|De Lorme, M|
Submitted to: Microbial Ecology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/19/2011
Publication Date: 2/22/2011
Citation: Eaton, H.L., De Lorme, M., Chaney, R.L., Craig, A.M. 2011. Identification of ovine ruminal microbes capable of biotransforming hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Microbial Ecology. 62:274-286. Interpretive Summary: Residues of explosives such as TNT and RDX in the environment cause harm to both wildlife and humans. Thus, inexpensive methods are needed to detoxify/remediate soils contaminated with such compounds. Our research has been developing a new approach for bioremediation of soil organic compounds which uses plant uptake of the contaminants and rumen microbes to degrade or otherwise detoxify these compounds (“Phyto-Ruminal Bioremediation”). In the present study, the fate of RDX in rumen microcosms was examined, and the microbial species and strains which accomplished the transformation of RDX were characterized. When RDX was added to rumen microcosms, the RDX was rapidly transformed to bound residues or biodegraded such that little or none of the parent compound or usual metabolites remained in the solution phase. The method of enrichment culture was used to select for microbial strains which metabolized RDX. After selection, microbial populations were identified which very rapidly transformed all of the added RDX. By using 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting, the species which accomplished effective RDX transformation were characterized. Both common ruminal microbes and unusual ruminal microbes were capable of rapid transformation of RDX. This increased rate of metabolism can be induced in rumen microbes by continuous exposure, or by selection of inoculant microbes or plasmids to transfer the ability to ruminnats which will be used to graze the plant biomass at a phytoremediation site with RDX contaminated soils. Additional studies with 14C-RDX are needed to further characterize the RDX transformation vs. biodegradation during normal rumen metabolism by sheep and other ruminants.
Technical Abstract: Bioremediation is of great interest in the detoxification of soil contaminated with residues from explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although there are numerous forms of in situ and ex situ bioremediation, ruminants would provide the option of an in situ bioreactor that could be transported to the site of contamination. Bovine rumen fluid has been previously shown to transform 2,4,6-trinitrotoluene (TNT), a similar compound, in four hours. In this study, RDX incubated in whole ovine rumen fluid was nearly eliminated within four hours. Whole ovine rumen fluid was then inoculated into five different types of media to select for archaeal and bacterial organisms capable of RDX biotransformation. Cultures containing 30 µg mL-1 RDX were transferred each time the RDX concentration decreased to 5 µg mL-1 or less. Time point samples were analyzed for RDX biotransforma-tion by HPLC. The two fastest transforming enrichments were in methanogenic and low nitrogen basal media. After 21 days, DNA was extracted from all enrichments able to partially or completely transform RDX in seven days or less. To understand microbial diversity, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted. Cloning and sequencing of partial 16S rRNA fragments were performed on both low nitrogen basal and methanogenic media enrichments. Phylogenetic analysis revealed similar homologies to eight different bacterial and one archaeal genera classified under the phyla Firmicutes, Actinobacteria, and Euryarchaeota. After continuing enrichment for RDX- degraders for one year, two consortia remained: one that transformed RDX in 4 days, and one which had slowed after two months of transfers without RDX. DGGE comparison of the slower transforming consortium to the faster one showed identical banding patterns except one band. Homology matches to clones from the two consortia identified the same uncultured Clostridia genus in both; Sporanaerobacter acetigenes was identified only in the consortia able to completely transform RDX. This is the first study to examine the rumen as a potential bioremediation tool for soils contaminated with RDX, as well as to discover S. acetigenes in the rumen and its potential ability to metabolize this energetic compound.