Fermentation Optimization Of Vibrio Sp. QY102for Alginate Lyase Production

Marine Vibrio sp. QY102isolated from surface of sargassum by Ocean University of China efficiently produces an extracellular alginate lyase with high alginate-degrading activity. The Iyase is stable in wide range of pH and shows the polymannuronic acid specificity. At present large-scale production of alginate lyase with high activity and substrate specificity still faces challenges despite many potential applications of the oligosaccharides depolymerized from alginate has been discovered as well as the alginate lyase. This project aims to substantially improve the production of the enzyme by fermentation optimization and process control, develop a fermentation process for large-scale production of alginate lyase by marine Vibrio sp. QY102to promote the industrial application of the new enzyme.In this work, medium components were first confirmed by single-factor experiments. Further optimization of medium was carried out by using statistical methodologies including orthogonal design, Plackett-Burman design, steepest ascent method and response surface methodology. In the optimized medium of alginate6.0g/L, soluble starch5.0g/L, yeast extract2.0g/L, tryptone4.7g/L, KH2PO41.7g/L, MgSO4·7H2O0.10g/L, NaCl30g/L and initial pH5, enzyme activity reached11.39U/ml at48h which was enhanced by65%as compared to that in the original medium. Then culture conditions were optimized. The optimal cultivation temperature was30℃. The growth and enzyme production was not sensitive to shear stress. Moreover, increasing dissolved oxygen could improve the growth of cell, but not the enzyme production.Growth kinetics on starch and alginate as sole carbon source were then investigated in5L batch cultures. The results showed that alginate was more easily utilized by Vibrio sp. QY102, while starch as the carbon source not repressing enzyme expression extended the growth phase and weakened the violent pH fluctuations during the fermentation process. Then an optimized alginate feeding strategy was proposed as that pulse feeding solid alginate which could extend the production phase and enhance the production. The process follows:Vibrio sp. QY102grows on starch solely until the end of the logarithmic growth phase, then solid alginate is added as1g/L every3h. The initial pH was set as5and no pH control was performed during the whole process. By this way, the maximum alginate lyase production was obtained as52.8U/ml, which was665%,363%and329%higher than that in shake flask culture with original medium, new medium and5-L batch culture, respectively. Then effects of pH control on the fermentation were performed in5L bioreactor. When controlling constant pH at5,6,7and8, the maximum alginate lyase productions were8.6、34.2、36.7and13.9U/ml respectively, which were84%、35%、30%and74%lower than that without pH control. High pH improved starch utilization but decreased the alginate lyase production. Finally, fermentation scale-up was performed in30L bioreactor using the above strategy. The maximum alginate lyase production of46.8U/ml was obtained at23h. The average enzyme production rate reached2.9U/(ml·h), which was44%higher than that in5L bioreactor (2.0U/(ml·h)).