Andhra Pradesh, India. Received ; revised

Transcription

1 Isolation and identification of bacteria from marine biofilm on the artificial plat forms (iron panels) from Visakhapatnam coast, India G.sravankumar 1*, Y.V.K. Durgaprasad 1,T.Ramana 2, Harasreeramulu 1 & C.S.L.V. Devi 1 1 Department of Biotechnology Centre for research, Dr.V.S.Krishna. Govt. P.G. College. Visakhapatnam, Andhra Pradesh, India. 2 Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, India. [ sravang2010@gmail.com] Received ; revised Visakhapatnam coast is natural and colossal, the fishing harbor, Iron ore jetty of Visakhapatnam port, and Naval dockyard canal areas are studied for the locally associated biofilms. In the present study, Culturable bacteria forming marine biofilms were isolated and identified on artificial substrate (iron panels) according to Bergey, from Visakhapatnam coastal area, Northern Andhra Pradesh. Mainly 3 genera of 10 species were identified, they are Bacillus weihenstephanensis, Bacillus flexus, Serratia nematodiphila, Bacillus halotolerans, Bacillus aerius, Vibrio natriegens, Bacillus vallismortis, Bacillus endophyticus, Bacillus thuringiensis, Vibrio hepatarius. Where bacillus sps. are dominant and commonly found in all the three stations, Vibrio and Serratia were found in two stations. [Key word: microorganisms, bacteria, biofilms, nutrient media ] Introduction Biofilms are species rich, partly due to highly efficient powers of dispersal of the microorganisms and have wide tolerance to marine environmental conditions. Characteristically are the first organisms to respond to and recover from stress 1.Micro fouling organisms include bacteria, algal zoospores, diatoms and other colonizing microorganism. In which bacteria secrete exopolysaccharides (EPS) 2. Bacterial cells can encase themselves in a hydrated matrix of polysaccharides and proteins and form a slimy layer known as biofilm 3. These Biofilms in the Marine environment with diverse microbial populations cause lot of damage to the substratum like the hull of the marine vessels 4. This phenomenon naturally reduces the mechanical advantage of the vessel thereby reducing the maritime activity. This results in the frequent dry docking of the vessels for clearing the biofilm settlements. Present study is to identify the new microbial organisms which were totally different from the earlier microbial populations 5, and to test the tolerance of the growth of these new organisms with different kinds of growth

2 inhibiting substances and device an antifouling material that can inhibit the formation of microbial biofilms on the marine vessels 6, 7. In this study we performed 1) isolation of bacteria that form biofilm on artificial iron panels 8 and 2) identification of isolated bacteria, from three different stations of Visakhapatnam coast of northern Andhra Pradesh for further investigations. Materials and Methods The bacteria were isolated from three different stations, fishing harbor (station1). Iron ore jetty Visakhapatnam port (station 2), and Naval dockyard canal (station3). Iron panels of size 2x1 inches were placed in water tied to nylon threads along the berths 9, 10 in the three stations of visakhapatnam17.43n 83.7 E. After 5 days, the iron panels were collected in sterile polythene zip lock covers and brought to laboratory for further investigations. The artificial iron panels were washed with sterile physiological saline to remove vegetative cells, then the panel is swabbed on nutrient media, the obtained culture is mixed in distilled water and are serially diluted. Biofilm bacteria were isolated by standard spread plate method using Zobell marine agar 4 after two days incubation at 28+/-2 0 c. 11 Inoculated serially diluted samples in sterile Zobell marine agar plate 12 and kept for incubation at 28+/-2 0 c. After 24 hrs of incubation,the plates showed clear colonies, morphology of the colonies (size, color, form, surface, texture) were studied. Micro morphologic characteristics such as (gram staining, and endospore staining, motility) were studied 13. The morphology and results were recorded by observing under microscopically for 100 X oil immersion. Test cultures were analyzed for motility test in normal physiological saline and in distilled water by Hanging Drop preparation using a high power dry objective and reduced illumination. Smears were made on clear slides from the pure isolates and stained by Grams staining procedure and observed for their morphology and gram s characteristic 14.

3 The isolated pure colonies were sub cultured and then grown on basal media(nutrient agar, ZoBell marine agar,) differential media( MacConkey agar, EMB agar. ) and selective media( Mannitol salt agar, Cetrimide agar, TCBS agar IMViC, catalase, oxidase, urease, nitrate, casein hydrolysis, starch utilization, H 2 S, Hemolysin of selected isolates were studied and identified by adopting standard procedure 15. Results and Discussion Visakhapatnam sea coast has a natural harbor of high maritime activity and is subjected to limited exposure in the field of biofilm study (micro fouling 16 ). Visakhapatnam harbor is a major commercial port with round the clock exports and imports, Hence there is need to study the locally associated biofilm forming bacteria so that this could become the reference in future research. The bacteria isolated were identified based on traditional morphological methods and bergeys manual of systematic bacteriology In the present study biofilms were collected from three different stations namely, station 1. Fishing harbor, station 2. Iron ore jetty, Visakhapatnam port and station 3 naval dockyard canal. Samples were placed on Zobell Agar medium for isolation of bacteria,.4 isolates from station 1, 3 isolates from station 2 and 3 isolates from station3.totally 10 isolates were obtained from 3 stations and named as M1, M2, M3, M4, M5, M6, M7, M8, M9, M10. All the isolates showed good growth at 6% NaCl which confirmed their marine origin. All isolates were motile, the gram positive were bacillus and the gram negative were serratia and Vibrio sps. Morphological (table1) and Biochemical characteristics of biofilms were studied and represented in (table 2). The identified species show biochemical and morphological variations. In this present investigation, a long chained giant bacillus sps. Was identified almost 42 chained length from naval dockyard canal of Visakhapatnam coast and is identified as Bacillus thuringiensis 21,22 to confirm the occurrence of the observed strains in the Visakhapatnam harbor waters,biochemical tests (table1&2), differential media and selective media observations were performed (table3)..

4 Conclusion The identified species are M1 Bacillus weihenstephnensis, M2 Bacillus flexus, M3 Serratia nematodiphila, M4 Brevibacterium, M5 Bacillus aerius, M6 Vibrio natriegens, M7 Bacillus vallismortis, M8 Bacillus endophyticus, M9 Bacillus thuringiensis, and M10 Vibrio hepatarius. In station 1 fishing harbour isolated four bacteria species Bacillus weihenstephnensis, Bacillus flexus, Serratia nematodiphila, Brevibacterium, adhered to the iron panel, In station 2 Iron ore jetty three bacterial species Bacillus aerius, Vibrio natriegens, Bacillus vallismortis, were isolated and In station 3 naval dockyard canal bacterial species, Bacillus endophyticus, Bacillus thuringiensis, Vibrio hepatarius were isolated. In all the three stations the commonly found and dominant micro fouling bacteria is Bacillus sps. (Table 4). Acknowledgement Author greatly acknowledges the UGC, India for the financial support and thankful to the Commissioner of Collegiate Education, Hyderabad. and to the principal Dr. J.V.V.S.N. Murthy for laboratory facilities. References 1. Lowe R.L and Pan Y. (1996). Benthic algal communities as biological monitors. In: Algal Ecology: Freshwater Benthic Ecosystems (eds R. J. Stevenson, M. L. Bothwell and R. L. Lowe) Academic Press, San Diego Lawrence.J.R, D.R. Korber, B.D.Hoyle, J.W.Costerton and D.E Caldwell,(1991), Optical sectioning of microbial biofilms. Jour.Bacteriol,173e: Augustin, M., Vehams, A.T., Assessment of enzymatic cleaning agents and disinfectants against bacterial biofilms. J. pharm. Pharmaceut. Sci., 7(1), (2000)

5 4. Beveridge, T.J., Makin, S.A Kadurugamuwa, J.L, and Li, Z Interactions between biofilms and the environments, FEMSMicroiol.Rev.20 (1997), Beattie, E. J., & Lindow, S.E., Bacterial colonization of leaves: A spectrum of Strategies, Phytopathology, 89 (1999): Armstrong, E., Boyd, K G., Burgess, JG. Prevention of marine biofouling using natural compounds from marine organisms. Biotech. Ann.Rev. 6(2000) Armstrong, E., Kenneth, L. Y., Boyd, G., Wright, P. C. & Burgess, J. G., the symbiotic role of marine microbes on living surfaces, hydrobiologia, 461,(3) (2001): D Souza, F., & Bhosle, N. B.Analysis of microfouling products formed on metallic surfaces exposed in a marine environment. Biofouling, (2003) 19: Geesey, G,G., and D.C. White.. Determination of bacterial growth and activity at solid-liquid interfaces.ann.rev. Microbiol. 44(1990), Geesey, G.G., Lewandowski Z.and Flemming H C (eds.).. Biofouling and Biocorrosion in industrial water systems Lewis publishers. (1994). 11. Cappuccino And Sherman. Microbiology: A Laboratory Manual.(ed): Dorling. 7 th edn. (Kindersley India pvt. Limited) Zobell CE, Allen EC (1935) The Significance of marine bacteria in the fouling of submerged surfaces. J Bacteriol 29(3), Nicholson, W. L., Munakata, N., Horneck, G., Melosh, H. J., & Setlow, P., Resistance of Bacillus endospores to extreme terrestrial and extra terrestrial environments, microbial.mol.biol.rev.64(2000) Allegrucci, M., Sauer, K, Characterization of colony morphology variants isolated from streptococcus pnesumoniae biofilms. J. bacterial., 189(5)(2007), Holt J.G., Krieg N. R., Sneath P.H.A. and Williams S.T., Bergey s manual of systematic bacteriology, 9 th edition Baltimore: Williams and Wilkins, (1994).

6 16. Characklis, W.G. & Cooksey, K.E. (1983) Biofilm and microbial fouling. Advances in Applied Microbiology 29, Schaal, KP. (1985), Identification of clinically significant actinomycetes and related bacteria using chemical techniques. In chemical methods in bacterial systematics. M.Goodfellow and D.E. Minnikin (eds.), Academic press, NewYork Boone, D.R., Castenholz, R.W., Garrity, G.M., Bergey s Manual of Systematic Bacteriology,( 2 nd Edn).Vol. 1. (2001) Springer, Newyork Sakazaki R. Shimad T. Vibrio species as causative Agent of Food-Borne infection. In: Development of Food Microbiology, Robinson, R.K.London, Elesvier, 2(1986) Garrity, G., Boone, M., David, R., Castenolz, R. W. Bergeys Manual of systematic Bacteriology, (2001) XXI,721pp. 21. Kwon K K, Lee H S, Jung S Y, Yim J H, Lee J H,& Lee H K, Isolation and identification of biofilmforming marine bacteria on glass surface in Dae-Ho Dike, Korea, Journal of microbiology, 40(2002) Oguntoyinbo, F. A. Monitoring of Marine Bacillus diversity among the bacteria community of sea water. African journal of Biotechnology, 6(2007),(2): Shirling E B &Gottileb D, Methods for characterization of Streptomyces species, International Journal of Systematic Bacteriology, 6 (1996) Virginia Siqueira. M., Helena Oliveira, M.B., Filamentous fungi in Drinking water, particularly in Relation to Biofilm Formation Int. J Environ, res and Public Health. 8(2011) Muller, T., Ulrich, A., Ott, M. E. & Muller, M, Identification of plant associated environments, Microb.Ecol.47, (2004):

7 Table 1: Biochemical characteristics of biofouling bacteria. Identification test M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 Shape bacilli bacilli rod Cocobacilli rod rod bacilli bacilli bacilli rod Gram staining Motility Endosporestaining Indole Methyl red Vogesproskauer Citrate Starch Oxidase Catalase Urease H 2 S O/F A A F F A F F F A F Salt tolerance 0% % % Positive, - Negative, F- facultative, An -anaerobic, A - aerobic. OF- oxidative fermentation

8 Table 2: Biochemical characteristics M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 Carbohydrate utilization Dextrin Raffinose Sucrose Lactose Maltose Trehalose Cellobiose Mellibiose Arabinose Xylose Rhamminose Mannose Galactose Sugar Alcohols Adonitol Mannitol Dulcitol Inositol Aminoacid&derv Lysine Arginine Ornithine Carbohydrate utilization table, + indicates utilization indicates un utilization Table3: Characteristics of biofilm forming bacteria on differential and selective media

9 Selective media M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 NA NSA EMB - - Pink mucoid MSA Yellow colonies TCBS yellow colonies Yellow colonies CETRIMIDE Macconkey M.Blood.A Alpha. H Good growth, + growth,- No growth, NLF Non Lactose Fermenting, Alpha.H- Alpha haemolysis, M Blood.A Marine Blood agar, NSA Nutrient sporulating agar, Table 4: Fishing harbor ( Station1) Bacillus weihenstephnensis Bacillus flexus Serratia nematodiphila Brevibacterium Visakhapatnam port (Station2) Bacillus aerius, Vibrio natriegens, Bacillus vallismortis Naval dockyard canal (Station3) Bacillus endophyticus, Bacillus thuringiensis, Vibrio hepatarius

10 Fig. 1: Microscopic gram s stained photographs of the isolated marine bacteria 1.Bacillus weihenstephanensis 2.Bacillus flexus 3.Serratia nematodiphilia 4.Brevibacterium 5.Bacillus aerius 6.Vibrio natriegens 7.Bacillus vallismortis 8.Bacillus endophyticus

11 9.Bacillus thuringiensis 10.Vibrio hepatarius