| In appropriate conditions, some algae can dramatically increase or aggregation, then cell density sharply increases in seawater,and the water color changes and may cause death of marine organisms, which is so-called red tide phenomenon. In addition to algae, other some single cells can form red tide.Marine eutrophication is the direct cause of red tide. Red tide research at home and abroad has continued for decades, the predecessors used physical, chemical and biological· methods to governance red tide but got no ideal conditions. Thus the use of microorganisms for biological control of algae has aroused great concern.Currently, algae-lysing microorganisms include algicidal bacteria, actinomycetes lysing, lysing fungi and cyanophage as well as protozoa. Because of their easy propagation, lysing effect and host-specific characteristics, they have attracted much attention as one of the most promising biological control methods.Algicidal bacteria (Algicidal bacteria) refers to directly or indirectly inhibit or kill algae, bacteria dissolve algal cells collectively.1924algicidal bacteria were first discovered.In microalgae, Gymnodinium is more common in the southern seas of red tide outbreaks reported dominant species.Gymnodinium is single-celled algae, cell walls bare or, with an inter-karyotype nucleus. Yokomizo obvious, the flagellum, the plant is divided into horizontal groove on the lower conical portion. Most heterotrophic life is aquatic bait. Blooms in the water reflects red-brown. Gymnodinium excessively grow would form harmful aquatic "red tide." It does’t only produce a variety of toxins, poisonous to fish and shellfish, etc., but also hazardous to the human body. While at home and abroad threre are relatively little research on red tide caused by Gymnodinium.In this study, we respectively collected water samples from China Laizhou Bay, Jiaozhou Bay and Sanya City, Hongsha Bay,and used liquid infection method, which was adding isolated single colonies to Gymnodinium culture and co-cultured, and ultimately isolated marine bacterium HSB01and HSB07from seawater of Hongsha Bay of Sanya, South China Sea. Otherwise, bacterium HSB07performed stronger inhibitory effect to algae than HSB01.Based on the16S rRNA gene sequence and biochemical characters, the isolated strain HSB01was identified as Staphylococcus sp. and HSB07was Halomonas sp.In the bioactive prescreening of the two bacteria and their fermentation product, the crude extract of strain HSB07with ethyl acetate showed moderate inhibitory effect activity against Gymnodinium sp.20L LB medium scale fermentation were carried for bacterial HSB07and obtained14g crude extract. By silica gel column chromatography and thin-layer chromatography two fractions B and C were obtained.Dissolving fractions B and C in DMSO (dimethyl sulfoxide) respectively, and using liquid infection method, which is the same to the above. Then we found that fraction B behaved significant algaal-lysing effect, and fraction C also behaved certain algaal-lysing effect.Fractions B, and C were taken to further gel column chromatography and separation of the liquid phase, a pure component was separated from the C that is, component#12. NMR and mass spectral analyzes used to determine molecular weight of#12to414. Specific structures need to be further analyzed and identified.Given20L scale fermentation of biomass is not ideal, thus the bacteria HSB07fermentation conditions were optimized. Considering20L scale fermentation conditions28℃, pH7.5, salinity is3, fermentation time24～48h. With the fermentation broth pH. salinity and fermentation time as the three factors affecting extracellular main parameters of the active substances. Selectting the three levels of each factor orthogonal experiment, the choice of L9(34) orthogonal experimental was designed. The fermentation broth was centrifuged (6500rpm) for10min, then bacteria was isolated from the upper and extracellular bacterial metabolites from the lower, collectted the supernatant.15ml bacterial culture with extracellular metabolites in it was added to120ml algae culture. Taking algae-lysing rate as the evaluation indicator of fermentation conditions. The main results are as follows:1. Separation and identification of algae-lysing bacteria 1) Seven strains of bacteria were isolated from seawater of Hongsha Bay of Sanya, South China Sea. Two of them showed more obvious inhibitory effect to Gymnodinium sp., which were respectively named HSB01、HSB07and for further study. HSB07performed stronger inhibitory effect.2) The almost-complete16S rRNA gene sequence of strain HSB01and HSB07were obtained and used for initial BLAST searches in Genbank. Considering biochemical and morphological characteristics, HSB01and HSB07were respectively identified as Staphylococcus sp. and Halomonas sp. The Genbank number was respectively KC832320and KC832321.3) Observe morphology of two algicidal bacteria colonies:bacterium HSB01colonies are pale yellow, small and round, convex surface, silty and smooth edges; bacterium HSB07colonies are transparent, larger, round, moist and sticky, not easily picken, not so smooth edges. Electron microscopy shows that two strains had no flagella.2. The mechanism of bacteria lysing algae1) When Gymnodinium growed in the14th day, which is the late logarithmic phase, two bacteria were added to algae culture. Significance test of difference was conducted: there are Significant differences in density of algal culture adding HSB07broth, Sig value of less than0.05; while adding HSB01difference was not significant, Sig value of0.27>0.05, when cultured in the next period, it demonstrated a significant difference. This indicates that two strains HSB01and HSB07inhibited the growth of algae obviously. Adding some bacteria culture to algae culture late at the logarithmic phase could accelerate it into death phase, which was more obvious when adding HSB07medium.2) Study for the mechanism of the two algae-lysing bacteria, we found that the inhibiting factor was not the bacterium but the secondary metabolism product. In another word, it inhibits the algae in indirect way.3. Separation and purification of bioactive substance from algae-lysing bacteria1) After large-scale fermentation of HSB07and extract crude by ethyl acetate, we put crude to some algae culture to study its inhibitory effect. The crude showed moderate inhibitory effect and could be purified further.2) Separated by silica gel column chromatography and thin layer chromatography, fractions B and C with inhibitory activity were separated.3) Dissolving fractions B and C in DMSO (dimethyl sulfoxide) respectively to conduct algae-lysing test. Then we found that fraction B behaved significant algae-lysing effect, and fraction C also behaved certain algae-lysing effect.4) Fractions B and C were taken further gel column chromatography, and separation of the liquid phase, a pure component was separated from the component C, that is. component#12.5) Component#12nuclear magnetic resonance and mass spectral analyzes were used to determine its molecular weight to414. Specific structures need to be further analyzed and identified.6) By orthogonal experiments we found that:the medium pH8, salinity of3.5, fermentation time36h optimum fermentation conditions for the relative. In this condition, extracellular secretion of bacterial HSB07showed relatively strongest algal activity. |
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