Isolation Of Antifouling Compounds And Fermentation Optimization Of Coral-derived Symbiotic Microorganisms From The South China Sea

Marine biofouling did harm to many human activities,including shipping industry,marine engineering and aquaculture.Using antifouling paint is the most effective method to prevent and remove marine biofouling.Antifouling agent,the kernel of antifouling paint,can prevent marine biofouling organisms adhering to the surface of marine facilites through constantly release.After traditional heavy metal antifouling agents such as TBT and Cu02 have been banned or restricted becatuse of their toxicity to marine ecological environment,developing environmentally friendly antifouling agent is in need.Many soft corals and gorgonians have smooth surfaces,and their symbiotic microorganisms are believed to produce natural antifouling substances to participate in the host's chemical defense,which is a potential source for the development of environmentally friendly antifouling agents.Because microorganisrms have a short growth cycle and are easy to be cultured,we can solve the problem of yield limitation,which is often encountered in the development of natural antifouling substances from marine animals and plants,through microbial fermentation*.In this paper,different culture mediums were used to isolated microorganisms from surface and coelom of soft corals and gorgonians from the South China Sea.The target strain Aspergillus sydowii with good antifouling activity and abundant metabolites were screened out Compounds were isolated from the fermentation broth of A.sydowii,and then their structures and antifouling activities were detected.At the end,different fermentation conditions were conducted to study their effects on compounds yields and to determine the optimum fermentation condition for increasing the yield of antifouling compounds.The main results are shown as follow.(1)12 fungi,including 4 strains of Penicilliun,6 strains of Aspergillus,1 strain of Rhinocladiella,1 strain of Acremonium,and 6 actinomycetes of Streptomyces were isolated from soft corals and gorgonians in the South China Sea.Balanus albicostatus and Perna viridis were used as biological models to detect antifouling activities of strains.Extracts of 10 fungi,including Penicillium christenseniae,had significant inhibitory effects on the attachement of barnacle cyprids,meanwhile extracts from Streptomyces pactum.Streptomyces koyangensi??Streptomyces griseorubens and Streptomyces tirandamycinicus also had significant inhibitory effects on the attachment of B.albicostatus cyprids.Extract of Aspergillus penicillioides and Streptomyces griseorubens had significant inhibitory effect on the byssus thread production of the P.viridis.HPLC was used to detect the compound diversity of different fungal fermentation broth extracts,and the result showed that the extract of fermentation broth of A.sydowii had the most abundant of compounds.Therefore,A.sydowii from the surface of Euplexaura robusta was selected as the target strain to isolate and purify of compounds.(2)The target strain A.sydowii was fermented and a new compound 3,7-dimethyl-l,5-dioxocane-2,6-dione with 4 known compouds,including p-Hydroxybenzaldehyde,p-Hydroxybenzoic acid,4-Hydroxyphenylacetic acid and diorcinol,were isolated from the extract of its fermentation broth.Diorcinol had inhibitory effect on Ulva.tepida spore attachment with EC50 of 43.82 p.g/mL.p-hydroxybenzoic acid,p-hydroxyphenylacetic acid,diorcinol and 3,7-dimethyl-1,5-dioxocane-2,6-dione had inhibitory effects on the attachment of larvae of Bugula neritina with EC50 of 36.00,42.50,37.02 and 37.01p.g/mL,respectively.And the carboxyl groups in p-Hydroxybenzoic acid and p-Hydroxyphenylacetic acid played an important role in antifouling activities according to the structure-activity analysis of 3 phenols.(3)Optimizing fermentation conditions of A.sydowii for increase of yields of 2 hypotoxicity antifouling compounds,diorcinol and p-Hydroxybenzoic acid.The result showed that the optimal initial pH,the optimal inoculum size,the optimal medium volume,the optimal seawater proportion,the optimal fermentation time and the optimal fermentation temperature of fermentation for diorcinol yield are 8,4%,200 mL,0%,Czapek-Dox Medium,10 days and 22 0C respectively.And the maximum yield of diorcinol is 16708.25 ?g/L.The optimal in:itial pH,the optimal inoculum size,the optimal medium volume,the optimal seawater proportion,the optimal fermentation time and the optimal fermentation temperature of fermentation for p-Hydroxybenzoic yield are 9,3%,300 mL,0%,MY Medium,15 days and 28? respectively.And the maximum yield of diorcinol is 149.97 ?g/L.