Current Microbiology

, Volume 65, Issue 2, pp 195–201 | Cite as

Biochemical and Microbial Analysis of Ovine Rumen Fluid Incubated with 1,3,5-Trinitro-1,3,5-triazacyclohexane (RDX)



In this study, the rumen was assessed for its potential to detoxify RDX using molecular microbial ecology as well as analytical chemistry techniques. Results indicated significant loss (P < 0.05) of RDX in <8-h post incubation, and qualitative LC-MS/MS analysis showed evidence for the formation of 1-NO-RDX (M–O + HCOO) and methylenedinitramine metabolites. A total of 1106 16S rRNA-V3 clones were sequenced, and most sequences associated with either the phyla Bacteroidetes or Firmicutes. A LibCompare analysis for the RDX treatment showed an enrichment (P < 0.01) of the genus Prevotella. From these results, it can be concluded that the rumen is capable of detoxifying RDX, and the members of the genus Prevotella are linked to this detoxification.



The research was supported by a jointly funded grant by the Oregon Agricultural Experiment Station project ORE00871 and by the U.S. Department of Agriculture under project number 6227-21310-007-00D agreement nos. 58-6227-8-044 and 58-1265-6-076. Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. The authors would like to thank Ms. Karen Walker for her help with HPLC, Lia Murty during the LC-MS/MS analysis, and Ms. Zelda Zimmerman for editorial assistance.

Supplementary material

284_2012_144_MOESM1_ESM.doc (44 kb)
Supplementary material 1 (DOC 43 kb)


  1. 1.
    Adrian NR, Arnett CM (2006) Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) serves as a carbon and energy source for a mixed culture under anaerobic conditions. Curr Microbiol 53:129–134PubMedCrossRefGoogle Scholar
  2. 2.
    Agency for Toxic substances and Disease Registry (1995) Toxicological profile for RDX: U.S. Department of Health and Human Services, Public Health Service, Atlanta, GAGoogle Scholar
  3. 3.
    Allison MJ, Cook HM, Dawson KA (1981) Selection of oxalate degrading rumen bacteria in continuous cultures. J Anim Sci 53:810–816Google Scholar
  4. 4.
    An D, Dong X, Dong Z (2005) Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses. Anaerobe 11:207–215PubMedCrossRefGoogle Scholar
  5. 5.
    Anderson RC, Rasmussen MA, DiSpirito AA, Allison MJ (1997) Characteristics of a nitropropanol-metabolizing bacterium isolated from the rumen. Can J Microbiol 43:617–624PubMedCrossRefGoogle Scholar
  6. 6.
    Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW (2010) GenBank. Nucleic Acids Res 38:D46–D51PubMedCrossRefGoogle Scholar
  7. 7.
    Bhushan B, Trott S, Spain JC, Halasz A, Paquet L, Hawari J (2003) Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22. Appl Environ Microbiol 69:1347–1351PubMedCrossRefGoogle Scholar
  8. 8.
    Chunlong Z, George NB (2005) Biodegradation of xenobiotics by anaerobic bacteria. Appl Microbiol Biotechnol V67:600–618Google Scholar
  9. 9.
    Claesson MJ, O’Sullivan O, Wang Q, Nikkila J, Marchesi JR, Smidt H, de Vos WM, Ross RP, O’Toole PW (2009) Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine. PLoS ONE 4:e6669PubMedCrossRefGoogle Scholar
  10. 10.
    Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, O’Toole PW (2010) Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38:e200PubMedCrossRefGoogle Scholar
  11. 11.
    Crocker FH, Indest KJ, Fredrickson HL (2006) Biodegradation of the cyclic nitramine explosives RDX, HMX, and CL-20. Appl Microbiol Biotechnol 73:274–290PubMedCrossRefGoogle Scholar
  12. 12.
    De Lorme M, Craig M (2009) Biotransformation of 2,4,6-trinitrotoluene by pure culture ruminal bacteria. Curr Microbiol 58:81–86PubMedCrossRefGoogle Scholar
  13. 13.
    Dominguez-Bello MG, Lovera M, Rincon MT (1997) Characteristics of dihydroxypyridine-degrading activity in the rumen bacterium Synergistes jonesii. FEMS Microbiol Ecol 23:361–365CrossRefGoogle Scholar
  14. 14.
    Drummond AJ, Ashton B, Cheung M, Heled J, Kearse M, Moir R, Stones-Havas S, Thierer T, Wilson A (2007) Geneious v3.7. (
  15. 15.
    Eaton HL, De Lorme M, Chaney RL, Craig AM (2011) Ovine ruminal microbes are capable of biotransforming hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Microb Ecol 62:274–286PubMedCrossRefGoogle Scholar
  16. 16.
    Edwards JE, McEwan NR, Travis AJ, John R (2004) 16S rDNA library-based analysis of ruminal bacterial diversity. Antonie Van Leeuwenhoek 86:263–281CrossRefGoogle Scholar
  17. 17.
    Elbing K, Brent R (2002) Media preparation and bacteriological tools. Curr Protoc Mol Biol 59:1.1.1–1.1.7Google Scholar
  18. 18.
    Engebrecht J, Brent R, Kaderbhai MA (2000) Alkaline lysis in 96 well microtiter dishes. Curr Protoc Mol Biol 15:1.6.2–1.6.3Google Scholar
  19. 19.
    Godoy-Vitorino F, Ley RE, Gao Z, Pei Z, Ortiz-Zuazaga H, Pericchi LR, Garcia-Amado MA, Michelangeli F, Blaser MJ, Gordon JI, Dominguez-Bello MG (2008) Bacterial community in the crop of the hoatzin, a neotropical folivorous flying bird. Appl Environ Microbiol 74:5905–5912PubMedCrossRefGoogle Scholar
  20. 20.
    Hawari J, Halasz A, Sheremata T, Beaudet S, Groom C, Paquet L, Rhofir C, Ampleman G, Thiboutot S (2000) Characterization of metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) with municipal anaerobic sludge. Appl Environ Microbiol 66:2652PubMedCrossRefGoogle Scholar
  21. 21.
    Hawari J, Halasz A, Groom C, Deschamps S, Paquet L, Beaulieu C, Corriveau A (2002) Photodegradation of RDX in aqueous solution: a mechanistic probe for biodegradation with Rhodococcus sp. Environ Sci Technol 36:5117–5123PubMedCrossRefGoogle Scholar
  22. 22.
    Hobson PN, Stewart CS (1988) The rumen microbial ecosystem. Chapman & Hall, New YorkGoogle Scholar
  23. 23.
    Huse SM, Dethlefsen L, Huber JA, Mark Welch D, Relman DA, Sogin ML (2008) Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing. PLoS Genet 4:e1000255PubMedCrossRefGoogle Scholar
  24. 24.
    Koike S, Yoshitani S, Kobayashi Y, Tanaka K (2003) Phylogenetic analysis of fiber-associated rumen bacterial community and PCR detection of uncultured bacteria. FEMS Microbiol Lett 229:23–30PubMedCrossRefGoogle Scholar
  25. 25.
    Lou J, Dawson KA, Strobel HJ (1996) Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola. Appl Environ Microbiol 62:1770PubMedGoogle Scholar
  26. 26.
    McCormick NG, Cornell JH, Kaplan AM (1981) Biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine. Appl Environ Microbiol 42:817–823PubMedGoogle Scholar
  27. 27.
    Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700PubMedGoogle Scholar
  28. 28.
    Nelson KE, Zinder SH, Hance I, burr P, Odongo D, Wasawo D, Odenyo A, Bishop R (2003) Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche. Environ Microbiol 5:1212–1220PubMedCrossRefGoogle Scholar
  29. 29.
    Paquet L, Monteil-Rivera F, Hatzinger PB, Fuller ME, Hawari J (2011) Analysis of the key intermediates of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) in groundwater: occurrence, stability and preservation. J Environ Monit 13:2304–2311PubMedCrossRefGoogle Scholar
  30. 30.
    Perumbakkam S, Craig AM (2011) Design and in vitro evaluation of new rpoB-DGGE primers for ruminants. FEMS Microbiol Ecol 76:156–169PubMedCrossRefGoogle Scholar
  31. 31.
    Perumbakkam S, Mitchell EA, Morrie Craig A (2011) Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet. Antonie Van Leeuwenhoek 99:231–240PubMedCrossRefGoogle Scholar
  32. 32.
    Rattray RM, Craig AM (2007) Molecular characterization of sheep ruminal enrichments that detoxify pyrrolizidine alkaloids by denaturing gradient gel electrophoresis and cloning. Microb Ecol 54:264–275PubMedCrossRefGoogle Scholar
  33. 33.
    Russell JB, Rychlik JL (2001) Factors that alter rumen microbial ecology. Science 292:1119–1122PubMedCrossRefGoogle Scholar
  34. 34.
    Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541PubMedCrossRefGoogle Scholar
  35. 35.
    Smith DJ, Craig AM, Duringer JM, Chaney RL (2008) Absorption, tissue distribution, and elimination of residues after 2,4,6-trinitro[14C]toluene administration to sheep. Environ Sci Technol 42:2563–2569PubMedCrossRefGoogle Scholar
  36. 36.
    Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored “rare biosphere”. PNAS 103:12115PubMedCrossRefGoogle Scholar
  37. 37.
    Spain JC, Hughes JB, Knackmuss HJ (2000) Biodegradation of nitroaromatic compounds and explosives. CRC Press, Boca RatonGoogle Scholar
  38. 38.
    Tajima K, Aminov RI, Nagamine T, Matsui H, Nakmura M, Benno Y (2001) Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Appl Environ Microbiol 67:2766–2774PubMedCrossRefGoogle Scholar
  39. 39.
    Turnbaugh PJ, Ley RE, Mahowald MA, Vincent Mardis ER, Gordon JI (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027–1131PubMedCrossRefGoogle Scholar
  40. 40.
    Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI (2007) The human microbiome project. Nature 449:804–810PubMedCrossRefGoogle Scholar
  41. 41.
    U.S. Environmental Protection Agency (2006) Method 8330B (SW-846): Nitroaromatics, nitramines, and nitrate esters by high performance liquid chromatography (HPLC). U.S. Environmental Protection Agency, Washington, DC, p 60Google Scholar
  42. 42.
    Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267PubMedCrossRefGoogle Scholar
  43. 43.
    Whitford MF, Forster RJ, Beard CE, Gong J, Teather RM (1998) Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes. Anaerobe 4:153–163PubMedCrossRefGoogle Scholar
  44. 44.
    Williams LR, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon DI, Braga MF (2011) RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures. Environ Health Perspect 119:357–363PubMedCrossRefGoogle Scholar
  45. 45.
    Zhao JS, Manno D, Hawari J (2008) Regulation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolism in Shewanella halifaxensis HAW-EB4 by terminal electron acceptor and involvement of c-type cytochrome. Microbiology 154:1026–1037PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Environmental and Molecular ToxicologyOregon State UniversityCorvallisUSA
  2. 2.Department of Veterinary MedicineOregon State UniversityCorvallisUSA

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