Department of Civil and Environmental Engineering, Graduate School of Engineering, Gunma University, Kiryu, Japan.
ISME J. 2011 Dec;5(12):1844-56. doi: 10.1038/ismej.2011.59. Epub 2011 May 12.
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.
通过基于放射性同位素和稳定同位素的功能分析、放射性标记-荧光原位杂交 (MAR-FISH) 和 16S rRNA 的稳定同位素探测 (RNA-SIP)，鉴定和量化了产甲烷厌氧消化污泥中主要的乙酸利用细菌和古菌种群，这些方法可以将 16S rRNA 系统发育直接与原位代谢功能联系起来。首先，用（14）C-乙酸进行 MAR-FISH 表明，在消化污泥中，只有两个主要群体——未鉴定的细菌细胞和类似于 Methanosaeta 的丝状古菌细胞——能够显著利用乙酸。为了鉴定利用乙酸的未鉴定细菌，进行了 RNA-SIP 实验，以（13）C（6）-葡萄糖和（13）C（3）-丙酸盐作为唯一的碳源，随后对 16S rRNA 进行了系统发育分析。我们发现，属于协同菌属 4 组的细菌在从用（13）C-葡萄糖和（13）C-丙酸盐培养的污泥中获得的 16S rRNA 克隆文库中都有普遍检测到。为了确认该细菌群可以利用乙酸，我们新设计并应用了针对协同菌属 4 组的特定 FISH 探针，用于用（14）C-乙酸进行 MAR-FISH。MAR-FISH 结果表明，属于协同菌属 4 组的细菌显著地摄取了乙酸，其活性种群大小与该污泥中的 Methanosaeta 相当。此外，由于属于协同菌属 4 组的细菌对乙酸的 K(m)值较高，最大利用速率较高，因此在高乙酸浓度（2.5-10 mM）下，它们比 Methanosaeta 更具竞争优势。据我们所知，这是首次报道未培养的属于协同菌属 4 组的细菌对乙酸的利用活性及其对产乙酸甲烷菌 Methanosaeta 的竞争意义。
