Applied Microbiology and Biotechnology

, Volume 86, Issue 2, pp 435–444 | Cite as

Nitrification and degradation of halogenated hydrocarbons—a tenuous balance for ammonia-oxidizing bacteria

  • Luis A. Sayavedra-Soto
  • Barbara Gvakharia
  • Peter J. Bottomley
  • Daniel J. Arp
  • Mark E. Dolan


The process of nitrification has the potential for the in situ bioremediation of halogenated compounds provided a number of challenges can be overcome. In nitrification, the microbial process where ammonia is oxidized to nitrate, ammonia-oxidizing bacteria (AOB) are key players and are capable of carrying out the biodegradation of recalcitrant halogenated compounds. Through industrial uses, halogenated compounds often find their way into wastewater, contaminating the environment and bodies of water that supply drinking water. In the reclamation of wastewater, halogenated compounds can be degraded by AOB but can also be detrimental to the process of nitrification. This minireview considers the ability of AOB to carry out cometabolism of halogenated compounds and the consequent inhibition of nitrification. Possible cometabolism monitoring methods that were derived from current information about AOB genomes are also discussed. AOB expression microarrays have detected mRNA of genes that are expressed at higher levels during stress and are deemed “sentinel” genes. Promoters of selected “sentinel” genes have been cloned and used to drive the expression of gene-reporter constructs. The latter are being tested as early warning biosensors of cometabolism-induced damage in Nitrosomonas europaea with promising results. These and other biosensors may help to preserve the tenuous balance that exists when nitrification occurs in waste streams containing alternative AOB substrates such as halogenated hydrocarbons.


Nitrification Chlorinated aliphatic hydrocarbons N. europaea Degradation Cometabolism 



The authors thank The National Science Foundation (Biocomplexity grant number 0412711) and the Oregon Agricultural Experimental Station for the funds provided.


  1. Abeliovich A (1987) Nitrifying bacteria in wastewater reservoirs. Appl Environ Microbiol 53:754–760Google Scholar
  2. Alpaslan Kocamemi B, Cecen F (2007) Kinetic analysis of the inhibitory effect of trichloroethylene (TCE) on nitrification in cometabolic degradation. Biodegradation 18:71–81CrossRefGoogle Scholar
  3. Alvarez-Cohen L, Speitel GE Jr (2001) Kinetics of aerobic cometabolism of chlorinated solvents. Biodegradation 12:105–126CrossRefGoogle Scholar
  4. Archibald F, Methot M, Young F, Paice MG (2001) A simple system to rapidly monitor activated sludge health and performance. Water Res 35:2543–2553CrossRefGoogle Scholar
  5. Arp DJ, Bottomley PJ (2006) Nitrifiers: more than 100 years from isolation to genome sequences. Microbe 1:229–234Google Scholar
  6. Arp DJ, Stein LY (2003) Metabolism of inorganic N compounds by ammonia-oxidizing bacteria. Crit Rev Biochem Mol Biol 38:471–495CrossRefGoogle Scholar
  7. Arp DJ, Yeager CM, Hyman MR (2001) Molecular and cellular fundamentals of aerobic cometabolism of trichloroethylene. Biodegradation 12:81–103CrossRefGoogle Scholar
  8. Arp DJ, Sayavedra-Soto LA, Hommes NG (2002) Molecular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea. Arch Microbiol 178:250–255CrossRefGoogle Scholar
  9. Arp DJ, Chain PS, Klotz MG (2007) The impact of genome analyses on our understanding of ammonia-oxidizing bacteria. Annu Rev Microbiol 61:503–528CrossRefGoogle Scholar
  10. Barney BM, LoBrutto R, Francisco WA (2004) Characterization of a small metal binding protein from Nitrosomonas europaea. Biochemistry 43:11206–11213CrossRefGoogle Scholar
  11. Bashyam MD, Hasnain SE (2004) The extracytoplasmic function sigma factors: role in bacterial pathogenesis. Infect Genet Evol 4:301–308CrossRefGoogle Scholar
  12. Betts JC, Lukey PT, Robb LC, McAdam RA, Duncan K (2002) Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 43:717–731CrossRefGoogle Scholar
  13. Booth IR, Ferguson GP, Miller S, Li C, Gunasekera B, Kinghorn S (2003) Bacterial production of methylglyoxal: a survival strategy or death by misadventure? Biochem Soc Trans 31:1406–1408CrossRefGoogle Scholar
  14. Bott CB, Love NG (2002) Investigating a mechanistic cause for activated-sludge deflocculation in response to shock loads of toxic electrophilic chemicals. Water Environ Res 74:306–315CrossRefGoogle Scholar
  15. Bott CB, Love NG (2004) Implicating the glutathione-gated potassium efflux system as a cause of electrophile-induced activated sludge deflocculation. Appl Environ Microbiol 70:5569–5578CrossRefGoogle Scholar
  16. Brandt KK, Pedersen A, Sorensen J (2002) Solid-phase contact assay that uses a lux-marked Nitrosomonas europaea reporter strain to estimate toxicity of bioavailable linear alkylbenzene sulfonate in soil. Appl Environ Microbiol 68:3502–3508CrossRefGoogle Scholar
  17. Capestany CA, Tribble GD, Maeda K, Demuth DR, Lamont RJ (2008) Role of the Clp system in stress tolerance, biofilm formation, and intracellular invasion in Porphyromonas gingivalis. J Bacteriol 190:1436–1446CrossRefGoogle Scholar
  18. Chain P, Lamerdin J, Larimer F, Regala W, Lao V, Land M et al (2003) Complete genome sequence of the ammonia-oxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J Bacteriol 185:2759–2773CrossRefGoogle Scholar
  19. Condon C (2006) Shutdown decay of mRNA. Mol Microbiol 61:573–583CrossRefGoogle Scholar
  20. Cui R, Chung WJ, Jahng D (2005) A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation. Biosens Bioelectron 20:1788–1795CrossRefGoogle Scholar
  21. Duncan AJ, Bott CB, Terlesky KC, Love NG (2000) Detection of GroEL in activated sludge: a model for detection of system stress. Lett Appl Microbiol 30:28–32CrossRefGoogle Scholar
  22. Egli K, Langer C, Siegrist HR, Zehnder AJ, Wagner M, van der Meer JR (2003) Community analysis of ammonia and nitrite oxidizers during start-up of nitritation reactors. Appl Environ Microbiol 69:3213–3222CrossRefGoogle Scholar
  23. El Sheikh AF, Klotz MG (2008) Ammonia-dependent differential regulation of the gene cluster that encodes ammonia monooxygenase in Nitrosococcus oceani ATCC 19707. Environ Microbiol 10:3026–3035CrossRefGoogle Scholar
  24. El Sheikh AF, Poret-Peterson AT, Klotz MG (2008) Characterization of two new genes, amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitrosococcus oceani ATCC 19707. Appl Environ Microbiol 74:312–318CrossRefGoogle Scholar
  25. Ely RL, Hyman MR, Arp DJ, Guenther RB, Williamson KJ (1995) A cometabolic kinetics model incoroporating enzyme inhibition, inactivation, and recovery: II. Trichloroethylene degradation experiments. Biotechnol Bioeng 46:232–245CrossRefGoogle Scholar
  26. Ely RL, Williamson KJ, Hyman MR, Arp DJ (1997) Cometabolism of chlorinated solvents by nitrifying bacteria: kinetics, substrate interactions, toxicity effects, and bacterial response. Biotechnol Bioeng 54:520–534CrossRefGoogle Scholar
  27. Ensign SA, Hyman MR, Arp DJ (1993) In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper. J Bacteriol 175:1971–1980Google Scholar
  28. Erguder TH, Boon N, Wittebolle L, Marzorati M, Verstraete W (2009) Environmental factors shaping the ecological niches of ammonia-oxidizing archaea. FEMS Microbiol Rev 33:855–869CrossRefGoogle Scholar
  29. Fetzner S (1998) Bacterial dehalogenation. Appl Microbiol Biotechnol 50:633–657CrossRefGoogle Scholar
  30. Field JA, Sierra-Alvarez R (2004) Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds. Rev Environ Sci Biotechnol 3:185–254CrossRefGoogle Scholar
  31. Gerdes K, Christensen SK, Lobner-Olesen A (2005) Prokaryotic toxin–antitoxin stress response loci. Nat Rev Microbiol 3:371–382CrossRefGoogle Scholar
  32. Gvakharia BO, Permina EA, Gelfand MS, Bottomley PJ, Sayavedra-Soto LA, Arp DJ (2007) Global transcriptional response of Nitrosomonas europaea to chloroform and chloromethane. Appl Environ Microbiol 73:3440–3445CrossRefGoogle Scholar
  33. Gvakharia BO, Bottomley PJ, Arp DJ, Sayavedra-Soto LA (2009) Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in response to co-oxidation of chloroform. Appl Microbiol Biotechnol 82:1179–1185CrossRefGoogle Scholar
  34. Hamamura N, Page C, Long T, Semprini L, Arp DJ (1997) Chloroform cometabolism by butane-grown CF8, Pseudomonas butanovora, and Mycobacterium vaccae JOB5 and methane-grown Methylosinus trichosporium OB3b. Appl Environ Microbiol 63:3607–3613Google Scholar
  35. Harms H, Wells MC, van der Meer JR (2006) Whole-cell living biosensors—are they ready for environmental application? Appl Microbiol Biotechnol 70:273–280CrossRefGoogle Scholar
  36. Hyman MR, Murton IB, Arp DJ (1988) Interaction of ammonia monooxygenase from Nitrosomonas europaea with alkanes, alkenes, and alkynes. Appl Environ Microbiol 54:3187–3190Google Scholar
  37. Hyman MR, Ensign SA, Arp DJ, Ludden PW (1989) Carbonyl sulfide inhibition of CO dehydrogenase from Rhodospirillum rubrum. Biochemistry 28:6821–6826CrossRefGoogle Scholar
  38. Hyman MR, Russell SA, Ely RL, Williamson KJ, Arp DJ (1995) Inhibition, inactivation, and recovery of ammonia-oxidizing activity in cometabolism of trichloroethylene by Nitrosomonas europaea. Appl Environ Microbiol 61:1480–1487Google Scholar
  39. Iizumi T, Mizumoto M, Nakamura K (1998) A bioluminescence assay using Nitrosomonas europaea for rapid and sensitive detection of nitrification inhibitors. Appl Environ Microbiol 64:3656–3662Google Scholar
  40. Jetten MS, Niftrik LV, Strous M, Kartal B, Keltjens JT, Op den Camp HJ (2009) Biochemistry and molecular biology of anammox bacteria. Crit Rev Biochem Mol Biol 26:1–20CrossRefGoogle Scholar
  41. Juliette LY, Hyman MR, Arp DJ (1993a) Mechanism-based inactivation of ammonia monooxygenase in Nitrosomonas europaea by allylsulfide. Appl Environ Microbiol 59:3728–3735Google Scholar
  42. Juliette LY, Hyman MR, Arp DJ (1993b) Inhibition of ammonia oxidation in Nitrosomonas europaea by sulfur compounds—thioethers are oxidized to sulfoxides by ammonia monooxygenase. Appl Environ Microbiol 59:3718–3727Google Scholar
  43. Kao CM, Prosser J (1999) Intrinsic bioremediation of trichloroethylene and chlorobenzene: field and laboratory studies. J Hazard Mater 69:67–79CrossRefGoogle Scholar
  44. Keener WK, Arp DJ (1993) Kinetic studies of ammonia monooxygenase inhibition in Nitrosomonas europaea by hydrocarbons and halogenated hydrocarbons in an optimized whole-cell assay. Appl Environ Microbiol 59:2501–2510Google Scholar
  45. Keener WK, Arp DJ (1994) Transformations of aromatic compounds by Nitrosomonas europaea. Appl Environ Microbiol 60:1914–1920Google Scholar
  46. Klotz MG, Norton JM (1995) Sequence of an ammonia monooxygenase subunit A-encoding gene from Nitrosospira sp. NpAV. Gene 163:159–160CrossRefGoogle Scholar
  47. Klotz MG, Arp DJ, Chain PS, El-Sheikh AF, Hauser LJ, Hommes NG et al (2006) Complete genome sequence of the marine, chemolithoautotrophic, ammonia-oxidizing bacterium Nitrosococcus oceani ATCC 19707. Appl Environ Microbiol 72:6299–6315CrossRefGoogle Scholar
  48. Kohlmeier S, Mancuso M, Deepthike U, Tecon R, van der Meer JR, Harms H, Wells M (2008) Comparison of naphthalene bioavailability determined by whole-cell biosensing and availability determined by extraction with Tenax. Environ Pollut 156:803–808CrossRefGoogle Scholar
  49. Love NG, Bott CB (2002) Evaluating the role of microbial stress response mechanisms in causing biological treatment system upset. Water Sci Technol 46:11–18Google Scholar
  50. Martens-Habbena W, Berube PM, Urakawa H, de la Torre JR, Stahl DA (2009) Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature 461:976–979CrossRefGoogle Scholar
  51. Murrell JC, Gilbert B, McDonald IR (2000) Molecular biology and regulation of methane monooxygenase. Arch Microbiol 173:325–332CrossRefGoogle Scholar
  52. Norton JM, Klotz MG, Stein LY, Arp DJ, Bottomley PJ, Chain PS et al (2008) Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment. Appl Environ Microbiol 74:3559–3572CrossRefGoogle Scholar
  53. Painter HA (1986) Nitrification in the treatment of sewage and waste-waters. In: Prosser JI (ed) Nitrification. IRL, Washington, pp 185–211Google Scholar
  54. Pandey DP, Gerdes K (2005) Toxin–antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res 33:966–976CrossRefGoogle Scholar
  55. Park S, Ely RL (2008a) Whole-genome transcriptional and physiological responses of Nitrosomonas europaea to cyanide: identification of cyanide stress response genes. Biotechnol Bioeng 102:1645–1653CrossRefGoogle Scholar
  56. Park S, Ely RL (2008b) Candidate stress genes of Nitrosomonas europaea for monitoring inhibition of nitrification by heavy metals. Appl Environ Microbiol 74:5475–5482CrossRefGoogle Scholar
  57. Park S, Ely RL (2008c) Genome-wide transcriptional responses of Nitrosomonas europaea to zinc. Arch Microbiol 189:541–548CrossRefGoogle Scholar
  58. Radniecki TS, Dolan ME, Semprini L (2008) Physiological and transcriptional responses of Nitrosomonas europaea to toluene and benzene inhibition. Environ Sci Technol 42:4093–4098CrossRefGoogle Scholar
  59. Radniecki TS, Semprini L, Dolan ME (2009a) Expression of merA, trxA, amoA, and hao in continuously cultured Nitrosomonas europaea cells exposed to cadmium sulfate additions. Biotechnol Bioeng 104:1004–1011CrossRefGoogle Scholar
  60. Radniecki TS, Semprini L, Dolan ME (2009b) Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions. Biotechnol Bioeng 102:546–553CrossRefGoogle Scholar
  61. Rasche ME, Hicks RE, Hyman MR, Arp DJ (1990) Oxidation of monohalogenated ethanes and n-chlorinated alkanes by whole cells of Nitrosomonas europaea. J Bacteriol 172:5368–5373Google Scholar
  62. Rasche ME, Hyman MR, Arp DJ (1991) Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: cometabolic inactivation of ammonia monooxygenase and substrate specificity. Appl Environ Microbiol 57:2986–2994Google Scholar
  63. Reeve CA, Bockman AT, Matin A (1984a) Role of protein degradation in the survival of carbon-starved Escherichia coli and Salmonella typhimurium. J Bacteriol 157:758–763Google Scholar
  64. Reeve CA, Amy PS, Matin A (1984b) Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12. J Bacteriol 160:1041–1046Google Scholar
  65. Ren S (2004) Assessing wastewater toxicity to activated sludge: recent research and developments. Environ Int 30:1151–1164CrossRefGoogle Scholar
  66. Ren S, Frymier PD (2003) Use of multidimensional scaling in the selection of wastewater toxicity test battery components. Water Res 37:1655–1661CrossRefGoogle Scholar
  67. Satoh H, Sasaki Y, Nakamura Y, Okabe S, Suzuki T (2005) Use of microelectrodes to investigate the effects of 2-chlorophenol on microbial activities in biofilms. Biotechnol Bioeng 91:133–138CrossRefGoogle Scholar
  68. Sayavedra-Soto LA, Hommes NG, Alzerreca JJ, Arp DJ, Norton JM, Klotz MG (1998) Transcription of the amoC, amoA and amoB genes in Nitrosomonas europaea and Nitrosospira sp, NpAV. FEMS Microbiol Lett 167:81–88CrossRefGoogle Scholar
  69. Schmidt I, Bock E (1997) Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha. Arch Microbiol 167:106–111CrossRefGoogle Scholar
  70. Schmidt I, Zart D, Bock E (2001) Gaseous NO2 as a regulator for ammonia oxidation of Nitrosomonas eutropha. Antonie Van Leeuwenhoek 79:311–318CrossRefGoogle Scholar
  71. Stein LY, Arp DJ, Berube PM, Chain PS, Hauser L, Jetten MS et al (2007) Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation. Environ Microbiol 9:2993–3007CrossRefGoogle Scholar
  72. Vannelli T, Logan M, Arciero DM, Hooper AB (1990) Degradation of halogenated aliphatic compounds by the ammonia-oxidizing bacterium Nitrosomonas europaea. Appl Environ Microbiol 56:1169–1171Google Scholar
  73. Wackett LP (1996) Co-metabolism: is the emperor wearing any clothes? Curr Opin Biotechnol 7:321–325CrossRefGoogle Scholar
  74. Wahman DG, Henry AE, Katz LE, Speitel GE Jr (2006) Cometabolism of trihalomethanes by mixed culture nitrifiers. Water Res 40:3349–3358CrossRefGoogle Scholar
  75. Wei XM, Yan TF, Hommes NG, Liu XD, Wu LY, McAlvin C et al (2006) Transcript profiles of Nitrosomonas europaea during growth and upon deprivation of ammonia and carbonate. FEMS Microbiol Lett 257:76–83CrossRefGoogle Scholar
  76. Yang L, Chang Y, Chou M (1999) Feasibility of bioremediation of trichloroethylene contaminated sites by nitrifying bacteria through cometabolism with ammonia. J Hazard Mater 69:111–126CrossRefGoogle Scholar
  77. Yeager CM, Bottomley PJ, Arp DJ (2001) Requirement of DNA repair mechanisms for survival of Burkholderia cepacia G4 upon degradation of trichloroethylene. Appl Environ Microbiol 67:5384–5391CrossRefGoogle Scholar
  78. Zellmeier S, Schumann W, Wiegert T (2006) Involvement of Clp protease activity in modulating the Bacillus subtilis sigmaw stress response. Mol Microbiol 61:1569–1582CrossRefGoogle Scholar
  79. Zhu MM, Skraly FA, Cameron DC (2001) Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxification by expression of the Pseudomonas putida glyoxalase I gene. Metab Eng 3:218–225CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Luis A. Sayavedra-Soto
    • 1
  • Barbara Gvakharia
    • 1
  • Peter J. Bottomley
    • 2
  • Daniel J. Arp
    • 1
  • Mark E. Dolan
    • 3
  1. 1.Department of Botany and Plant PathologyOregon State UniversityCorvallisUSA
  2. 2.Department of MicrobiologyOregon State UniversityCorvallisUSA
  3. 3.School of Chemical, Biological and Environmental EngineeringOregon State UniversityCorvallisUSA

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