Effects Of Sponge - Borne Microbes On The Adhesion Properties Of Benthic Diatoms

Biofouling causes lost with tens of billions of dollars in the global scope every year, and antifouling becomes worldwide serious problem. The toxic chemicals in traditional antifouling paints were more and more severely forbidden for using, because of their high toxicity and low degradation. Nowadays, it is an urgent issue to search for some effective and environmental friendly antifouling chemicals to replace traditional poisoning ones. Sponges possess chemical defending mechanisms against biofouling. More and more researches have proved that the producers of antifouling chemicals of sponges were their associated bacteria. Sponge-associated bacteria were promising resource for seeking effective and environmental friendly antifouling chemicals. Biofilms act as a key factor in antifouling processes and finally cause complex fouling organisms communities. If early biofilm forming processes were prevented effectively, the subsequent macro-organisms fouling were proved also to be prevented. The lost caused by biofilms and biofoulings would be saved at the same time.Actually in light reachable depth, the majority organism in biofilm is the microphyte of diatoms. Therefore, antidiatom has become an essential and fundamental step to avoid biofilm formation and subsequent macro-organism fouling. Developing antifouling strategy against multi-species targets including diatom is an inevitable way to solve biofouling problem. Current knowledge indicates that the raphe systems and extracellular polymeric substances (EPS) are important factors for diatoms to adhere on surfaces, playing crucial roles in biofilm forming and developing process.In this study, biofilms and EPS produced by diatoms were studied under the conditions treated by bacteria and their crude extracts with antidiatom activities. Diatoms adhesion performance was traced under disturbance of bacteria or crude extracts. Firstly, active bacteria were selected from sponge-associated-bacteria-bank depending on the results of antidiatom assays. Secondly, the antidiatom activities of various phases from bacterial culture were examined by bacteria-algae confronting tests. Thirdly, biofilm formation and EPS production of diatoms treated by active bacterial crude extracts were studied. Finally, above EPS were purified and analysed to clarify their monosaccharide compositions. Specific monosaccharides contributing to diatom adhesion performance were found. Details were listed as below:1. In this study,224 strains of bacteria isolated from sponge species collected in the sea of San Juan Island, USA were selected to test antidiatom activity. Diversity analysis for the genus of known samples showed they belonged to 4 phyla and 23 genera,85% samples belonged to Firmicutes and Proteobacteria, and Bacillus was the largest genus. At the concentration of 100 μg/mL, crude extract of 24 strains were found with strong activities of inhibiting the adhesion of at least four diatoms. Lower concentrations for 5 strains selected were tested with antidiatom activities against two diatoms Nitzschia closterium and Amphora sp.. Results showed that antidiatom activities reduced with concentrations reduced and EC50 against Amphora sp. were higher than against N. closterium. The differences of sensitivity to crude extracts of two diatoms maybe are relative to their raphe systems. Two active but unidentified strains were identified according to their cultural characteristics and 16S rDNA, UST050418-708 identified as Psychrobacter arcticus and UST050418-694 as Microbacterium testaceum.2. The relationship in direct confrontation between bacteria and diatoms was studied using active bacteria as tools. The direct confrontation tests were firstly conducted on 24 well plates between diatom Stauroneis sp. and each of four active bacteria strains. The results showed that antidiatom activity of bacteria became stronger under higher bacteria/diatom ratio and the supernatant of bacteria possessed the higher activity than living bacteria cells, dead bacteria cells, and total bacteria culture. Then diatom Stauroneis sp. was cultured with bacteria UST050418-708 for 16 days in triangular flasks for a direct confrontation test with larger scale and longer time. The results showed that living bacteria cells and total bacteria culture promoted diatom growth, dead bacteria cells showed no significant effect on diatom growth, and supernatant of bacteria culture inhibited diatom growth. At the same time, the distribution of diatom cells in biofilm and supernatant was changed by active bacteria. The supernatant of bacteria culture made the ratio of biofilm forming cells reduced, while cells of bacteria, especially the dead ones, made the ratio raised. The possible reason is conferred as that, some materials with antidiatom attachment activity were destroyed during high temperature sterilization process, and the nutritional ingredients or inducing diatom attachment existed in bacteria cells were released.3. Diatoms Stauroneis sp. and Amphora in large volume culture, being treated with crude extracts of active bacteria UST050418-708, were also studied in terms of growth and adhesion, EPS production, and monosaccharide compositions of EPS. The results showed that treatments affected diatom growth and distribution of algae cells in culture system, and the changes were also found in EPS. For Stauroneis sp., high concentration treatment of crude extract made EPS production per unit algae went up greatly. As for Amphora sp., the production per unit algae remained similar as control. But, TB-EPS productions of bio film-forming cells per unit algae were increased for two diatoms. It is conferred that TB-EPS maybe have close relation to cell’s adhesion. To a certain degree, the differences of reactions against crude extracts treatment of active bacteria were due to different living form of diatoms.4. The crude EPS of two diatoms were purified by degreasing by organic solvent washing, deproteinization by the method of Sevage, and desalination by dialysis. The purified EPS were completely hydrolyzed in TFA, derivatized by PMP, and then subjected to HPLC analysis. The monosaccharide compositions and the molar ratios between each monosaccharide were calculated based on the HPLC profiles. The monosaccharide compositions of total EPS of Stauroneis sp. were identified as Man, Glc-N, Rha, Glc-A, Glc-NAC, Glc, Gal, Xyl and Fuc in accordance with the order of retation time. The compositions of total EPS of Amphora sp. included three monosaccharides (Gal-A and two unknown ones) in addition to the nine of Stauroneis sp.. For a diatom species, the dominant monosaccharaide of EPS from different phases were different. For TB-EPS of Stauroneis sp., no matter coming from biofilm-forming cells or floating cells, the most dominant monosaccharide was Glc, with percentages of 38% or 59%, respectively. The second dominant monosaccharide was Gal for F-TB-EPS, Man for BF-TB-EPS. For diatom Amphora sp., the dominant monosaccharaides of TB-EPS of floating cells were GlcN and Xyl, especially Xyl with predominance; as for TB-EPS of biofilm-forming cells, the dominant monosaccharide was Gal,Glc-A and Man, Gal with predominance. For different diatom species, the dominant monosaccharaide of EPS were usually different, but some EPS from similar resources possessed some similarity. For SL-EPS, coming from all algae cells in the culture, the major monosaccharaides of both Stauroneis sp. and Amphora sp.were Glc-A and Xyl.The treatments of crude extracts made EPS changed. The smallest changes, which were with the numerical changes but without dominant monosaccharides changes, were found in SL-EPS of two diatoms. In general, the characteristic monosaccharaides of diatom Stauroneis sp. were 4 species, as Xyl, Glc, Man and Gal. The characteristic monosaccharaides species of diatom Amphora sp. were 5 species, as Xyl, Gal, Man, Glc-A, and GlcN. The dominant monosaccharaides always were hot sites, big changes took place sites, at nomal case. But some monosaccharaides, not being dominant at normal time, became hot sites after treatments.Analysis about the relationship between diatom adhesion performance and the monosaccharide composition indicated that the mole content (%) of "hexosamine+uronic acid", uronic acid, and Glc-A in the EPS of diatom Amphora sp. possessing strong adhesion performance were higher than those of diatom Stauroneis sp., which behaves with relatively weak adhesion. Especially, Glc-A is the absolutely dominant monosaccharides in the EPS of Amphora sp., and is relatively low but also dominant in Stauroneis sp.. When treated with the active crude extract, the reducions of mole content (%) of Glc-A were consistent with treatment concentrations and accompanied by the adhesion of the cells for both two species of diatoms. Therefore, at least for this two diatoms, Glc-A has great relevance with EPS viscidity and the cell adhesion performance.The results of this study will not only deepen our knowledge of chemical defense mechanisms of sponges, but also help us understanding fouling mechanisms of organisms, especially for diatoms, at the same time. In addition, they also provide supports for seeking resources for effective and environmental friendly chemicals with antifouling activity.