Optimization Of Fermentation Process For Production Of Scleroglucan By Sckerotium Rolfsii

Scleroglucan,a fungal polysaccharide,is synthesized and secreted by Sclerotium glucanicum,in which Sclerotium rolfsii is the most typical strain.In order to enhance the production of scleroglucan and reduce the cost,a wild type Sclerotium rolfsii WSH-G01screened in our previous works was used as a start strain in this study.The fermentation medium components and the fermentation conditions were optimized by single factor experiment in shake flasks.Combining with response surface methodology,the initial optimum fermentation medium composition was obtained.Then,the nutrition and culture conditions were further studied in batch fermentation process in a 3 L fermentor.Finally,the physicochemical properties and hydrolysis conditions was analyzed preliminarily.The main results in this thesis were described as follows:?1?Based on detectig the growth curve of Sclerotium rolfsii WSH-G01 in shake flasks,the optimum inoculum age was determined at 72 h.The key compositions of the medium were optimized,including the concentration of glucose,yeast extract,NaNO₃ and the culture time.The obtained optimum medium and culture conditions were composed of:glucose 55.0 g·L^-1,yeast extract 0.5 g·L^-1,NaNO₃ 2.5 g·L^-1,incubation time 5 d,initial pH 4.0,inoculum volume5%,liquid volume 50 mL in 250 m L flasks.The titer of scleroglucan was 11.8 g·L^-1 cultured at30?,220 r·min^-1 for 120 h.?2?Effects of the initial glucose concentration(35 g·L^-1,55 g·L^-1,75 g·L^-1,95 g·L^-1),stirring speed(200 r·min^-1,400 r·min^-1,600 r·min^-1),ventilation ratio?0.5 vvm,1 vvm,2 vvm,3 vvm?and feeding strategies?one-time feeding,constant flow feeding and constant concentration feeding?on scleroglucan production were investigated in 3 L fermentor.The results showed that the optimal initial glucose concentration was 75 g·L^-1,stirring speed was400 r·min^-1,ventilation ratio was 1 vvm,and the best feeding strategy is keeping the glucose concentration at 65 g·L^-11 for 32 h after 40 hours of fermentation.Under this condition,the titer of scleroglucan reached 34.5 g·L^-1,the biomass was 20.7 g·L^-1,and the productivity was 0.48g·L^-1·h^-1,the substrate utilization was 30%.?3?Preliminary analysis of scleroglucan was conducted,including the scleroglucan purity and scleroglucan structure.The results showed that the scleroglucan was actually a kind of dextran with?-D glucopyranosulfan structure?the purity was 96.2%,composed of glucose molecules and weight-average molecular weight was 1.9×10⁷ Da?.Then the hydrolysis process for scleroglucan applying the method of acid and?-1,3 glucanase was performed.The weight-average molecular weight distribution of the polysaccharide was 65185,5048,1061,217 Da after 0.1 M HCl hydrolysis,respectively.Four different sources of?-1,3 glucanases gene BGLM,PGLA,EXG1,and BGLF expression vectors were constructed to obtain the crude enzyme to hydrolyze the scleroglucan,the abtained weight-average molecular weight was about 20 kDa under 37?,while the abtained molecular weight was about 200 kDa at 30?.