Study On Enrichment And Activity Components Of Fermentation Process Of Sr18 Bacteria

Plant-parasitic nematodes cause great damage to agriculture and forestry in worldwide nowadays. Concerned about environment and human health, people pay more attention to investigate the production of new biologically nematicidal active metabolites from microorganism. The metabolites from filamentous fungus Sr18 showed very strong and wide nematicidial spectrum against some kinds of important plant-parasitic nematodes. The character of its soluble in water and high thermal stability made it has the great potential to practical application for nematode control. The dissertation focuses on the optimization and scale-up submerged fermentation condition of Sr18 and isolation of the neutral nematicidal constituents from the metabolites of Sr18. The results were summarized as follows:1. Scale-up submerged fermentation of nematicidal metabolites from filamentous fungus Sr18 on the scale of 30L, 200L and 5000L fermenter:The influence of different medium to the growth and nematicidal metabolites production of Sr18 on the basis of shaking flask was studied in this dissertation. The result shows that when potato was used as nitrogen source and 0.2% malt extract was added, Sr18 fungus will form smaller (dia. 2mm), smooth, ivory yellow pellets and its nematicidal activity is higher. By using peptone as nitrogen source, the pellets are bigger and rough, and the metabolites production of Sr18 has high nematicidal activity when the pH value was adjusted to 7.0.The fermentation optimization of Sr18 nematicidal metabolites production was carried out by 30L fermenter according to the optimal culture conditions in the shake flask and 5L fermenter. The optimum fermentation conditions for Sr18 in the scale of 30L fermenter was determined as follows: the seeds were cultured on the stationary condition for 24h, inoculum volume fraction was 4%, ventilation was 0.35～1VVM and dissolved oxygen(DO) was controlled among 20%~30%, incubation period was about 48h. The nematicidal mortality of one time diluted broth reached 95% by above conditions.This dissertation also deals with the relationship between the growth of Sr18 fungus and its nematicidal metabolites production during the different fermentation period by observing the morphology of the hyphae. The result showed that nematicidal activity of metabolites related closely to the morphology of Sr18 hyphae. The result can provide some morphologic proof for the further research to improve the nematicidal metabolites production.The fermentation of Sr18 nematicidal metabolites production in 200L and 5t fermenter was studied in this dissertation. By controling the condition and quality process of 200L and 5t fermenter, the nematicidal mortality of one time diluted broth reached 100%. And the nematicidal rate reached above 80% in the three time diluted fermentation solution which produced by 5t fermenter. The result make a contribution to the industrialize production of Sr18 nematicidal metabolites.2. Separation of neutral nematicidal constituents from the fermentation broth of Sr18 fungus:The fermentation broth of Sr18 was disacidified by ion exchange chromatography, the nematicidal rate of the neutral component obtained by the ion exchange experiment is 49.95%, the results shows that nematicidal bio-active constituents were composed of the neutral substance as well. And then the neutral components were separated by macroporous adsorbent chromatography, three bio-active constituents CI, EI and EII were obtained. Then CI, EI and EII were separated by means of molecular sieven chromatography in Sephacryl S-100 column, and three bio-active constituents (F I、F III、F VI) were obtained. And then the bio-active constituents FI was separated to three constituents (DI、D II、D III) by DEAE sepharose fast flow. The aim of this experiment is to separate the neutral nematicidal constituents of the metabolite and make a base of establishing the separating process of the bio-active substances.