Assessing the biodegradation of toluene, ethylbenzene and RDX and the identification of the microorganisms involved using stable isotope probing and high throughput amplicon sequencing

Contamination of groundwater by organic pollutants is a worldwide environmental problem. Bioremediation is a viable option for cleaning and reclaiming sites contaminated with pollutants amenable to microbial transformation. However, our understanding of microorganisms playing key roles in bioremediation is still developing. In this study, specific aspects of biodegradation of three organic pollutants namely toluene, ethylbenzene and the nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) were investigated. The overall objectives of this research were to 1) assess the biodegradation potential of toluene, ethylbenzene and RDX using soils from various sources, 2) identify the major groups of microorganisms responsible for the degradation of ethylbenzene and RDX in these samples and 3) assess the effect of a potential co-contaminant, isobutanol, on toluene biodegradation.;The first study examined the effect of isobutanol on the biodegradation of toluene under sulfate amended, nitrate amended or methanogenic conditions. The results indicated that toluene biodegradation was not greatly affected by isobutanol in five of the six experimental set-ups. However, toluene biodegradation was completely inhibited in one set of microcosms amended with sulfate and inocula from wastewater treatment plant activated sludge. This suggests that if co-contamination occurs, in some cases toluene degradation may be inhibited. In the second study, stable isotope probing (SIP) and high throughput sequencing were used with ethylbenzene degrading consortia to identify microorganisms benefiting from 13C label uptake from ethylbenzene (or metabolites). Several phylotypes were relatively more abundant in the heavy fractions from the labeled ethylbenzene amended soil microcosms compared to the controls indicating 13C label uptake. This included phylotypes within the families Oxalobacteraceae, Rhodospirillaceae, Xanthomonadaceae and Rhodocyclaceae (Proteobacteria) as well as the genus Gemmatimonas. This work indicates microorganisms not previously linked to ethylbenzene degradation could have significant roles in the carbon uptake from this pollutant.;The third and fourth studies involved applying SIP and high throughput sequencing to investigate RDX degrading microbial communities. In the third study, microbial communities obtained from four soils previously unexposed to explosives were investigated. Sequences from the total DNA extracts of all soils illustrated an increase in abundance of Brevundimonas and/or unclassified Bacillaceae 1 compared to the microbial communities in the initial soil or no RDX treatments. The fourth study investigated a RDX degrading community obtained from a Navy Base previously contaminated with explosives with and without glucose. The microbial communities in the total DNA samples indicated phylotypes classing as Pseudomonadaceae and Acinetobacter was more abundant in the presence and absence of glucose respectively. The SIP study also found that unclassified Pseudomonadaceae were primarily responsible for label uptake in both treatments. When glucose was present, Comamonas also increased in abundance following RDX degradation and was enriched in the heavy fractions, suggesting, for the first time, that this phylotype is also important for RDX removal. Overall, these data suggest both novel RDX degraders and previously reported RDX degraders were associated with RDX removal in these soils.