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For some environments such as planktonic/aqueous environments, the separation of bacteria cells from eukaryotic cells prior to DNA extraction using filtration is relatively straightforward. However, for woodchips, the bacteria are attached/embedded within the wood matrix, which prevents easy removal of bacterial cells. In this study, a method for the selective extraction of DNA from bacteria inhabiting Eucalyptus spp. woodchips has been developed. The objective was to compare milled and unmilled woodchips processed via three detachment methods, viz., sonication, vortexing and shaking followed by filtration using Teflon filters according to three relevant criteria: DNA yield, DNA purity and quality of DNA. Highest DNA yield was obtained by milling and vortexing for 10 min (77.50 ± 5.17 ng/μl), followed by milling and vortexing for 2 min (61.00 ± 6.56 ng/μl), unmilled and vortexing for 10 min (38.67 ± 5.17 ng/μl) and milled and shaking for 2 h (31.62 ± 5.17 ng/μl). The lowest DNA yield was obtained by using unmilled woodchips and 5 min of sonication treatment (7.00 ± 1.22 ng/μl). There was no significant difference in DNA purity for milled or unmilled woodchips processed via the three detachment methods. Duration of cell detachment treatment did not significantly influence DNA yield and purity. Following optimisation experiments, it was possible to extract bacterial DNA using milled woodchips and 10 minute vortexing devoid of DNA from the host background and other associated eukaryotes and of sufficient quality and quantity for metagenomic analysis.

Taxonomic and metabolic profiles of bacterial inhabitants of Eucalyptus spp. woodchips from storage piles were analysed using whole genome shotgun metagenomics sequencing. The majority of the bacterial species present in the metagenome from the Eucalyptus spp. woodchips are found within four bacterial phyla: Proteobacteria (77%), Bacteroidetes (15%), Acidobacteria (3%) and Actinobacteria (2%). The most abundant species in the metagenome were Burkholderia spp. (25%; Proteobacteria) and Chryseobacterium spp. (37%; Bacteroidetes). Other species prevalent in this bacterial metagenome are Rhodanobacter spp. (7%), Klebsiella spp. (6%) and Pseudomonas spp. (5%). Both PhyloSift and Metagenome Rapid Annotation Subsystem Technology tools to estimate taxonomic diversity showed that the phylum Proteobacteria was the predominant group among the bacteria in the bacterial metagenome from the Eucalyptus spp. woodchips, followed by Bacteroidetes, Acidobacteria and Actinobacteria. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism which are key to degradation of the lignocellulose were the most abundant SEED subsystems representing 14% and 11% of the bacterial metagenome, respectively. Approximately 8% of the sequences were grouped under “miscellaneous functions” and 3% under metabolism of aromatic compounds. One thousand two hundred forty-seven potential contigs encoding biomass-degrading enzymes including glycoside hydrolases (GH, 525 contigs), glycosyl transferases (GT, 452 contigs) carbohydrate binding modules (CBM, 149 contigs), carbohydrate esterases (CE, 70 contigs), polysaccharide lyases (PL, 16 contigs) and auxiliary activity (AA, 35 contigs) were identified. Both taxonomic and functional classifications suggest that the bacterial inhabitants of the Eucalyptus spp. woodchips present a broader metabolic potential than the degradation of plant biomass.