| Cellulose is a linear polymer of anhydroglucose units arranged together byβ-1, 4-glycosidic bonds. It is a major component of plant biomass and abundant in agricultural and domestic waste. As its large quantity, short-term renewable, low price, and no competitions for food with people, it has been considered as raw material to produce fuel ethanol, an environmental-friendly biofuel.The conversion process of cellulose to bioethanol includes three main process: pretreatment of lignocellulosic materials, the degradation of cellulose to fermentable sugars and fermentation of sugars with yeast. In the process, the high cost of cellulase is an important factor restricting the commercialization of cellulosic ethanol. The original strains screened from nature can not directly used for the cellulase commercial production because of its low enzyme secretion. The strains were often mutated by physical or chemical methods to increase its cellulase production capacity.Penicillium decumbens JUA10-1, a catabolic-repression-resistant mutant from a wild strain P. decumbens 114-2, has been used industrially for cellulase production. However, it is still necessary to improve its cellulase activity and productivity to reduce the costs of cellulase production. Current methods for improving industrial microorganisms'productivity range from the random approach of classical strain improvements to the highly rational methods of protein engineering or genetic engineering. As the genetic background of P. decumbens was not known yet and there was no appropriate genetic tool, it was difficult to design rational methods to improve its cellulase production. In addition, JUA10-1 is a mutant strain of P. decumbens 114-2 after many rounds of mutagenesis and carries many negative defects, so it may increase further decline in its growth and production capacity if we continue to make mutagenesis on it.As a method of microbial mutation breeding, ion beam implantation technology has been used in the breeding of a variety of crops, insects and microorganisms such as Aspergillus oiyzae and Aspergillus niger. Genome shuffling is a procedure which can combine multiple genetic advantages of different parent cells by recursive protoplast to generate new composite offspring although the genetic backgrounds were not known. In this study, ion beam implantation and genome shuffling were used in breeding of strain JUA10-1 to improve its cellulase productivity, and the crude cellulase from the mutants and fusants were used in enzymatic hydrolysis of pretreated wheat straw and in SSF (Simultaneous saccharification and fermentation) to evaluate its application in enzymatic degradation of cellulosic substrate and production of cellulosic ethanol. The resultswere as follows:(1) According to the survival rate of strain in different mutation doses, the optimal mutation dose of N+ ion beam for P.decumbens JUA10-1 was obtained.(2) Mutant of P.decumbens JUA10-1 was obtained by N+ ion beam implantation and screening with double-layer cellulose plate and shake flask liquor fermentation. The filter paper activity of crude cellulase from the mutant was at least 20% higher than that from strain JUA10-1 andβ-glucosidase activity also increased.(3)The method of protoplast fusion was found. The time of protoplast inactivation was decreased by increasing temperature of heat inactivation, which effectively shortened the time of protoplast fusion.(4) Corncob residue from diluted sulfuric acid pretreatment of corncob and wheat straw pretreated by hot water were used as carbon source for enzyme production respectively, and found that, the mutants and fusants can produce cellulase with high activities of filter paper andβ-glucosidase compare with the strain JUA10-1.(5) For ethanol production with SSF process using wheat straw pretreated by hot water as substrate, when the substrate concentration was 10%, the highest ethanol concentration was 34 g/L and conversion of cellulose to sugar reached to 99%. |
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