Screening, Identification and Biological Characterization of Highly Potent Tributyltin Chloride Resistant Marine Bacteria from West Coast of India (Goa)

revealed it to be catalase positive, oxidase positive, facultative aerobe, MR positive, Indole positive, casein and Tween 80 hydrolysis positive, nitrate reduction positive, sugar utilization (glucose, arabinose, xylose, galactose, cellubiose, melibiose, trehalose and saccharose) positive. It was tentatively identified to be an Alcaligenes sp. Its identity was confirmed by 16S rDNA gene amplification by polymerase chain reaction (PCR) as previously described by18. 16S rDNA was reverse primer R’ 1387- GCC CGG GAA CGT ATT CAC CG of Escherichia coli 16S rRNA sequence. Phylogenetic analyses using the BLAST program showed that strains S3 belonged to the gamma subdivision of the phylum Proteobacteria and that it was closely related to the genus Alkaligenes sp.2-6. The DNA sequence was determined using the dideoxy chain termination method32. A total of 1000 bp was sequenced. The sequence was compared with other bacteria available in GenBank (http://www.ncbi.nlm.nih.gov.)1. The sequence was then aligned with available 16S rRNA reference sequences. Similarities and alignments were obtained using the Basic Local Alignment Search Tool (BLAST) 2 algorithm to identify known sequences with a high degree of similarity. Two strains such as Pseudomonas aeruginosa USS25W and Pseudomonas aeruginosa PA01 were used as standard cultures for comparison. The characteristics of all the isolates and the phenogram (Figure 2) showed similarity among the isolates, which have been grouped as Alkaligenes sp. The biochemical characteristics of the isolate S3 led to the tentative identification as Alkaligenes sp. (fig. 2). Phenogram showing similarity among different isolates. The comparative study showed that bacterial isolates of WISL are more resistant than GSL. This may be due to the fact that MPT harbour being one among the oldest port of India (since 1934), receives many cargo and passenger vessels at its various berths, these ships dock for weeks, during which the TBT may be leached to the marine environment due to hydrolysis as early reported by14. WISL with its latest ship repairing systems is the only one of its kind in the west coast of India. Like wise GSL (commissioned in 1957) involves repairing and construction of Naval ships. In these process, the old paint that is being blasted out before applying the new antifouling paint ends up in to the marine environment, where TBT leaches into the marine environment as reported earlier14. The extensive use of TBTC as an antifoulant in ship paints, shipyards and harbours is considered to be the prime source of TBTC in the marine ecosystem. Chau and co-workers12 demonstrated that heavy contamination of coastal waters by TBT was associated with major commercial harbours. For example, the range of butyltin concentrations in five sediment samples collected from Hamilton Harbour was from 63 to 294 ng Sn g-1. It has been reported that organotin compounds are toxic to both Gram-negative and Gram-positive bacteria, but triorganotins are more active towards the Gram-positive bacteria than Gram-negative bacteria30, 31 have reported several organisms resistant to different organotin compounds, but bacteria utilizing TBTC as the sole source of carbon have not been reported so far31. Debutylation of TBT compounds to di- and mono- accumulating TBTC, thus contributing to the removal of TBT from marine environment13. The high lipid solubility of organotins ensures the interaction of TBTC with intracellular sites by penetration through cell wall and cell membrane 13. Although the degradation of organotins has been shown to be mediated by microorganisms, information is still limited in relation to the mechanism of degradation, tolerance mechanism of microbes and their relative significance, and also the role of anionic radicals in the degradation process in natural habitats17, 23, 24. Biotic processes have been demonstrated to be the most significant mechanisms for tributyltin degradation, both in soil as well as in freshwater, marine and estuarine environment6, 19. Ship repair activities involve removal of the old paint from the hull and application of a new coating. High amounts of TBT can be released due to wastewater discharge, wind transportation of dust and paint and mismanagement of waste. In areas where high TBT input had occurred, or is still occurring, the sediment acts as a TBT reservoir. There is a serious risk of re-introduction of this TBT to the aquatic ecosystem if the sediment is remobilized (e.g. dredging). Organotins are thus pollutants of anthropogenic origin. They make their way from a variety of industrial and agricultural sources into aquatic ecosystems, where they can be concentrated up to 10,000-fold in the surface microlayer and up to 4,000 times in oily sediments14, 16. Ecotoxicological effects of organotins include morphological and reproductive aberrations and metabolic disruption in a variety of nontarget organisms, including shellfish and finfish. They can be bioaccumulated in microorganisms, which are at the base of the food web, and from there into higher organisms.

1. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. Journal of Molecular Biology 215, pp. 403–410. 2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein data base search programs. Nucleic Acids Res 25: pp. 3389–3402 3. Alzieu, C.L., Sanjuan, J., Deltreil, J.P., Borel, M. (1986). Tin contamination in Arcachan Bay" Effect on oyster shell anomalies. Mar. Pollu. Bull. 17: pp. 494-498. 4. Aston, S.R., (1980). “Nutrients, Dissolved Gases and General Biogeochemistry in Estuaries”, (Eds. E. Olausson and Cato, I) John Wiley & Sons, New York. pp. 233 – 257. 6. Barug, D. (1981). Microbial degradation of bis (tributyltin) oxide. Chemosphere. 10: pp. 1145-1154. 7. Bauer AW, Kirby WMM, Sherris JC, Turck M. (1966). Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol.;5: pp. 493–6. 8. Beaumont, A. R. and Budd, M. D. (1984). Mar. Pollut. Bull.15, pp. 402. 9. Blunden, S.J., Hobbs, L.A. and Smith, P.J. (1984). The environmental chemistry of organoitn compounds. In: Environmental chemistry. Edited by H.J.Bowen Soc. Chem., London. pp. 46-77. 10. Bourgois, J.J., F. E. Sluse, F. Baguet, and J. Mallefet (2001). Kinetics of Light Emission and Oxygen Consumption by Bioluminescent Bacteria. Journal of Bioenergetics and Biomembranes, Vol. 33, No. 4. 11. Bryan, G. W., Gibbs, P. E. and Hummerstone, L. G. (1986). J. Mar. Biol. Assoc. UK, 66, pp. 611. 12. Chau, Y.K., Maguire, R.J., Brown, M., Yang, F. and Batchelar, S.P. (1997). Occurrence of organotin compounds in the Canadian aquatic environment. Water Qual. Res. J (Canada). 32: pp. 453- 521. 13. Clark, E.A., Sterritt, R.M and Lester, J.N. (1988). The fate of tributyltin in the aquatic environment, a look at the data. Environ. Sci. Technol. 22: pp. 600-604. 14. Cleary, J.J., and Stebbing, A.R.D. (1987). Organotin in the surface microlayer and subsurface waters of Southwest England. Mar. Pollu. Bull. 18: pp. 238-246. 15. Cooney, J.J. (1995). Organotin compounds and aquatic bacteria - a review. Helgoland Meeresunter. 49: pp. 663-677. 16. Cooney, J.J. and Wuertz, S. (1989). Toxic effect of tin compounds on microorganisms. J. Indus. Microbiol. 4: pp. 375-402. 17. Cooney, J.J. (1988). Microbial transformation of tin and tin compounds. J. Ind. Microbiol. 3: pp. 195-204. 18. Dauga, P.A.D. and Grimont, P.A.D. (1991) Nucleotide sequence of 16S rRNA from ten

19. de Mora, S.J., Stewart, C. and Phillips, D. (1995). Sources and rate of degradation of Tri(n-butyl) tin in marine sediments near Auckland , New Zealand. Mar. Pollut. Bull. 30: pp. 50-57. 20. de Mora, S.J. and Pelltier, E. (1997). Environmental tributyltin research: past, present and future. Environ. Technol. 18: pp. 1169-1177. 21. Dowson, P.H., Bubb, J.M. and Lester, J.N. (1996). Persistence and degradation pathways of tributyltin in freshwater and estuarine sediments. Estuarine, Coastal and Shelf Science. 42: pp. 551- 562. 22. Dowson, P.H., Bubb, J.M., Lester, J.N. (1993). Temporal distribution of organotins in the aquatic environment: five years after the 1987 UK retail ban on TBT based antifouling paints. Mar. Pollu. Bull. 26: pp. 487-494. 23. Gadd, G.M. (1993). Microbial formation and transformation of organometallic and organ metalloid compounds. FEMS Microbiol. Rev. 11: pp. 297-316. 24. Gadd, G.M. (2000). Microbial interaction with tributyltin compounds, detoxification, accumulation and environmental fate. Sci.Total Environ. 258: pp. 119-127. 25. Grasshoff, K. (1983). Determination of salinity. In Methods of seawater analysis. 2nd Ed. 26. Iwata, H. Tanabe, S. Sakai, N and Tatsukawa, R. (1993). Environ. Sci. Technol.27, pp. 1080. 27. Krieg, N. R. and Holt, J. G. (1984). Bergey’s Manual of Systemic Bacteriology, The Williams & Wilkins Co, Baltimore, USA, 1984, vol. 1. 28. Rajendran, K. Sampathkumar, P. Govindasamy, C. Ganesan, M. Kannan, R.and Kannan, L. (1993). Mar. Pollut. Bull., 26, pp. 283. 29. Riley, J.P. and Chester, R. (1980). “Introduction to marine chemistry”, Academic Press, London. pp. 152-179. 30. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, pp. 487–491. V.P. Singh, K.L. Garg). Publication Print House, New Delhi, India, pp. 297-316. 32. Sanger, F., Nicklen, S. and Coulso, A.R. (1977). Proc. Natl. Acad. Sci. USA. 74, pp. 5463. 33. Subramanyam, N.S. and Sambamurty, A.V. S. S. (2000). Marine Ecology. In Ecology Ed. Subramanyam, N.S. and Sambamurty, A.V. S. S. Narosa Publishing House, New Delhi. pp. 244-253. 34. White, J.S., Tobin, J.M. and Cooney, J.J. (1999). Organotin compounds and their interaction with microorganisms. Can. J. Microbiol. 45(7): pp. 541-554. 35. Wuertz, S. Miller, C.E., Pfister, R.M. and Cooney, J.J. (1991). Tributyltin-Resistant bacteria from estuarine and freshwater sediments. Appl. Environ. Microbiol. 57: pp. 2783-2789.

Dr. R. Krishna. Murthy thank Dr. Santosh Kumar Dubey, (JSPS Fellow) Professor and Head, Dept. of Microbiology, Goa University, research guide and mentor for his never ending support and Goa University for financial assistance in the form of research studentship.

Assistant Professor, P.G. Department of Microbiology, CPGS, Jain University, 18/3, 9th Main, Jayanagar 3rd Block, Bangalore

rkmurthy07@rediffmail.com