizing bacteria (Ito et al. 2011), and it is therefore possible that the observed increase in Synergistetes in reactor B (Fig. 3e, IV to V) resulted in a low accumulation of acetate in phase IV (Fig. 2e). Gammaproteobacteria, Deltaproteobacteria, and Betaproteobacteria were the major orders of Proteobacteria identified in the reactors. "
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    • "For instance, E. coli is known to β-oxidize LCFA anaerobically with nitrate as a terminal electron acceptor (Campbell, Morgan-Kiss and Cronan 2003); yet, the cause of the increase in relative abundance of Escherichia/Shigella-affiliated sequences to 5% in these methanogenic bioreactors (Fig. 5B) is not known. Moreover, increases in the relative abundance of sequences aligning with Synergistes and Levilinea (Figs 4 and 5), members of which have been respectively associated with syntrophic short-chain fatty acid (Ito et al. 2011) and protein degradation (Yamada and Sekiguchi 2009), suggested that the microbial oleic acid-degrading food web was potentially complex. "
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    • ". Briefly, 1 g of cesium chloride (CsCl) was added to 2 ml of the extracted DNA (5 mg), and 100 ml of ethidium bromide (10 mg ml À 1 ) was added to the DNA þ CsCl solution in an ultracentrifuge tube (11 Â 32 mm 2 , Quick-Seal PA tube; Beckman Coulter, Fullerton, CA, USA). Heavy and light DNAs were separated by centrifugation with Optima TLX (Beckman Coulter, Inc., Brea, CA, USA) at 201 458 g (68 000 r.p.m. using a Beckman rotor TLA-120.2 (Beckman Coulter, Fullerton, CA, USA)) for 36 h at 20 1C (Ito et al., 2011 "
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