A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor.

Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using 13C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse Archaea and Bacteria grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were anaerobic methanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and Gammaproteobacteria was the dominant bacterial lineage. Fluorescence in situ hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.

Treatment of low-strength wastewater in an anaerobic down-flow hanging sponge (AnDHS) reactor at low temperature.

The process performance of a novel anaerobic down-flow hanging sponge (AnDHS) reactor for the treatment of low strength wastewater was investigated. A lab-scale experiment was conducted in which 300-400 mgCOD L(-1) of artificial wastewater was fed in over 600 days. The reactor exhibited sufficient performance: 70-90% of total COD removal, and 60-90% of methane recovery were maintained at 20°C, with a hydraulic retention time (HRT) of 2 h. It was possible to maintain COD removal by extending the HRT to 4 h at 15°C and 10 h at 10°C. With regard to the wastewater feed, one-pass mode (without effluent recirculation) gave better performance in COD removal as compared with recirculation mode. The results of batch feeding experiments using single substrates (such as acetate, propionate or sucrose) indicated that acetate degradation was more strongly affected by decreasing operational temperature. In addition, the AnDHS reactor system had no significant problems related to sludge retention such as massive loss of sludge throughout the experiment. Microbial structure analysis of the retained sludge with respect to the domain Archaeal 16S rRNA gene showed the proliferation of relatives of both the acetate-utilizing genus Methanosaeta and the hydrogen-utilizing genus Methanolinea.

Development of 16S rRNA gene-targeted primers for detection of archaeal anaerobic methanotrophs (ANMEs).

Uncultured archaeal anaerobic methanotrophs (ANMEs) are known to operate the anaerobic oxidation of methane process, an important sink for the greenhouse gas methane in natural environments. In this study, we designed 16S rRNA gene-specific primers for each of the phylogenetic groups of ANMEs (ANME-1, Guaymas Basin hydrothermal sediment clones group within the ANME-1, ANME-2a, ANME-2b, ANME-2c and ANME-3) based on previously reported sequences. The newly designed primers were used for the detection of the various groups of ANMEs in the sulphate-limited anaerobic environmental samples, i.e. methanogenic sludges, rice field soils, lotus field sediments and natural gas fields. The ANME 16S rRNA gene sequences were detected only in a natural gas field sample among the environments examined in this study and were of the ANME-1 and -2c groups. In addition, the quantitative real-time PCR analysis using the designed primers showed that abundances of ANME-1 and -2c were estimated to be <0.02% of the total prokaryotic 16S rRNA gene community. The newly designed ANME group-specific primers in this study may be useful to survey the distribution and quantitative determination of ANMEs.

Quantification of mcrA by fluorescent PCR in methanogenic and methanotrophic microbial communities.

A quantitative fluorogenic PCR method for detecting methanogenic and methanotrophic orders was established using a refined primer set for the methyl coenzyme M reductase subunit A gene (mcrA). The method developed was applied to several microbial communities in which diversity and abundance of methanogens or anaerobic methanotrophs (ANMEs) was identified by 16S rRNA gene clone analysis, and strong correlations between the copy numbers of mcrA with those of archaeal 16S rRNA genes in the communities were observed. The assay can be applied to detecting and assessing the abundance of methanogens and/or ANMEs in anoxic environments that could not be detected by 16S rRNA gene sequence analyses.