Fermentation Optimization Of Renibacterium Sp. QD1for Chitosanase Production

Despite many potential applications of the chito-oligosaccharides depolymerized fromchitosan has been discovered. How to develop high production chitosan degradation enzymebecomes the bottleneck of limiting functional chito-oligosaccharides preparation Marine strainsRenibacterium sp. QD1isolated from the marine environment by our laboratory, efficientlyproduces an extracellular chitosanase known as Csn-A. The Csn-A with high chitosan-degradingactivity is stable in wide range of pH and shows the substrate specificity. This article aims todevelop a fermentation medium and process for large-scale production of chitosanase by marineRenibacterium sp. QD1by fermentation statistical optimization and fermentation engineeringprocess optimization,In this work, medium components were determined by single-factor experiments. Furtheroptimization of medium was carried out by using statistical methodologies including orthogonaldesign, Plackett-Burman design, steepest ascent method and response surface methodology. In theend, we developed a new type for large-scale fermentation culture medium: chitosan14.23g/L,soluble starch3.00g/L, yeast extract4.72g/L, KH2PO41.00g/L, K2HPO44.00g/L、MgSO40.70g/L and initial pH6.42. Enzyme activity reached608.11U/ml at40h which was enhanced nearly6times as compared to that initial chitosanase.Then culture conditions including temperature, rotate speed, shear stress were optimized. Theresult: optimal cultivation temperature was28°C, the optimal cultivation rotate speed was170rpm.,The growth and chitosanase was not sensitive to shear stress experiment.We optimized fermentation engineering process through5L fermentation reactor based on theoptimized new fermentation medium and culture conditions, chitosanase production droppedsignificantly under the condition of constant pH fermentation. The productive fermentationprocess: the initial pH was set as6.42and no pH control was performed during the whole process,dissolved oxygen remain above40%. The result: the maximum biomass and chitosanaseproduction was obtained at27h, a third time shortened, compared with the shake flaskfermentation. Then we feed batch fermentation experiments, acid soluble chitosan feeding experiments, acid soluble chitosan feeding experimental results show that the feeding experimentscan’t significantly increase the yield of chitosanase. Water-soluble chitosan feeding experimentsshow that chitosanase production increased nearly1.8times compared with the batch fermentationexperiments. This kind of phenomenon contribute to elucidating the chitosanase inductionmechanism has important theoretical significance.Based on this, fermentation scale-up was performed in50L bioreactor using the abovestrategy. In fermentation24h, chitosanase production reached476.63U/ml. Results showed thatthe bacteria growth and enzyme production is faster in50L reactor. The results show that marinestrains Renibacterium sp. QD1preliminary has the capability of the industrial application.