Insights into Organic-Solvent-Tolerant Bacteria and Their Biotechnological Potentials



Organic-solvent-tolerant bacteria (OSTB) are an amazing group of extremophiles that survive the massive membrane-disturbing effect produced by solvent accumulation. These bacteria possess novel adaptation mechanisms such as toluene efflux pumps, cis-trans isomerisation of membrane fatty acids, changes in cellular morphology such as decrease in cell surface to volume ratio, rapid membrane repair mechanisms, etc. In addition to the scientific significance, these microbes have tremendous potential in industrial and environmental biotechnology due to their enzymes retaining function in the presence of organic solvents.

In the present study, estuarine sediment samples from the Mandovi Estuary of Goa were used for the isolation of OSTB. SB 1 and BC 1 were the predominant isolates obtained by butanol enrichment and growth on cholesterol agar respectively and identified to belong to the genus Bacillus on the basis of morphological and biochemical characteristics. Changes in cell surface hydrophobicity, reduction in cell size from 3 to 1.8 µm in length, filamentation in solvent saturation conditions along with thickening of the cell, production of exopolymer and the presence of a capsule were certain phenomena observed in Bacillus subtilis SB1. Bacillus megaterium BC1 effectively transformed cholesterol into cholest-4-ene-3,6-dione in a biphasic system of 50 % chloroform and phosphate buffer. Since they withstand extreme solvent stress and survive in solvent-saturated environments, they would be good candidates for biotechnological applications.


Extremophiles Organic-solvent-tolerant bacteria Butanol Bacillus subtilis Cholesterol transformation Bacillus megaterium 


  1. Alex, R., & Bhat, S. (2012). Isolation and characterization of a solvent-tolerant alkalophilic marine bacterium. International Journal of Scientific Engineering and Research, 3, 1–5.Google Scholar
  2. Alvarez, H. (2012). Biology of Rhodococcus. New York: Springer.Google Scholar
  3. Aono, R., & Kobayashi, K. (1997). Cell surface properties of organic solvent tolerant mutants of E.coli K 12. Applied and Environmental Microbiology, 63, 3637–3642.Google Scholar
  4. Aono, R., Doukyu, N., Kobayashi, H., Nakajima, H., Horikoshi, K. (1994). Oxidative bioconversion of cholesterol by Pseudomonas sp. Strain ST-200 in water-organic solvent 2-phase system. Applied and Environmental Microbiology, 60, 2598–2523.Google Scholar
  5. Arima, K., Nagasawa, M., Bae, M., Tamura, G. (1969). Microbiological transformation of sterols, part I: decomposition of cholesterol by micro-organisms. Agricultural Biological Chemistry, 33, 1636–1643.CrossRefGoogle Scholar
  6. Arinbasarova, A., & Koskcheenko, K. (1983). Use of water-organic systems in enzyme-steroid transformation. Applied Microbiology Biotechnology, 19, 16–27.Google Scholar
  7. Asako, H., Nakajima, K., Kobayashi, M., Kobayashi, K., Aono, R. (1997). Organic solvent ­tolerance and antibiotic resistance increased by overexpression of marA in E.coli. Applied and Environmental Microbiology, 63, 1428–1433.Google Scholar
  8. Banerjee, S., Yalkowsky, S., Valvanic, S. (1980). Water solubility and octanol water partition ­coefficient s of organics: limits of solubility partition coefficient co-relation. Environmental Science and Technology, 14, 1227–1229.CrossRefGoogle Scholar
  9. Bar, R. (1987). Phase toxicity in water-solvent 2-liquid phase microbial system. In C. Laane, J. Tramper, & M. Lilly (Eds.), Biocatalysis in organic media (pp. 147–153). Amsterdam: ­Elsevier.Google Scholar
  10. Bowles, L., & Ellefson, W. (1985). Effects of butanol on Clostridium acetobutylicum. Applied Environmental Microbiology, 50, 1165–1170.Google Scholar
  11. Brink, L., & Tramper, J. (1985). Optimisation of organic solvents in multiphase biocatalysis. ­Biotechnology Bioengineering, 27, 1258–1269.CrossRefGoogle Scholar
  12. Cartwright (1986). Ethanol dissipates the p.m.f. across the plasma membrane of Saccharomyces cerevisiae. Journal of General Microbiology, 132, 369–377.Google Scholar
  13. Cruden, D., Wolfram, J., Rogers, R., Gibson, D. (1992). Physiological properties of a ­Pseudomonas strain which grows with p-xylene in a 2-phase organic-aqueous medium. Applied Environmental Microbiology, 58, 2723–2729.Google Scholar
  14. De Bont, J. (1998). Solvent tolerant bacteria in bio-catalysis. Trends in Biotechnology, 16, ­493–499.CrossRefGoogle Scholar
  15. Doukyu, N., & Aono, R. (1998). Purification of an extracellular cholesterol oxidase with high activity in presence of organic solvents from Pseudomonas sp. strain ST-200. Applied Environmental Microbiology, 64, 1929–1932.Google Scholar
  16. Doukyu, N., Ishikawa, K., Watanabe, R., Ogino, H. (2012). Improvement in organic solvent tolerance by double disruption of proV and mar R genes in E.coli. Journal of Applied Microbiology, 112, 464–474.CrossRefGoogle Scholar
  17. Edwards, A., Melchias, G., Vishwanathan, D., Kumar, S., Ajithan, C. (2012). Role of efflux pump in affording organic solvent tolerance to bacterial strains from petroleum contaminated soil. Middle East Journal of Scientific Research, 12, 80–87.Google Scholar
  18. Fei, T., Yaling, S., Ziqui, F., Zhi, T., Ding, X. (2012). Genome sequence of P. putida S12, a potential platform strain for industrial production of valuable chemicals. Journal of Bacteriology, 194, 5985–5986.CrossRefGoogle Scholar
  19. Heipeiper, H., Weber, F., Sikkema, J., Keweloh, H., de Bont, J. (1994). Mechanisms behind resistance of whole cells to toxic organic solvents. Trends in Biotechnology, 12, 409–415.Google Scholar
  20. Horikoshi, K., Antranikian, G., Bull, A. T., Robb, F. T., Steller, K. (2011). Extremophiles Handbook. (Eds.) XXIV, 1247 p, Springer.Google Scholar
  21. Huertas, M., & Duque, E. (1998). Survival in soil of different toluene degrading Pseudomonas strains after solvent shock. Applied Environmental Microbiology, 64, 38–42.Google Scholar
  22. Ingram, L.O., & Buttke, T. (1984). Effects of alcohols on micro-organisms. Advances in Microbial Physiology, 25, 253–300.CrossRefGoogle Scholar
  23. Inoue, A., & Horikoshi, K. (1989). A Pseudomonas putida thrives in high concentrations of ­toluene. Nature, 338, 264–266.CrossRefGoogle Scholar
  24. Inoue, A., & Horikoshi, K. (1991). Effects of solvent tolerance on bacteria by the solvent ­parameter log P. Journal of Fermentation and Bioengineering, 71, 194–196.CrossRefGoogle Scholar
  25. Isken, S., & de Bont, J. (1998a). Bacteria tolerant to organic solvents. Extremophiles, 2, 229–238.CrossRefGoogle Scholar
  26. Isken, S., & de Bont, J. (1998b) Active efflux of organic solvents by Pseudomonas putida is influenced by solvents. Journal of Bacteriology, 180, 6769–6772.Google Scholar
  27. Islam, R., Cicek, N., Sparling, R., Levin, D. (2009). Influence of initial cellulose concentration on the carbon flow distribution during batch fermentation by Clostridium thermocellum. ATCC 27405. Applied Environmental Microbiology, 829, 141–148.Google Scholar
  28. Janozka, T., Wilkazynski, K., Trypien, C. (2000). Analysis of oxygenated cholesterol derivatives by planar chromatography. Journal of Planar Chromatography, 13, 447–442.Google Scholar
  29. Kadavy, D., Hornby, J., Haverbast, T., Nickerson, K. (2000). Natural antibiotic resistance of ­bacteria isolated from the larvae of the oilfly. Applied Environmental Microbiology, 66, ­4615–4619.CrossRefGoogle Scholar
  30. Kato, C., Inoue, A., Horikoshi, K. (1996). Isolating and characterizing deep sea marine ­micro-organisms. Trends in Biotechnology, 14, 6–12.CrossRefGoogle Scholar
  31. Kobayashi, H., Yamamoto, M., Aono, R. (1998). Appearance of a stress response protein, phage shock protein A in E.coli exposed to hydrophobic organic solvents. Microbiology, 144, ­353–354.CrossRefGoogle Scholar
  32. Krist, M., Hancock, V., Klemm, P. (2008). Inactivation of efflux pumps abolishes bacterial biofilm production. Applied Environmental Microbiology, 74, 7376–7382.CrossRefGoogle Scholar
  33. Li, X., Xang, L., Poole, K. (1998). Role of the multi-drug efflux system of Pseudomonas ­aeruginosa in organic solvent tolerance. Journal of Bacteriology, 118, 2987–2991.Google Scholar
  34. Liszka, M., Clark, M., Schneider, E., Clark, D. (2012). Nature v/s nurture: developing enzymes that function under extreme stress conditions. Annual Reviews of Chemical and Biomolecular Engineering, 3, 77–102.CrossRefGoogle Scholar
  35. Moriya K., Yanigitani S., Usami R., Horikoshi K.(1995). Isolation and some properties of an ­organic solvent tolerant marine bacterium degrading cholesterol. Journal of Marine ­Biotechnology, 2, 131–133.Google Scholar
  36. Nicolau, S., Gaida, S., Papoutsakis, E. (2012). A comparative view of metabolite and substrate stress and tolerance in microbial processing: from biofuels and chemicals to biocatalysis and bioremediation. Metabolic Engineering, 12, 307–331.CrossRefGoogle Scholar
  37. Oeithinger, W., Kern, V., Goldman, J. (1998). Association of organic solvent tolerane and ­fluoroquinolone resistance in clinical isolates of E.coli. Journal of. Antimicrobial Chemotherapy, 41, 111–114.CrossRefGoogle Scholar
  38. Ogino, H., Yasui, K., Shiotani, T. (1995). Organic solvent tolerant bacterium which secretes an organic solvent stable proteolytic enzyme. Applied Environmental Microbiology, 61, 4258–4262.Google Scholar
  39. Oh, K., Lee, J., Kim, O. (2012). Increase of organic solvent tolerance of E.coli by the deletion of 2 regulator genes fadR and mar R. Applied Microbiology Biotechnology, 96, 1619–1627.CrossRefGoogle Scholar
  40. Paje, C., Neilan, B., Couperwhite, I. (1997). A Rhodococcus that thrives on media saturated with liquid benzene. Microbiology, 143, 2975–2981.CrossRefGoogle Scholar
  41. Pandey, S., & Singh, S. (2012). Organic solvent tolerance of an alpha amylase from haloalkalophilic bacteria as a function of pH, temp, salt conc. Applied Biochemistry Biotechnology, 166, 1747–1757.CrossRefGoogle Scholar
  42. Pinkart, C., Wolfram, J., Rogers, R., White, D. (1996). Cell and envelope changes in solvent ­tolerant and solvent sensitive Pseudomonas putida strain following exposure to o-xylene. ­Applied Environmental Microbiology, 62, 1129–1132.Google Scholar
  43. Ramos, J., Duque, E., Rodriguez, H., Godoy, P., Haidour, A., Reyes, F., Fernandez, B. (1997). Mechanisms of solvent tolerance in bacteria. Journal of Biological Chemistry, 272, 3887–3890.CrossRefGoogle Scholar
  44. Ramos, J., Duque, E., Godoy, P., Segura, A. (1998). Efflux pumps involved in the toluene tolerance of Pseudomonas putida Dot-T1E. Journal of Bacteriology, 180, 3323–3329.Google Scholar
  45. Sardessai, Y., & Bhosle, S. (2002a). Organic solvent tolerant bacteria in mangrove ecosystem. Current Science, 821, 622–623.Google Scholar
  46. Sardessai, Y., & Bhosle, S. (2002b). Tolerance of bacteria to organic solvents. Research in ­Microbiology, 153, 263–268.CrossRefGoogle Scholar
  47. Sardessai, Y., & Bhosle, S. (2003). Isolation of an organic solvent tolerant cholesterol transforming Bacillus species BC1 from coastal sediment. Marine Biotechnology, 5, 1–3.CrossRefGoogle Scholar
  48. Sardessai, Y., & Bhosle, S. (2004). Industrial potential of organic solvent tolerant bacteria. ­Biotechnology Progress, 20, 655–660.CrossRefGoogle Scholar
  49. Sarkar, P., & Ghosh, S. (2012). Bioremediation potential of a newly isolated strain Bacillus ­thermophilus PSII. Journal of Bioscience and Biotechnology, 1, 141–147.Google Scholar
  50. Segura, A., Molina, L., Fillet, S., Krell, T., Bernal, P., Munoz-Rojaz, J., Ramos, J. (2012). Solvent tolerance in gram-negative bacteria. Current Opinion in Biotechnology, 23, 415–421.CrossRefGoogle Scholar
  51. Sikkema, J., de Bont, J., Poolman, B. (1995). Mechanisms of solvent toxicity of hydrocarbons. Microbiological Reviews, 59, 201–222.Google Scholar
  52. Smith, L. (1984). Steroids. In H. J. Rehm & G. Reed (Eds.), Biotechnology : Vol. 6a Biotransformations (pp. 32–77). (Voted. Kieslich). Weinheim: Chemie.Google Scholar
  53. Torres, S., Pandey, A., Castro, G. (2011). Organic solvent adaptation of gram-positive bacteria: applications and biotechnological potential. Biotechnology Advances, 29, 442–45.CrossRefGoogle Scholar
  54. Trivedi, N., Gupta, V., Kumar, M., Kumar, P., Reddy, C., Jha, B. (2011). Solvent tolerant marine bacterium Bacillus aquimarius secreting organic solvent stable alkaline cellulase. Chemosphere, 83, 706–712.CrossRefGoogle Scholar
  55. Vessal, M., & Rasti, M. (1998). Estradiol antagonistic effects of winter cherry extract on pituitary and hypothalamic G6PD activities. Iranian Journal of Medical Sciences, 20, 152–158.Google Scholar
  56. Watanabe, R., & Doukyu, N. (2012). Contribution of mutations in acrR and mar R genes to organic solvent tolerance in E.coli. AMB Express, 12, 2–58.Google Scholar
  57. Weber, F., Ooijkaas, L., Schemen, R., Hartman, S., de Bont, J. (1993). Adaptation of Pseudomonas putida S12 to high concentrations of styrene and other organic solvents. Applied Environmental Microbiology, 59, 3502–3504.Google Scholar
  58. Zingaro, K., & Papoutsakis, E. (2012). Towards a semi-synthetic stress response system to engineer microbial solvent tolerance. mBio $(3), e00308-12, published online Oct 2, 2012. doi:10.1128/ mBio 00308-12.Google Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Goa College of PharmacyPanajiIndia

Personalised recommendations