Skip to main content
Log in

Anaerobic oxidation of toluene (analogues) to benzoate (analogues) by whole cells and by cell extracts of a denitrifying Thauera sp.

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Toluene and related aromatic compounds can be mineralized to CO2 under anoxic conditions. Oxidation requires new dehydrogenase-type enzymes and water as oxygen source, as opposed to the aerobic enzymatic attack by oxygenases, which depends on molecular oxygen. We studied the anaerobic process in the denitrifying bacterium Thauera sp. strain K172. Toluene and a number of its fluoro-, chloro- and methyl-analogues were transformed to benzoate and the respective analogues by whole cells and by cell extracts. The transformation of xylene isomers to methylbenzoate isomers suggests that xylene degradation is similarly initiated by oxidation of one of the methyl groups. Toluene oxidation was strongly, but reversibly inhibited by benzyl alcohol. The in vitro oxidation of the methyl group was coupled to the reduction of nitrate, required glycerol for activity, and was inhibited by oxygen. Cells also contained benzyl alcohol dehydrogenase (NAD+), benzaldehyde dehydrogenase (NADP+), benzoate-CoA ligase (AMP-forming), and benzoyl-CoA reductase (dearomatizing). The toluene-oxidizing activity was induced when cells were grown anaerobically with toluene and also with benzyl alcohol or benzaldehyde, suggesting that benzyl alcohol or benzaldehyde acts as inducer. The other enzymes were similarly active in cells grown with toluene, benzyl alcohol, benzaldehyde, or benzoate. This is the first in vitro study of anaerobic oxidation of an aromatic hydrocarbon and of the whole-cell regulation of the toluene-oxidizing enzyme.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Altenschmidt U, Fuchs G (1991) Anaerobic degradation of toluene in denitrifying Pseudomonas sp: indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate. Arch Microbiol 156:152–158

    Google Scholar 

  • Altenschmidt U, Fuchs G (1992) Anaerobic toluene oxidation to benzyl alcohol and benzaldehyde in a denitrifying Pseudomonas strain. J Bacteriol 174:4860–4862

    Google Scholar 

  • Altenschmidt U, Oswald B, Fuchs G (1991) Purification of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp. J Bacteriol 173: 5494–5501

    Google Scholar 

  • Anders H-J, Kaetzke A, Kämpfer P, Ludwig W, Fuchs G (1995) Taxonomic position of aromatic degrading denitrifying pseudomonad strains K172 and KB740. Description of a new member of the genus Thauera, Thauera aromatica sp. nov. and of the genus Azoarcus, Azoarcus evansii sp. nov., members of the beta subclass of proteobacteria. Int J Sytem Bacteriol (in press)

  • Beller HR, Grbić-Galić D, Reinhard M (1992) Microbial degradation of toluene under sulfate-reducing conditions and the influence of iron on the process. Appl Environ Microbiol 58:786–793

    Google Scholar 

  • Biegert T, Altenschmidt U, Eckerskorn C, Fuchs G (1993) Enzymes of anaerobic metabolism of phenolic compounds: 4-hydroxybenzoate-CoA ligase from a denitrifying Pseudomonas species. Eur J Biochem 213:555–561

    Google Scholar 

  • Biegert T, Altenschmidt U, Eckerskorn C, Fuchs G (1995) Purification and properties of benzyl alcohol dehydrogenase from a denitrifying Thauera sp. Arch Microbiol accompanying paper

  • Bossert ID, Whited G, Gibson DT, Young LY (1989) Anaerobic oxidation of p-cresol mediated by a partially purified methylhydroxylase from a denitrifying bacterium. J Bacteriol 171: 2956–2962

    Google Scholar 

  • Brackmann R, Fuchs G (1993) Enzymes of anaerobic metabolism of phenolic compounds: 4-hydroxybenzoyl-CoA reductase (dehydroxylating) from a denitrifying Pseudomonas species. Eur J Biochem 213:563–571

    Google Scholar 

  • Bradford M M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Chalmers RM, Scott AJ, Fewson CA (1990) Purification of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 of Pseudomonas putida MT53 and their preliminary comparison with benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase I and II from Acinetobacter calcoaceticus. J Gen Microbiol 136:637–643

    Google Scholar 

  • Dagley S (1984) Microbial degradation of aromatic compounds. Dev Ind Microbiol 25:53–65

    Google Scholar 

  • Dangel W, Brackmann R, Lack A, Mohammed M, Koch J, Oswald B, Seyfried B, Tschech A, Fuchs G (1991) Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA. Arch Microbiol 155:256–262

    Google Scholar 

  • Dolfing J, Zeyer J, Binder-Eicher P, Schwarzenbach RP (1990) Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen. Arch Microbiol 154:336–341

    Google Scholar 

  • Edwards EA, Grbić-Galić D (1994) Anaerobic degradation of toluene and o-xylene by a methanogenic consortium. Appl Environ Microbiol 60:313–322

    Google Scholar 

  • Edwards EA, Wills LE, Reinhard M, Grbić-Galić D (1992) Anaerobic transformation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions. Appl Environ Microbiol 58:794–800

    Google Scholar 

  • Evans PJ, Mang DT, Kim KS, Young LY (1991) Anaerobic degradation of toluene by a denitrifying bacterium. Appl Environ Microbiol 57:1139–1145

    Google Scholar 

  • Evans PJ, Ling W, Goldschmidt B, Ritter ER, Young LY (1992) Metabolites formed during anaerobic transformations of toluene and o-xylene and their proposed relationship to the initial steps of toluene mineralization. Appl Environ Microbiol 58:496–501

    Google Scholar 

  • Fries MR, Zhou J, Chee-Sanford J, Tiedje JM (1994) Isolation characterization and distribution of denitrifying toluene-degraders from a variety of habitats. Appl Environ Microbiol 60:2801–2810

    Google Scholar 

  • Fuchs G, Mohamed M, Altenschmidt U, Koch J, Lack A, Brackmann R, Lochmeyer C, Oswald B (1994) Biochemistry of anaerobic biodegradation of aromatic compounds. In: Ratledge C (ed) Biochemistry of microbial degradation, Kluwer, Dordrecht, pp 513–553

    Google Scholar 

  • Gibson DT, Subramanian V (1984) Microbial degradation of organic compounds. In: Gibson DT (ed) Microbial degradation of organic compounds. Dekker, New York, pp 181–252

    Google Scholar 

  • Gibson J, Dispensa M, Fogg GC, Evans DT, Harwood CS (1994) 4-Hydroxybenzoate-Coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation. J Bacteriol 176:634–641

    Google Scholar 

  • Grbić-Galić D, Vogel TM (1987) Transformation of toluene and benzene by mixed methanogenic cultures. Appl Environ Microbiol 3:254–260

    Google Scholar 

  • Hopper DJ, Bossert ID, Rhodes-Roberts ME (1991) p-Cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol. J Bacteriol 173:1298–1301

    Google Scholar 

  • Jüttner F, Henatsch JJ (1986) Anoxic hypolimnion is a significant source of biogenic toluene. Nature 323:797–798

    Google Scholar 

  • Koch J, Fuchs G (1992) Enzymatic reduction of benzoyl-CoA to alicyclic compounds: a key reaction in anaerobic aromatic reduction. Eur J Biochem 205:195–202

    Google Scholar 

  • Koch J, Eisenreich W, Bacher A, Fuchs G (1993) Products of enzymatic reduction of benzoyl-CoA: a key reaction in anaerobic aromatic metabolism. Eur J Biochem 211:649–661

    Google Scholar 

  • Kuhn EP, Colberg PJ, Schnoor JL, Wanner O, Zehnder AJB, Schwarzenbach RP (1985) Microbial transformation of substituted benzenes during infiltration of river water to groundwater: laboratory column studies. Environ Sci Technol 19:961–968

    Google Scholar 

  • Kuhn EP, Zeyer J, Eicher P, Schwarzenbach RP (1988) Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns. Appl Environ Microbiol 54:490–496

    Google Scholar 

  • Lovley DR, Lonergan DJ (1990) Anaerobic oxidation of toluene, phenol, and p-cresol by the dissimilatory iron-reducing organism, GA-15. Appl Environ Microbiol 56:1858–1864

    Google Scholar 

  • Lovley DR, Baedecker MJ, Lonergan DJ, Cozzarelli IM, Philips EJP, Siegel DI (1989) Oxidation of aromatic contaminants coupled to microbial iron reduction. Nature 339:297–300

    Google Scholar 

  • MacKintosh RW, Fewson CA (1988a) Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus: purification and preliminary characterization. Biochem J 250:743–751

    Google Scholar 

  • MacKintosh RW, Fewson CA (1988b) Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus: substrate specificities and inhibition studies. Biochem J 255:653–661

    Google Scholar 

  • Macy JM, Rech S, Auling G, Dorsch M, Stackebrandt E, Sly LJ (1993) Thauera selenatis gen. nov., sp. nov. a member of the beta subclass of proteo bacteria with a novel type of anaerobic respiration. Int J Syst Bacteriol 213:135–142

    Google Scholar 

  • Rabus R, Nordhaus R, Ludwig W, Widdel F (1993) Complete oxidation of toluene under strictly anoxic conditions by a new sulfate-reducing bacterium. Appl Environ Microbiol 59: 1444–1451

    Google Scholar 

  • Rudolphi A, Tschech A, Fuchs G (1991) Anaerobic degradation of cresols by denitrifying bacteria. Arch Microbiol 155:238–248

    Google Scholar 

  • Schocher RJ, Seyfried B, Vazquez F, Zeyer J (1991) Anaerobic degradation of toluence by pure cultures of denitrifying bacteria. Arch Microbiol 157:7–12

    Google Scholar 

  • Seyfried B, Glod G, Schocher R, Tschech A, Zeyer J (1994) Initial reactions in the anaerobic oxidation of toluene and m-xylene by denitrifying bacteria. Appl Environ Microbiol 60:4047–4052

    Google Scholar 

  • Shaw JP, Harayama S (1990) Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida. Eur J Biochem 191:705–714

    Google Scholar 

  • Tschech A, Fuchs G (1987) Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying Pseudomonas. Arch Microbiol 148:213–217

    Google Scholar 

  • Vogel TM, Grbić-Galić D (1986) Incorporation of oxygen from water into toluene and benzene during anaerobic fermentative transformation. Appl Environ Microbiol 52:200–202

    Google Scholar 

  • Wilson BH, Smith GB, Rees JH (1986) Biotransformations of selected alkylbenzenes and halogenated aliphatic hydrocarbons in methanogenic aquifier material: a microcosm study. Environ Sci Technol 20:997–1002

    Google Scholar 

  • Zeyer J, Kuhn EP, Schwarzenbach RP (1986) Rapid microbial mineralization of toluene and 1,3-dimethylbenzene in the absence of molecular oxygen. Appl Environ Microbiol 52:944–947

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Dedicated to Prof. Achim Trebst

Rights and permissions

Reprints and permissions

About this article

Cite this article

Biegert, T., Fuchs, G. Anaerobic oxidation of toluene (analogues) to benzoate (analogues) by whole cells and by cell extracts of a denitrifying Thauera sp.. Arch. Microbiol. 163, 407–417 (1995). https://doi.org/10.1007/BF00272129

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00272129

Key words

Navigation