Microbial degradation of contaminants using heavy water stable isotope probing

The effect of carbon source on the buoyant density of DNA during H 218O stable isotope probing (SIP) was determined. It was assumed that DNA from organisms grown on carbon sources lacking oxygen would be heavier than from cultures grown on carbon sources containing oxygen. H218O DNA-SIP was performed by incubating pure cultures of taxonomically distant bacteria with either glucose or toluene as a sole carbon source. After ultracentrifugation, the density of DNA from toluene cultures was ~0.02 g/mL heavier than glucose cultures. Based on the above information, a second objective was to characterize the active microbial community response to crude oil in pristine soils. H218O DNA SIP of soil fungal and bacterial amplicons was performed after 10 and 20 day incubations with and without crude oil, as well as with and without a label. DNA was pooled into heavy and light fractions for all samples and sequenced on an Illumina MiSeq with subsequent analysis using Quantitative Insights into Microbial Ecology (QIIME). Significant differences in microbial community structure and richness were detected, and some organisms such as Perlucidibaca not previously associated with crude oil degradation were identified. In addition, the efficacy of the H218O SIP method in resolving active community structure was confirmed by comparing heavy labeled fractions with light unlabeled fractions.