A BIOFILM-FORMING MARINE BACTERIUM PRODUCING PROTEINS AS

Any surface immersed in an aqueous environment is rapidly colonized by microorganisms, building up a biofilm. Biofilm consists on bacteria embedded into their slime composed essentially of extracellular polymeric substances (EPS) such as polysaccharides or proteins. Searching on marine biofilm formation will be helpful in fighting against marine biofouling. AIMS: EPS biochemical characterization and observation by epifluorescence microscopy. Development of a marine biofilm formation model onto a polystyrene microtiter plate using the “O ’ Toole and Kolter method ” [1] adapted with the well known fluorescent DNA staining DAPI. EPS Biochemical characterization Bacterial Model: Pseudoalteromonas sp.D41 marine bacteria was isolated from a natural biofilm on Teflon immersed for 24 hours in the Atlantic Ocean at Brest. EPS Production: The EPS production was performed in fermentor as described by Raguénès et al. [2]. Two soluble EPS were isolated from the fluid supernatant recovered from the medium by high-speed centrifugation (20 000g, 2h, 4°C). EPS2 fraction was obtained by ultrafiltration of the supernatant onto 100kDa membrane and EPS1 fraction was further obtained by ultrafiltration of the EPS2 onto a 10kDa membrane. Non soluble EPS3 fraction was recovered from the cell pellet by dialysis. Model of marine biofilm formation in a microtiter plate Microtiter plate biofilm formation: Pseudoalteromonas sp. D41 was inoculated at approximately 2.109 cfu/ml in sterile natural sea water in a sterile black polystyrene microtiter plate and incubated from 45 min. to 24 h. at 20°C under horizontally shaking. After washings, wells were stained with 4µg/ml DAPI for 20 min. and washed again