Preliminary Exploration Of The Algicidal Mechanism Of Bacteria F5-2and Preliminary Identification Of The Algicidal Substance

Because of the severe economic and public health effects of the harmful algal blooms, extensive researches have been undertaken to control and finally deal with the problem.Physical and chemical techniques have some adverse effects, so the biological techniques are most promising.Our laboratory isolated an algicidal bacteria, strain F5-2, the supernatant has algicidal activity against Heterosigma akashiwo,Prorocentrum minimu, Chrysophyta, Scrippsiella trochoidea and Skeletonema costatum, has certain inhibitionon the growth of Chaetoceros muelleri; has algicidal activity against Microcysis aeruginosa and Chlorella pyrenoidosa. We find that alga cells have swum to the bottom, then it become xanthic and be killed.In addition, VC has certain inhibition on the growth of M. aeruginosa, and the algal cells are precipitated. So we initially explored the mechanism of the broth supernatant and VC against M. aeruginosa. Broth supernatant (v/v=1:50) and VC (w/v=1:5000) against the M. aeruginosa, firstly observe the differences of the phenomenons, supernatant against M. aeruginosa with significant phenomenon of the precipitation and xanthic, VC against M. aeruginosa with significant phenomenon of the precipitation; Secondly count the all algal cells and suspended algae cells, calculate the changes in the number of precipitated cells, found that supernatant against M. aeruginosa with the precipitation and xanthic, result in the drastically reduce of cells, VC against M. aeruginosa with significant phenomenon of the precipitation, but the algal cells is keep unchanged essentially; By the microscopic observation, found that breakdown fastly and the number reduce significantly of the algae cells with supernatant, the status and the number unchange essentially of the algae cells with VC. Compare the GSH-Px,SOD activity and MDA content of F5-2supernatant with VC.The results show that, their lysing mechanisms are different greatly, the inhxibition of the F5-2may be related to the accumulation of reactive oygen species and free radicals, accompanied the death in the precipitation process, the VC is unrelated, the algal cells are only precipitated, can not be killed. Strain F5-2, that produced some extracellular products have algicidal activity against the Heterosigma akashiwo. In order to explore the nature and the structure of the substance, we implemented the stability, protease and nuclease, dialysis, alcohol precipitation and extraction tests, then separated by both HPLC and the TLC methods, and preliminary identified by mass spectrometry, IR and elemental analysis. The results show that the active substance is basically stable, the influence of the temperature and storage time is few; the active substance is not polysaccharide,nucleic acid and protein; molecular weight less than2KD; lipophilic and has weak polarity; The HPLC parameters has been identified by optimization:The chromatogram was detected at Eclipse XDB-C18column, detection wavelength at300nm,25℃, the mobile phase V(water):V(methanol)=60:40; The TLC parameters has been identified by optimization:The solvent V(ethyl acetate):V(petroleum ether)=1:1, Rf is0.8033-0.927, second the solvent V(ethyl acetate):V(petroleum ether)=4:1, obtain the pure material components of the lytic activity; After the series of identification, algicidal active substance molecular weight is about260Da, its structure does not contain N, P, S and unsaturated bonds, may contain at least of Ar-,-OH,-C=O and-CH2-, the number of C is11, the number of H is15.