| Bioethanol, a clean and renewable liquid transportation fuel, is receiving increasing attention and application, mainly due to its majorenvironmental benefits.Bioethanol can be used by ethanol-fuel or flex-fuel. The amount of carbon dioxide (CO2) from ethanol combustion is approximately equal to the amount of CO2 absorbed by the biomass to produce ethanol, that is, the net CO2 emission is equal to zero.It can be produced from different kinds of renewable feedstock such as e.g.sugarcane, corn, wheat, cassava and sweet sorghum(first generation),cellulose biomass (second generation) and algal biomass(third generation).We improved bioethanol production from three angles, (i)applying Very High Gravity (VHG) fermentationin cassava bioethanol by optimizing viscosity-reducing enzyme cocktails; (ii) Constructing the effective xylose pathway based on CO2-fixation pathway in Sacchora,myces cerevisiae;(iii) Constructing cellulose and xylose-fermentingS. cerevisiae.High ethanol concentration after fermentation is a good foundation for saving the energy cost of bioethanol production. But high viscous nature of cassava tuber slurry impedes VHG fermentation of cassava. The simplest way was cofermentation of corn and cassava for ethanol production. When corn accounted for more than 40% (w/w), the ethanol concentration and yield were 109.3 g·L-1 and 87 %, which were 9.4% and 11% higher than sole cassava fermentation. In order to complete VHG cassava fermentation, viscosity-reducing enzyme cocktails were designed and optimized to reduce the viscosity of the slurry. The enzyme cocktail in mixing section was: 12U·g-1 ?-amylase, 10 U·g-1 cellulase,5U·g-1 xylanase and 8 U·g-1 acid pectinase, while enzyme cocktail in fermentation section was 150 U·g-1 glucoamylase, 0.08 U·g-1 pullulanase,10 U·g-1 acid protease, 10 U·g-1cellulase and5U·g-1 xylanase.By adopting the batch simultaneous saccharification and fermentation (SSF), the final utilization rate of starch, ethanol concentration, and the average productivity of 38% (w/v) cassava slurry were 94.47%, 140 g·L-1 and 2.86 g·L-1·h-1. Although input cost of enzymes was increased, reduced cassava cost decreased the total cost.Xylose is the second abundant sugar of lignocelluloses hydrolysates,but its commercial-scale conversion to ethanol by fermentation is challenged by incomplete and inefficient utilization of xylose. Firstly, we obtained a xylose-fermenting yeaststrain YSX4with co-expression of wild-typeXYL1 (coding for XR), mutant XYL1 (coding for XR (R276H)),XYL2 (coding forXDH) and XKS1 (coding forXK) in S. cerevisiaeYS58.YSX4 exhibited a higher efficiency of xylose utilization (0.70 g/L/h)when grown on xylose and maltose than grown on xylose and glucose(0.57 g/L/h). Furthermore, we engineered S. cerevisiaeYS58 with a Form-? based CO2 fixation system by co-expression of cbbM(Form-?Rubisco), sPRK(PRK), and GroEL-GroES (chaperone), resulting ina strain named YSC111. Similarly, in another yeast strain called YSC222,cbbL1-cbbS1 (Form-? Rubisco) andcfxP1(PRK) from Ralstonia eutropha H16, and HSP60-HSP10 (chaperone) constructed a Form-? based CO2 fixation system. The xylose consumption rates of YSX4C111 and YSX4C222 arrived to 0.97 and 1.1 g/L/h respectively. The total sugar consumption rate and the ethanol yield of YSX4C222 reached 3.1 g/L/h and 0.47 g/g sugars,which was 63% and 15% higher than YSX4C000.YSX4C222 cell extract also showed the highest carboxylation activity.The MFI h-C02 values ofthese strains at different times were calculated to evaluate the relative C02 flux, and the results indicatedthat over 8% of Ru5P from xylose was converted to G3P through the C02-fixation pathway in YSX4C111 orYSX4C222. The C02-fixation rate in the engineered yeasts was also calculated. YSX4C111 and YSX4C222were able to fix C02 at a rate of 336.6 and 436.3 mg C02/L/h,significantly exceeding the natural or the engineered microbes (5.8 to 147.0 mg C02/L/h) in previous reports.Consolidated bioprocessing (CBP) of cellulose mixed with fermentable sugar(s) is considered as a promising alternative to the use of cellulose as sole substrate for bioethanol production. Our research metabolically engineered Saccharomyces cerevisiae to allow for the co-conversion of cellulose and either sucrose or xylose to bioethanol. Constitutive promoter substitution and xylose metabolic pathway integration were carried out in a strain previously modified to express both bifunctional minicellulosomes by galactose induction and a cellodextrin pathway.StrainEBY101-CC, engineeredfor the co-fermentation of cellulose and sucrose, produced 4.3 g·L-1 ethanol from 10 g·L-1 carboxymethyl cellulose (CMC) and batch fed sucrose with an ethanol yield of 0.43 g·g-1.Strains modified for co-fermentation of xylose and cellulose,EBY101-X5CC and EBY101-X5CP were able to produce 2.9 g·L-1 cellulosic ethanol from 10 g·L-1 CMC and 1.2 g·L-1 from 10 g·L-1 phosphoric acid-swollen cellulose (PASC) respectively when xylose was depleted. |
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