Abstract
Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future.
Highlights
Biogas digesters can efficiently degrade lignocellulose and have long been used in the treatment of animal manure and agriculture residues[1]
In addition to these microorganisms, uncultured cellulolytic bacteria from Clostridia group 4 were identified as dominant cellulolytic bacteria in a landfill-derived biogas digester through DNA-stable isotope probing (DNA-SIP) and fluorescence in situ hybridization (FISH) techniques [10]
Using a similarity threshold of !99% for operational taxonomic unit (OTU) determination, there were 225 and 151 bacterial OTUs, and 14 and 8 archaeal OTUs observed for Z7 and Z8, respectively (S4 Table; S2 File and S3 File)
Summary
Biogas digesters can efficiently degrade lignocellulose and have long been used in the treatment of animal manure and agriculture residues[1]. The cellulolytic bacteria Clostridium thermocellum, Clostridium stercorarium, Clostridium cellulolyticum, and Bacteroides cellulosolvens were detected in various biogas digesters by culture-independent methods (i.e., 16S rRNA gene-based methods), suggesting that these microorganisms could be the dominant cellulolytic bacteria in many biogas digesters [4,5,6,7,8,9]. In addition to these microorganisms, uncultured cellulolytic bacteria from Clostridia group 4 were identified as dominant cellulolytic bacteria in a landfill-derived biogas digester through DNA-stable isotope probing (DNA-SIP) and fluorescence in situ hybridization (FISH) techniques [10]. Due to the absence of corresponding isolates, the glycoside hydrolase (GH) genes and lignocellulose degradation mechanisms of several dominant cellulolytic bacteria are completely unknown
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