Ovine ruminal microbes are capable of biotransforming hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

TitleOvine ruminal microbes are capable of biotransforming hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).
Publication TypeJournal Article
Year of Publication2011
AuthorsEaton HL, De Lorme M, Chaney RL, Craig AM
JournalMicrobial ecology
Volume62
Issue2
Pagination274-86
Date Published2011 Aug
ISSN1432-184X
KeywordsAnimals, Archaea, Bacteria, Biodegradation, Environmental, Chromatography, High Pressure Liquid, Culture Media, Denaturing Gradient Gel Electrophoresis, DNA, Archaeal, DNA, Bacterial, Genes, rRNA, Genetic Variation, Male, Microbial Consortia, Nitrogen, Phylogeny, Rumen, Sequence Homology, Nucleic Acid, Sheep, Time Factors, Triazines
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 4 h. In this study, RDX incubated in whole ovine rumen fluid was nearly eliminated within 4 h. 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 biotransformation 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 7 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 1 year, two consortia remained: one that transformed RDX in 4 days and one which had slowed after 2 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.

Alternate JournalMicrob. Ecol.