| When any substrate surface is immersed in seawater, | | | | were identified as Pseudomonas sp., Salmonella sp., |
| adhesion of many microorganisms is observed within a | | | | Vibrio sp. and Proteus sp. In stainless steel coupons, |
| short time. This initial covering of exposed surface is | | | | the identified genera of Gram-positive bacteria were |
| called the slim, biofilm or primary film. Once this | | | | Bacillus sp., Staphylococcus sp. and Micrococcus sp., |
| attachment process is initiated, a succession of settling | | | | while in Gram-negative group showed all the isolates |
| of organisms is observed leading to the development | | | | except Salmonella sp. In brass Gram-negative strains |
| microbial population. The predominant organisms are | | | | were found to dominate over the Gram-positive by |
| bacteria, algae, unicellular fungi and protozoa in a matrix | | | | the genre Pseudomonas sp., Salmonella sp., Vibrio sp. |
| of detritus, which provides a continuous source of | | | | and Proteus sp., while Bacillus was the only |
| nourishment1. The epiphytic nature of marine bacteria | | | | Gram-positive group on brass. Titanium showed all the |
| was first studied by Zobell and this bacterial adhesion | | | | Gram-positive isolates like, Bacillus sp., csp. and |
| leads the surfaces to corrosion of the material2. | | | | Micrococcus sp., and all the Gram-negative isolates like. |
| Fouling refers to the undesirable formation of deposits | | | | Pseudomonas sp., Salmonella sp, Vibrio sp. and |
| on equipment surfaces, which significantly decreases | | | | Proteus sp. In copper coupons, the genera identified |
| equipment’s performance and/or its useful life. | | | | under Gram-positive included Bacillus sp., and |
| Several types of fouling and their combinations may | | | | Staphylococcus sp. Micrococcus was not registered |
| occur, including biological, corrosion, particulate and | | | | from copper. In Gram-negative isolates were identified |
| precipitation fouling. In general, more than one type of | | | | as Pseudomonas sp., and Vibrio sp., in copper coupons |
| fouling will be occurring simultaneously3. | | | | Salmonella sp. and Proteus sp. will absent. |
| A sequence of discrete events occurs in the | | | | The generic composition of manganese-oxidizing |
| development of biofilms on clean surfaces when they | | | | heterotrophic bacteria isolated from the coupons of |
| are immersed in the marine environment4. | | | | PVC, stainless steel, brass, titanium and copper was |
| 1. Adsorption of an abiotic conditioning film. | | | | shows in the Table: 3. all the tested coupons were |
| 2. Approach of bacteria to the conditioned surface | | | | recorded with both Gram-positive and Gram-negative |
| through water movement, diffusion and/or motility. | | | | groups of bacteria. The common contaminant Bacillus |
| 3. Reversible adhesion of both motile and non-motile | | | | sp. was commonly encountered in all the tested |
| bacteria. | | | | materials. Staphylococcus  sp. and Micrococcus sp. |
| 4. Irreversible adhesion of bacteria mediated by | | | | were the other Gram-positive isolates from the |
| bacteria polymers, and | | | | coupons. Copper coupons did not show the presence |
| 5. Development of a secondary microflora. | | | | of Staphylococcus  sp. The Gram-negative MHB |
| Adhesion of microorganisms and subsequent | | | | isolates from different coupons contains |
| development of surface colonies has been the subject | | | | Pseudomonas sp., E. coli and Vibrio sp. When |
| of extensive research5, 6, 7, 8, 9, and 10. Subsequently, | | | | comparing HB isolates Salmonella sp. and Proteus sp |
| several workers collected data on this aspect from | | | | were absent in MHB and E. coli is present. The |
| the important commercial harbors of India1. | | | | presence of E. coli registered only from PVC and SS |
| Sulphate-reducing bacteria and iron-oxidizing bacteria | | | | coupons. In general the bacterial strain isolated from all |
| have long been considered as major contributors of | | | | the coupons showed that Gram- positive groups were |
| corrosion. Recently, manganese-oxidizers have also | | | | dominant. |
| been identified as major contributors to corrosion11, 12. | | | | Table: 3. Generic composition of Manganese-oxidizing |
| Dickinson et al,13 first to identify the deposition of | | | | heterotrophic bacterial isolates and their present |
| MnO2 as the likely causative agent of corrosion It was | | | | occurrence in immersed coupons like, PVC, Stainless |
| found that the ennoblement effect on the stainless | | | | Steel, Brass, Titanium and Copper. |
| steel could be reproduced by coating the coupons with | | | | The Manganese (Mn) concentration in sea water and |
| an alternative for the cathodic reduction of MnO2 and | | | | biofilm formed in different material were showed in |
| such involvement was reported by Linhardt 14 | | | | Table: 4. Among the five types of coupons tested |
| Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â MnO2Â +Â H2O + | | | | PVC exhibited highest Mn value of 5543 ng/g, while |
| e-Â Â Â Â Â ? MnOOH + OH- | | | | lowest Mn value 309 ng/g was recorded from brass |
|                 MnO2 + 2 H2O + | | | | coupons. Sea water showed 0.78µg/l concentration |
| 2e- ? Mn2+Â +4 OH- | | | | of Mn. |
| Considering the importance of MnOB essentially | | | | Table: 4.Heavy metal concentration in sea water (µg |
| heterotrophic bacteria and its microbial induced | | | | l) and biofilm (ng/g) on PVC, Stainless Steel, Brass, |
| corrosion in different material, and lack of information | | | | Titanium and Copper coupons. |
| on the genera involved in manganese deposition | | | | Bacterial slim samples generated on the exposed |
| oxidation with analyzing the physical properties of sea | | | | coupons were scrapped using sterile brush and |
| water were studied. Present study is aimed to identify | | | | immediately transferred to sterile saline water. All the |
| HB and MHB bacteria in biofilm of various materials | | | | samples were serially diluted. For quantitative |
| exposed to Rameswaram (Latitude 9o 10’ and 9 o | | | | examination of the bacterial colonies, the samples |
| 20’ and longitude 79 o 13’  79 o 27’ E) | | | | were inoculated by spread plate method. The Zobell |
| coastal waters (Palk Bay) Southeast coast of India. | | | | Marine Agar medium (2216E) was used to enumerate |
| PVC coupons (15 cm x 6 cm), Stainless steel coupons | | | | the heterotrophic bacteria (HB) and K-medium was |
| (30mm x 15 mm x 2mm), Titanium coupons (15 cm x 3 | | | | used to enumerate the Manganese-oxidizing |
| cm), Brass coupons (15 cm x 10 cm) and Copper | | | | heterotrophic bacteria (MHB). The pure cultures were |
| coupons (15 cm x 10 cm). All the coupons were | | | | maintained in slants for bacterial characterization. The |
| polished, washed with detergents and rinsed with | | | | isolated bacterial strains were characterized up to |
| distilled water and stored under sterile conditions until | | | | generic level was done to the key described in the |
| use. The coupons of PVC, Titanium, Brass, Copper | | | | Bergey’s manual of determinative bacteriology |
| and Stainless Steel were immersed one meter below | | | | (8thedition and other developed schemes)17. The |
| water surface, using wooden rafts. The metal | | | | identification of isolated bacterial strain was done by |
| coupons, Brass, Titanium and Brass were exposed for | | | | inoculating each strain in to nutrient broths and nutrient |
| a period of six months (October 2005 to March 2006). | | | | agar plates and incubated for 24-48 hours at 37 oC. |
| The PVC and SS were suspended in the sea for two | | | | The developed cultures were subjected to further |
| months (February and March 2006). Sea water | | | | microscopic, physiological and biochemical analysis was |
| samples were also collected from the study area | | | | shown on Table: 6. |
| using water sampler to estimate the physiochemical | | | | Table: 6.Morphology and biochemical characterization |
| parameters such as salinity, air and water temperature, | | | | of isolated bacterial strains from various materials. |
| hydrogen-ion concentration (pH) and dissolved oxygen. | | | | ACKNOWLEDGEMENT: We thank the Co-ordinators, |
| The sea water nutrients were analysed. | | | | Department of Oceanography and Coastal Area |
| Table: 1 shows the average population of | | | | Studies, Alagappa University, Karaikudi, Thondi Campus, |
| Heterotrophic bacterial (HB) and Manganese-oxidizing | | | | Thondi for permission to communicate the results. |
| heterotrophic bacterial (MHB) count in immersed | | | | References |
| coupons like, PVC, Stainless Steel, Brass, Titanium and | | | | 1.     Nagabhushanam, R., and Sarojini, R., An |
| Copper. The population of HB and MHB on PVC was | | | | overview of Indian Research Efforts on Marine |
| registered as 3.62 x 107 CFU/cm2 and 2.87 x 107 CFU | | | | Wood-boring and Fouling Organisms, (eds |
| cm2, respectively while on Stainless Steel the | | | | Nagabhushanam, R and Thompson, M.E.), Fouling |
| population density of HB and MHB was recorded as | | | | organisms of the Indian Ocean Biology and Control |
| 3.79 x 105 CFU/cm2 and 1.34 x 105 CFU/cm2. The | | | | Technology, Oxford & IBH publishing Co. Pvt. |
| lower population density of Manganese-oxidizing | | | | Ltd., New Delhi, 1997, pp. 1-79. |
| heterotrophic bacteria at Stainless Steel was due to | | | | 2.     Costerton, J. W. and Lewandowski, Z., |
| the nature of the surface of the material11. On titanium | | | | Annu. Rev. Microbiol., 1995, 49, 711-745. |
| coupons the count of HB was 3.86 x 105 CFU/cm2 | | | | 3.     Videla, H.A. and Characklis W.G., Biofouling |
| and MHB was 2.86 x105 CFU/cm2, while the counts | | | | and Microbially Influenced Corrosion, Inter. |
| HB and MHB of copper were recorded as 1.42 x 103 | | | | Biodeterioration and Biodegradation, 1992, 29, 195 – |
| CFU/cm2  and 1.10 x103 CFU/cm2. Brass | | | | 212. |
| coupons recorded the population density of HB and | | | | 4.     Mitchell, R., and Kirchman, D., The microbial |
| MHB were 4.03 x 106 CFU/cm2 and 2.02 x 106 CFU | | | | ecology of marine surfaces. In : J.D. Costlow and R.C. |
| cm2. The PVC and titanium coupons were recorded | | | | Tipper, (eds.) Marine Biodeteriation An Interdisciplinary |
| relatively higher values comparing with other coupons, | | | | Study, Naval Institute Press, Annapolis, 1984, pp. 49-56. |
| and it may be due to the non-toxic nature of the | | | | 5.     Absolom, D.R., Lamberti, F.W., Policova, Z., |
| substratum. Brass also recorded higher bacterial | | | | Zingg, W., Van Oss, D. J., and Neumann, A.W., Surface |
| population density compared to copper. Videla et al.3 | | | | thermodynamics of bacterial adhesion, Appl. Environ. |
| reported that the maximum number of bacterial | | | | Microbio, 1983, 46, 90 – 97. |
| colonization were found in titanium coupons and it | | | | 6.     Barier, R.E., Adhesion in the biologic |
| served as an ideal substratum for bacterial colonization | | | | environment, J. Biomed. Mater. Res. 1984,12, 123-160. |
| this may be due to the corrosion resistant nature of | | | | 7.     Barier, R. E., Meyer, A. E., Natiella, R. R., and |
| the material. The least population density observed in | | | | Carter, J. M., Surface properties determine bioadhesive |
| copper coupons could be due to it toxic nature. | | | | outcomes: Methods and results, J. Biomed. Mater. |
| Ponmariappan et al15 reported less HB population | | | | Res,1984,18, 337 – 355. |
| density on copper than Monel in Tuticorn harbour | | | | 8.     Marshall, K.C., (Ed), Microbias adhesion and |
| waters. Venugopal et al.16 studied that bacteria and | | | | aggregation, Dehlem Konferezen Springer Verlag, |
| diatoms constitute two major groups of | | | | Berlin, 1984. |
| microorganisms that colonize solid surface immersed in | | | | 9.     Savage, D.C., and Fletcher, M., (eds), |
| Kalppakkam coastal waters. The population density of | | | | Bacterial adhesion, Planum Press, New York, 1985. |
| Manganese-oxidizing heterotrophic bacteria was more | | | | 10. Hamilton, H. A., Biofilm: Microbial interaction and |
| or less same as heterotrophic bacteria in all coupons | | | | metabolic activities, In : M. Fletcher, T.R.G. Gray and J.G. |
| tested. It indicates that most of the heterotrophic | | | | Jones (Eds.), Ecology of Microbial Communities, |
| bacterial strains isolated were act as manganese | | | | Society for general microbiology symposium 41, |
| depositors after oxidation. | | | | Cambridge University Press, Cambridge, 1987, pp. |
| Â | | | | 361-387. |
| Table: 2. Generic composition of heterotrophic bacterial | | | | 11. Palanichamy. S., Maruthaumuthu, S., Manickam, S. T., |
| isolates and their present occurrence in immersed | | | | and Rajendran, A., Microfouling of manganese oxidizing |
| coupons like, PVC, Stainless Steel, Brass, Titanium and | | | | bacteria in Tuticorin harbour waters. Curr. Sci, 2002, 82 |
| Copper. | | | | (7), 865 – 869. |
| Table: 2 shows the generic composition of | | | | 12. Dexter, S. C., and Maruthamuthu, Corrosion, Paper |
| heterotrophic bacterial strains isolated from biofilm | | | | No.01256, 2001, pp. 01256/1-01256/15. |
| samples scrapped from various coupons. Both | | | | 13. Dickinson, W. B., Caccavo, F., and Lewandoski, Z., |
| Gram-positive and Gram-negative groups were noted | | | | Corros. Sci. 1996, 38, 1407-1422. |
| on all the materials studied. The notable thing was that | | | | 14. Linhardt, P., Abstract paper 011-9, International |
| Gram-positive group was fond to be dominant than | | | | Society of Electrochemistry, Portugal, 1995. |
| Gram-negative on all materials studied. When | | | | 15. Ponmariappan, S., Kanabiran, S., Stella, C., |
| comparing with other coupons the Brass showed | | | | Mathiarasu, J., and Maruthamuthu, S., Indian. J. Microbiol., |
| higher population density of Gram-negative group than | | | | 1998, 39, 113-118. |
| Gram-positive. The similar findings were reported | | | | 16. Venugopal, V. P., Rao, T. S., Sargunam, C. A., and |
| earlier by Palanichamy et al11. The generic composition | | | | Nair, K. V. K., in Some observations on the biological |
| and microbial load were found to be varying from | | | | and biochemical aspects of biofilm development in |
| material to material. The genera identified under | | | | Kalpakkam coastal waters. (eds Thompson, M. F., |
| Gram-positive were Bacillus sp., hlococcus sp. and | | | | Nagabhushanam, R., Sarojini, R., and Fingerman, M.), |
| Micrococcus sp. and the Gram-negative strains | | | | Recent developments in biofouling control, Oxford and |
| identified as Pseudomonas sp., Salmonella sp., Vibrio sp. | | | | IBH Publishing Co. Pvt. Ltd., New Delhi,1994. |
| and Proteus sp. In PVC, the genera identified under | | | | 17. Bergeys`s Manual of Determinative Bacteriology, |
| Gram-positive included Bacillus sp., Stphylococcus sp. | | | | 8th edn, Warely Press Inc., The Williams and Wilkins |
| and Micrococcus sp., while the Gram-negative groups | | | | Company, Baltimore,1997. |