Role of Clostridial Nitroreductases in Bioremediation

  • Razia KuttyEmail author
  • George N. Bennett


The use of clostridial enzymes in industry, environment, medical and economical aspects has been of immense significance and is well documented. Over decades the research has been focused on developing efficient strains and modified enzymes to be used in the bio-butanol production. The research interest in clostridial nitroreductase enzyme has increased recently due to its application in cancer treatment and bioremediation. To date, the report on the nitroreductases from Clostridium species is scarce. The function of these nitroreductases in bioremediation has been demonstrated. The structure of nitroreductases from Escherichia coli and Bacillus subtilis has been determined; however, very little documentation is available on clostridial nitroreductases. The information on the nitroreductase genes and proteins has become available from genomic and protein databases. However the significance of clostridial nitroreductases in its metabolism is not well understood. In this chapter, we discuss the findings on clostridial nitroreductases on the avenue for bioremediation and its applications in medical and industrial areas. The literature also aims to have comprehensive analysis of clostridial physiology based on the existing information.


Bacillus Clostridium Biogas Bioremediation Digestion Environment 


  1. Angermaier L, Simon H (1983) On the reduction of aliphatic and aromatic nitrocompounds by Clostridia, the role of ferredoxin and its stabilization. Hoppe Seylers Z Physiol Chem 364:961–975. CrossRefPubMedGoogle Scholar
  2. Angermaier L, Hein F, Simon H (1981) Investigations on the reduction of aliphatic and aromatic nitrocompounds by Clostridia species and enzyme systems. In: Bothe H, Trebst A (eds) Biology of inorganic nitrogen and sulfur, proceedings in life sciences. Berlin, Springer, pp 266–275CrossRefGoogle Scholar
  3. Baffert C, Sybirna K, Ezanno P, Lautier T, Hajj V, Meynial-Salles I (2012) Covalent attachment of FeFe hydrogenases to carbon electrodes for direct electron transfer. Anal Chem 84:7999–8005. CrossRefPubMedGoogle Scholar
  4. Baffert C, Fourmond V, Leger C, Meynial-Salles I, Soucaille P, Sybirna K (2014) Covalent attachment of FeFe hydrogenases to graphite electrode and inhibition studies. J Biol Chem 19:S280–S280Google Scholar
  5. Batstone DJ, Virdis B (2014) The role of anaerobic digestion in the emerging energy economy. Curr Opin Biotechnol 27:142–149. CrossRefPubMedGoogle Scholar
  6. Bohutskyi P, Ketter B, Chow S, Adams KJ, Betenbaugh MJ, Allnutt FC, Bouwer EJ (2015) Anaerobic digestion of lipid-extracted Auxenochlorella protothecoides biomass for methane generation and nutrient recovery. Bioresour Technol 183:229–239. CrossRefPubMedGoogle Scholar
  7. Boopathy R, Wilson M, Kulpa CM (1993) Anaerobic removal of 2,4,6-trinitrotoluene (TNT) under different electron accepting conditions: laboratory study. Water Environ Res 65:271–275. CrossRefGoogle Scholar
  8. Boopathy R, Wilson M, Montemagno CD, Manning JF, Kulpa CF (1994) Biological transformation of 2,4,6-trinitrotoluene (TNT) by soil bacteria isolated from TNT-contaminated soil. Bioresour Technol 47:19–24. CrossRefGoogle Scholar
  9. Bradley PC, Chapelle FH, Landmeyer JE, Schumacher JG (1997) Potential for intrinsic bioremediation of a DNT-contaminated aquifer. Ground Water 35:12–17. CrossRefGoogle Scholar
  10. Caballero A, Lazaro JJ, Ramos JL, Esteve-Nunez A (2005) PnrA, a new nitroreductase-family enzyme in the TNT-degrading strain Pseudomonas putida JLR11. Environ Microbiol (8):1211–1219. CrossRefGoogle Scholar
  11. Daun GL, Reuss M, Knackmuss HJ (1998) Biological treatment of TNT-contaminated soil Anaerobic cometabolic reduction and interaction of TNT and metabolites with soil components. Environ Sci Technol 32:1956–1963. CrossRefGoogle Scholar
  12. Esteve-Nunez A, Caballero A, Ramos JL (2001) Biological degradation of 2,4,6-Trinitrotoluene. Microbiol Mol Biol Rev 3:335–352. CrossRefGoogle Scholar
  13. Fernández M, Duque E, Pizarro-Tobías P, Van Dillewijn P, Wittich R, Ramos JL (2009) Microbial responses to xenobiotic compounds. Identification of genes that allow Pseudomonas putida KT2440 to cope with 2,4,6-trinitrotoluene. Microb Biotechnol 2:287–294. CrossRefPubMedPubMedCentralGoogle Scholar
  14. FitzGerald JA, Allen E, Wall DM, Jackson SA, Murphy JD, Dobson AD (2015) Methanosarcina play an important role in anaerobic co-digestion of the seaweed Ulva lactuca: taxonomy and predicted metabolism of functional microbial communities. PLoS One 10(11):e0142603. eCollection 2015CrossRefPubMedPubMedCentralGoogle Scholar
  15. Fontaine L, Meynial-Salles I, Girbal L, Yang X, Croux C, Soucaille P (2002) Molecular characterization and transcriptional analysis of adhE2, the gene encoding the NADH-dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum ATCC 824. J Bacteriol 184:821–830. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fourmond V, Baffert C, Ezanno P, Leger C, Greco C, Bruschi M (2014) The oxidative inactivation of FeFe hydrogenase reveals the plasticity of the H-cluster. J Biol Inorg Chem 19:S232–S232. CrossRefGoogle Scholar
  17. Fuess LT, Garcia ML (2015) Bioenergy from stillage anaerobic digestion to enhance the energy balance ratio of ethanol production. J Environ Manag 162:102–114. CrossRefGoogle Scholar
  18. Girbal L, Soucaille P (1998) Regulation of solvent production in Clostridium acetobutylicum. Trends Biotechnol 16:11–16. CrossRefGoogle Scholar
  19. Girbal L, von Abendroth G, Winkler M, Benton PMC, Meynial-Salles I (2005) Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl Environ Microbiol 71:2777–2781. CrossRefPubMedPubMedCentralGoogle Scholar
  20. González-Pajuelo M, Meynial-Salles I, Mendes F, Andrade JC (2005) Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1, 3-propanediol from glycerol. Metab Eng 7:329–336. CrossRefPubMedGoogle Scholar
  21. Gorwa MF, Croux C, Soucaille P (1996) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J Bacteriol 178:2668–2675. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Guerrini O, Burlat B, Léger C, Guigliarelli B, Soucaille P, Girbal L (2008) Characterization of two 2 [4Fe4S] ferredoxins from Clostridium acetobutylicum. Curr Microbiol 56:261–267. CrossRefPubMedGoogle Scholar
  23. Heap JT, Theys J, Ehsaan M, Kubiak AM, Dubois L, Paesmans K, Mellaert LV, Knox R, Kuehne SA, Lambin P, Minton NP (2014) Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo. Oncotarget 5:1761–1769. 10.18632/oncotarget.1761 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Huang S, Lindahl PA, Wang C, Bennett GN, Rudolph FB, Hughes JB (2000) 2,4,6-Trinitrotoluene reduction by carbon monoxide dehydrogenase from Clostridium thermoaceticum. Appl Environ Microbiol 66:1474–1478. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524CrossRefGoogle Scholar
  26. Ju K, Parales RE (2010) Nitroaromatic compounds, from synthesis to biodegradation. Microbiol Mol Biol Rev 74:250–272. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kivisaar M (2011) Evolution of catabolic pathways and their regulatory systems in synthetic nitroaromatic compounds degrading bacteria. Mol Microbiol 82:265–268. CrossRefPubMedGoogle Scholar
  28. Kim BH, Zeikus JG (1985) Importance of hydrogen metabolism in regulation of solventogenesis by Clostridium acetobutylicum. Dev Ind Microbiol 26:1–14Google Scholar
  29. 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). Can J Microbiol 46:278–282CrossRefGoogle Scholar
  30. Kobori T, Sasaki H, Lee WC, Zenno S, Saigo K, Murphy ME, Tanokura M (2000) Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds. J Biol Chem 276:2816–2823. CrossRefPubMedGoogle Scholar
  31. Kröger M, Schumacher ME, Risse H, Fels G (2004) Biological reduction of TNT as part of a combined biological–chemical procedure for mineralization. Biodegradation 15:241–248. CrossRefPubMedGoogle Scholar
  32. Kutty R, Bennett GN (2005) Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from Clostridium acetobutylicum ATCC 824 over expressed in E coli. Arch Microbiol 184:158–167. CrossRefPubMedGoogle Scholar
  33. Kutty R, Bennett GN (2006) Studies on inhibition of transformation of 2,4,6-trinitrotoluene catalyzed by Fe-only hydrogenase from Clostridium acetobutylicum. J Ind Microbiol Biotechnol 33:368–376. CrossRefPubMedGoogle Scholar
  34. Kutty R, Bennett GN (2007) Characterization of a novel ferredoxin with N-terminal extension from Clostridium acetobutylicum ATCC 824. Arch Microbiol 187:161–169. CrossRefPubMedGoogle Scholar
  35. Lenke HW, Warrelmann J, Daun G, Hund K, Steglen U, Walter U, Knackmuss HJ (1998) minated soil. 2 Biologically induced immobilization of the contaminants and full-scale application. Environ Sci Technol 32:1964–1971. CrossRefGoogle Scholar
  36. Leungsakul T, Johnson GR, Wood TK (2006) Protein engineering of the 4-Methyl-5-nitrocatechol monooxygenase from Burkholderia sp. Strain DNT for enhanced degradation of nitroaromatics. Appl Environ Microbiol 72:3933–3939. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Li RW, Giarrizzo JG, Wu S, Li W, Duringer JM, Craig AM (2014) Metagenomic insights into the RDX-degrading potential of the ovine rumen microbiome. PLoS One 9:e110505. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Liu SC, Ahn GO, Kioi M, Dorie MJ, Patterson AV, Brown JM (2008) Optimized clostridium-directed enzyme prodrug therapy improves the antitumor activity of the novel DNA cross-linking agent PR-104. Cancer Res 68:7995–8003. CrossRefPubMedPubMedCentralGoogle Scholar
  39. McAnulty MJ, Yen J, Freedman BG, Senger RS (2012) Genome-scale modeling using flux ratio constraints to enable metabolic engineering of clostridial metabolism in silico. BMC Syst Biol 6:42. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Nölling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R (2001) Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183:4823–4838. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Padda RS, Wang C, Hughes JB, Kutty R, Bennett GN (2003) Mutagenicity of nitroaromatic degradation compounds. Environ Toxicol Chem 22:2293–2297. CrossRefPubMedGoogle Scholar
  42. Peters JW (1998) X-ray crystal structure of the Fe-Only Hydrogenase (CpI) from Clostridium pasteurianum to 1.8 Angstrom Resolution. Science 282:1853–1858. CrossRefPubMedGoogle Scholar
  43. Peres CM, Agathos SN (2000) Biodegradation of nitroaromatic pollutants: from pathways to remediation. Biotechnol Annu Rev 6:197–220. CrossRefPubMedGoogle Scholar
  44. Rafii F, Cerniglia CE (1993) Comparison of the azoreductase and nitroreductase from Clostridium perfringens. Appl Environ Microbiol 59:1731–1734CrossRefGoogle Scholar
  45. Rafii F, Coleman T (1999) Cloning and expression in Escherichia coli of an azoreductase gene from Clostridium perfringens and comparison with azoreductase genes from other bacteria. J Basic Microbiol 39:29–35CrossRefGoogle Scholar
  46. Reeve BW, Reid SJ (2016) Glutamate and histidine improve both solvent yields and the acid tolerance response of Clostridium beijerinckii NCP 260. J Appl Microbiol 120:1271–1281. CrossRefPubMedGoogle Scholar
  47. Reeve JN, Beckler GS, Cram DS, Hamilton PT, Brown JW, Krzycki JA, Kolodziej AF, Alex L, Orme-Johnson WH, Walsh CT (1989) A hydrogenase-linked gene in Methanobacterium thermoautotrophicum strain ΔH encodes a polyferredoxin. Proc Natl Acad Sci U S A 86:3031–3035CrossRefGoogle Scholar
  48. 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. CrossRefGoogle Scholar
  49. Rieger P, Meier H, Gerle M, Vogt U, Groth T, Knackmuss H (2002) Xenobiotics in the environment: present and future strategies to obviate the problem of biological persistence. J Biotechnol 94:101–123. CrossRefPubMedGoogle Scholar
  50. Ryan A, Kaplan E, Laurieri N, Lowe E, Sim E (2011) Activation of nitrofurazone by azoreductases: multiple activities in one enzyme. Sc Rep 1:63. CrossRefGoogle Scholar
  51. Schmidt O, Drake HL, Horn MA (2010) Hitherto unknown [Fe-Fe]-Hydrogenase gene diversity in anaerobes and anoxic enrichments from a moderately acidic fen. Appl Environ Microbiol 76:2027–2031. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Seravalli J, Ragsdale SW (2008) 13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase. Biochemist 47:6770–6781. CrossRefGoogle Scholar
  53. Soucaille P (2009) Metabolic engineering of Clostridium acetobutylicum for the production of butanol at high yield. New Biotechnol 25:S324CrossRefGoogle Scholar
  54. Steigerwald VJ, Pihl TD, Reeve JN (1992) Identification and isolation of the polyferredoxin from Methanobacterium thermoautotrophicum strain delta H. Proc Natl Acad Sci U S A 89:3031–3035CrossRefGoogle Scholar
  55. Tanner JJ, Lei B, Tu S, Krause KL (1996) Flavin reductase P: structure of a dimeric enzyme that reduces flavin. Biochemist 42:13531–13539. CrossRefGoogle Scholar
  56. Theys J, Pennington O, Dubois L, Anlezark G, Vaughan T, Mengesha A, Landuyt W, Anné J, Burke PJ, Dûrre P, Wouters BG, Minton NP, Lambin P (2006) Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo. Br J Cancer 95:1212–1219. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Vorholt JA, Vaupel M, Thauer RK (1996) A polyferredoxin with eight [4Fe–4S] clusters as a subunit of molybdenum formylmethanofuran dehydrogenase from Methanosarcina barkeri. Eur J Biochem 236:309–317. CrossRefPubMedGoogle Scholar
  58. Watrous MM, Clark S, Kutty R, Huang S, Rudolph FB, Hughes JB, Bennett GN (2003) 2,4,6 trinitrotoluene reduction by an Fe-only hydrogenase in Clostridium acetobutylicum. Appl Environ Microbiol 69:1542–1547. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Weiss DS, Thauer RK (1993) Methanogenesis and the unity of biochemistry. Cell 72:819–822CrossRefGoogle Scholar
  60. Xie B-T, Liu Z-Y, Tian L, Li F-L, Chen X-H (2015) Physiological response of Clostridium ljungdahlii DSM 13528 of ethanol production under different fermentation conditions. Bioresour Technol 177:302–307. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  1. 1.Department of MicrobiologySavitribai Phule Pune UniversityPuneIndia
  2. 2.Department of Biochemistry & Cell Biology MS-140Rice UniversityHoustonUSA

Personalised recommendations