Study On Lipid Fermentation Strategies Of Corn Stover Pretreated With Alkaline-methanol Solution

Microbial lipid prepared from lignocellulosic biomass still suffers from the low lipid yield and high cost,which hinders the industrial success.High density fermentation is one of the important ways to help the industrialization of microbial lipids.In this thesis,a variety of fermentation strategies have been investigated using the regenerated corn stover pretreated with NaOH-methanol solution by the oleaginous yeast Cutaneotrichosporon oleaginosum ATCC 20509.The research results were summarized as follows:Firstly,the enzymatic hydrolysis and lipid fermentation performance of the NaOH-methanol pretreated corn stover were investigated.The monosaccharide conversions of glucan and xylan reached 88.7% and 79.2%,respectively,when the enzymatic hydrolysis was conducted at a solid-to-liquid ratio of 10%(w/v).The total reducing sugar(TRS)yield reached 0.82 g/g.Lipid titer and yield reached the highest of 13.8 g/L and 0.21 g/g,respectively,when 0.5 g/L(NH4)2SO4 was supplemented to the hydrolysates.This yield corresponded to 0.17 g lipid per gram of regenerated corn stover.The highest lipid yield of 0.14 g/g regenerated corn stover was obtained through the simultaneous saccharification and lipid fermentation with an optimal enzyme loading of 7.5 FPU/g.Secondly,a fed-batch enzymatic hydrolysis of the regenerated corn stover was successfully performed at a high solid loading of 47%(w/v).The concentrations of glucose,xylose and TRS reached 180.0 g/L,54.0 g/L and 299.5 g/L,respectively.In addition,21.3 g/L cello-oligosaccharides and 29.8 g/L xylo-oligosaccharides were inevitably co-produced,which accounted for 17.1% of the total sugars in the hydrolysates.The conversion of glucan and xylan were 96.8% and 79.0%,respectively.Some culture strategies including the fed-batch fermentation,two-stage fed-batch fermentation,and the fed-batch simultaneous saccharification and fermentation were investigated and compared using the resultant hydrolysates containing high concentration of sugars.The fed-batch fermentation was found superior to other strategies for lipid production.Lipid titer,content,and yield reached37.6 g/L,57.1%,and 0.17 g/g,respectively.At last,a two-step washing strategy based on quantitative calculation was developed after the fed-batch enzymatic hydrolysis to obtain three hydrolysates with different sugar concentrations suitable for fed-batch fermentation.The sugar loss issue was addressed and the recovery of TRS increased 43.8%.A turbid liquid hydrolysate was collected during solid-liquid separation process and serious sugar loss would take place in the process of heating mediated clarification.The effects of the different feeding media including turbid hydrolysate,heated turbid hydrolysate,and clear hydrolysate on the fed-batch lipid fermentation was compared.The highest lipid yield was obtained when the heated hydrolysate without solid-liquid separation was fed,which was 1.37 times and 1.29 times,respectively,high than those feeding with the clear and untreated hydrolysate.Cello-oligosaccharides(82.2%)and xylo-oligosaccharides(68.7%)were assimilated by C.oleaginosum during the fed-batch fermentation.