The Anti-diatom Attachment Activities Of Sponge-associated Bacteria And The Study Of Active Compounds

Marine biofouling is a common worldwide problem. Current toxic antifouling compounds have serious harm to marine environment and human health. Natural non-toxic antifouling compound development has been a main way to solve this environmental problem. Because of the specific conditions in marine environment, metabolites produced by marine microbes have great differences from those produced by terrigenous ones. These marine microbes could produce natural metabolites with unique structures and diverse bioactivities. Marine sponges have become an important source of natural antifouling compounds, due to their unique chemical defense mechanisms and their diverse associated microbes, which could produce significant antifouling metabolites.From120strains isolated from8sponge species in San Juan Island, USA,11strains were found with strong activities of restraining diatom attachment. Two active strains of them, No.683and No.333, were identified. After optimization of fermentation conditions for both strains, No.683was chosen for further active compounds separation and identification. The results of this study will provide supports for seeking sponge-associated microorganisms with anti-diatom attachment activity.The main results of this study are as follows:1. In this study, by using optical microscopic counting,11strains were found with strong activities of restraining diatom attachment from120sponge-associated bacteria. The results showed that their inhibition rates at the concentration of100μg/mL against5diatom species were all larger than90.0%. The active strains occupied9.2%of total test strains. Among active strains, strain No.683showed the highest anti-diatom attachment activity with an average inhibitory rate up to98.0%; strain No.333had a secondary activity with an average inhibitory rate up to96.3%.2. According to16S rDNA sequence analysis, the similarity between strain No.683and Bacillus pumilus JQ428828is99%, and the one between strain No.333and Bacillus pumilus GU191914is74%. Therefore, in combination with the morphological observation, strain No.683and No.333are identified as Bacillus pumilus.3. Orthogonal tests were carried out to optimize the cultural conditions of strain No.683. The test was designed as four factors and three levels of orthogonal experiments. It has found that these four factors:peptone concentration, NaCl concentration, pH, and fermentation temperature, remarkably affected the strain activity. The order of the effect was peptone concentration> NaCl concentration> pH> fermentation temperature. As a result, the optimum fermentation conditions of strain No.683were fermentation temperature25℃, pH7.5, NaCl concentration of19.45g/L, and peptone concentration of5g/L.4. Orthogonal tests were also carried out to optimize the cultural conditions of strain No.333. The test was designed as four factors and three levels of orthogonal experiments. It has found that these four factors:peptone concentration, NaCl concentration, pH, fermentation temperature remarkably affected the strain activity. The order of the effect was peptone concentration> pH> NaCl concentration> fermentation temperature. As a result, the optimum fermentation conditions of strain No.333were fermentation temperature25℃, pH9.5, NaCl concentration of24.45g/L, and peptone concentration of7g/L.5. Strain No.683was fermented in large scale (100L) under optimized conditions, and9.9g crude extracts were obtained from the fermentation broth by ethyl acetate-acetone extraction. The separation processes for crude extracts of strain No.683were as follows.1) The preliminary separation:base on their polarity, the extracts were primarily separated into three parts, n-hexane phase, dichloromethane (DCM) phase, and65%methanol aqueous phases. Microscopic counting was used to test the anti-diatom attachment activity of three fractions. The results showed that the DCM phase was the most effective one.2) Solvent system selection for silica gel column separation:solvent system, solvent ratio, and polarity range were selected by Thin-Layer Chromatography (TLC) tests. The results showed that DCM and methonal acting as flowing solvents achieved best isolation.3) Silica gel column separation:the elution gradient was pure DCM,50:1,40:1,30:1,20:1,15:1,10:1,5:1,1:1, pure methanol. The40:1eluant had the strongest activity.4) Reverse phase decompression column separation:the elution gradient of10%,30%,45%,60%,80%,95%and100%methonal in distilled water was employed to separate the active fraction. The95%methonal eluant had the strongest activity.5) Separation by semi-preparative high performance liquid chromatography (HPLC):95%methonal eluant was eluted by constant90%methanol and was separated into three fragments. Frag.1and Frag.2had higher quantities and purities. The activity results showed that these all three fragments had strong activities.6. Structure identification of Frag.1and Frag.2:Frag.l and Frag.2could not be separated thoroughly because of similar low polarity. The fractions were subjected to Nuclear Magnetic Resonance (NMR) and Gas Chromatography-Mass Spectrometer (GC-MS) analysis. Based on GC-MS library search, some possible compounds were deduced and then put into activity verification. Combined with GC-MS and NMR results, they were probably fatty acid compounds. In comparison with the standard fatty acids, we proved the active compounds produced by strain No.683are fatty acids.