No abstract

The potential for bioaugmentation with aerobic explosive degrading bacteria to remediate hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) contaminated aquifers was demonstrated. Repacked aquifer sediment columns were used to examine the transport and RDX degradation capacity of the known RDX degrading bacterial strains Gordonia sp. KTR9 (modified with a kanamycin resistance gene) Pseudomonas fluorescens I-C, and a kanamycin resistant transconjugate Rhodococcus jostii RHA1 pGKT2:Km+. All three strains were transported through the columns and eluted ahead of the conservative bromide tracer, although the total breakthrough varied by strain. The introduced cells responded to biostimulation with fructose (18 mg L(-1), 0.1 mM) by degrading dissolved RDX (0.5 mg L(-1), 2.3 µM). The strains retained RDX-degrading activity for at least 6 months following periods of starvation when no fructose was supplied to the column. Post-experiment analysis of the soil indicated that the residual cells were distributed along the length of the column. When the strains were grown to densities relevant for field-scale application, the cells remained viable and able to degrade RDX for at least 3 months when stored at 4 °C. These results indicate that bioaugmentation may be a viable option for treating RDX in large dilute aerobic plumes.

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.

The ability of ruminal microorganisms to degrade octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (high melting explosive, HMX) as consortia from whole rumen fluid (WRF), and individually as 23 commercially available ruminal strains, was compared under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for delineation of the metabolic pathway. In WRF, 30 μM HMX was degraded to 5 μM HMX within 24 h. Metabolites consistent with m/z 149, 193 and 229 were present throughout the incubation period. We propose that peaks with an m/z of 149 and 193 are arrived at through reduction of HMX to nitroso or hydroxylamino intermediates, then direct enzymatic ring cleavage to produce these HMX derivatives. Possible structures of m/z 229 are still being investigated and require further LC-MS/MS analysis. None of the 23 ruminal strains tested were able to degrade HMX as a pure culture when grown in either a low carbon or low nitrogen basal medium over 120 h. We conclude that microorganisms from the rumen, while sometimes capable as individuals in the bioremediation of other explosives, excel as a community in the case of HMX breakdown.

Background/Aims: Termites have an important role in the carbon and nitrogen cycles despite their reputation as destructive pests. With the assistance of microbial endosymbionts, termites are responsible for the conversion of complex biopolymers into simple carbon substrates. Termites also rely on endosymbionts for fixing and recycling nitrogen. As a result, we hypothesize that termite bacterial endosymbionts are a novel source of metabolic pathways for the transformation of nitrogen-rich compounds like explosives. Methods: Explosives transformation capability of termite (Reticulitermes flavipes)-derived endosymbionts was determined in media containing the chemical constituents nitrotriazolone (NTO) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) that comprise new insensitive explosive formulations. Media dosed with 40 µg/ml of explosive was inoculated with surface-sterilized, macerated termites. Bacterial isolates capable of explosives transformation were characterized by 16S rRNA sequencing. Results: Termite-derived enrichment cultures demonstrated degradation activity towards the explosives NTO, RDX, as well as the legacy explosive 2,4,6-trinitrotoluene (TNT). Three isolates with high similarity to the Enterobacteriaceae(Enterobacter, Klebsiella) were able to transform TNT and NTO within 2 days, while isolates with high similarity to Serratia marcescens and Lactococcus lactis were able to transform RDX. Conclusion: Termite endosymbionts harbor a range of metabolic activities and possess unique abilities to transform nitrogen-rich explosives.