Hydrolysis Intensification, Recovery And Reaction Mechanism Of Cellulase

In order to dissolve the low efficiency of cellulase and high cost of bio-ethanol, several researches were carried on including the study of cellulase reaction mechanism, hydrolysis enhance using design enzyme complex, high concentration ethanol produced in high-solids simultaneous saccharification and fermentation and co-fermentation (SSF and SSCF), cellulase recycling by re-adsorption, and usage of membrane bioreactor (MBR). The main aspects and conclusions were displayed as follow:1. Reaction mechanism: (1) Pure cellulose: Focused on the insoluble residues of microcrystalline cellulose and filter paper cellulose, the hydrolysis process of cellulose was characterized and reaction mechanism was studied using different chromatography and spectroscopy technologies. We found that cellobiohydrolase (CBH) digestion follows a layer-by-layer solubilization manner, which might be the major reason for slow enzymatic hydrolysis of cellulose. Endoglucanase (EG) digestion follows a random scission manner. The chain stiffness of cellulose became stronger, as well as the inter/intramolecular hydrogen bonds. (2) Lignocellulose: In micro-scale, the release kinetics profiles of glucose and xylose were analyzed; in meso-scale, the molecular weight and size distribution, crystallility index and strength of hydrogen bonds were also measured.2. Enzyme complexes: The optimized cellulase (Spezyme CP) andβ-glucosidase (Novozyme 188) were determined to be 30 FPU/g glucan and 30 CBU/g glucan, respectively. The supplementation of xylanase and pectinase can increase the conversion of cellulose and hemicellulose significantly and pectinase seemed more effective. The supplementation of pectinase with 0.12mg protein/g glucan could increase the yields of glucose and xylose by 12.9% and 29.3%, respectively.3. Fed-batch SSCF at high dry matter. A xylose-utilizing strain of Z. mobilis CP4 was used to co-ferment glucose and xylose. The recombinant strain was then used in SSCF corncob at high dry matter (DM) of 15%. The suitable operation condition was determined, which was Z.mobilis loading of 0.3 g/L at pH 5.5 and 30 oC, when the final ethanol concentration was 49.24 g/L, corresponding to 85.18% of theoretic ethnol yield. Higher final ethanol concentrations, 60.52 g/L, were obtained for 10% DM supplementation giving a total DM of 25%.4. High concentration ethanol produced from high dry matter corncob using fed-batch SSF after combined pretreatment. Treatment with two steps of acid and alkali removed most of the non-cellulosic material, that increased cellulose content and bulk density, which was benefit for SSF at high-solids concentrations. High ethanol concentration of 84.7 g/L was obtained after 96 h SSF of H2SO4-NaOH corncob with adding fresh substrate of 6% into 19% initial dry matter, and exceeds the limits of feasible alcohol distillation.5. Enzyme recovery and recycling after high-solids SSF. Before readsorption, the cellulase adsorption/desorption behavior was measured. Then the readsorption conditions were optimized. The second round cellulase recovery was higher with the DM concentration of 15% at a cellulase loading of 30 FPU/g cellulose, pH 5.0, when the cellulose digested and ethanol produced in the reactor increased to 2.1-fold and 1.8-fold than those in single round, respectively. When the cellulase loading was 45 FPU/g cellulose, near 42 g/L ethanol, were obtained after cellulase recycled for a next round.6. The build and application of semi-continuous MBR in enzymatic hydrolysis of lignocellulose. The semi-continuous MBR was used in both ammonia aqueous pretreated corncob and H2SO4-NaOH corncob. Glucose produced in the reactor was 37.5 g/L and 121.5 g/L, which are 1.52 and 1.74 fold of conventional fed-batch reactor, and 2.07 and 2.84 fold of batch MBR.