Pseudomonas pp 229-240 | Cite as

Physiological Evidence for Respiration of TNT by Pseudomonas sp. JLR11

  • Juan-Luis Ramos
  • Antonio Caballero
  • Estrella Duque
  • Pieter van Dillewijn
  • María del Mar González-Pérez
  • Abraham Esteve-Núñez


The aim of this review is to summarize our current understanding of the anaerobic respiration of 2,4,6-trinitrotoluene (TNT) by a pure bacterial culture of Pseudomonas sp. JLR11 from a physiological and biochemical point of view. The article consists of four main sections. Section 1 presents background issues related to TNT pollution and a brief description of TNT metabolism by certain strict anaerobes. Section 2 describes a bacterial strain belonging to the genus Pseudomonas, isolated for its capability to use TNT as an N-source. This section also deals with enrichment methodologies. Section 3 examines anaerobic respiration as an emerging new biochemical process, and describes details of the physiological system. Section 4 is devoted to biotechnological applications of anaerobic TNT respiration by Pseudomonas sp. JLR11 and its role in bioremediation. Some general considerations are also discussed.


Nitro Group Pseudomonas Putida Anaerobic Respiration Physiological Evidence Nitroaromatic Compound 
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  1. 1.
    Achtnich, C., Femandes, E., Bollag, J.M., Knackmuss, HJ., and Lenke, H., 1999, Covalent binding of reduced metabolites of 15N-TNT to soil organic matter during a bioremediation process analyzed by 15N NMR spectroscopy. Environ. Sci. Technol., 33:4448–4456.CrossRefGoogle Scholar
  2. 2.
    Achtnich, C. and Lenke, H., 2001, Stability of immobilized 2,4,6-trinitrotoluene metabolites in soil under long-term leaching conditions. Environ. Toxicol. Chem., 20:280–283.PubMedCrossRefGoogle Scholar
  3. 3.
    Achtnich, C., Lenke, H., Klaus, U., Spiteller, M., and Knackmuss, HJ., 2000, Stability of immobilized TNT derivatives in soil as a function of nitro group reduction. Environ. Sci. Technol., 34:3698–3704.CrossRefGoogle Scholar
  4. 4.
    Achtnich, C., Pfortner, P., Weiler, M.G., Niessner, R., Lenke, H., and Knackmuss, H.J., 1999, Reductive transformation of bound trinitrophenyl residues and free TNT during a bioremediation process analyzed by immunoassay. Environ. Sci. Technol., 33:3421–3426.CrossRefGoogle Scholar
  5. 5.
    Ahmad, F. and Hughes J.B., 2000, Anerobic transformation of TNT by Clostridium, In J. Spain, J.N. Hughes, and H.-J. Knackmuss (eds), Biodegradation of Nitroaromatic Compounds and Explosives, pp. 185–212. Lewin Publishers, Boca Raton, FL.Google Scholar
  6. 6.
    Aranda-Olmedo, I., Tobes, R., Manzanera, M., Ramos, J.L., and Marqués, S., 2002, Species-specific repetitive extragenic palyndromic (REP) sequences in Pseudomonas putida. Nucl. Acids. Res., 30:1826–1833.PubMedCrossRefGoogle Scholar
  7. 7.
    Banerjee, H.N., Verma, M., Hou, L.H., Ashraf, M., and Dutta, S.K., 1999, Cytotoxicity of TNT and its metabolites. Yale J. Biol. Med., 72:1–4.PubMedGoogle Scholar
  8. 8.
    Berthe-Corti, L., Jacobi, H., Kleihauer, S., and White, I., 1998, Cytotoxicity and mutagenicity of a 2,4,6-trinitrotoluene (TNT) and hexogen contaminated soil in Salmonella typhimurium and mammalian cells. Chemosphere, 37:209–218.PubMedCrossRefGoogle Scholar
  9. 9.
    Boopathy, R. and Kulpa, CF., 1992, Trinitrotoluene as a sole nitrogen source for a sulfate-reducing bacterium Desulfovibrio sp. (B. strain) isolated from an anaerobic digester. Curr. Microbiol., 25:235–241.PubMedCrossRefGoogle Scholar
  10. 10.
    Boopathy, R., Manning, J., and Kulpa, CF., 1997, Optimization of environmental factors for the biological treatment of trinitrotoluene-contaminated soil. Arch. Environ. Contam. Toxicol., 32:94–98.PubMedCrossRefGoogle Scholar
  11. 11.
    Boopathy, R., Manning, J., and Kulpa, CF., 1998, A laboratory study of the bioremediation of 2,4,6-trinitrotoluene-contaminated soil using aerobic/anoxic soil slurry reactor. Water Environ. Res., 70:80–86.CrossRefGoogle Scholar
  12. 12.
    Boopathy, R., Wilson, M., and Kulpa, C.F., 1993, Anaerobic removal of 2,4,6-trinitrotoluene (TNT) under different electron accepting conditions: Laboratory study. Water Environ. Res., 65:271–275.CrossRefGoogle Scholar
  13. 13.
    Brooks, L.R., Jacobson, R.W., Warren, S.H., Kohan, M.J., Donnelly, K.C., and George, S.E., 1997, Mutagenicity of HPLC-fractionated urinary metabolites from 2,4,6-trinitrotoluene-treated Fischer 344 rats. Environ. Mol. Mutagen., 30:298–302, 337.PubMedCrossRefGoogle Scholar
  14. 14.
    Bruns-Nagel, D., Bretung, J., von Löw, E., Steinbach, K., Borontzy, T., Kahl, M., Blotevogel, K.-H., and Gemsa, D., 1996, Microbial transformation of 2,4,6-trinitrotoluene in aerobic soil columns. Appl. Environ. Microbiol., 62:2651–2656.PubMedGoogle Scholar
  15. 15.
    Bruns-Nagel, D., Steinbach, J.K., Gemsa, D., and von Löw, E., 2000, Composting (humification) of nitroaromatic compounds, In J. Spain, J.B. Hughes, and H.-J. Knackmuss (eds), Biodegradation of Nitroaromatic Compounds and Explosives, pp. 357–393. Lewin Publishers, Boca Raton, FL.Google Scholar
  16. 16.
    Cash, G.G., 1998, Prediction of chemical toxicity to aquatic microorganism: ECOSAR vs. Microtox assay. Environ. Toxicol. Water Qual., 132:211–216.CrossRefGoogle Scholar
  17. 17.
    Daun, G., Lenke, H., Reuss, M., and Knackmuss, HJ., 1998, Biological treatment of TNT-contaminated soil. 1. Anaerobic cometabolic reduction and interaction of TNT and metabolites with soil components. Environ. Sci. Technol., 32:1956–1963.CrossRefGoogle Scholar
  18. 18.
    Drzyzga, D., Bruns-Nagel, D., Gorontzy, T., Blotevogel, K.H., Gemsa, D., and van Löw, E., 1998, Mass balance studies with 14C-labeled 2,4,6-trinitrotoluene (TNT) mediated by an anaerobic Desulfovibrio species and an aerobic Serratia species. Curr. Microbiol., 37:380–386.PubMedCrossRefGoogle Scholar
  19. 19.
    Drzyzga, O., Bruns-Nagel, D., Gorontzy, T., Blotevogel, K.H., Gemsa, D., and von Low, E., 1998, Incorporation of 14C-labeled 2,4,6-trinitrotoluene metabolites into different soil fractions after anaerobic and anaerobic-aerobic treatment of soil/molasses mixtures. Environ. Sci. Technol., 32:3529–3535.CrossRefGoogle Scholar
  20. 20.
    Drzyzga, O., Bruns-Nagel, D., Gorontzy, T., Blotevogel, K.H., and von Löw, E., 1999, Anaerobic incorporation of the radiolabeled explosive TNT and metabolites into the organic soil matrix of contaminated soil after different treatment procedures. Chemosphere, 38:2081–2095.PubMedCrossRefGoogle Scholar
  21. 21.
    Duque, E., Haïdour, A., Godoy, E, and Ramos, J.L., 1993, Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene. J. Bacteriol., 175:2278–2283.PubMedGoogle Scholar
  22. 22.
    Ederer, M.M., Lewis, T.A., and Crawford, R.L., 1997, 2,4,6-Trinitrotoluene (TNT) transformation by Clostridia isolated from a munition-fed bioreactor: Comparison with non-adapted bacteria. J. Ind. Microbiol. Biotechnol., 18:82–88.PubMedCrossRefGoogle Scholar
  23. 23.
    Elovitz, M.S. and Weber, E.J., 1999, Sediment-mediated reduction of 2,4,6-trinitrotoluene and fate of the resulting aromatic (poly)amines. Environ. Sci. Technol., 33:2617–2625.CrossRefGoogle Scholar
  24. 24.
    Espinosa-Urgel, M., Költner, R., and Ramos, J.L., 2002, Root colonization by Pseudomonas putida: Love at first sight. Microbiology, 148:341–343.PubMedGoogle Scholar
  25. 25.
    Esteve-Nuñez, A., Caballero, A., and Ramos, J.L., 2001, Biological degradation of 2,4,6-trinitrotoluene. Microbiol. Mol. Biol. Rev., 65:335–352.PubMedCrossRefGoogle Scholar
  26. 26.
    Esteve-Núñez, A., Lucchesi, G., Philipp, B., Schink, B., and Ramos, J.L., 2000, Respiration of 2,4,6-trinitrotoluene by Pseudomonas sp. strain JLR11. J. Bacteriol., 182:1352–1355.PubMedCrossRefGoogle Scholar
  27. 27.
    Esteve-Núñez, A. and Ramos, J.L., 1998, Metabolism of 2,4,6-trinitrotoluene by Pseudomonas sp. JLR11. Environ. Sci. Technol., 32:3802–3808.CrossRefGoogle Scholar
  28. 28.
    Fernando, T.J., Bumpus J.A., and Aust, S.D., 1990, Biodegradation of TNT (2,4,6-trinitrotoluene) by Phanerochaete chrysosporium. Appl. Environ. Microbiol., 56:1666–1671.PubMedGoogle Scholar
  29. 29.
    Funk, S.B., Roberts, DJ., Crawford, D.L., and Crawford, R.L., 1993, Initial-phase optimization for bioremediation of munition compound-contaminated soils. Appl. Environ. Microbiol., 59:2171–2177.PubMedGoogle Scholar
  30. 30.
    George, S.E., Huggins-Clark, G., and Brooks, L.R., 2001, Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations. Mutat. Res., 490:45–56.PubMedCrossRefGoogle Scholar
  31. 31.
    Gong, P., Wilke, B., and Fleischmann, S., 1999, Soil-based phytotoxiciry of 2,4,6-trinitrotoluene (TNT) to terrestrial higher plants. Arch. Environ. Contam. Toxicol., 36:152–157.PubMedCrossRefGoogle Scholar
  32. 32.
    Haïdour, A. and Ramos, J.L., 1996, Identification of products resulting from the biological reduction of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene by Pseudomonas sp. Environ. Sci. Technol., 30:2365–2370.CrossRefGoogle Scholar
  33. 33.
    Hawaii, J., Halasz, A., Paquet, L, Zhou, E., Spencer, B., Ampleman, G., and Thiboutot, S., 1998, Characterization of metabolites in the biotransfomation of 2,4,6-trinitrotoluene with anaerobic sludge: Role of triaminotoluene. Appl. Environ. Microbiol., 64:2200–2206.Google Scholar
  34. 34.
    Hofstetter, T.B., Heijman, CG., Haderlein, S.B., Holliger, C., and Schwarzenbach, R.P., 1999, Complete reduction of TNT and other (poly)nitroaromatic compounds under iron-reducing subsurface conditions. Environ. Sci. Technol., 33:1479–1487.CrossRefGoogle Scholar
  35. 35.
    Honeycutt, M.E., Jarvis, A.S., and McFarland, VA., 1996, Cytotoxicity and mutagenicity of 2,4,6-trinitrotoluene and its metabolites. Ecotoxicol. Environ. Saf., 35:282–287.PubMedCrossRefGoogle Scholar
  36. 36.
    Hughes, J.B., Wong, C.H.Y., and Zhang, C.H., 1999, Anaerobic biotransformation of 2,4-dinitrotoluene and 2,6-dinitrotoluene by Clostridium acetobutylicum: A pathway through dihydroxylamino intermediates. Environ. Sci. Technol., 33:1065–1070.CrossRefGoogle Scholar
  37. 37.
    Knackmuss, H.-J., 1996, Basis of knowledge and perspectives of bioelimination of xenobiotic compounds. J. Biotechnol., 51:287–295.CrossRefGoogle Scholar
  38. 38.
    Knicker, H., Bruns-Nagel, D., Drzyzga, O., von Low, E., and Steinbach, K., 1999, Characterization of 15N-TNT residues after an anaerobic/aerobic treatment of soil/molasses mixtures by solid-state 15N NMR spectroscopy. 1. Determination and optimization of relevant NMR spectroscopy parameters. Environ. Sci. Technol., 33:343–349.CrossRefGoogle Scholar
  39. 39.
    Lenke, H., Warrelmann, J., Daun, G., Hund, K. Sieglen, and Knackmuss, HJ., 1998, Biological treatment of TNT-contaminated soil. 2. Biological induced immobilization of the contaminants and full-scale application. Environ. Sci. Technol., 32:1964–1971.CrossRefGoogle Scholar
  40. 40.
    Lewis, T.A., Ederer, M.M., Crawford, R.L., and Crawford, D.L., 1997, Microbial transformation of 2,4,6-trinitrotoluene. J. Ind. Microbiol. Biotechnol., 18:89–96.CrossRefGoogle Scholar
  41. 41.
    Li, A.Z., Marx, K.A., Walker, J., and Kaplan, D.L., 1997, Trinitrotoluene and metabolites binding to humic acid. Environ. Sci. Technol., 31:584–589.CrossRefGoogle Scholar
  42. 42.
    Molina, L., Ramos, C., Duque, E., Ronchel, M.C., Garcia, J.M., Wyke, L., and Ramos, J.L., 2000, Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under greenhouse and environmental conditions. Soil Biol. Biochem., 32:315–321.CrossRefGoogle Scholar
  43. 43.
    Pak, J.W., Knoke, K.L., Noguera, D.R., Fox, B.G., and Chambliss, G.H., 2000, Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C. Appl. Environ. Microbiol., 66:4742–4750.PubMedCrossRefGoogle Scholar
  44. 44.
    Preuss, A., Fimpel, J., and Diekert, G., 1993, Anaerobic transformation of 2,4,6-trinitrotoluene (TNT). Arch. Microbiol., 159:345–353.PubMedCrossRefGoogle Scholar
  45. 45.
    Preuss, A. and Rieger, P.G., 1995, Anaerobic transformation of 2,4,6-TNT and other nitroaromatic compounds, In J.C. Spain (ed.), Biodegradation of Nitroaromatic Compounds, pp. 69–85. Plenum, New York, NY.Google Scholar
  46. 46.
    Ravatn, R., Zehnder, A.J.B., and van der Meer, J.R., 1998, Low-frequency horizontal transfer of an element containing the chlorocatechol degradation genes from Pseudomonas sp. strain B13 to Pseudomonas putida Fl and to indigenous bacteria in laboratory-scale-activated sludge microorganisms. Appl Environ. Microbiol., 64:2126–2132.PubMedGoogle Scholar
  47. 47.
    Rieger, P. and Knackmuss, HJ., 1995, Basic knowledge and perspectives on biodegradation of 2,4,6-trinitrotoluene and related nitroaromatic compounds in contaminated soil. In J.C. Spain (ed.), Biodegradation of Nitroaromatic Compounds, pp. 1–18. Plenum, New York, NY.Google Scholar
  48. 48.
    Robidoux, P.Y., Hawaii, J., Thiboutot, S., Ampleman, G., and Sunahara, G.I., 1999, Acute toxicity of 2,4,6-trinitrotoluene in earthworm (Eiseniaandrei). Ecotoxicol Environ. Saf, 44:311–321PubMedCrossRefGoogle Scholar
  49. 49.
    Rodgers, J.D. and Bunce, N.J., 2001, Treatment methods for the remediation of nitroaromatic explosives. Water Res., 35:2101–2111.PubMedCrossRefGoogle Scholar
  50. 50.
    Ronchel, M.C., Ramos-Diaz, M.A., and Ramos, J.L., 2000, Retrotransfer of DNA in the rhizosphere. Env. Microbiol., 2:319–323.CrossRefGoogle Scholar
  51. 51.
    Salyers, A.A., Shoemaker, N.B., Stevens, A.M., and Li, L.Y., 1995, Conjugative transposons: An unusual and diverse set of integrated gene transfer elements. Microbiol Rev., 59:579–590.PubMedGoogle Scholar
  52. 52.
    Scheibner, K., Hofrichter, M., Herre, A., Michels, J., and Fritsche, W., 1997, Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene. Appl. Microbiol. Biotechnol., 47:452–457.PubMedCrossRefGoogle Scholar
  53. 53.
    Schnell, S. and Schinck, B., 1991, Anaerobic aniline degradation via reductive deamination of 4-aminobenzoyl-CoA in Desulfobacterium anilini. Arch. Microbiol., 155:183–190.CrossRefGoogle Scholar
  54. 54.
    Selim, H.M., Xue, S.K., and Iskandar, I.K., 1995, Transport of 2,4,6-trinitrotoluene and hexahydro-l,3,5-trinitro-l,3,5-triazine in soils. Soil Sci., 160:328–339.CrossRefGoogle Scholar
  55. 55.
    Sembries, S. and Crawford, R.L., 1997, Production of Clostridium bifermentans spores as inoculum for bioremediation of nitroaromatic contaminants. Appl. Environ. Microbiol., 63: 2100–2104.PubMedGoogle Scholar
  56. 56.
    Siciliano, S.D., Roy, R., and Greer, C.W., 2000, Reduction in denitrification activity in field soils exposed to long term contamination by 2,4,6-trinitrotoluene (TNT). FEMS Microbiol Ecol., 32:61–68.PubMedCrossRefGoogle Scholar
  57. 57.
    Spanggord, R.J., Mortelmans, K.E., Griffin, A.F., and Simmon, VE., 1982, Mutagenicity in Salmonella typhimurium and structure-activity relationships of waste water components emanating from the manufacture of trinitrotoluene. Environ. Mutagen., 4:163–179.PubMedCrossRefGoogle Scholar
  58. 58.
    Styles, J.A. and Cross, M.F., 1983, Activity of 2,4,6-TNT in an in vitro mammalian gene mutation assay. Cancer Lett., 20:103–108.PubMedCrossRefGoogle Scholar
  59. 59.
    Tadros, M.G., Crawford, A., Mateo-Sullivan, A., Zhang, C., and Hughes, J.B., 2000, Toxic effects of hydroxylamino intermediates from microbial transformation of trinitrotoluene and dinitrotoluenes on algae Selenastrum capricornutum. Bull Environ. Contam. Toxicol, 64:579–585.PubMedCrossRefGoogle Scholar
  60. 60.
    Tan, EX., Ho, C.H., Griest, W.H., and Tyndall, R.L., 1992, Mutagenicity of trinitrotoluene and its metabolites formed during composting. J. Toxicol. Environ. Health, 36:165–175.PubMedCrossRefGoogle Scholar
  61. 61.
    Vaatanen, A.R., 1997, Spectrum of spontaneous and 2,4,6-trinitrotoluene (TNT)-induced mutations in Salmonella typhymurium strains with different nitroreductase and o-acetyltransferase activities. Mutat. Res., 379:185–190.PubMedCrossRefGoogle Scholar
  62. 62.
    Widrig, D.L., Boopathy, R., and Manning, J.F., 1997, Bioremediation of TNT-contaminated soil: A laboratory study. Environ. Toxicol. Chem., 16:1141–1148.CrossRefGoogle Scholar
  63. 63.
    Wikstrom, P., Andersson, A.C., Nygren, Y., Sjostrom, J., and Forsman, M., 2000, Influence of TNT transformation on microbial community structure in four different lake microcosms. J.Appl. Microbiol., 89:302–308.PubMedCrossRefGoogle Scholar
  64. 64.
    Won, W.D. and Disalvo, L.H.N.J., 1976, Toxicity and mutagenicity of 2,4,6-trinitrotoluene and its microbial metabolites. Appl. Environ. Microbiol., 31:576–580.PubMedGoogle Scholar
  65. 65.
    Zhou, J.Z. and Tiedje, J.M., 1995, Gene transfer from a bacteria injected into an aquifer to an indigenous bacterium. Mol. Ecol., 4:613–619.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2004

Authors and Affiliations

  • Juan-Luis Ramos
    • 1
  • Antonio Caballero
    • 1
  • Estrella Duque
    • 1
  • Pieter van Dillewijn
    • 1
  • María del Mar González-Pérez
    • 1
  • Abraham Esteve-Núñez
    • 1
  1. 1.Department of Biochemistry and Molecular and Cell Biology of PlantsEstación Experimental del ZaidínGranadaSpain

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