Skip to main content

Advertisement

SpringerLink
Log in

Ovine Ruminal Microbes Are Capable of Biotransforming Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX)

  • Environmental Microbiology
  • Published:
Microbial Ecology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. 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  PubMed  CAS  Google Scholar 

  2. Agency for Toxic substances and Disease Registry. Toxicological profile for RDX. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service, 1995.

  3. Alfreider A, Vogt C (2007) Bacterial diversity and aerobic biodegradation potential in a BTEX-contaminated aquifer. Water Air Soil Pollut 183:415–426

    Article  CAS  Google Scholar 

  4. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  5. An D, Cai S, Dong X (2006) Actinomyces ruminicola sp. nov., isolated from cattle rumen. Int J Syst Evol Microbiol 56:2043–2048

    Article  PubMed  CAS  Google Scholar 

  6. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL (2005) GenBank. Nucleic Acids Res 34:D16–D20

    Article  Google Scholar 

  7. Bhadra R, Wayment DG, Williams RK, Barman SN, Stone MB, Hughes JB, Shanks JV (2001) Studies on plant-mediated fate of the explosives RDX and HMX. Chemosphere 44:1259–1264

    Article  PubMed  CAS  Google Scholar 

  8. Burken JG. Phytoremediation/wetlands treatment at the Iowa Army Ammunition Plant. Columbus, OH: McGraw-Hill Companies, 2001. http://www.mhhe.com/biosci/pae/environmentalscience/casestudies/case12.mhtml.

  9. Chaucheyras F, Fonty G et al (1995) In vitro H2 utilization by a ruminal acetogenic bacterium cultivated alone or in combination with an Archaea methanogen is stimulated by a probiotic strain of Saccharomyces cerevisiae. Appl Environ Microbiol 6(1)

  10. Coppotelli BM, Ibarrolaza A, Del Panno MT, Morelli IS (2008) Effects of the inoculant strain Sphingomonas paucimobilis 20006FA on soil bacterial community and biodegradation in phenanthrene-contaminated soil. Microb Ecol 55:173–183

    Article  PubMed  CAS  Google Scholar 

  11. Daniels JI, Knezovich JP (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

  12. De Lorme M, Craig AM (2008) Biotransformation of 2,4,6-trinitrotoluene by pure culture ruminal bacteria. Curr Microbiol 58:81–86

    Google Scholar 

  13. Delong EF (1992) Archaea in coastal marine environments. Proceedings of the National Academy of Sciences 89:5685–5689

  14. Dworkin M, Falkow S, Rosenburg E, Schleifer KH, Stackebrandt E (2006) The Prokaryotes: Ecophysiology and Biochemistry. New York, NY, Springer

  15. Engebrecht J, Brent R, Kaderbhai M (2001) Minipreps of plasmid DNA. Current protocols in molecular biology. F. M. Ausubel, R. Brent, R. E. Kingston et al., John Wiley & Sons, Inc. doi:10.1002/0471142727.mb0106s15

  16. Felenstein J (2004) PHYLIP (Phylogeny Inference Package) version 3.6. Seattle, WA: J. Felenstein, Department of Genome Sciences, University of Washington

  17. 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  PubMed  CAS  Google Scholar 

  18. 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  PubMed  CAS  Google Scholar 

  19. Greening R, Leedle J (1989) Enrichment and isolation of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen. Archives of Microbiology 151(5):399–406

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

    Article  PubMed  CAS  Google Scholar 

  21. 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 66:2652–2657

    Article  PubMed  CAS  Google Scholar 

  22. Hernandez-Eugenio G, Fardeau ML, Cayol JL, Patel BKC, Thomas P, Macarie H, Garcia JL, Ollivier B (2002) Sporanaerobacter acetigenes gen. nov., sp. nov., a novel acetogenic, facultatively sulfur-reducing bacterium. Int J Syst Evol Microbiol 52:1217–1223

    Article  PubMed  CAS  Google Scholar 

  23. Hobson PN, Stewart CS (1997) The rumen microbial ecosystem. Blackie Academic and Professional, New York, NY, p 709

    Google Scholar 

  24. Hwang K, Shin SG, Kim J, Hwang S (2008) Methanogenic profiles by denaturing gradient gel electrophoresis using order-specific primers in anaerobic sludge digestion. Appl Microbiol Biotechnol 80:269–276

    Article  PubMed  CAS  Google Scholar 

  25. Joblin KN (1999) Ruminal acetogens and their potential to lower ruminant methane emissions. Aust J Agric Res 50:1307–1313

    Article  Google Scholar 

  26. Krumholz LR, Bryant MP (1985) Clostridium pfennigii sp. nov. Uses Methoxyl Groups of Monobenzenoids and Produces Butyrate. Int J Syst Bacteriol 35(4):454–456

    Google Scholar 

  27. Krumholz LR, Bryant MP (1986) Syntrophococcus sucromutans sp. nov. gen. nov. uses carbohydrates as electron donors and formate, methoxymonobenzenoids or Methanobrevibacter as electron acceptor systems. Archives of microbiology 143(4):313–318

  28. Liu C, Finegold SM, Song Y, Lawson PA (2008) Reclassification of Clostridium coccoides, Ruminococcus hansenii, Ruminococcus hydrogenotrophicus, Ruminococcus luti, Ruminococcus productus and Ruminococcus schinkii as Blautia coccoides gen. nov., comb. nov., Blautia hansenii comb. nov., Blautia hydrogenotrophica comb. nov., Blautia luti comb. nov., Blautia producta comb. nov., Blautia schinkii comb. nov. and description of Blautia wexlerae sp. nov., isolated from human faeces. Int J Syst Evol Microbiol 58:1896–1902

    Article  PubMed  CAS  Google Scholar 

  29. Long R, Lappin-Scott H, Stevens J (2009) Enrichment and identification of polycyclic aromatic compound-degrading bacteria enriched from sediment samples. Biodegradation 20:521–531

    Article  PubMed  CAS  Google Scholar 

  30. Ozutsumi Y, Tajima K, Takenaka A, Itabashi H (2005) The effect of protozoa on the composition of rumen bacteria in cattle using 16S rRNA gene clone libraries. Bioscience, Biotechnol and Biochem 69:499–506

    Article  CAS  Google Scholar 

  31. Pearson WR (1991) Searching protein sequence libraries: comparison of the sensitivity and selectivity of the Smith–Waterman and FASTA algorithms. Genomics 11:635–650

    Article  PubMed  CAS  Google Scholar 

  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 54:264–275

    Article  PubMed  CAS  Google Scholar 

  33. Rieu-Lesme F, Fonty G et al (1995) Isolation and characterization of a new hydrogen-utilizing bacterium from the rumen. FEMS Microbiology Letters 125(1):77–82

    Google Scholar 

  34. Rieu-Lesme F, D C et al (1996a) Acetogenic coccoid spore-forming bacteria isolated from the rumen. Research in Microbiology 147(9):753–764

  35. Rieu-Lesme F, Morvan B et al (1996b) A new H2 / CO2-using acetogenic bacterium from the rumen: description of Ruminococcus schinkii sp. nov. FEMS Microbiology Letters 140(2-3):281–286

  36. Rieu-Lesme F, Dauga C et al (1998) Isolation from the rumen of a new acetogenic bacterium phylogenetically closely related to Clostridium difficile. Anaerobe 4(2):89–94

    Google Scholar 

  37. Roling WFM, Couto de Brito IR, Swannell RPJ, Head IM (2004) Response of archaeal communities in beach sediments to spilled oil and bioremediation. Appl Environ Microbiol 70:2614–2620

    Article  PubMed  Google Scholar 

  38. Rylott EL, Jackson RG, Edwards J, Womack GL, Seth-Smith HMB, Rathbone DA, Strand SE, Bruce NC (2006) An explosive-degrading cytochrome p450 activity and its targeted application for the phytoremediation of RDX. Nat Biotechnol 24:216–219

    Article  PubMed  CAS  Google Scholar 

  39. Saia F, Damianovic M, Cattony E, Brucha G, Foresti E, Vazoller R (2007) Anaerobic biodegradation of pentachlorophenol in a fixed-film reactor inoculated with polluted sediment from Santos-São Vicente Estuary. Brazil Appl Microbiol Biotechnol 75:665–672

    Article  CAS  Google Scholar 

  40. Sejrsen K, Hvelplund T, Nielsen MO (2006) Ruminant physiology: digestion, metabolism and impact of nutrition on gene expression, immunology and stress. Wageningen Academic Publishers, Wageningen, The Netherlands, p 600

    Google Scholar 

  41. 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 42:2563–2569

    Article  PubMed  CAS  Google Scholar 

  42. Smith TF, Waterman MS (1981) Identification of common molecular subsequences. J Mol Biol 147:195–197

    Article  PubMed  CAS  Google Scholar 

  43. Sunahara GI, Guilherme L, Kuperman RG, Hawari J (2009) Ecotoxicology of explosives. CRC Press, Boca Raton, FL, p 325

    Google Scholar 

  44. Tajima K, Aminov RI, Nagamine T, Matsui H, Nakamura M, Benno Y (2001) Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Appl Environ Microbiol 67:2766–2774

    Article  PubMed  CAS  Google Scholar 

  45. Tajima K, Aminov RI, Nagamine T, Ogata K, Nakamura M, Matsui H, Benno Y (1999) Rumen bacterial diversity as determined by sequence analysis of 16S rDNA libraries. FEMS Microbiol Ecol 29:159–169

    Article  CAS  Google Scholar 

  46. Talley JW, Sleeper PM (1997) Roadblocks to the implementation of biotreatment strategies. Annals New York Academy of Sciences 829:16–29

    Google Scholar 

  47. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  48. U. S. Environmental Protection Agency (2006) Method 8330B (SW-846): Nitroaromatics, Nitramines, and Nitrate Esters by High Performance Liquid Chromatography (HPLC). Washington, DC: EPA, p. 60

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

  50. Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chater KF, van Sinderen D (2007) Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum. Microbiol Mol Biol Rev 71:495–548

    Article  PubMed  CAS  Google Scholar 

  51. Vila M, Lorber-Pascal S, Laurent F (2007) Fate of RDX and TNT in agronomic plants. Environ Pollut 148:148–154

    Article  PubMed  CAS  Google Scholar 

  52. Vila M, Mehier S, Lorber-Pascal S, Laurent F (2007) Phytotoxicity to and uptake of RDX by rice. Environ Pollut 145:813–817

    Article  PubMed  CAS  Google Scholar 

  53. 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  PubMed  CAS  Google Scholar 

  54. Zrafi-Nouira I, Guermazi S, Chouari R, Safi N, Pelletier E, Bakhrouf A, Saidane-Mosbahi D, Sghir A (2009) Molecular diversity analysis and bacterial population dynamics of an adapted seawater microbiota during the degradation of Tunisian zarzatine oil. Biodegradation 20:467–486

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Paul Munson for his technical assistance and Dr. Jennifer Duringer for her editorial expertise. This study was supported in part by a gift from Ruminant Solutions, UC (New Mexico), the Oregon Agricultural Experiment Station project ORE00871 and USDA, Agriculture Research Service project 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 U.S. Department of Agriculture.

Author information

Authors and Affiliations

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

    H. L. Eaton

  2. Department of Biosystems and Agriculture Engineering, Oklahoma State University, Stillwater, OK, 74075, USA

    M. De Lorme

  3. USDA-ARS Environmental Management and By-Product Utilization Laboratory, Building 007, 10300 Baltimore Avenue, BARC-West, Beltsville, MD, 20705, USA

    R. L. Chaney

  4. College of Veterinary Medicine, Oregon State University, Corvallis, OR, 97331, USA

    A. M. Craig

Authors
  1. H. L. Eaton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. De Lorme
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. L. Chaney
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. M. Craig
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. M. Craig.

Electronic Supplementary Materials

Below is the link to the electronic supplementary material.

ESM 1

(RTF 38 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Eaton, H.L., De Lorme, M., Chaney, R.L. et al. Ovine Ruminal Microbes Are Capable of Biotransforming Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX). Microb Ecol 62, 274–286 (2011). https://doi.org/10.1007/s00248-011-9809-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-011-9809-8

Keywords

Search

Navigation