Development of Biomarkers for Assessing In Situ RDX Biodegradation Potential.

摘要

This project addresses the problem of widespread RDX contamination at DoD sites. The objective was to identify the microorganisms responsible for the biodegradation of RDX in complex, mixed culture samples through the application of stable isotope probing (SIP). This approach identifies microorganisms responsible for a particular function without cultivation therefore, RDX degradation can ultimately be studied under conditions more similar to those at contaminated sites. Additionally, only active organisms are targeted. For this, RDX degrading microcosms were exposed to labeled RDX and after an incubation period DNA was extracted ultracentrifuged (to separate the labeled nucleic acid from the unlabeled background nucleic acid) and finally molecular analysis steps (terminal restriction fragment length polymorphism TRFLP, 16S rRNA gene sequencing) were performed to identify the organisms responsible for label uptake from RDX. Two RDX concentrations were examined (10 and 20 ppm), however only the higher concentration resulted in a significant SIP signal. In these ultracentrifugation fractions only one TRFLP fragment (260 bp) showed a reliable trend of label uptake. Specifically, this fragment was of higher relative abundance in the heavier fractions from labeled samples compared to the heavier fractions from the unlabeled control samples. Partial 16S rRNA gene sequencing indicated the organisms represented by fragment 260 bp belonged to either the Sphingobacteria or the Acidobacteria. In conclusion, the proof-of-concept was achieved and the methods could be applied to other RDX transforming cultures or environmental samples to determine additional RDX degraders in complex samples and thus biomarkers for assessing the feasibility of natural attenuation.