Pretreatment Of Rice Straw By Deep Eutectic Solvents And Its High-value Utilization

Rice straw?RS?is one of the most abundant lignocellulosic biomass in China,and its traditional disposal methods always cause waste of resource and severe environmental pollution.With the rising prices and depletion of fossil fuels,as well as many environmental issues,more studies have been focused on sustainable bioenergy.Therefore,it is of great significance to obtain high value-added products such as bioenergy and high-energy phosphate from lignocellulose raw materials.However,the key step in the high-value utilization of lignocellulosic biomass is the reduction of recalcitrant structures.In this thesis,a novel pretreatment method by deep eutectic solvent?DES?was applied to utlize polysaccharides in RS for butanol production and ATP-free in vitro biosynthesis of fructose 1,6-diphosphate through enzymatic reactions.?1?A two-stage pretreatment method using choline chloride:formic acid:acetic acid?Foraceline?,a dihydrogen-bonding deep eutectic solvents?DES?,and 0.5%?w/v?Na₂CO₃ was developed for RS pretreatment.The highest glucose and total sugar concentrations of 38.8 g·L^-1 and 41.9 g·L^-1 were obtained after hydrolysis of 24 h using 50 FPU·g^-1_{tot a l-solid} of cellulase.The RS attached cellulase was recycled for 5 cycles.Cellulase loading was reduced from 50 FPU·g^-1_total-solid?Cycle I?to 30 FPU·g^-1_{tot al-solid}?Cycle V?,resulting in a 40%enzyme saving.The total sugar concentrations were 41.3 g L^-1 and 42.6 g L^-1,respectively.Hydralysate I and V were used for the butanol fermentation of C.saccharobutylicum DSM 13864 and butanol concentration and yield of 9.5 g.L^-11 and 0.25 g?g^-1_{tot a l sugar} were obtained,similar to that of glucose medium.?2?Another novel DES was synthesized using ethylamine hydrochloride?EAC?as the hydrogen bond acceptor and lactic acid as the hydrogen bond donor for RS pretreatment.Under above pretreatment and enzymatic conditions,DES_1E:1L exhibited more significant effect than that of Foraceline.The pretreatment effects of fresh and recovered DES_1E:1L on RS were compared.Using 5^th recovery DES_1E:1LE:1L to pretreat RS,24.14 g·L^-1 of glucose was obtained,which was only 5.96 g·L^-1 lower than that of fresh DES_1E:1L.This result suggests recovered DES_1E:1L could be used in the pretreatment of RS.Then,RS_DES1E:1L was soaked in 2%?w/v?NaClO in the dark for 12 h at room temperature,a total sugar concentration of 59.2 g·L^-1 was achieved,and the removal rates of hemicellulose and lignin?95.6%and 82.8%?were 1.31-and1.48-fold that of RS_DES1E:1L,respectively.The structure of the treated rice straw was further characterized,and the mechanisms and pretreatment effects of different pretreatment methods were illucidated.Using above prepared RS hydrolysate(RS_CombDN),10.16 g·L^-1 butanol was produced and 46.73 g·L^-11 total sugar was consumed,demonstrating the feasibility of this newly developed pretreatment protocol.?3?In vitro ATP-free enzymatic pathway for the synthesis of FDP was constructed utilizing cellobiose in RS_CombDN hydrolysate as a substrate,similar to glycolytic metabolic pathway.Under the participation of cellobiose phosphorylase?CBP?,phosphoglucomutase?PGM?,phospho-glucose isomerase?PGI?and pyrophosphate-dependent phosphofructokinase?PPi-PFK?,four enzymtic reactions were carried out sequentially.Except for the relative lower 65.8%conversion in the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate,all other steps reached more than 96%.Our result demonstrates the ATP-free biosynthesis of fructose 1,6-diphosphate by in vitro cascade enzymatic approach is feasible.