Studies On The Production Of The Fuel Ethanol By The Fermentation Of Rice Straw Cellulose

Cellulosic material is the most abundant renewable carbon source in the world. The annual output of rice straw is mor than three hundreds million tons in China. Rice straw cellulose may be hydrolyzed by cellulase to produce glucose, and then glucose can be used for the production of fuel ethanol. The utilization of renewable biomass can not only save the foodstuff but also reduce the environmental pollution. Based on the screening and breeding of strains, the researches investigated the characters of the production of cellulase by the strains and the kinetic process of the fermentation, built the batch fermentation technologies, separated and purified the cellulase, constructed the engineering yeast strain with the cellobiose metabolizing pathway, studied the fermentation with immobilized engineering yeast cells, discussed the cellulase hydrolysis technology of rice straw, and finally investigated the production of fuel ethanol using the two-step coupling bio-reactor. The research results are as follows:1. A strain named YT01 (Penicillium) with high cellulase activity was screened, and then it was mutated by ultraviolet with protoplast and optimized by liquid fermentation, which is named as YT02. The medium with rice straw powder as carbon source, bean powder and (NH₄)₂SO₄ as nitrogen source was optimal and the maximum cellulase activity was reached in the conditions of 29℃and origin pH 6.0 when cultivated for 120 h. The CMCase activity, filter paper activity andβ-glucosidase activity were 3.86 IU/mL, 207.41 IU/mL and 1.40 IU/mL respectively in shaking flask.2. Response surface methodology was used to optimize the medium for cellulase production by YT02. The optimized composition of fermentation medium was (g/L): rice straw powder, 41.95; bean powder, 24.83; bran powder, 22.16; (NH₄)₂SO₄, 4; KH₂PO₄, 4; MgSO₄, 0.5. The CMCase activity, filter paper activity andβ-glucosidase activity were 8.8967 IU/mL, 357.41 IU/mL and 3.704 IU/mL respectively after the fermentation in the optimized medium for 120 h. All results are higher than before.3. Since the production of cellulase was the major contribution to the bioconversion process, the DO, temperature and pH value of submerged fermentation by YT02 was studied. The fermentation was scaled up in a 5 L stirred fermenter, and the technical conditions of batch fermentation are as follows: fermentation temperature at 0-32 h was 32℃, DO 70 %; fermentation temperature at 32-120 h was 29℃, DO 50 %; origin pH value of fermentation broth was 6.0. The CMCase activity , filter paper activity andβ-glucosidase activity were 11.13 IU/mL, 465.24 IU/mL and 4.08 IU/mL respectively after 4 days, which were higher than the values in shaking fermentation. The dynamic results of batch fermentation showed that the growth of YT02 and the cellulase activity were coupled partially.4. CMCase andβ-glucosidase were purified by DEAE Sephadex A-25 and Sephadex G-75. The purification multiple was 13.48-fold and 18.62-fold to homogeneity by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography with a recovery yield of 10.54 % and 8.62 % respectively. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 73 kDa, 43 kDa and 57.8 kDa. N-end amino acid sequences and MALDI-TOF analysis of the cellulases were also performed.5. The strategy for direct integration of a cellobiase gene (BGL1) into the Saccharomyces cerevisia chromosome is an effective method for the stable expression of cellobiase in the industrial strain of Saccharomyces cerevisia NAN-27, using an integrating vector pYMIKP which containing a rDNA portion as a homologous recombination sequence to obtain multicopy integrants and PGK1 promoter and terminator, and with the G418 resistance gene (KanMX) as dominant selection marker. This integrating vector is an ideal vector for construction of the genetically engineered S. cerevisiae that used industrial strain as the host. The strategy expended the substrate for fuel ethanol production, and reduced the inhibitor of cellobiose to cellulase hydrolysis. It was found that the engineering Saccharomyces cerevisia NAN-28 cells were immobilized efficiently and rich in cellobiase entrapped into calcium alginate gels. Comparing with the traditional immobilization of pure enzyme protein, this new method was more convenient and economical. The activity of enzyme was not destroyed, and the immobilized cells were quite stable with a long half-life and could accelerate the synergetic hydrolysis process of cellulosic biamass. Comparing with free cells, the fermentation term of immobilized cells was shortened, and the yield of fuel ethanol was improved. The immobilized cells could utilize cellulosic hydrolysate to produce fuel ethanol efficiently.6. During the saccharification of cellulosic material, the key influence factors including character and concentration of substrate, enzyme dosage, temperature and pH were investigated. Since the cellulase system from YT02 was poor in cellobiase (CB/FPA was 0.38), the yield of rice straw residue to ethanol was only 18 %. When under the synergetic reaction of YT02 cellulase and immobilized engineering Saccharomyces cerevisia NAN-28 cells, the yield of rice straw residue to ethanol was raised to 26 %, while the yield of rice straw residue to ethanol in free cells was just 20 %. All results were significant for the elucidation of synergistic degradation mechanism of cellulase.7. A two-step coupling bioreactor was set up by coupling the cellulose hydrolysis, the immobilized cells and the immobilized cells producing cellobiase together. In this coupling bioreactor, the feedback inhibition to cellulase reaction caused by the accumulation of cellobiose and glucose was eliminated, and the hydrolysis of cellulosic material was promoted. The yield of fuel ethanol from cellulose reached 25.5 g/L at 40 h for fermentation, the conversion rate was 43.0 % (the theoretical conversion rate was 56.61 %), and the production efficiency was 0.64 g/( L·h) which was the 1.65 times of SSF in free cells. The new reactor was stable and efficient, and the immobilized cells could be repeatedly used for a long time. Under fed-batch process, the final concentration of cellulosic substrate and ethanol were increased to 250 g/L and 66.51 g/L respectively. The utilization of cellulase and the productive efficiency of ethanol were both improved. The bioreactor showed a good performance in synergetic saccharification and fermentation. This research work simplified the equipment, facilitated the automatic operation, and was important for cost-saving.