World Journal of Microbiology and Biotechnology

, Volume 31, Issue 2, pp 371–377 | Cite as

Biodegradation of hexachlorobenzene by a constructed microbial consortium

  • Da-Zhong YanEmail author
  • Ling-Qi Mao
  • Cun-Zhi Li
  • Jun Liu
Original Paper


A consortium comprised of an engineered Escherichia coli DH5α and a natural pentachlorophenol (PCP) degrader, Sphingobium chlorophenolicum ATCC 39723, was assembled for degradation of hexachlorobenzene (HCB), a persistent organic pollutant. The engineered E. coli strain, harbouring a gene cassette (camA + camB + camC) that encodes the F87W/Y96F/L244A/V247L mutant of cytochrome P-450cam (CYP101), oxidised HCB to PCP. The resulting PCP was then further completely degraded by ATCC 39723. The results showed that almost 40 % of 4 μM HCB was degraded by the consortium at a rate of 0.033 nmol/mg (dry weight)/h over 24 h, accompanied by transient accumulation and immediate consumption of the intermediate PCP, detected by gas chromatography. In contrast, in the consortium comprised of Pseudomonas putida PaW340 harbouring camA + camB + camC and ATCC 39723, PCP accumulated in PaW340 cells but could not be further degraded, which may be due to a permeability barrier of Pseudomonas PaW340 for PCP transportation. The strategy of bacterial co-culture may provide an alternative approach for the bioremediation of HCB contamination.


Biotransformation Cytochrome P-450cam Hexachlorobenzene Pentachlorophenol Sphingobiumchlorophenolicum ATCC 39723 








This work was supported by the Natural Science Foundation of Hubei Province of China (2008CDB067) and the National Natural Science Foundation of China (NSFC, Project No. 31270112). We thank Luet-Lok Wong for the gift of plasmid pCWSGB-camC and Luying Xun for providing Sphingobium chlorophenolicum ATCC 39723.


  1. Arfmann H, Timmis KN, Wittich R (1997) Mineralization of 4-chlorodibenzofuran by a consortium consisting of Sphingomonas sp. strain RW1 and Burkholderia sp. strain JWS. Appl Environ Microbiol 63:3458–3462Google Scholar
  2. Bell SG, Harford-Cross CF, Wong LL (2001) Engineering the CYP101 system for in vivo oxidation of unnatural substrates. Protein Eng 14:797–802CrossRefGoogle Scholar
  3. Bosma T, Damborsky J, Stucki G, Janssen DB (2002) Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene. Appl Environ Microbiol 68:3582–3587CrossRefGoogle Scholar
  4. Boyer HW, Roulland-Dussoix D (1969) A complementation of analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472CrossRefGoogle Scholar
  5. Cai M, Xun L (2002) Organization and regulation of pentachlorophenol-degrading genes in Sphingobium chlorophenolicum ATCC 39723. J Bacteriol 184:4672–4680CrossRefGoogle Scholar
  6. Chang BV, Su CJ, Yuan SY (1998) Microbial hexachlorobenzene dechlorination under three reducing conditions. Chemosphere 36:2721–2730CrossRefGoogle Scholar
  7. Chen X, Christopher A, Jones JP, Bell SG, Guo Q, Xu F, Rao Z, Wong LL (2002) Crystal structure of the F87W/Y96F/V247L mutant of cytochrome P-450cam with 1,3,5-trichlorobenzene bound and further protein engineering for the oxidation of pentachlorobenzene and hexachlorobenzene. J Biol Chem 277:37519–37526CrossRefGoogle Scholar
  8. Dai M, Copley SD (2004) Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Appl Environ Microbiol 70:2391–2397CrossRefGoogle Scholar
  9. de Lorenzo V, Eltis L, Kessler B, Timmis KN (1993) Analysis of Pseudomonas gene products using lacI q /Ptrp-lac plasmids and transposons that confer conditional phenotypes. Gene 123:17–24CrossRefGoogle Scholar
  10. Fathepure BZ, Tiedje JM, Boyd SA (1988) Reductive dechlorination of hexachlorobenzene to tri- and dichlorobenzenes in anaerobic sewage sludge. Appl Environ Microbiol 54:327–330Google Scholar
  11. Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652CrossRefGoogle Scholar
  12. Gilbert ES, Walker AW, Keasling JD (2003) A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion. Appl Microbiol Biotechnol 61:77–81CrossRefGoogle Scholar
  13. Haro MA, de Lorenzo V (2001) Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene. J Biotechnol 85:103–113CrossRefGoogle Scholar
  14. Jacoff FS, Scarberry R, Rosa D (1986) Source assessment of hexachlorobenzene from the organic chemical manufacturing industry. IARC Sci Publ 77:31–37Google Scholar
  15. Jayachandran G, Görisch H, Adrian L (2003) Dehalorespiration with hexachlorobenzene and pentachlorobenzene by Dehalococcoides sp. strain CBDB1. Arch Microbiol 180:411–416CrossRefGoogle Scholar
  16. Jeenes DJ, Williams PA (1982) Excision and integration of degradative pathway genes from TOL plasmid pWW0. J Bacteriol 150:188–194Google Scholar
  17. Kunisue T, Someya M, Kayama F, Jin Y, Tanabe S (2004) Persistent organochlorines in human breast milk collected from primiparae in Dalian and Shenyang, China. Environ Pollut 131:381–392CrossRefGoogle Scholar
  18. Li L, Yang C, Lan W, Xie S, Qiao C, Liu J (2008) Removal of methyl parathion from artificial off-gas using a bioreactor containing a constructed microbial consortium. Environ Sci Technol 42:2136–2141CrossRefGoogle Scholar
  19. Pieper DH, Reineke W (2000) Engineering bacteria for bioremediation. Curr Opin Biotech 11:262–270CrossRefGoogle Scholar
  20. Saber DL, Crawford RL (1985) Isolation and characterization of Flavobacterium strains that degrade pentachlorophenol. Appl Environ Microbiol 50:1512–1518Google Scholar
  21. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  22. Takagi K, Iwasaki A, Kamei I, Satsuma K, Yoshioka Y, Harada N (2009) Aerobic mineralization of hexachlorobenzene by newly isolates pentachloronitrobenzene-degrading Nocardioides sp. strain PD653. Appl Environ Microbiol 75:4452–4458CrossRefGoogle Scholar
  23. Walker AW, Keasling JD (2002) Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source. Biotechnol Bioeng 78:715–721CrossRefGoogle Scholar
  24. Williams PA, Murray K (1974) Metabolism of benzoate and the methylbenzoates by Pseudomonas putida (arvilla) mt-2: evidence for the existence of a TOL plasmid. J Bacteriol 120:416–423Google Scholar
  25. Xu D, Zhong W, Deng L, Chai Z, Mao X (2004) Regional distribution of organochlorinated pesticides in pine needles and its indication for socioeconomic development. Chemosphere 54:743–752CrossRefGoogle Scholar
  26. Yan DZ, Liu H, Zhou NY (2006) Conversion of Sphingobium chlorophenolicum ATCC 39723 to a hexachlorobenzene degrader by metabolic engineering. Appl Environ Microbiol 72:2283–2286CrossRefGoogle Scholar
  27. Yeh DH, Pavlostathis SG (2001) Development of hexachlorobenzene-dechlorinating mixed cultures using polysorbate surfactants as a carbon source. Water Sci Technol 43:43–50Google Scholar
  28. Zhou Y, Tigane T, Li X, Truu M, Truu J, Mander U (2013) Hexachlorobenzene dechlorination in constructed wetland mesocosms. Water Res 47:102–110CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Da-Zhong Yan
    • 1
    Email author
  • Ling-Qi Mao
    • 1
  • Cun-Zhi Li
    • 1
  • Jun Liu
    • 1
  1. 1.School of Biology and Pharmaceutical EngineeringWuhan Polytechnic UniversityWuhanChina

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