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Anaerobic bioremediation of RDX by ovine whole rumen fluid and pure culture isolates

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Abstract

The ability of ruminal microbes to degrade the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in ovine whole rumen fluid (WRF) and as 24 bacterial isolates was examined under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography analysis, followed by liquid chromatography–tandem mass spectrometry identification of metabolites. Organisms in WRF microcosms degraded 180 μM RDX within 4 h. Nitroso-intermediates hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) were present as early as 0.25 h and were detected throughout the 24-h incubation period, representing one reductive pathway of ring cleavage. Following reduction to MNX, peaks consistent with m/z 193 and 174 were also produced, which were unstable and resulted in rapid ring cleavage to a common metabolite consistent with an m/z of 149. These represent two additional reductive pathways for RDX degradation in ovine WRF, which have not been previously reported. The 24 ruminal isolates degraded RDX with varying efficiencies (0–96 %) over 120 h. Of the most efficient degraders identified, Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. desulfuricans degraded RDX when medium was supplemented with both nitrogen and carbon, while Anaerovibrio lipolyticus, Prevotella ruminicola, and Streptococcus bovis IFO utilized RDX as a sole source of nitrogen. This study showed that organisms in whole rumen fluid, as well as several ruminal isolates, have the ability to degrade RDX in vitro and, for the first time, delineated the metabolic pathway for its biodegradation.

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References

  • Adrian NR, Arnett CM, Hickey RF (2003) Stimulating the anaerobic biodegradation of explosives by the addition of hydrogen or electron donors that produce hydrogen. Water Res 37:3499–3507

    Article  CAS  Google Scholar 

  • Agency for Toxic Substances and Disease Registry (1995) Toxicological profile for RDX. US Department of Health and Human Services, Public Health Service, Atlanta

    Google Scholar 

  • Arnett CM, Adrian NR (2009) Cosubstrate independent mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a Desulfovibrio species under anaerobic conditions. Biodegradation 20:15–26

    Article  CAS  Google Scholar 

  • Axtell C, Johnston CG, Bumpus JA (2000) Bioremediation of soil contaminated with explosives at the Naval Weapons Station Yorktown. Soil Sediment Contam 9:537–548

    Article  CAS  Google Scholar 

  • Banerjee HM, Verma M, Hou L-H, Ashraf M, Dutta SK (1999) Cytotoxicity of TNT and its metabolites. Yale J Biol Med 72:1–4

    CAS  Google Scholar 

  • Bayman P, Ritchey SD, Bennett JW (1995) Fungal interactions with the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). J Ind Microbiol 15:418–423

    Article  CAS  Google Scholar 

  • Best EPH, Sprecher SL, Larson SL, Fredrickson HL, Bader DF (1999) Environmental behavior of explosives in groundwater from the Milan army ammunition plant in aquatic and wetland plant treatments. Removal, mass balances and fate in groundwater of TNT and RDX. Chemosphere 38:3383–3396

    Article  CAS  Google Scholar 

  • Boopathy R, Manning J, Kulpa CF (1998) Biotransformation of explosives by anaerobic consortia in liquid culture and in soil slurry. Int Biodeterior Biodegrad 41:67–74

    Article  CAS  Google Scholar 

  • Borton C, Olson L (2006) Trace level analysis of explosives in ground water and soil. Application note: explosive analysis. Forster City, CA, Applied Biosystems, pp 1–6

  • Brooks LR, Jacobson RW, Warren SH, Kohan MJ, Donnelly KC, George SE (1997) Mutagenicity of HPLC-fractionated urinary metabolites from 2,4,6-trinitrotoluene-treated Fischer 344 rats. Environ Mol Mutag 30:298–302

    Article  CAS  Google Scholar 

  • Clausen J, Robb J, Curry D, Korte N (2004) A case study of contaminants on military ranges: Camp Edwards, Massachusetts, USA. Environ Pollut 129:13–21

    Article  CAS  Google Scholar 

  • Coleman NV, Nelson DR, Duxbury T (1998) Aerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as a nitrogen source by a Rhodococcus sp., strain DN22. Soil Biol Biochem 30:1159–1167

    Article  Google Scholar 

  • Crocker FH, Thompson KT, Szecsody JE, Fredrickson HL (2005) Biotic and abiotic degradation of CL-20 and RDX in soils. J Environ Qual 34:2208–2216

    Article  CAS  Google Scholar 

  • Crocker FH, Indest KJ, Fredrickson HL (2006) Biodegradation of the cyclic nitramine explosives RDX, HMX, and CL-20. Appl Microbiol Biotechnol 73:274–290

    Article  CAS  Google Scholar 

  • Daniels J.I. and Knezovich J.P. (1994) Human health risks from TNT, RDX, and HMX in environmental media and consideration of the U.S. Regulatory Environment. In International Symposium on the Rehabilitation of Former Military Sites and Demilitarization of Explosive Ordnance. Kirchberg, Luxembourg: Proceedings Demil '94

  • De Lorme M. and Craig A.M. (2009) Biotransformation of 2,4,6-trinitrotoluene by pure culture ruminal bacteria. Curr Microbiol 58: 81–86

    Google Scholar 

  • Duringer JM, Craig AM, Smith DJ, Chaney RL (2010) Uptake and transformation of soil [14-C]-trinitrotoluene by cool-season grasses. Environ Sci Technol 44:6325–6330

    Article  CAS  Google Scholar 

  • Eaton H.L., De Lorme M., Chaney R. and Craig A.M. (2011) Ovine ruminal microbes are capable of biotransforming hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Microb Ecol 62:274–286

    Google Scholar 

  • Environmental Protection Agency US (1993) Risk estimate for carcinogenicity and reference dose for oral exposure for 2, 4, 6-trinitrotoluene. Office of Health and Environmental Assessment, US EPA, Washington, DC

    Google Scholar 

  • Fleischmann TJ, Walker KC, Spain JC, Hughes JB, Morrie Craig A (2004) Anaerobic transformation of 2,4,6-TNT by bovine ruminal microbes. Biochem Biophys Res Commun 314:957–963

    Article  CAS  Google Scholar 

  • Fournier D, Trott S, Hawari J, Spain J (2005) Metabolism of the aliphatic nitramine 4-nitro-2,4-diazabutanal by Methylobacterium sp. strain JS178. Appl Environ Microbiol 71:4199–4202

    Article  CAS  Google Scholar 

  • Fuller ME, Lowey JM, Schaefer CE, Steffan RJ (2005) A peat moss-based technology for mitigating residues of the explosives TNT, RDX, and HMX in soil. Soil Sediment Contam 14:373–385

    Article  CAS  Google Scholar 

  • Fuller ME, McClay K, Hawari J, Paquet L, Malone TE, Fox BG, Steffan RJ (2009) Transformation of RDX and other energetic compounds by xenobiotic reductases XenA and XenB. Appl Microbiol Biotechnol 84:535–544

    Article  CAS  Google Scholar 

  • Gust KA, Pirooznia M, Quinn MJ Jr, Johnson MS, Escalon L, Indest KJ, Guan X, Clarke J, Deng Y, Gong P, Perkins EJ (2009) Neurotoxicogenomic investigations to assess mechanisms of action of the munitions constituents RDX and 2,6-DNT in Northern Bobwhite (Colinus virginianus). Toxicol Sci 110:168–180

    Article  CAS  Google Scholar 

  • Hawari J, Beaudet S, Halasz A, Thiboutot S, Ampleman G (2000a) Microbial degradation of explosives: biotransformation versus mineralization. Appl Microbiol Biotechnol 54:605–618

    Article  CAS  Google Scholar 

  • Hawari J, Halasz A, Sheremata T, Beaudet S, Groom C, Paquet L, Rhofir C, Ampleman G, Thiboutot S (2000b) Characterization of metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) with municipal anaerobic sludge. Appl Environ Microbiol 66:2652–2657

    Article  CAS  Google Scholar 

  • Hesselmann RMX, Stenstrom MK (1994) Treatment concept for RDX-containing wastewaters using activated carbon with offline solvent biological regeneration. CA University of California, Los Angeles, Los Angeles

    Google Scholar 

  • Hobson PN, Stewart CS (1997) The rumen microbial ecosystem. Blackie Academic and Professional, New York

    Book  Google Scholar 

  • Krishnan IS, Singhal RK, Dua RD (1986) Purification and characterization of glutamine synthetase from Clostridium pasteurianum. Biochemistry (Mosc) 25:1589–1599

    Article  CAS  Google Scholar 

  • Kumada Y, Benson DR, Hillemann D, Hosted TJ, Rochefort DA, Thompson CJ, Wohlleben W, Tateno Y (1993) Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proc Natl Acad Sci 90:3009–3013

    Article  CAS  Google Scholar 

  • Kwon MJ, Finneran KT (2006) Microbially mediated biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine by extracellular electron shuttling compounds. Appl Environ Microbiol 72:5933–5941

    Google Scholar 

  • McCormick NG, Feeherry FE, Levinson HS (1976) Microbial transformation of 2,4,6-trinitrotoluene and other nitroaromatic compounds. Appl Environ Microbiol 31:949–958

    CAS  Google Scholar 

  • Prescott LM, Harley JP, Klein DA (2005) Microbiology. McGraw-Hill, New York

    Google Scholar 

  • Qasim MM, Moore B, Taylor L, Honea P, Gorb L, Leszczynski J (2007) Structural characteristics and reactivity relationships of nitroaromatic and nitramine explosives—a review of our computational chemistry and spectroscopic research. Int J Mol Sci 8:1234–1264

    Article  CAS  Google Scholar 

  • Sejrsen K, Hvelplund T, Nielsen MO (2006) Ruminant physiology: digestion, metabolism and impact of nutrition on gene expression, immunology and stress. Wageningen Academic, Wageningen

    Google Scholar 

  • Smith DJ, Craig AM, Duringer JM, Chaney RL (2008) Absorption, tissue distribution, and elimination of residues after 2,4,6-trinitro[14 C]toluene administration to sheep. Environ Sci Technol 42:2563–2569

    Article  CAS  Google Scholar 

  • Stenuit BA, Agathos SN (2010) Microbial 2,4,6-trinitrotoluene degradation: could we learn from (bio)chemistry for bioremediation and vice versa? Appl Microbiol Biotechnol 88:1043–1064

    Article  CAS  Google Scholar 

  • Sunahara GI, Guilherme L, Kuperman RG, Hawari J (2009) Ecotoxicology of explosives. CRC, Boca Raton

    Google Scholar 

  • Talley JW, Sleeper PM (1997) Roadblocks to the implementation of biotreatment strategies. Ann New York Acad Sci 829:16–29

    Article  CAS  Google Scholar 

  • Turley SD, Burton DT (1995) Reduction of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) toxicity to the Cladoceran Ceriodaphniadubia following photolysis in sunlight. Bull Environ Contam Toxicol 55:89–95

    Article  Google Scholar 

  • U. S. Environmental Protection Agency (2007) Method 8330A (SW-846): Nitroaromatics and nitramines by high performance liquid chromatography (HPLC). EPA, Washington, DC

    Google Scholar 

  • U.S. Environmental Protection Agency (2009) Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Environmental Protection Agency, Washington, DC

    Google Scholar 

  • Williams RT, Ziegenfuss PS, Sisk WE (1992) Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions. J Ind Microbiol 9:137–144

    Article  Google Scholar 

  • Zhang C, Hughes JB (2003) Biodegradation pathways of hexahydro-1,3,5-trinitro- 1,3,5-triazine (RDX) by Clostridium acetobutylicum cell-free extract. Chemosphere 50:665–671

    Article  CAS  Google Scholar 

  • Zhao JS, Paquet L, Halasz A, Hawari J (2003) Metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine through initial reduction to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine followed by denitration in Clostridium bifermentans HAW-1. Appl Microbiol Biotechnol 63:187–193

    Article  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Michael Wiens for his technical assistance. This research was supported in part by a gift from Ruminant Solutions, LLC (New Mexico), the Oregon Agricultural Experiment Station project no. ORE00871, and the US Department of Agriculture, Agriculture Research Service project no. 50-1265-6-076. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the US Department of Agriculture.

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Authors and Affiliations

  1. Department of Microbiology, Oregon State University, Corvallis, OR, 97331, USA

    H. L. Eaton

  2. Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, 97331, USA

    J. M. Duringer

  3. Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR, 97331, USA

    L. D. Murty

  4. Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, 101 Magruder Hall, Corvallis, OR, 97331, USA

    A. M. Craig

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  1. H. L. Eaton
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Correspondence to A. M. Craig.

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Eaton, H.L., Duringer, J.M., Murty, L.D. et al. Anaerobic bioremediation of RDX by ovine whole rumen fluid and pure culture isolates. Appl Microbiol Biotechnol 97, 3699–3710 (2013). https://doi.org/10.1007/s00253-012-4172-3

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