Skip to main content
SpringerLink
Log in

Biodegradation of 2-Chlorobenzoate by Recombinant Burkholderia Cepacia Expressing Vitreoscilla Hemoglobin Under Variable Levels of Oxygen Availability

  • Published:
Biodegradation Aims and scope Submit manuscript

Abstract

The influence of bacterial hemoglobin, VHb, on dechlorinationand degradation of 2-chlorobenzoate (2-CBA) by recombinantBurkholderia sp. under variable oxygen availability with an initial dissolved oxygenconcentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture. Abilityto express VHb was provided to recombinant Burkholderia by transformationwith the VHb gene, vgb, on plasmid pSC160. 100% of 0.5 mM CBA was degraded incultures with 85% and 70% of total volume as headspace air in closed reactorsby both wild type and recombinant Burkholderia. The recombinant cultures were able todechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 °Ccompared to more than 120 hours for wild type cultures. The rate and extent of CBAdegradation by recombinant cultures with 40% of total volume as headspace air was higher than thoseachieved by wild type cells at the end of the 168 hours of incubation period, 98and 73%, respectively. The chloride released: CBA degraded molar ratio for cultures with 40%of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinantstrains, whereas it was non-stoichiometric (molar ratio = 0.24)for wild type cells. The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorinationand degradation pathway for recombinant cells under hypoxic conditions.The degradation and dechlorination ability of both types of cells was alsoinvestigated in continuous reactor studies by varying the dilution rate under hypoxicconditions. Regarding potential of the recombinant strain for 2-CBA degradation in eitheropen ecosystems or closed bioreactor bioremediation systems, the stability of the plasmidcontaining vgb in the recombinant cells was also studied; the plasmid was100% stable at 0.025 h-1 dilution rate (∼1.7 d hydraulic retention time),even after one month.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Arensdorf JJ & Focht DD (1994) Formation of chlorocatechol meta cleavage products by a Pseudomonad during metabolism of monochlorbiphenyls. Appl. Environ. Microbiol. 60: 2884–2889

    Google Scholar 

  • Arensdorf JJ & Focht DD (1995) A meta cleavage pathway for 4-Chlorbenzoate, an intermediate in the metabolism of 4-chlorobiphenyl by Pseudomonas cepacia P166. Appl. Environ. Microbiol. 61: 443–447

    Google Scholar 

  • Balajee S & Mahadevan A (1990) Utilization of chloroaromatic substances by Azotobacter chroococcum. Syst. Appl. Microbiol. 13: 194–198

    Google Scholar 

  • Bartels I, Knackmuss HJ & Reineke W (1984) Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols. Appl. Environ. Microbiol. 47: 500–505

    Google Scholar 

  • Champagne P, Van Geel, PJ & Parker WJ (1998) A proposed transient model for cometabolism in biofilm systems. Biotechnol. Bioeng. 60: 541–550

    Google Scholar 

  • Chung JW, Webster DA, Pagilla KR & Stark BC (2001) Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability. J. Ind. Microbiol. Biotechnol. 27: 27–33

    Google Scholar 

  • Deweerd KA & Bedard DL (1999) Use of halogenated benzoates and other halogenated aromatic compounds to stimulate the microbial dechlorination of PCBs. Environ. Sci. Technol. 33: 2057–2063

    Google Scholar 

  • Dikshit KL & Webster DA (1988) Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene 70: 377–386

    Google Scholar 

  • Dikshit KL, Dikshit, RP & Webster DA (1990) Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion. Nucl. Acid Res. 18: 4149–4155

    Google Scholar 

  • Fish PA, Webster DA & Stark BC (2000) Vitroeoscilla hemoglobin enhances the first step in 2,4-dinitrotoluene degradation in vitro and at low aeration in vivo. J. Mol. Cat. B 9: 75–82

    Google Scholar 

  • Flanagan WP & May RJ (1993) Metabolite detection as evidence for naturally occurring aerobic PCB biodegradation in Hudson River sediments. Environ. Sci. Technol. 27: 2207–2212

    Google Scholar 

  • Harkness MR, McDermott JB, Abramowicz DA, Salvo JJ, Flanagan WP, Stephens ML, Mondello FJ, May RJ, Lobos JH, Carroll KM, Brenna MJ, Bracco AA, Fish KM, Warner GL, Wilson PR, Dietrich DK, Lin DT, Morgan, CB & Gately WL (1993) In situ stimulation of aerobic PCB degradation in Hudson river sediments. Science 259: 503–507

    Google Scholar 

  • Joshi M & Dikshit KL (1994) Oxygen dependent regulation of Vitreoscilla globin gene: evidence for positive regulation by FNR. Biochem. Biophys. Com. 202: 535–542

    Google Scholar 

  • Khosla C & Bailey JE (1988a) The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence, and genetic expression in Escherichia coli. Mol. Gen. Genetics. 214: 158–161

    Google Scholar 

  • Khosla C & Bailey JE (1988b) Heterologous expression of a bacterial hemoglobin improves the growth properties of recombinant Escherichia coli. Nature 331: 633–635

    Google Scholar 

  • Kröckel L & Focht DD (1987) Construction of chlorobenzene utilizing recombinants by progentive manifestation of a rare event. Appl. Environ. Microbiol. 53: 2470–2475

    Google Scholar 

  • Krooneman J, Wieringa EBA, Moore ERB, Gerritse J, Prins RA & Gottschal, JC (1996) Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols. Appl. Environ. Microbiol. 62: 2427–2434

    Google Scholar 

  • Krooneman J, Moore ERB, van Velzen JCL, Prins RA, Forney, LJ & Gottschal, JC (1999) Isolation of Alcaligens sp. train L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (Chloro) catechols. Appl. Environ. Microbiol. 62: 2427–2434

    Google Scholar 

  • Lin JM, Stark BC & Webster DA (in press) Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (DNT) dioxygenase activity of Burkholderia and on DNT degradation in two-phase bioreactors. J. Ind. Microbiol. Biotechnol.

  • Liu SC, Webster DA & Stark BC (1995) Cloning and expression of Vitreoscilla hemoglobin gene in Pseudomonas: Effects on cell growth. Appl. Microbiol. Biotechnol. 44: 419–424

    Google Scholar 

  • Mars AE, Kasberg T, Kaschabek SR, van Agteren MH, Janssen DB & Reineke W (1997) Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene. J. Bacteriol. 179: 4530–4537

    Google Scholar 

  • Müller R, Deckwer WD & Hecht V (1996) Degradation of chloroand methyl-substituted benzoic acids by a genetically modified microorganism. Biotechnol. Bioeng. 51: 528–537

    Google Scholar 

  • Nasr MA, Hwang KW, Akbas M, Webster DA & Stark BC (2001) Effects of culture conditions on enhancement of 2,4-dinitrotoluene degradation by Burkholderia engineered with the Vitreoscilla hemoglobin gene. Biotechnol. Prog. 17: 359–361

    Google Scholar 

  • Niedan V & Schöler HF (1997) Natural formation of chlorobenzoic acids (CBA) and distinction between PCB degraded bacteria. Chemosphere 35: 1233–1241

    Google Scholar 

  • Oldenhius R, Kuijk L, Lammers A, Janssen DB & Witholt B (1989) Degradation of chlorinated and nonchlorinated aromatic solvents in soil suspensions by pure bacterial cultures. Appl. Microbiol. Biotechnol. 30: 211–217

    Google Scholar 

  • Oltmanns HG, Rast RH & Reineke W (1988) degradation of 1,4-dichlorobenzene and toluene by a Pseudomonas strain. Appl. Environ. Microbiol. 57: 157–162

    Google Scholar 

  • Park KW, Kim KJ, Howard AJ, Stark BC & Webster DA (2002) Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases. J. Biol. Chem. 277: 33334–33337

    Google Scholar 

  • Patel SM, Stark BC, Hwang KW, Dikshit KL & Webster DA (2000) Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation. Biotechnol. Prog. 16: 26–30

    Google Scholar 

  • Rainer, BW (1990) Oxygen transfer in bioreactors. Chem. Biochem Eng. Q4. 4: 185–196

    Google Scholar 

  • Ramandeep, Hwang KW, Raje M, Kim KJ, Stark BC, Dikshit KL & Webster DA (2001) Vitreoscilla hemoglobin: Intracellular localization and binding to membranes. J. Biol. Chem. 276: 24781–24789

    Google Scholar 

  • Reineke W & Knackmuss HJ (1984) Microbial metabolism of haloaromatics: Isolation and properties of a chlorobenzene degrading bacterium. Appl. Environ. Microbiol. 47: 395–402

    Google Scholar 

  • Romanov V & Hausinger RP (1994) Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism of 2,4-dichloro-and 2-chlorobenzoate. J. Bacteriol. 176: 3368–3374

    Google Scholar 

  • Schmidt E & Knackmuss H J (1980) Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid. Biochem. J. 192: 339–347

    Google Scholar 

  • Seeger M, Zielinski M, Timmis KN & Hofer B (1999) Regiospecifity of dioxygenation of di-to pentachlorobiphenyls and their degradation to chlorobenzoates by the bph-encoded catabolic pathway of Burkholderia sp. strain LB400. Appl. Environ. Microbiol. 65: 3614–3621

    Google Scholar 

  • Spanggord RJ, Spain JC, Nishino SF & Mortelmans KE (1991) Biodegradation of 2,4-dinitrotoulene by a Pseudomonas sp. Appl. Environ. Microbiol. 57: 3200–3205

    Google Scholar 

  • Tross ME, Schraa G & Zehinder AJB (1996) Transformation of low concentrations of 3-chlorobenzoate by Pseudomonas sp. strain B13: Kinetics and residual concentrations. Appl. Environ. Microbiol. 62: 437–442

    Google Scholar 

  • Tsai PS, Hatzimanikatis V & Bailey JE (1996) Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherchia coli carbon and energy metabolism. Biotechnol. Bioeng. 49: 139–150

    Google Scholar 

  • Tsoi TV, Plotnikova EG, Cole J, Guerin WF, Bagdasarian M & Tiedje J (1999) Cloning, expression and nucleotide sequence of the Pseudomonas aeruginosa 142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates. Appl. Environ. Microbiol. 65: 2151–2162

    Google Scholar 

  • Van Der Woude BJ (1996) Extent of reductive dechlorination of chlorobenzoates in anoxic sediment slurries depends on the sequence of chlorine removal. Environ. Science Technol. 30: 1352–1357

    Google Scholar 

  • Van Der Woude BJ, Gottschal JC & Prins RA (1995) Degradation of 2,5-dichlorobenzoic acid by Pseudomonas aeruginosa JB2 at low oxygen tensions. Biodegradation 6: 39–46

    Google Scholar 

  • Viliesid F & Lilly MD (1992) Influence of dissolved oxygen tension on the synthesis of catechol 1,2-dioxygenase by Pseudomonas putida. Enzyme Microbiol. Technol. 14: 561–565

    Google Scholar 

  • Vollmer MD & Schlomann M (1995) Conversion of 2-chloro-cis,cis-muconate and its metabolites 2-chloro-and 5-chloromuconolactone by chloromuconate cycloisomerases of pJP4 and pAC27. J. Bacteriol. 177: 2938–2941

    Google Scholar 

  • Wakabayashi S, Matsubara H & Webster DA (1986) Primary sequence of a dimeric bacterial hemoglobin from Vitreoscilla. Nature 322: 481–483

    Google Scholar 

  • Webster D (1987) Structure and function of bacterial hemoglobin and related proteins. In: Eichorn GC & Marzilli LG (Eds) Advances in Inorganic Biochemistry, Vol 7 (pp 245–265). Elsevier, New York

    Google Scholar 

  • Zaitsev GM, Uotila JS, Tsitko IV, Lobanok AG & Salonen MSS (1995) Utilization of halogenated benzenes, phenols and benzoates by Rhodococcus opasus GM-14. Appl. Environ. Microbiol. 61: 4191–4201

    Google Scholar 

  • Zaitsev GM & Karasevich YN (1984) Utilization of 2-chlorobenzoic acid by Pseudomonas cepacia. Mikrobiologiya 53: 75–80

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemical and Environmental Engineering, Illinois Institute of Technology Chicago, IL, 60616, USA

    Meltem Urgun-Demirtas & Krishna R. Pagilla

  2. Department of Biological, Chemical and Physical Sciences, Illinois Institute of Technology Chicago, IL, 60616, USA

    Benjamin C. Stark & Dale Webster

Authors
  1. Meltem Urgun-Demirtas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Krishna R. Pagilla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Benjamin C. Stark
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dale Webster
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Krishna R. Pagilla.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Urgun-Demirtas, M., Pagilla, K.R., Stark, B.C. et al. Biodegradation of 2-Chlorobenzoate by Recombinant Burkholderia Cepacia Expressing Vitreoscilla Hemoglobin Under Variable Levels of Oxygen Availability. Biodegradation 14, 357–365 (2003). https://doi.org/10.1023/A:1025672528291

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1025672528291

Search

Navigation