The identification of the microorganisms and the functional genes responsible for the biodegradation of vinyl chloride and RDX using samples from contaminated sites

Contamination of soil, sediments and groundwater by organic pollutants is a current problem that threatens both human and environmental health. The use of microorganisms to remediate pollution (bioremediation) is a viable option for site remediation. However, limited knowledge exists concerning the key microorganisms involved in the degradation of many pollutants. In this study, specific aspects of the biodegradation of two organic pollutants namely vinyl chloride (VC) and hexahydro- 1,3,5-trinitro-1,3,5-triazine (RDX) were investigated. The overall objectives were to 1) identify the key microorganisms responsible for the degradation of VC and RDX in samples derived from a number of contaminated sites, 2) design specific primers to quantify the key phylotypes involved in VC degradation and 3) design specific primers towards the functional genes associated with RDX degradation.;The first study examined the microorganisms involved in the degradation of VC in a mixed culture derived from a VC contaminated site. This culture degraded VC slowly (120 mumol in ∼45 days). Using stable isotope probing (SIP) and high throughput sequencing (Illumina MiSeq), VC degraders were putatively identified as belonging to the genera Nocardioides (previously linked to VC degradation), Gp4, Brevundimonas, Tissierella, Sediminibacterium and Rhodoferax (genera not previously linked to VC degradation). The results suggest that previous work involving isolations may not accurately represent active VC assimilators in mixed communities.;In a second study, another mixed culture derived from contaminated groundwater was able to degrade VC rapidly (∼120 mumol in 7 days). Stable isotope probing and high throughput sequencing were again used to identify the dominant VC degraders and in this culture the resulting key genera included Nocardioides, Sediminibacterium, Aquabacterium and Variovorax. Specific primers were then designed towards the novel VC degrading phylotypes (Sediminibacterium, Aquabacterium and Variovorax) and quantitative PCR (qPCR) was used to confirm label uptake by these microorganisms. Both studies indicated that microorganisms previously linked to VC degradation as well as novel genera could have significant roles in the carbon uptake from this pollutant.;The third study investigated the microorganisms and functional genes (xenA, xenB and xplA) linked to RDX biodegradation in microcosms composed of sediments or groundwater from two RDX-contaminated Navy sites. Sediment samples from five depths (5 ft to 30 ft) at two wells were studied from one Navy site. Also, groundwater upstream and downstream of an emulsified biobarrier were examined from another Navy site. The study found that phylotypes from Firmicutes, Actinobacteria, Proteobacteria, Acidobacteria and Bacteroidetes benefited from RDX degradation. A notable trend was the increase in xplA and xenB gene copies in the majority of sediment microcosms derived from one Navy site compared to the controls. Gene copies of xenA increased in a smaller number of the treatments. Interestingly, Pseudomonas (previously associated with xenA and xenB) and Rhodococcus (associated with xplA) also illustrated a high level of enrichment in many of these RDX degrading microcosms. The data provide insight into the microorganisms linked to in situ RDX degradation. Further, the functional gene primers designed in this study could be used to facilitate the prediction of RDX biodegradation rates at contaminated sites.