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Stable isotope probing reveals the importance of Comamonas and Pseudomonadaceae in RDX degradation in samples from a Navy detonation site

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Abstract

This study investigated the microorganisms involved in hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation from a detonation area at a Navy base. Using Illumina sequencing, microbial communities were compared between the initial sample, samples following RDX degradation, and controls not amended with RDX to determine which phylotypes increased in abundance following RDX degradation. The effect of glucose on these communities was also examined. In addition, stable isotope probing (SIP) using labeled (13C3, 15N3-ring) RDX was performed. Illumina sequencing revealed that several phylotypes were more abundant following RDX degradation compared to the initial soil and the no-RDX controls. For the glucose-amended samples, this trend was strong for an unclassified Pseudomonadaceae phylotype and for Comamonas. Without glucose, Acinetobacter exhibited the greatest increase following RDX degradation compared to the initial soil and no-RDX controls. Rhodococcus, a known RDX degrader, also increased in abundance following RDX degradation. For the SIP study, unclassified Pseudomonadaceae was the most abundant phylotype in the heavy fractions in both the presence and absence of glucose. In the glucose-amended heavy fractions, the 16S ribosomal RNA (rRNA) genes of Comamonas and Anaeromxyobacter were also present. Without glucose, the heavy fractions also contained the 16S rRNA genes of Azohydromonas and Rhodococcus. However, all four phylotypes were present at a much lower level compared to unclassified Pseudomonadaceae. Overall, these data indicate that unclassified Pseudomonadaceae was primarily responsible for label uptake in both treatments. This study indicates, for the first time, the importance of Comamonas for RDX removal.

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References

  • Adrian NR, Arnett CM (2004) Anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Acetobacterium malicum strain HAAP-1 isolated from a methanogenic mixed culture. Curr Microbiol 48:332–340

    Article  CAS  Google Scholar 

  • Adrian NR, Chow T (2001) Identification of hydroxylamino-dinitroso-1,3,5-triazine as a transient intermediate formed during the anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine. Environ Toxicol Chem 20:1874–1877

    Article  CAS  Google Scholar 

  • Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    CAS  Google Scholar 

  • Andeer P, Stahl DA, Lillis L, Strand SE (2013) Identification of microbial populations assimilating nitrogen from RDX in munitions contaminated military training range soils by high sensitivity stable isotope probing. Environ Sci Technol 47:10356–10363

    CAS  Google Scholar 

  • Beller HR (2002) Anaerobic biotransformation of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by aquifer bacteria using hydrogen as the sole electron donor. Water Res 36:2533–2540

    Article  CAS  Google Scholar 

  • Bernstein A, Ronen Z (2012) Biodegradation of the explosives TNT, RDX and HMX. Microbial Degradation of Xenobiotics 135–176

  • Bernstein A, Adar E, Nejidat A, Ronen Z (2011) Isolation and characterization of RDX-degrading Rhodococcus species from a contaminated aquifer. Biodegradation 22:997–1005

    Article  CAS  Google Scholar 

  • Bhushan B, Trott S, Spain JC, Halasz A, Paquet L, Hawari M (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 69:1347–1351

    Article  CAS  Google Scholar 

  • Bhushan B, Halasz A, Thiboutot S, Ampleman G, Hawari J (2004) Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2. Biochem Biophys Res Commun 316:816–821

    Article  CAS  Google Scholar 

  • Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, Fierer N, Knight R (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci USA 108:4516–4522

    Article  CAS  Google Scholar 

  • Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621–1624

    Article  CAS  Google Scholar 

  • Cho YS, Lee BU, Oh KH (2008) Simultaneous degradation of nitroaromatic compounds TNT, RDX, atrazine, and simazine by Pseudomonas putida HK-6 in bench-scale bioreactors. J Chem Technol Biotechnol 83:1211–1217

    Article  CAS  Google Scholar 

  • Cho KC, Lee DG, Roh H, Fuller ME, Hatzinger PB, Chu K-H (2013) Application of 13C-stable isotope probing to identify RDX-degrading microorganisms in groundwater. Environ Pollut 178:350–360

    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 

  • Coleman NV, Spain JC, Duxbury T (2002) Evidence that RDX biodegradation by Rhodococcus strain DN22 is plasmid-borne and involves a cytochrome p-450. J Appl Micro 93:463–472

    Article  CAS  Google Scholar 

  • Cupples AM (2013) RDX degrading microbial communities and the prediction of microorganisms responsible for RDX bioremediation. Int Biodeter Biodegr 85:260–270

    Article  CAS  Google Scholar 

  • EPA (2014) Technical Fact Sheet: hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX). http://www2.epa.gov/sites/production/files/2014–03/documents/ffrrofactsheet_contaminant_rdx_january2014_final.pdf

  • Fournier D, Halasz A, Spain J, Fiurasek P, Hawari J (2002) Determination of key metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp strain DN22. Appl Environ Microbiol 68:166–172

    Article  CAS  Google Scholar 

  • Fuller ME, Steffan RJ (2008) Groundwater chemistry and microbial ecology effects on explosives biodegradation

  • Fuller ME, McClay K, Higham M, Hatzinger PB, Steffan RJ (2010a) Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) bioremediation in groundwater: are known RDX-degrading bacteria the dominant players? Biochem J 14:121–134

    CAS  Google Scholar 

  • Fuller ME, Perreault N, Hawari J (2010b) Microaerophilic degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains. Lett Appl Micro 51:313–318

    Article  CAS  Google Scholar 

  • Halasz A, Manno D, Strand SE, Bruce NC, Hawari J (2010) Biodegradation of RDX and MNX with Rhodococcus sp. strain DN22: new insights into the degradation pathway. Environ Sci Technol 44:9330–9336

    Article  Google Scholar 

  • Indest KJ, Jung CM, Chen HP, Hancock D, Florizone C, Eltis LD, Crocker FH (2010) Functional characterization of pGKT2, a 182-kilobase plasmid containing the xplAB genes, which are involved in the degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine by Gordonia sp. strain KTR9. Appl Environ Microbiol 76:6329–6337

    Article  CAS  Google Scholar 

  • Jayamani I, Manzella MP, Cupples AM (2013) RDX degradation potential in soils previously unexposed to RDX and the identification of RDX-degrading species in one agricultural soil using stable isotope probing. Water Air Soil Poll 224:1745. doi:10.1007/s11270-013-1745-4

  • Kitts CL, Cunningham DP, Unkefer PJ (1994a) Isolation of 3 hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading species of the family Enterobacteriaceae from nitramine explosive-contaminated soil. 60: 4608–4611

  • Kitts CL, Cunningham DP, Unkefer PJ (1994b) Isolation of three hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading species of the family Enterobacteriaceae from nitramine explosive-contaminated soil. Appl Environ Microbiol 60:4608–4611

    CAS  Google Scholar 

  • Kitts CL, Green CE, Otley RA, Alvarez MA, Unkefer PJ (2000) Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). 46: 278–282

  • Kwon MJ, Finneran KT (2008) Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) biodegradation kinetics amongst several Fe (III)-reducing genera. Soil Sediment Contam 17:189–203

    Article  CAS  Google Scholar 

  • Kwon MJ, Finneran KT (2010) Electron shuttle-stimulated RDX mineralization and biological production of 4-nitro-2,4-diazabutanal (NDAB) in RDX-contaminated aquifer material. Biodegradation 21:923–937

    Article  CAS  Google Scholar 

  • Kwon MJ, O’Loughlin EJ, Antonopoulos DA, Finneran KT (2011) Geochemical and microbiological processes contributing to the transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in contaminated aquifer material. Chemosphere 84:1223–1230

    Article  CAS  Google Scholar 

  • Livermore J, Oh Jin Y, LePuil M, Arnseth R, Mattes TE (2013) Microbial community dynamics during acetate biostimulation of RDX-contaminated groundwater. Environ Sci Technol 47:7672–7678

    Article  CAS  Google Scholar 

  • McCormick NG, Cornell JH, Kaplan AM (1981) Biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine. 42: 817–823

  • Michalsen MM, Weiss R, King A, Gent D, Dmedina VF, Istok JD (in press) Push-pull tests for estimating RDX and TNT degradation rates in groundwater. Ground Monitor Rem

  • Moshe SSB, Ronen Z, Dahan O, Weisbrod N, Groisman L, Adar E, Nativ R (2009) Sequential biodegradation of TNT, RDX and HMX in a mixture. Environ Pollut 157:2231–2238

    Article  Google Scholar 

  • Nejidat A, Kafka L, Tekoah Y, Ronen Z (2008) Effect of organic and inorganic nitrogenous compounds on RDX degradation and cytochrome P-450 expression in Rhodococcus strain YH1. 19: 313–320

  • Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Gloeckner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. 35: 7188–7196

  • Pudge IB, Daugulis AJ, Dubois C (2003) The use of Enterobacter cloacae ATCC 43560 in the development of a two-phase partitioning bioreactor for the destruction of hexahydro-1,3,5-trinitro-1,3,5-s-triazine (RDX). J Biotechnol 100:65–75

    Article  CAS  Google Scholar 

  • Regan KM, Crawford RL (1994) Characterization of Clostridium bifermentans and its biotransformation of 2,4,6-trinitrotoluene (TNT) and 1,3,5-triaza-1,3,5-trinitrocyclohexane (RDX). Biotechnol Lett 16:1081–1086

    Article  CAS  Google Scholar 

  • Ringelberg D, Richmond M, Foley K, Reynolds C (2008) Utility of lipid biomarkers in support of bioremediation efforts at army sites. J Microbiol Methods 74:17–25

    Article  CAS  Google Scholar 

  • Roh H, Yu CP, Fuller ME, Chu KH (2009) Identification of hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading microorganisms via 15N-stable isotope probing. Environ Sci Technol 43:2505–2511

    Article  CAS  Google Scholar 

  • Ronen Z, Yanovich Y, Goldin R, Adar E (2008) Metabolism of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a contaminated vadose zone. Chemosphere 73:1492–1498

    Article  CAS  Google Scholar 

  • Schloss PD (2009) A high-throughput DNA sequence aligner for microbial ecology studies. 4

  • Schloss PD (2013) MiSeq SOP. In: Schloss PD (Hrsg.), Mothur wiki, pp. This is a page describing the standard operating procedure for analysing MiSeq data using Mothur

  • Seth-Smith HMB, Rosser SJ, Basran A, Travis ER, Dabbs ER, Nicklin S, Bruce NC (2002) Cloning, sequencing, and characterization of the hexahydro-1,3,5-trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous. Appl Environ Microbiol 68:4764–4771

    Article  CAS  Google Scholar 

  • Seth-Smith HMB, Edwards J, Rosser SJ, Rathbone DA, Bruce NC (2008) The explosive-degrading cytochrome P450 system is highly conserved among strains of Rhodococcus spp. Appl Environ Microbiol 74:4550–4552

    Article  CAS  Google Scholar 

  • Sherburne LA, Shrout JD, Alvarez PJJ (2005) Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation by Acetobacterium paludosum. Biodegradation 16:539–547

    Article  CAS  Google Scholar 

  • Singh R, Soni P, Kumar P, Purohit S, Singh A (2009) Biodegradation of high explosive production effluent containing RDX and HMX by denitrifying bacteria. World J Microbiol Biotechnol 25:269–275

    Article  CAS  Google Scholar 

  • Sun WM, Cupples AM (2012) Diversity of five anaerobic toluene-degrading microbial communities investigated using stable isotope probing. Appl Environ Microbiol 78:972–980

    Article  CAS  Google Scholar 

  • Sun WM, Xie SG, Luo CL, Cupples AM (2010) Direct link between toluene degradation in contaminated-site microcosms and a Polaromonas strain. Appl Environ Microbiol 76:956–959

    Article  CAS  Google Scholar 

  • Sun WM, Sun XX, Cupples AM (2012) Anaerobic methyl tert-butyl ether-degrading microorganisms identified in wastewater treatment plant samples by stable isotope probing. Appl Environ Microbiol 78:2973–2980

    Article  CAS  Google Scholar 

  • Thompson KT, Crocker FH, Fredrickson HL (2005) Mineralization of the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine by Gordonia and Williamsia spp. Appl Environ Microbiol 71:8265–8272

    Article  CAS  Google Scholar 

  • Xie SG, Sun WM, Luo CL, Cupples AM (2010) Stable isotope probing identifies novel m-xylene degraders in soil microcosms from contaminated and uncontaminated sites. Water Air Soil Pollut 212:113–122

    Article  CAS  Google Scholar 

  • Xie SG, Sun WM, Luo CL, Cupples AM (2011) Novel aerobic benzene degrading microorganisms identified in three soils by stable isotope probing. Biodegradation 22:71–81

    Article  CAS  Google Scholar 

  • Young DM, Unkefer PJ, Ogden KL (1997) Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a prospective consortium and its most effective isolate Serratia marcescens. 53: 515–522

  • Zhang CL, 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, Halasz A, Paquet L, Beaulieu C, Hawari J (2002) Biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine and its mononitroso derivative hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine by Klebsiella pneumoniae strain SCZ-1 isolated from an anaerobic sludge. Appl Environ Microbiol 68:5336–5341

    Article  CAS  Google Scholar 

  • Zhao JS, Paquet L, Halasz A, Hawari J (2003a) 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 

  • Zhao JS, Spain J, Hawari M (2003b) Phylogenetic and metabolic diversity of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-transforming bacteria in strictly anaerobic mixed cultures enriched on RDX as nitrogen source. FEMS Micro Ecol 46:189–196

    Article  CAS  Google Scholar 

  • Zhao JS, Greer CW, Thiboutot S, Ampleman G, Hawari J (2004) Biodegradation of the nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine in cold marine sediment under anaerobic and oligotrophic conditions. Can J Microbiol 50:91–96

    Article  Google Scholar 

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Acknowledgments

This research was supported by a grant to A.M. Cupples from Strategic Environmental Research and Development Program (SERDP Project ER1606). Thanks to Mark E. Fuller and Paul Hatzinger (CB & I Federal Services) for providing samples from the Navy base site.

Conflict of interest

The authors have no conflicts of interest with the research described in this manuscript.

Compliance with ethical standards

This research did not involve research with human participants or animals.

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Authors and Affiliations

  1. A135 Research Engineering Complex, Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, 48824, USA

    Indumathy Jayamani & Alison M. Cupples

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  1. Indumathy Jayamani
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  2. Alison M. Cupples
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Correspondence to Alison M. Cupples.

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Responsible editor: Gerald Thouand

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Figure 1

Buoyant density (g mL−1) of each fraction following ultracentrifugation of DNA extracted from the labeled and unlabeled RDX amended microcosms, with and without glucose. (DOCX 44 kb)

Table 1

Summary of sample names, amendments and time for DNA extraction. (DOCX 14 kb)

Table 2

T-test (two-tailed) results from a comparison of the relative abundance of five phylotypes with and without RDX. Also, results are shown from comparing the relative abundance of the three enriched phylotypes in fractions from the labeled and unlabeled RDX amended microcosms (SIP results). (DOCX 15 kb)

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Jayamani, I., Cupples, A.M. Stable isotope probing reveals the importance of Comamonas and Pseudomonadaceae in RDX degradation in samples from a Navy detonation site. Environ Sci Pollut Res 22, 10340–10350 (2015). https://doi.org/10.1007/s11356-015-4256-6

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