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Biodegradation of benzene by pure and mixed cultures of Bacillus spp.

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Abstract

Benzene has a wide range of industrial applications, but it is also a major source of environmental pollution. The most eco-friendly/cost-effective method of remediation is biodegradation. In the present study, we used a variety of microbial strains in different combinations on a selection of substrate concentrations to determine the most effective degradation processes. Bacterial strains of pure culture (L4, N3, and N6) were isolated from oil sludge in both Luria–Bertani buffer (LB) and nutrient broth media, and identified by 16S-rRNA analysis (≥98% similarity). The degradation experiments were performed using different combinations of bacterial strains (L4, N3, N6, L4 + N3, L4 + N6, N3 + N6, and L4 + N3 + N6) in modified carbon-free media with different concentrations of benzene as a carbon source (60, 100, and 160 mg l−1) at 30 °C. The isolates of L4 (Acc no: FJ686821), N3 (FJ686825) and N6 (FJ868628) were identified as Bacillus spp. using 16S-rRNA gene sequence analysis. All combinations of isolates have the capacity to degrade benzene. However, the L4 + N3 combination was more efficient than the other mixed or single cultures. In the presence of N6 isolate, the degradation rate of benzene decreased, possibly due to inter- and/or intra species interaction amongst the bacteria. The kinetic parameters ‘K m’ of the Lineweaver–Burk regressions conducted as part of this experiment showed that the lower the level of K m was, the better the biodegradation achieved. The results of this study showed that the use of Bacillus strains in benzene decomposition is feasible. In addition, different strain combinations exhibited different degradation patterns, which are attributed to the most efficient mixed cultures of Bacillus spp.

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Abbreviations

μ:

Specific growth rate

μmax :

The maximum specific growth rate

[S]:

Substrate concentration (mg l−1)

K m :

Kinetic constant characteristic of the particular enzyme and substrate

References

  • Alvarez PJJ, Vogel TM (1991) Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries. Appl Environ Microbiol 57:2981–2985

    CAS  Google Scholar 

  • Baldwin BR, Mesarcit MB, Nies L (2000) Broad substrate specificity of naphthalene- and biphenyl-utilizing bacteria. Appl Microbiol Biotechnol 53:748–753

    Article  CAS  Google Scholar 

  • Camper AK, McFeters GA (1979) Chlorine injury and the enumeration of waterborne coliform bacteria. Appl Environ Microbiol 37:633–641

    CAS  Google Scholar 

  • Chang MK, Voice TC, Criddle CS (1993) Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and pxylene by two pseudornonas isolates. Biotechnol Bioeng 41:1057–1065

    Article  CAS  Google Scholar 

  • Collins LD, Daugulis AJ (1999a) Benzene/toluene/p-xylene degradation. Part I. Solvent selection and toluene degradation in a two-phase partitioning bioreactor. Appl Microbial Biotechnol 52:354–359

    Article  CAS  Google Scholar 

  • Collins LD, Daugulis AJ (1999b) Benzene/toluene/p-xylene degradation. Part II. Effect of substrate interactions and feeding strategies in toluene/benzene and toluene/p-xylene fermentations in a partitioning bioreactor. Appl Microbiol Biotechnol 52:360–365

    Article  CAS  Google Scholar 

  • Cubitto MA, Morán AC, Commendatore M, Chiarello MN, Baldini MD, Siñeriz F (2004) Effects of Bacillus subtilis O9 biosurfactant on the bioremediation of crude oil-polluted soils. Biodegradation 15:281–287

    Article  CAS  Google Scholar 

  • Das K, Mukherjee AK (2007) Crude petroleum-oil biodegradation efficiency of bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil contaminated soil from north-east India. Bioresour Technol 98:1339–1345

    Article  CAS  Google Scholar 

  • Dowd RM (1984) Leaking underground storage tanKm. Environ Sci Technol 18:309A

    Article  Google Scholar 

  • Dubey RC, Maheshwari DK (2005) Practical microbiology. 1st edn (Reprint). S. Chand and Company Ltd., New Delhi, pp 10–25

    Google Scholar 

  • Furukawa K, Simon JR, Chakrabarty AM (1983) Common induction and regulation of biphenyl, xylene/toluene, and salicylate catabolism in Pseudomonas paucimobilis. J Bacteriol 154:1356–1362

    CAS  Google Scholar 

  • Grady CPL Jr, Smets BF, Barbeau DS (1996) Variability in kinetic parameter estimates: a review of possible causes and a proposed terminology. Water Res 30:742–748

    Article  CAS  Google Scholar 

  • Gunaseelan A, Cowan R (2003) Substrate inhibition kinetics for toluene and benzene degrading pure cultures and a method for collection and analysis of respirometric data for strongly inhibited cultures. Biotechnol Bioeng 83:798–809

    Article  Google Scholar 

  • Hyman MR, Sansome-Schmith AW, Shears JH, Wood PM (1985) A kinetic study of benzene oxidation to phenol by whole cells of Nitrosomonas europea and evidence for the further oxidation of phenol to hydroquinone. Arch Microbiol 143:302–306

    Article  CAS  Google Scholar 

  • Jannasch H, Egli T (1993) Microbial growth kinetics: a historical perspective. Antonie van Leeuwenhoek 63:213–224

    Article  CAS  Google Scholar 

  • Jean J-S, Tsai C-L, Ju S-H, Tsao C-W, Wang S-M (2002) Biodegradation and transport of benzene, toluene, and xylenes in a simulated aquifer: comparison of modeled and experimental results. Hydrol Process 16:3151–3168

    Article  Google Scholar 

  • Jean J-S, Lee M-K, Wang S-M, Chattopadhyay P, Maity JP (2008) Effects of inorganic nutrient levels on the biodegradation of benzene, toluene, and xylene (BTX) by Pseudomonas spp. in a laboratory porous media sand aquifer model. Bioresour Technol 99:7807–7815

    Article  CAS  Google Scholar 

  • Kim SB, Park CH, Kim DJ, Jury WA (2003) Kinetics of benzene biodegradation by Pseudomonas aeruginosa: parameter estimation. Environ Toxicol Chem 22:1038–1045

    CAS  Google Scholar 

  • Kobal VM, Gibson DT, Davis RE, Garza A (1973) X-ray determination of the absolute stereochemtstry of the initial oxidation product formed from toluene by Pseudomonas putida 39/D. J Am Chem Soc 95:4420–4421

    Article  CAS  Google Scholar 

  • Kova′rova′-Kovar K, Egli T (1998) Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics. Microbiol Mol Biol Rev 62:646–666

    Google Scholar 

  • Lee EY, Jun YS, Cho KM, Ryu HW (2002) Degradation characteristics of toluene, benzene, ethylbenzene, and xylene by Stenotrophomonas maltophilia T3-c. J Air Waste Manage Assoc 52:400–406

    CAS  Google Scholar 

  • Mallakin A, Ward O (1996) Degradation of BTEX compounds in liquid media and in peat biofilters. J Ind Microbiol 16:309–318

    Article  CAS  Google Scholar 

  • Marr EK, Stone RW (1961) Bacterial oxidation of benzene. J Bacteriol 18:425–430

    Google Scholar 

  • Medina-Moreno SA, Huerta-Ochoa S, Gutierrez-Rojas M (2005) Hydrocarbon biodegradation in oxygen-limited sequential batch reactors by consortium from weathered, oil-contaminated soil. Can J Microbiol 51:231–239

    Article  CAS  Google Scholar 

  • Mulligan CN, Yong RN, Gibbs BF (2001) Surfactant-enhanced remediation of contaminated soil: a review. Eng Geol 60:371–380

    Article  Google Scholar 

  • Natarajan MR, Lu Z, Oriel P (1994) Cloning and expression of a pathway for benzene and toluene from Bacillus stearothermophilus. Biodegradation 5:77–82

    Article  CAS  Google Scholar 

  • Oh YS, Bartha R (1997) Construction of a bacterial consortium for the biofiltration of benzene, toluene and xylene emissions. World J Microbiol Biotechnol 13:627–632

    Article  CAS  Google Scholar 

  • Oh YS, Choi SC (1997) Characterization of BTX-degrading bacteria and identification of substrate interactions during their degradation. J Microbiol 35:193–199

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Olsen RH, Kukor JJ, Kaphammer B (1994a) A novel toluene-3-monooxygenase pathway cloned from Pseudornonas pickettii PKO1. J Bacteriol 176:3749–3756

    CAS  Google Scholar 

  • Olsen RH, Mikesell MD, Kukor JJ (1994b) Enumeration and characterization of BTEX-degrading bacteria from hypoxic environments functional with mixed electron acceptors. Res Microbiol 145:47–49

    Article  CAS  Google Scholar 

  • Olsen RH, Mikesell MD, Kukor JJ, Byrne AM (1995) Physiological attributes of microbial BTEX degradation in oxygen-limited environments. Environ Health Perspect 103:49–51

    Article  CAS  Google Scholar 

  • Paje MLF, Neilan BA, Couperwhite L (1997) A Rhodococcus species that thrives on medium saturated with liquid benzene. Microbiology 143:2975–2981

    Article  CAS  Google Scholar 

  • Reardon KF, Mosteller DC, Rogers JB (2000) Biodegradation kinetics of benzene, toluene, and phenol as single and mixed substrates for Pseudomonas putida F1. Biotechnol Bioeng 69:385–400

    Article  CAS  Google Scholar 

  • Reardon KF, Mosteller DC, Rogers JB, DuTeau NM, Kim K-H (2002) Biodegradation kinetics of aromatic hydrocarbon mixtures by pure and mixed bacterial cultures. Environ Health Perspect 110:1005–1011

    CAS  Google Scholar 

  • Shim H, Yang ST (1999) Biodegradation of benzene, toluene, ethylbenzene, and o-xylene by a coculture of pseudomonas putida and pseudomonas fluorescens immobilized in a fibrous-bed bioreactor. J Biotechnol 67:99–112

    Article  CAS  Google Scholar 

  • Shim H, Yang ST (2002) BTEX removal from contaminated groundwater by a co-culture of pseudomonas putida and pseudomonas fluorescens immobilized in a continuous fibrous-bed bioreactor. J Chem Technol Biotechnol 77:1308–1315

    Article  CAS  Google Scholar 

  • Shim H, Yang ST (2006) Kinetics of BTEX biodegradation by a coculture of Pseudomonas putida and Pseudomonas fluorescens. J Environ Eng Manage 16:309–317

    CAS  Google Scholar 

  • Shim H, Shin E, Yang ST (2002) A continuous fibrous-bed bioreactor for BTEX biodegradation by a co-culture of Pseudomonas putida and Pseudomonas fluorescens. Adv Environ Res 7:203–216

    Article  CAS  Google Scholar 

  • Shim H, Hwang B, Lee SS, Kong SH (2005) Kinetics of BTEX biodegradation by a coculture of Pseudomonas putida and Pseudomonas fluorescens under hypoxic conditions. Biodegradation 16:319–327

    Article  CAS  Google Scholar 

  • Shirai K (1986) Screening microorganisms for catechol production from benzene. Agric Biol Chem 50:2875–2880

    CAS  Google Scholar 

  • Tan HM, Mason JR (1990) Cloning and expression of the plasmid-encoded benzene dioxygenase genes from P. putida ML2. Fems Microbtol Lett 72:259–264

    Article  CAS  Google Scholar 

  • Toledo FL, Gonzalez-Lopez J, Calvo C (2008) Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture. Bioresour Technol 99:8470–8475

    Article  CAS  Google Scholar 

  • Turner LH (1995) Vapor-liquid equilibrium of highly non-ideal aqueous organic system. Ph.D. thesis, Rutgers university, New jersey, USA

  • Weelink SAB (2008) Degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria. Ph.D. thesis, Wageningen university, Wageningen, Netherlands

  • Whang LM, Liu PW, Ma CC, Cheng SS (2008) Application of biosurfactants, rhamnolipid, and surfactin, for enhanced biodegradation of diesel-contaminated water and soil. J Hazard Mater 151:155–163

    Article  CAS  Google Scholar 

  • Winstanley C, Taylor SC, Williams PA (1987) pWW174: a large plasmid from Acinetobacter calcoaceticus encoding benzene catabolism by the ß-ketoadipate pathway. Mol Microbiol 1:219–227

    Article  CAS  Google Scholar 

  • Yasuda Y, Katoh S, Inoue Y, Shiomi N (2003) A bioremediation method based on self-immobilization of cells in shallow layer of soil. J Chem Eng Jpn 36:216–219

    Article  CAS  Google Scholar 

  • Yu H, Kim BJ, Rittmann BE (2001a) The roles of intermediates in biodegradation of benzene, toluene, and p-xylene by Pseudomonas putida F1. Biodegradation 12:455–463

    Article  CAS  Google Scholar 

  • Yu H, Kim BJ, Rittmann BE (2001b) A two-step model for the kinetics of BTX degradation and intermediate formation by Pseudomonas putida F1. Biodegradation 12:465–475

    Article  CAS  Google Scholar 

  • Ziffer H, Jerina DM, Gibsox DT, Kobal Val M (1973) Absolute stoichiochemistry of the (+)-cts-l, 2-dihydroxy-3-methylcyclohexa-3.5-diene produced from toluene by Pseudomonas putida. J Am Chem Soc 95:4048–4049

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the National Science Council in Taiwan (NSC No. NSC 96-2116-M-006-003).

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Authors and Affiliations

  1. Department of Earth Sciences, National Cheng Kung University, 1 University Road, Tainan City, Tainan, 70101, Taiwan (ROC)

    Jiann-Hong Liu, Jyoti Prakash Maity, Jiin-Shuh Jean & Sin-Yi Ho

  2. Department of Earth and Environmental Sciences, National Chung Cheng University, Ming-Shung, Chiayi County, Taiwan (ROC)

    Chien-Yen Chen

  3. Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung City, Taiwan (ROC)

    Chien-Cheng Chen

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  1. Jiann-Hong Liu
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  2. Jyoti Prakash Maity
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Correspondence to Jiin-Shuh Jean.

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Liu, JH., Maity, J.P., Jean, JS. et al. Biodegradation of benzene by pure and mixed cultures of Bacillus spp.. World J Microbiol Biotechnol 26, 1557–1567 (2010). https://doi.org/10.1007/s11274-010-0331-9

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