Graduate Thesis Or Dissertation
 

Anaerobic biodegradation of the cyclic nitramines, RDX and HMX, by ovine ruminal microbes

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  • Cyclic nitramines released into all environmental compartments through anthropogenic activities are toxic and possibly carcinogenic and mutagenic. Soils on military ranges, located throughout the world in various climatic regions and close to human activities, are especially susceptible to cyclic nitramine contamination. The properties of soils on military ranges will directly affect the cyclic nitramine contamination and biodegradation; however, studies in this area are limited. Previous related research was mainly focused on monitoring the temporal and spatial changes in nitrate explosive concentrations and various in situ and ex situ remediation techniques. Anaerobic biodegradation of cyclic nitramines in soils is scarce, but appears to be a more efficient means of bioremediation than aerobic techniques; however, most published research has not been applied in situ as there are numerous hindrances to applications in the field. The present research therefore aims to study the feasibility of ruminal bioremediation as an inexpensive, ecologically conscious, and viable means to remediate soils on military ranges contaminated with cyclic nitramines by (1) determining if RDX degradation in whole ovine rumen fluid occurs and isolating and identifying organisms capable of degradation through enrichments; (2) evaluating the ability of 24 commonly isolated bacteria from the rumen to degrade RDX and determining the metabolite pathway by capable isolates, as well as by consortia in whole rumen fluid; and (3) exploring HMX degradation in whole ovine rumen fluid and identifying the HMX-degradation pathway in whole rumen fluid and by capable isolates tested. 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) finger- printing 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 microbes to utilize the explosive compound RDX, in both ovine whole rumen fluid and 24 individual bacterial isolates from the rumen was examined. Compound degradation was determined by high performance liquid chromatography (HPLC) analysis, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of metabolites. Organisms in whole rumen fluid microcosms were able to degrade 180 μM RDX within 4 h. In whole rumen fluid, the concentrations of all mono-, di-, and tri-nitroso intermediates formed due to reduction of the nitro groups on RDX amounted to approximately 9 uM at 24 hours, which represented one pathway to ring cleavage. The appearance of peak m/z 175, hexahydro-1,3-dinitro-1,3,5-triazine, represented a second pathway that RDX followed after reduction to the first nitroso intermediate, MNX. Ruminal isolates were able to degrade RDX between ranges of 34 to 256 μM in 120 hours to unidentified ring cleavage metabolites. Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. desulfuricans were able to degrade RDX when it was supplemented in addition to nitrogen and carbon. Anaerovibrio lipolytica, Prevotella ruminicola and Streptococcus bovis IFO were able to degrade RDX when supplemented as a sole source of nitrogen. This data indicated that while several rumen bacterial strains may have the individual ability to degrade RDX, bioremediation in whole rumen fluid was more efficient. We proposed a pathway of RDX degradation by ruminal microbes under anaerobic conditions that involved the reduction of RDX to MNX, with trace amounts further reduced to DNX and TNX. The majority of the MNX was further degraded via a second pathway to hexahydro- 1,3-dinitro-1,3,5-triazine (peak m/z 175), which appeared to be an unstable molecule that resulted in rapid ring cleavage and degradation to unidentified metabolites. This was the first study to demonstrate that whole rumen fluid would be a more efficient means of bioremediation than individual ruminal isolates, based on the speed and completion of degradation of RDX; and that several ruminal isolates had the ability to degrade RDX in vitro. The octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) molecule is an eight-membered ring of alternating carbon and nitrogen atoms, with a nitro group attached to each nitrogen atom. The ability of ruminal microbes to degrade HMX as consortia in whole rumen fluid and as 23 common ruminal isolates was examined by LC-MS/MS analysis. The initial concentration of HMX was 27 μM and whole rumen fluid was incubated with HMX for 24 hours under anaerobic conditions in the dark, while the isolates were incubated with 17 μM HMX for 120 hours, along with negative controls. All experiments were repeated in triplicate. Our results demonstrated that HMX was nearly completely degraded in whole rumen fluid in four hours. Peaks at m/z 149 and m/z 193 suggest ring cleavage through the mono-nitroso intermediate, (1-NO-HMX), reduction pathway; and via hydroxylamino-HMX derivatives, as a second pathway. None of the 23 ruminal isolates tested were able to degrade HMX when supplemented in either a low carbon basal medium or low nitrogen basal medium in pure culture in 120 hours under the conditions specified. This is the first study to prove ruminal microbes can degrade HMX and to propose metabolic pathways, which could lead to the application of ruminal bioremediation on contaminated military soils. In summary, the present research revealed that ruminal organisms were not only capable of degrading the cyclic nitramines, RDX and HMX, in less than 24 hours, but the efficiency of degradation in the strongly reduced environment of the rumen surpassed other means of remediating explosive compounds through aerobic or alternate anaerobic techniques.
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