The antifouling potential of commercial hydrolases, four proteases, seven glycosidases and one lipase was evaluated on the adhesion of marine Pseudoalteromonas sp. D41. The experimental method, adapted to screen antifouling agents, was based on bacterial adhesion in natural sterile sea water in a microtiter plate and on total biomass quantification by the fluorescent dye DAPI (4[prime]6-diamidino-2-phenylindole). Savinase (subtilisin) was the most effective hydrolase in both the prevention of bacterial adhesion and the removal of adhered bacteria. However, some enzymatic preparations tested such as Amano protease were not only ineffective but also increased the number of adhered bacterial cells. Enumeration using epifluorescence microscopy of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and DAPI stained adhered D41 cells confirmed these observations. Overall, these results demonstrated that hydrolases could either prevent adhesion and remove adhered bacterial cells effectively, or conversely increase bacterial adhesion, depending on enzymatic concentrations and the type of enzymes tested.
Aims: To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid and quantitative microplate fluorescent test.
Methods and Results: Our experimental method is based on a natural biofilm formed by mono‐incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96‐well polystyrene microplate. The 4′6‐diamidino‐2‐phenylindole dye was used to quantify adhered bacteria in each well. The total measured fluorescence in the wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 μm2 and quantifying 2 × 107 to 2 × 108 bacteria adhered per cm2. The antifouling properties of three commercial surface‐active agents and chlorine were tested by this method in the prevention of adhesion and also in the detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on the agent concentration showed a sigmoid shaped dose‐response curve.
Conclusions: This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation.
Significance and Impact of the Study: In contrast to the usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on the antifouling properties of the products, especially the initial steps of marine biofilm formation.
Aims: The nature of exopolymers involved in the adhesion of a marine biofilm‐forming bacterium Pseudoalteromonas sp. D41 was investigated to evaluate and understand the antifouling potential of subtilisin.
Methods and Results: The exopolymers of D41 produced by fermentation were analysed by FTIR and SDS‐PAGE showing the presence of polysaccharides, glycoproteins and proteins. A high content of proteins was detected both in soluble and capsular fractions. The microscopic observations of fluorescamine and calcofluor stained adhered D41 indicated mainly the presence of proteins in exopolymers produced during adhesion. Subtilisin, the broad spectrum protease, tested in natural sea water and in polystyrene microplates showed that antifouling activity was higher in the prevention of bacterial adhesion than in the detachment of adhered D41 cells.
Conclusions: Overall, these results demonstrate the involvement of proteins in Pseudoalteromonas sp. D41 adhesion and confirm the high antifouling potential of subtilisin.
Significance and Impact of the Study: This study emphasizes the major role of proteins instead of polysaccharides, thus extending our knowledge regarding the nature of extracellular polymers involved in bacterial adhesion. Furthermore, the high antifouling potential of subtilisin evaluated in the very first stages of fouling, bacterial adhesion, could lead to less toxic compounds than organometallic compounds in antifouling paint.
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