Annals of Microbiology

, Volume 69, Issue 3, pp 267–277 | Cite as

Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT

  • Oanh Thi Nguyen
  • Duc Danh HaEmail author
Original Article


The cooperation of Bacillus subtilis strain DKT and Comamonas testosteroni KT5 was investigated for biofilm development and toluenes and chlorobenzenes degradation. Bacillus subtilis strain DKT and C. testosteroni KT5 were co-cultured in liquid media with toluenes and chlorobenzenes to determine the degradation of these substrates and formation of dual-species biofilm used for the degradation process. Bacillus subtilis strain DKT utilized benzene, mono- and dichlorinated benzenes as carbon and energy sources. The catabolism of chlorobenzenes was via hydroxylation, in which chlorine atoms were replaced by hydroxyl groups to form catechol, followed by ring fission via the ortho-cleavage pathway. The investigation of the dual-species biofilm composed of B. subtilis DKT and C. testosteroni KT5 (a toluene and chlorotoluene-degrading isolate with low biofilm formation) showed that B. subtilis DKT synergistically promoted C. testosteroni KT5 to develop biofilm. The bacterial growth in dual-species biofilm overcame the inhibitory effects caused by monochlorobenzene and 2-chlorotoluene. Moreover, the dual-species biofilm showed effective degradability toward the mixture of these substrates. This study provides knowledge about the commensal relationships in a dual-culture biofilm for designing multispecies biofilms applied for the biodegradation of toxic organic substrates that cannot be metabolized by single-organism biofilms.


Bacillus subtilis DKT Chlorobenzenes Ortho-cleavage pathway Comamonas testosteroni KT5 Dual-species biofilm Biodegradation 



This work was supported by Dong Thap University for the research group of environmental science. The authors are very thankful for all the support provided to do this research.


This research was funded by the Dong Thap University and authors’ own families.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts of interest.

Research involving human participants and/or animals

This article does not involve human participants or animals performed by any of the authors.

Informed consent

All individual participants in the study have obtained the informed consent.


  1. Bassler BL, Greenberg EP, Stevens AM (1997) Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179:4043–4045. CrossRefGoogle Scholar
  2. Bielefeldt AR, Stensel HD (1999) Modeling competitive inhibition effects during biodegradation of BTEX mixtures. Water Res 33:707–714. CrossRefGoogle Scholar
  3. Brunsbach FR, Reineke W (1994) Degradation of chlorobenzenes in soil slurry by a specialized organism. Appl Microbiol Biotechnol 42(2–3):415–420CrossRefGoogle Scholar
  4. Chang M-K, Voice TC, Criddle CS (1993) Kinetics of competitive inhibition and co-metabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates. Biotechnol Bioeng 41:1057–1065. CrossRefGoogle Scholar
  5. Cowan SE, Gilbert E, Liepmann D, Keaslin JD (2000) Commensal interactions in a dual-species biofilm exposed to mixed organic compounds. Appl Environ Microbiol 66:4481–4485. CrossRefGoogle Scholar
  6. Duc HD (2017) Degradation of chlorotoluenes by Comamonas testosteroni KT5. Appl Biol Chem 60(4):457–465. CrossRefGoogle Scholar
  7. Giaouris E, Chorianopoulos N, Doulgeraki A, Nychas GJ (2013) Co-culture with Listeria monocytogenes within a dual-species biofilm community strongly increases persistence of Pseudomonas putida to benzalkonium chloride. PLoS One 8(10):e77276. CrossRefGoogle Scholar
  8. Groschen GE, Arnold TL, Harris MA, Dupre DH, Fitzpatrick FA, Scudder BC, Morrow WS, Terrio PJ, Warner KL, Elizabeth A (2004) Murphy water quality in the upper Illinois River basin, Illinois, Indiana, and Wisconsin. U.S. Geological Survey, Reston, pp 1999–2001Google Scholar
  9. Haigler BE, Nishino SF, Spain (1988) Degradation of 1,2-dichlorobenzene by a Pseudomonas sp. Appl Environ Microbiol 54(2):294–301Google Scholar
  10. Lee CL, Song HJ, Fang MD (2005) Pollution topography of chlorobenzenes and hexachlorobutadiene in sediments along the Kaohsiung coast, Taiwan—a comparison of two consecutive years’ survey with statistical interpretation. Chemosphere 58:1503–1516. CrossRefGoogle Scholar
  11. Leriche V, Briandet R, Carpentier B (2003) Ecology of mixed biofilms subjected daily to a chlorinated alkaline solution: spatial distribution of bacterial species suggests a protective effect of one species to another. Environ Microbiol 5:64–71. CrossRefGoogle Scholar
  12. Li M, Peng L, Ji Z, Xu J, Li S (2008) Establishment and characterization of dual-species biofilms formed from a 3,5-dinitrobenzoic-degrading strain and bacteria with high biofilm-forming capabilities. FEMS Microbiol Lett 278(1):5–21. Google Scholar
  13. Li C, Li Y, Cheng X, Feng L, Xi C, Zhang Y (2013) Immobilization of Rhodococcus rhodochrous BX2 (an acetonitrile-degrading bacterium) with biofilm-forming bacteria for wastewater treatment. Bioresour Technol 131:390–396. CrossRefGoogle Scholar
  14. Lin C-W, Cheng Y-W, Tsai S-L (2007) Multi-substrate biodegradation kinetics of MTBE and BTEX mixtures by Pseudomonas aeruginosa. Process Biochem 42(8):1211–1217. CrossRefGoogle Scholar
  15. Liu JH, Maity JP, Jean JS, Chen CY, Chen CC, Ho SY (2010) Biodegradation of benzene by pure and mixed cultures of Bacillus spp. World J Microbiol Biotechnol 26(9):557–1567. CrossRefGoogle Scholar
  16. Makovcova J, Babak V, Kulich P, Masek J, Slany M, Cincarova L (2017) Dynamics of mono- and dual-species biofilm formation and interactions between Staphylococcus aureus and gram-negative bacteria. Microb Biotechnol 10(4):819–832. CrossRefGoogle Scholar
  17. Monferrán MV, Echenique JR, Wunderlin DA (2005) Degradation of chlorobenzenes by a strain of Acidovorax avenae isolated from a polluted aquifer. Chemosphere 61:98–106. CrossRefGoogle Scholar
  18. Mottaleb MA, Abedin MZ, Islam MS (2003) Determination of benzene, toluene, ethylbenzene and xylene in river water by solid-phase extraction and gas chromatography. Anal Sci 19(10):1365–1369. CrossRefGoogle Scholar
  19. Mukherjee AK, Bordoloi NK (2012) Biodegradation of benzene, toluene, and xylene (BTX) in liquid culture and in soil by Bacillus subtilis and Pseudomonas aeruginosa strains and a formulated bacterial consortium. Environ Sci Pollut Res 19(8):3380–3388. CrossRefGoogle Scholar
  20. Namkung E, Rittmann BE (1997) Estimating volatile organic compound emission from publicly owned treatment works. J Water Pollut Control Fed 59:670–678Google Scholar
  21. Nikolaou AD, Golfinopoulos SK, Kostopoulou MN, Kolokythas GA, Lekkas TD (2002) Determination of volatile organic compounds in surface waters and treated wastewater in Greece. Water Res 36(11):2883–2890. CrossRefGoogle Scholar
  22. Nishino SF, Spain JC, Belcher LA, Litchfield CD (1992) Chlorobenzene degradation by bacteria isolated from contaminated groundwater. Appl Environ Microbiol 58(5):1719–1726Google Scholar
  23. Oh YS, Shareefdeen Z, Baltzis BC, Bartha R (1994) Interactions between benzene, toluene and p-xylene (BTX) during their biodegradation. Biotechnol Bioeng 44:533–538. CrossRefGoogle Scholar
  24. Pandey G, Jain RK (2002) Bacterial chemotaxis toward environmental pollutants: role in bioremediation. Appl Environ Microbiol 68:5789–5795. CrossRefGoogle Scholar
  25. Paul D, Pandey G, Pandey J, Jain RK (2005) Accessing microbial diversity for bioremediation and environmental restoration. Trends Biotech 23:135–142. CrossRefGoogle Scholar
  26. Perumbakkam S, Hess TF, Crawford RL (2006) A bioremediation approach using natural transformation in pure-culture and mixed-population biofilms. Biodegradation 17:545–557. CrossRefGoogle Scholar
  27. Popp P, Brueggemann L, Keil P, Thuß U, Weiß H (2000) Chlorobenzenes and hexachlorocyclo-hexanes (HCHs) in the atmosphere of Bitterfeld and Leipzig (Germany). Chemosphere 41:849–855. CrossRefGoogle Scholar
  28. Potrawfke T, Timmis KN, Wittich R-M (1998) Degradation of 1,2,3,4-tetrachlorobenzene by Pseudomonas chlororaphis RW71. Appl Environ Microbiol 64:3798–3806Google Scholar
  29. Rapp P (2001) Multiphasic kinetics of transformation of 1,2,4-trichlorobenzene at nano- and micromolar concentrations by Burkholderia sp. strain PS14. Appl Environ Microbiol 67:3496–3500. CrossRefGoogle Scholar
  30. Rapp P, Gabriel-Jürgens LHE (2003) Degradation of alkanes and highly chlorinated benzenes, and production of biosurfactants, by a psychrophilic Rhodococcus sp. and genetic characterization of its chlorobenzene dioxygenase. Microbiology 149:2879–2890. CrossRefGoogle Scholar
  31. Rapp P, Timmis KN (1999) Degradation of cholorobenzenes at nanomolar concentrations by Burkholderia sp. strain PS14 in liquid cultures and in soil. Appl Environ Microbiol 65:2547–2552. Google Scholar
  32. Reardon KF, Mosteller DC, Rogers JDB (2000) Biodegradation kinetics of benzene, toluene and phenol as single and mixed substrates for Pseudomonas putida F1. Biotechnol Bioeng 69:385–400. CrossRefGoogle Scholar
  33. Rehfuss M, Urban J (2005) Rhodococcus phenolicus sp. nov., a novel bioprocessor isolated actinomycete with the ability to degrade chlorobenzene, dichlorobenzene and phenol as sole carbon sources. Syst Appl Microbiol 28:695–701. CrossRefGoogle Scholar
  34. Rickard AH, Leach SA, Hall LS, Buswell CM, High NJ, Handley PS (2002) Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria. Appl Environ Microbiol 68:3644–3650. CrossRefGoogle Scholar
  35. Rickard AH, Gilbert P, Handley PS (2004) Influence of growth environment on coaggregation between freshwater biofilm bacteria. J Appl Microbiol 96:1367–1373. CrossRefGoogle Scholar
  36. Robinson K, Flanagan S, Ayotte J, Campo K, Chalmers A, Coles JF, Cuffney TF (2004) Water quality in the New England coastal basins, Maine, New Hampshire, Massachusetts, and Rhode Island. U.S. Geological Survey, Reston, pp 1999–2001Google Scholar
  37. Sander P, Wittich RM, Fortnagel P, Wilkes H, Francke W (1991) Degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains. Appl Environ Microbiol 57(5):1430–1440Google Scholar
  38. Schraa G, Boone ML, Jetten MSM, van Neerven ARW, Colberg PJ, Zehnder AJB (1986) Degradation of 1,4-dichlorobenzene by Alcaligenes sp. strain A175. Appl Environ Microbiol 52:1374–1381Google Scholar
  39. Shadi AM, Yaghmaei S, Vafaei F, Khataee AR, Hejazi MS (2015) Degradation of benzene, toluene, and xylene (BTX) from aqueous solution by isolated bacteria from contaminated sites. Res Chem Intermediates 41(1):265–275. CrossRefGoogle Scholar
  40. Singh R, Paul D, Jain R (2006) Biofilms: implications in bioremediation. Trends Microbiol 14:389–397. CrossRefGoogle Scholar
  41. Slaine DD, Barker JF (1990) The detection of naturally occurring BTX during a hydrogeologic investigation. Ground Water Monit Remediat 10:89–94. CrossRefGoogle Scholar
  42. Spain JC, Nishino SF (1987) Degradation of 1,4-dichlorobenzene by a Pseudomonas sp. Appl Environ Microbiol 53:1010–1019Google Scholar
  43. Spiess E, Sommer C, Gorisch H (1995) Degradation of 1,4-dichlorobenzene by a Xanthobacter flavus 14p1. Appl Environ Microbiol 61:3884–3888Google Scholar
  44. van der Meer JR, Roelofsen W, Schraa G, Zehnder AJB (1987) Degradation of low concentrations of dichlorobenzenes and 1,2,4-trichlorobenzene by Pseudomonas sp. strain P51 in nonsterile soil columns. FEMS Microbiol Ecol 45:333–341. CrossRefGoogle Scholar
  45. van der Meer JR, van Neerven ARW, de Vries EJ, de Vos WM, Zhender AJB (1991) Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51. J Bacteriol 17:6–15. CrossRefGoogle Scholar
  46. Vyas TK, Murthy SR (2015) Chlorobenzene degradation by Bacillus sp. TAS6CB: a potential candidate to remediate chlorinated hydrocarbon contaminated sites. J Basic Microb 55(3):382–388. CrossRefGoogle Scholar
  47. Waddell KM, Gerner S, Thiros SA, Giddings EM, Baskin RL, Cederberg JR, Albano CM (2004) Water quality in the Great Salt Lake basins, Utah, Idaho, and Wyoming. U.S. Geological Survey, Reston, pp 1998–2001Google Scholar
  48. Wang MJ, Jones KC (1994) Uptake of chlorobenzens by carrots from spiked and sewage sludge-amended soil. Environ Sci Technol 28:1260–1267. CrossRefGoogle Scholar
  49. Wang MJ, McGrath SP, Jones KC (1995) Chlorobenzenes in field soil with a history of multiple sewage sludge application. Environ Sci Technol 29:1360–1367. Google Scholar
  50. Wang L, Zhou Q, Zhang BS, Li ZL, Chua H, Ren DM (2003) The biodegradation of 1,3-dichlorobenzene by an adapted strain Bacillus cereus PF-11 derived from town-gas industrial effluent. J Environ Sci Health A 38(9):1837–1848. CrossRefGoogle Scholar
  51. Yadav JS, Wallace RE, Reddy CA (1995) Mineralization of mono- and dichlorobenzenes and simultaneous degradation of chloro- and methyl-substitued benzenes by the white rot fungus Phanerochaete chrysosporium. Appl Environ Microbiol 61:677–680Google Scholar
  52. Yoshida S, Ogawa N, Fujii T, Tsushima S (2009) Enhanced biofilm formation and 3-chlorobenzoate degrading activity by the bacterial consortium of Burkholderia sp. NK8 and Pseudomonas aeruginosa PAO1. J Appl Microbiol 106:790–800. CrossRefGoogle Scholar
  53. Zhang J, Zhao W, Pan J, Qiu L, Zhu Y (2005) Tissue-dependent distribution and accumulation of chlorobenzenes by vegetables in urban area. Environ Int 31:855–860. CrossRefGoogle Scholar
  54. Zhang LL, Leng SQ, Zhu RY, Chen JM (2011) Degradation of chlorobenzene by strain Ralstonia pickettii L2 isolated from a biotrickling filter treating a chlorobenzene-contaminated gas stream. Appl Microbiol Biotechnol 91(2):407–415. CrossRefGoogle Scholar
  55. Ziagova M, Liakopoulou-Kyriakides M (2007) Comparison of cometabolic degradation of 1,2-dichlorobenzene by Pseudomonas sp. and Staphylococcus xylosus. Enzym Microb Technol 40(5):1244–1250. CrossRefGoogle Scholar
  56. Zolezzi M, Cattaneo C, Tarazona JV (2005) Probabilistic ecological risk assessment of 1,2,4-trichlorobenzene at a former industrial contaminated site. Environ Sci Technol 39:2920–2926. CrossRefGoogle Scholar

Copyright information

© Università degli studi di Milano 2019

Authors and Affiliations

  1. 1.Center for Chemical AnalysisDong Thap UniversityCao LanhVietnam
  2. 2.Faculty of Engineering-TechnologyDong Thap UniversityCao LanhVietnam

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