The Isolation And Identification For Compounds With Anti-diatom Attachment Activities From A Sponge-associated Bacillus Sp.

Because of their unique living environment, marine microorganisms can produce metabolites with great novelty and diversity. We can seek natural products with specific activity from them, which is an important research direction to the development of marine resources. Marine biofouling is always a thorny problem, the existing anti-fouling techniques are temporary, which use compounds relatively harmless to the environment, does not solve the problem fundamentally. Developing the efficient, non-toxic, and environmentally friendly active compounds, is widely recognized solution. More and more evidences suggest that the unique chemically anti-fouling mechanisms of sponges are likely depending on its symbiotic microorganisms. Therefore, looking for natural anti-fouling active substances from the sponge symbiotic microorganisms is an important and viable option to solve marine biofouling.In this study, a sponge-associated microorganism of UST050418-715, which was found possessing strong activities against the settlement of diatoms, was examined. The classification and identification of the strains, optimization of fermentation conditions, the extraction from fermentation culture, isolation and structure identification for the active compounds under the guidance of the activity were studied. The main findings were as follows:(1) Screening for active strain and its identificationPrevious study has found several strains with strong activities of restraining diatom attachment. Four of these strains (UST050418-315, UST050418-694, UST050418-708 and UST050418-715) were selected to retest the activities with three kinds of diatoms (Nitzschia closterium, Amphora coffeaeformis, and Nitzschia frustulum). The results showed that the four tested bacterias have the strong activities against the three test diatom cells attachment. The average inhibition rate is more than 90%. Among them, UST050418-715 has the strong activities against the settlement of three tested diatoms, the inhibition rates of which are all more than 90% with an average of 92.1%. Therefore, UST050418-715 was selected for further study.The test strain UST050418-715 was identified as Bacillus pumilus based on the 16S rDNA sequence analysis, cell morphology, and colony characteristics.(2) Fermentation conditions of strain UST050418-715.Orthogonal tests of strain UST050418-715 were carried out with four factors and three levels. The order of the effect was yeast concentration> peptone concentration> NaCl concentration> pH.By orthogonal test, combined with activity of the crude extract, crude extract yields and growth of the strain, each fermentation condition has a significant impact on the production of active substances. As a result, the optimum fermentation conditions of UST050418-715 were peptone concentration of 7.5 g/L, yeast concentration of 3 g/L, NaCl concentration was 10.45 g/L,pH9.5.(3) Isolation and identification of the active compounds produced by strain UST050418-715.We launched a large-scale fermentation of strain UST050418-715 and obtained its crude extracts, which was separated and purified under the guidance of bioassay. The separation steps were as follows:1) The preliminary separation, based on their polarity, the extracts were primarily separated into five parts by solvent extraction, petroleum ether phase, dichloromethane phase, ethyl acetate phase, butyl alcohol phase and water phase, marked Fr-1 to Fr-5. Test the anti-diatom attachment activity of five fractions by microscopic counting, analysis each fraction by HPLC. The results showed that the dichloromethane phase (Fr-2) was the most effective one, and the polarity of ethyl acetate phase for separation is moderate.2) Semi-preparative HPLC separation. Fr-3 was eluted by constant 40% methanol and was separated into ten fragments, Fr-3.1 to Fr-3.7. Among them Fr-3.2*, Fr-3.5*, Fr-3.6 and Fr-3.7* had higher quantities and purities, and Fr-3.3 had stronger activities. Fr-2 was separated into ten fragments, Fr-2.1 to Fr-2.8, with 40% methanol. Fr-2.1, Fr-2.3* and Fr-2.5* had higher quantities and purities, and Fr-2.4, Fr-2.6, Fr-2.7 and Fr-8 had stronger activities.3) Semi-preparative HPLC for further separation. Separate the fractions with stronger activities:Fr-3.3, Fr-2.4, Fr-2.6 and Fr-2.7. Get the following fractions:Fr-3.3.1 to Fr-3.3.8, Fr-2.4.1 to Fr-2.4.6, Fr-2.6.1 to Fr-2.6.6 and Fr-2.7.1 to Fr-2.7.3. Further separate Fr-2.6.2, obtain Fr-2.6.2.1 to Fr-2.6.2.4. Through the above separation, select relatively pure or active 34 fractions to conducte GC-MS test. The results showed that 12 fractions were purer, which were subjected to 1H-NMR analysis.According to the molecular weight analysis of GC-MS and the NMR profile, the major ingredient of Fr-2.3*, Fr-2.4.2 and Fr-3.5* were identified for the same compound as the Cyclo(L-Leu-L-Pro), the inhibition rates of them were 12.7%±8.4%,28.5%±3.5% and 30.7%±5.0%. The planar structure of Fr-2.4.3 is Cyclo(Val-Pro), the inhibition rate was 28.0%±10.4%. Fr-2.6.5 was identified as the Cyclo(D-Pro-L-Leu), the inhibition rate was 28.0%±12.6%. Fr-2.7.1 was identified as the Cyclo(L-Pro-D-Leu), the inhibition rate was 18.0%±2.8%.Fr-3.2* was identified as the Cyclo(Gly-D-Pro), the inhibition rate was 46.3%±10.8%. Fr-3.6was identified as the Cyclo(4R-D-Hydroxypro-L-Phe), the inhibition rate was 28.7%±11.3%.Fr-3.7* was identified as the Cyclo(4R-L-Hydroxypro-L-Phe), the inhibition rate was 11%±5.5%.