The consortia of microorganisms responsible for the hydrolysis of cellulose in situ are at present poorly characterized. Nonetheless, the importance of these communities is underscored by their capacity for converting biomass to greenhouse gases such as carbon dioxide and methane. The metabolic capacities of these organisms is particularly alarming considering the volume of biomass that is projected to re-enter the carbon cycle in Arctic tundra soil environments as a result of a warming climate. Novel cold-adapted cellulase enzymes also present enormous opportunities for a broad range of industries. DNA stable-isotope probing (DNA-SIP) is a powerful tool for linking the phylogenetic identity and function of cellulolytic microorganisms by the incorporation of isotopically labelled substrate into nucleic acids. By providing 13C-enriched glucose and cellulose to soil microcosms, it was possible to characterize the communities of microorganisms involved in the metabolism of these substrates in an Arctic tundra soil sample from Resolute Bay, Canada. A protocol for generating 13C-enriched cellulose was developed as part of this thesis, and a visual DNA-SIP protocol was generated to demonstrate the experimental outline. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone libraries were used to visualize changes in community structure and to identify prevalent, active phylotypes in the SIP incubations. Notably, predominant phylotypes changed over time and clustered based on substrate metabolism. Labelled nucleic acids identified by sequenced DGGE bands and 16S rRNA gene clone libraries provided converging evidence indicating the predominance of Clostridium and Sporolactobacillus in the 13C-glucose microcosms, and Betaproteobacteria, Bacteroidetes, and Gammaproteobacteria in the 13C-cellulose microcosms. Active populations consuming glucose and cellulose were distinct based on principle coordinate analysis of “light” and “heavy” DNA. A large portion of the recovered sequences possessed no close matches in the GenBank database, reflecting the paucity of data on these communities of microorganisms.

The consortia of microorganisms responsible for the hydrolysis of cellulose in situ are at present poorly characterized. Nonetheless, the importance of these communities is underscored by their capacity for converting biomass to greenhouse gases such as carbon dioxide and methane. The metabolic capacities of these organisms is particularly alarming considering the volume of biomass that is projected to re-enter the carbon cycle in Arctic tundra soil environments as a result of a warming climate. Novel cold-adapted cellulase enzymes also present enormous opportunities for a broad range of industries. DNA stable-isotope probing (DNA-SIP) is a powerful tool for linking the phylogenetic identity and function of cellulolytic microorganisms by the incorporation of isotopically labelled substrate into nucleic acids. By providing 13C-enriched glucose and cellulose to soil microcosms, it was possible to characterize the communities of microorganisms involved in the metabolism of these substrates in an Arctic tundra soil sample from Resolute Bay, Canada. A protocol for generating 13C-enriched cellulose was developed as part of this thesis, and a visual DNA-SIP protocol was generated to demonstrate the experimental outline. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA clone libraries were used to visualize changes in community structure and to identify prevalent, active phylotypes in the SIP incubations. Notably, predominant phylotypes changed over time and clustered based on substrate metabolism. Labelled nucleic acids identified by sequenced DGGE bands and 16S rRNA gene clone libraries provided converging evidence indicating the predominance of Clostridium and Sporolactobacillus in the 13C-glucose microcosms, and Betaproteobacteria, Bacteroidetes, and Gammaproteobacteria in the 13C-cellulose microcosms. Active populations consuming glucose and cellulose were distinct based on principle coordinate analysis of “light” and “heavy” DNA. A large portion of the recovered sequences possessed no close matches in the GenBank database, reflecting the paucity of data on these communities of microorganisms.

Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial ???indicator species??? for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.