The high computational cost of direct phylogenetic annotation of protein coding reads, assemblies like contigs/ORFs were still used for phylogenetic quantification in many studies, for example the metagenomic characterization of EBPR by Albertsen et al. [15], which apparently requires some sort of correction. In order to correct the ORFs annotation as well as to overcome the defects of reads annotation, an alternative method based on the annotation of ORFs and mapping reads to ORFs was applied in the present study. The result of this combined method was in consistency with classification based on 16S/18S in the taxon down to order level (Figure S3). The discrepancy at lower levels of family and genus might be in part explained by the unavoidable phylogenetic ambiguity of functional genes. About 39.8 of the reads were ML-281 assigned by this method, which was 4 times higher than the direct taxonomic annotation of short reads.converting the polysaccharide and resulted oligosaccharides (Polysaccharides and 18325633 Di- and oligosaccharides metabolism, Figure S4) into mono-sugars which could enter the central carbohydrate metabolism where via glycolysis to release energy to the consortium as well as provide NADH (Nicotinamide adenine dinucleotide) for the following anaerobic fermentation, while methanogens (main part of Archaea) further anaerobically oxidize fermentation intermediates to methane (Methanogenesis, Figure S4 insert) to achieve final oxidation of carbohydrate and remove inhibitory products for Bacteria metabolism (Figure 24272870 S4). Both genera of Clostridium and Thermoanaerobacterium had been reported to be able to metabolize lignocellulosic feedstock [2], however, our previous study found that growth of Thermoanaerobacterium over Clostridium under acidic condition (pH ,6.0) will significantly reduce the cellulose degrading capacity of the consortia [16]. This phenomenon could be explained by the results shown in Figure S6 that genus Thermoanaerobacterium of the sludge Castanospermine metagenome displayed deficient capacity towards polysaccharides, and Di- and oligosaccharides metabolism comparing to Clostridium.Functional AnalysisIt is not surprising to find that more functional information could be covered by the assembly results like ORFs, for example the “acetate to methane” and “coenzyme M synthesis” modules which were undetectable by short reads, were revealed in ORF annotation (Figure 3). However, since the current version of MEGAN software package was unable to parse the reads to ORFs alignment result into functional systems like SEED subsystem or KEGG pathway, the functional comparison between different taxonomic units showed below was based on the direct annotation of short reads using MG-RAST at E-value cutoff of 1E-5. Cooperation between Bacteria and Archaea was demonstrated in the metagenome that Bacteria initiated metabolism of cellulose byMining of Thermo-stable Carbohydrate-active Genes in the Sludge MetagenomeLignocellulose degradation requires a broad array of enzymes and associated proteins. Most of the enzymes involved in the process are GH (glycoside hydrolase) families which hydrolyze the glycosidic bond between carbohydrates or between a carbohydrate and a non-carbohydrate moiety [18]. Additionally, the CBMs, bringing the biocatalyst into intimate and prolonged association with its recalcitrant substrate, determine the rate of catalysis [2]. Therefore, the present study mainly focused on the GH families and CBM families. The CAZy databa.The high computational cost of direct phylogenetic annotation of protein coding reads, assemblies like contigs/ORFs were still used for phylogenetic quantification in many studies, for example the metagenomic characterization of EBPR by Albertsen et al. [15], which apparently requires some sort of correction. In order to correct the ORFs annotation as well as to overcome the defects of reads annotation, an alternative method based on the annotation of ORFs and mapping reads to ORFs was applied in the present study. The result of this combined method was in consistency with classification based on 16S/18S in the taxon down to order level (Figure S3). The discrepancy at lower levels of family and genus might be in part explained by the unavoidable phylogenetic ambiguity of functional genes. About 39.8 of the reads were assigned by this method, which was 4 times higher than the direct taxonomic annotation of short reads.converting the polysaccharide and resulted oligosaccharides (Polysaccharides and 18325633 Di- and oligosaccharides metabolism, Figure S4) into mono-sugars which could enter the central carbohydrate metabolism where via glycolysis to release energy to the consortium as well as provide NADH (Nicotinamide adenine dinucleotide) for the following anaerobic fermentation, while methanogens (main part of Archaea) further anaerobically oxidize fermentation intermediates to methane (Methanogenesis, Figure S4 insert) to achieve final oxidation of carbohydrate and remove inhibitory products for Bacteria metabolism (Figure 24272870 S4). Both genera of Clostridium and Thermoanaerobacterium had been reported to be able to metabolize lignocellulosic feedstock [2], however, our previous study found that growth of Thermoanaerobacterium over Clostridium under acidic condition (pH ,6.0) will significantly reduce the cellulose degrading capacity of the consortia [16]. This phenomenon could be explained by the results shown in Figure S6 that genus Thermoanaerobacterium of the sludge metagenome displayed deficient capacity towards polysaccharides, and Di- and oligosaccharides metabolism comparing to Clostridium.Functional AnalysisIt is not surprising to find that more functional information could be covered by the assembly results like ORFs, for example the “acetate to methane” and “coenzyme M synthesis” modules which were undetectable by short reads, were revealed in ORF annotation (Figure 3). However, since the current version of MEGAN software package was unable to parse the reads to ORFs alignment result into functional systems like SEED subsystem or KEGG pathway, the functional comparison between different taxonomic units showed below was based on the direct annotation of short reads using MG-RAST at E-value cutoff of 1E-5. Cooperation between Bacteria and Archaea was demonstrated in the metagenome that Bacteria initiated metabolism of cellulose byMining of Thermo-stable Carbohydrate-active Genes in the Sludge MetagenomeLignocellulose degradation requires a broad array of enzymes and associated proteins. Most of the enzymes involved in the process are GH (glycoside hydrolase) families which hydrolyze the glycosidic bond between carbohydrates or between a carbohydrate and a non-carbohydrate moiety [18]. Additionally, the CBMs, bringing the biocatalyst into intimate and prolonged association with its recalcitrant substrate, determine the rate of catalysis [2]. Therefore, the present study mainly focused on the GH families and CBM families. The CAZy databa.
