Identification of cellulolytic bacteria in soil by stable isotope probing

Abstract

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. (13)C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the (13)C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.

Quantitative Microbial Ecology through Stable Isotope Probing

“…This so-called priming effect is common and quantitatively significant (16) but remains mechanistically inscrutable. In past priming studies employing 13 C-SIP, some components of the microbial community were found to utilize as growth substrates the 13 C-labeled compounds added to initiate priming, though inferences about the organisms responsible for priming-i.e., degrading native soil organic matter-were weak, because no independent marker could validate their activity (62)(63)(64). Combining isotope tracers (using both 13 C and 18 O) can help by distinguishing microorganisms that respond to the original substrate pulse from those that respond indirectly by degrading soil organic matter (11), an approach useful for testing hypotheses about which groups of microorganisms contribute to priming.…”

DNA Stable-Isotope Probing (DNA-SIP)

“…nutrient supplementation, moisture, light) will vary depending on the type of sample that is incubated and the nature of the substrate. DNA-SIP experiments have been successfully performed using a variety of single carbon compounds 2,3 , multi-carbon compounds 4,5,6 , and using labelled nitrogen 7,8 or oxygen 9 . However, a drawback to using 15 N-or 18 O-labelled compounds is the decreased physical separation of labelled nucleic acid, primarily due to the presence of fewer nitrogen and oxygen atoms in DNA and RNA relative to carbon atoms.…”

Genomic Potential for Polysaccharide Deconstruction in Bacteria

“…These polysaccharides represent the major source of energy for most heterotrophs, including many microbes (e.g., bacteria and fungi). These heterotrophs produce glycoside hydrolases (GHs) to degrade polysaccharides and to release monosaccharides (e.g., glucose, fructose, N-acetylglucosamine, and xylose) that are key resources for microbial growth (2)(3)(4). However, not all microorganisms are directly involved in polysaccharide deconstruction.…”