Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as "Candidatus Cloacamonas acidaminovorans." "Candidatus Cloacamonas acidaminovorans" is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.The use of molecular techniques over the past few decades has shown the extent of microbial diversity and that the majority of archaeal and bacterial phyla still lack a cultivable representative (18,24). The recent advent of whole-community genome sequencing, or metagenomics, is rapidly changing this view and will revolutionize our understanding of the functional diversity of complex environments. For instance, a recent study has surprisingly revealed that ammonia-oxidizing Crenarchaeota are very abundant in soils (20). However, apart from the pioneering work in the Sargasso Sea (31) and the recent addition of millions of sequences from the Global Ocean Sampling expedition that revealed the extent of the ocean microbial diversity (25), most of the community sequencing programs have focused on relatively simple ecosystems. However, most microbial ecosystems are complex. Anaerobic digestion is a complex biological process that involves several metabolic pathways for the decomposition of organic matter into methane and carbon dioxide. The overall reactions-depolymerization, primary and secondary fermentation, acidogenesis, acetogenesis, and methanogenesis-are performed by a complex microbial community. Despite the industrial, technological, economic, and ecological importance of this community, little is known about the roles and activities of the microorganisms that inhabit anaerobic niches. The anaerobic digestion of organic matter involved in wastewater processing represents a good example of a complex and active microflora. During exploration of the bacterial diversity of an anaerobic mesophilic digester, a new bacterial candidate division called WWE1 was discovered (8). It was found that WWE1 bacteria could represent up to 10% of the bacterial microflora and thus could be a subdominant group. Using metagenomic sequence data and a specific genome assembly procedure, we were able to reconstruct the genome of a representative bacterium of the WWE1 division. We have for the first time obtained the complete genome sequence from a complex environment and from a bacterial candidate division with no cultivated representative. Because the metabolic pathways of anaerobic bacteria are ...

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anaerobic sludge digester. Two 16S rRNA gene libraries were constructed using total genomic DNA, and amplified by polymerase chain reaction (PCR) using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 246 and 579 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was performed using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota and Crenarchaeota. Interestingly, we detected a novel monophyletic group of 164 clones representing 66.6% of the archaeal library. Culture enrichment and probe hybridization show that this group grows better under formate or H2-CO2. Within the bacterial library 95.6% of the operational taxonomic units (OTUs) represent novel putative phylotypes never described before, and affiliated with eight divisions. The Bacteroidetes phylum is the most abundant and diversified phylogenetic group representing 38.8% of the OTUs, followed by the gram-positives (27.7%) and the Proteobacteria (21.3%). Sequences affiliated with phylogenetic divisions represented by few cultivated representatives such as the Chloroflexi, Synergistes, Thermotogales or candidate divisions such as OP9 and OP8 are represented by <5% of the total OTUs. A comprehensive set of 15 16S and 23S rRNA-targeted oligonucleotide hybridization probes was used to quantify these major groups by dot blot hybridization within 12 digester samples. In contrast to the clone library, Firmicutes and Actinobacteria together accounted for 21.8 +/- 14.9% representing the most abundant phyla. They were surprisingly followed by the Chloroflexi representing 20.2 +/- 4.6% of the total 16S rRNA. The Proteobacteria and the Bacteroidetes group accounted for 14.4 +/- 4.9% and 14.5 +/- 4.3%, respectively, WWE1, a novel lineage, accounted for 11.9 +/- 3.1% while Planctomycetes and Synergistes represented <2% each. Using the novel set of probes we extended the coverage of bacterial populations from 52% to 85.3% of the total rRNA within the digester samples.