Screening Of Antifouling Functional Marine Bacteria And Their Active Metabolites

Biofouling is the colonization of man-made surfaces by sessile marine organisms (such as barnacles, mussels, tubeworms and seaweeds). With the continuous development of marine industry, the harmful effects of biofouling become more serious. Organic tin and cuprous oxide antifouling paints pose a great damage to the marine environment in the past few decades. As they have been banned or restricted, it is necessary to develop environment-friendly marine antifouling agent. The secondary metabolites of marine organisms used for chemical defense is a potential source to develop environment-friendly antifouling agent. However, the content of antifouling active compounds is so low in marine organisms, that it is difficult to supply, which is the main problem of the development of environment-friendly antifouling agent. Marine microorganisms which have an the ecological effects on fouling organisms, can be supplied in large-scale by fermentation and gene manipulation. Therefore marine microorganisms have huge development potential for antifouling.In this paper we separated marine bacteria from Enteromorpha sp., Ulva sp., Mycale sp. and Culcita novaeguineae. Then we detected antifouling activity of crude extract to screen out the best active strain. Then the strain was identified and fermented. The fermented liquid was extracted with different phases and detected for antifouling activity. To isolate the antifouling compounds, separation and purification was guide by the antifouling activity. Pure compounds was detected for antifouling activity using a variety of marine fouling organisms. The main results were as follows:1. A total of14strains were isolated from Enteromorpha sp., Ulva sp. and Mycale sp.. Combined with12strains of laboratory storage, we received23ethyl acetate extracts and11methanol extracts. Through the activity detection,9strains were screened out. Vibrio alginolyticus and Vibrio gallicus had the best antifouling activity for inhibiting the barnacle larvae, Whose EC50was1.92and1.60μg/ml respectively.2. Active substances of V. alginolyticus and V. gallicus were extracted with different phase. With the active detection, the methylene chloride extract and ethyl acetate extract showed the best antifouling activity. The EC50of V. alginolyticus was3.9and2.82μg/ml respectively. The EC50of V. gallicus was3.27and9.57μg/ml respectively. With HPLC analysis of active component showed that V. alginolyticus dichloride methane phase contained rich of active substances.3.4housekeeping gene (rpoD, toX, rctB, gyrB) sequences of the V. alginolyticus were determined. By comparing the Gene homology with Vibrio alginolyticus ATCC17749, the similar rates of rpoD、toX, rctB、gyrB were99%、98%、99%、99%respectively. The phylogenetic trees constructed based on16S rRNA, rpoD, rctB, gyrB gene sequences showed that V. alginolyticus and Vibrio alginolyticus ATCC17749had the closest relationship. With the reference to 《Bergey’s Manual of Systematic Bacteriology》 Vo12the antifouling activity of strains was identificatied as V. alginolyticus.4. Two anti-fouling active compounds were isolated from V. alginolyticus. They were Indole and L-Pro-D-Leu. Other compounds isolated from V. alginolyticus were3-Formylindole and mixture of p-Hydroxybenzoic acid and Dibutyl phthalate.