Insights into networks of functional microbes catalysing methanization of cellulose under mesophilic conditions

DNA-SIP (stable isotope probing) was conducted on anaerobic municipal solid waste samples incubated with (13)C-cellulose, (13)C-glucose and (13)C-acetate under mesophilic conditions. A total of 567 full-length bacterial and 448 1100-bp-length archaeal 16S rRNA gene sequences were analysed. In the clone libraries derived from 'heavy' DNA fractions, the most abundant sequences were affiliated with the phyla Firmicutes, Bacteroidetes, the gamma-subclass of Proteobacteria and methanogenic orders Methanomicrobiales and Methanosarcinales. Sequences related to the genus Acetivibrio (phylum Firmicutes) were recovered only in the 'heavy' DNA fraction derived from the (13)C-cellulose incubation. An oligonucleotide probe (UCL284) targeting specifically Acetivibrio was designed and used for fluorescent in situ hybridization (FISH) experiments. Interestingly, hybridization of the probe was detected in microorganisms aggregated around cellulose fibres, strengthening the conclusion that these microorganisms were major cellulose degraders. Sequences related to genus Clostridium (phylum Firmicutes) and to the family Porphyromonadaceae (phylum Bacteroidetes) were retrieved in large numbers from the 'heavy' DNA library of (13)C-Glucose incubation, suggesting their involvement in saccharide fermentation. Design and hybridization of specific FISH-probes confirmed the abundant representation of Clostridium (CLO401, CLO1248) and Porphyromonadaceae (BAC1040), which were mostly observed in the planktonic phase. Surprisingly, in the (13)C-acetate experiment, the 'heavy' DNA archaeal library was dominated by sequences related to the strictly hydrogenotrophic methanogenic genus Methanoculleus. One single operational taxonomic unit containing 70 sequences, affiliated to the gamma-subclass of Proteobacteria, was retrieved in the corresponding bacterial library. FISH observations with a newly designed specific probe (UGA64) confirmed the dominance of this bacterial group. Our results show that combination of DNA-SIP and FISH applied with a series of functionally connected substrates can shed light on the networks of uncultured microbes catalysing the methanization of the most abundant chemical renewable energy source on Earth.