High-Efficient Lipid Production Via Integrating Consolidated Bioprocessing Of Starch And Simultaneous Saccharification And Fermentation Of Cellulose

The production of microbial lipid from low-cost substrates such as lignocellulose biomass has significant implications for addressing the current shortage of oil resources in China.However,the current technology route for producing lipid from lignocellulose has prominent issues such as low raw material utilization rate,complex process,and high enzyme costs,which greatly hinder its commercial production.Based on the enzyme producing characteristics of Lipomyces starkeyi AS 2.1560,an oleaginous yeast that produces its own amylase and β-glucosidase,this paper explores the feasibility and advantages of integration strategy of consolidated bioprocessing of starch and simultaneous saccharification and fermentation of cellulose.The aim is to significantly simplify the production process,reduce enzyme costs,and enhance lipid production efficiency through process integration.The results of the study are as follows:(1)Firstly,the problem of inadequate sugar supply in the early stage of cellulose simultaneous saccharification and fermentation was solved by adding simple or complex carbon sources.It was found that the addition of soluble starch could increase the lipid yield to 90% of that in separate fermentation.After optimizing the combined fermentation medium formula,the effect of β-glucosidase on the fermentation results under the integration strategy of consolidated bioprocessing and simultaneous saccharification and lipid production at an enzyme loading of 15 FPU/g of L.starkeyi was investigated.It was found that no additional β-glucosidase was required,and after 120 hours of fermentation,the lipid titer and lipid yield reached16.28 g/L and 0.20 g/g,respectively,which significantly reduced enzyme costs.(2)After further verifying that the integration strategy can strengthen cellulose simultaneous saccharification and fermentation for lipid fermentation,the amount of cellulase was optimized,and 5 FPU/g was selected as the best enzyme loading.In the fermentation of 40 g/L soluble starch mixed with 40 g/L cellulose,it was found that starch was rapidly hydrolyzed within 24 h to provide sugar.After 144 h,the lipid titer reached 16.40 g/L,and the highest amylase activity was 3.55 U/m L.The study also investigated the enzymatic hydrolysis effect of alkali-pretreated corn stover under different enzyme loadings,as well as its lipid production effect when combined with cassava starch.The highest lipid productivity reached 3.88 g/L/d.At last,synergistic effect for achieving higher lipid production by combined utilization of corn stover and cassava starch at relatively low enzyme dosage was realized,in comparison with the separate utilization of these two substrates.(3)Starch was quickly hydrolyzed by the amylolytic enzymes secreted by L.starkeyi to provide adequate fermentable sugars at the initial stage of culture.The low activity of glycosidase enzymes in the T-cellulase used were investigated.The effects of high concentration of cellulose and starch degradation component(glucose,cellobiose,maltose and dextrin)and combination media(soluble starch and cellulose)on lipid production were studied.When the concentration was between 80 g/L and200 g/L,the biomass and lipid titer showed an overall downward trend as the substrate concentration increased,with the most obvious trend in glucose medium.The results showed that the substrate inhibition of monomer was stronger than that of polymer at equal molar carbon concentration,and polysaccharides were beneficial to high substrate fermentation,providing a research basis for fed-batch fermentation with high substrate concentration.