Cellulase Overproduction By Trichoderma Reesei Through Batch-feeding Of Biosynthesized Soluble Inducer

Biofiiels and biochemicals produced from lignocellulosic biomass are renewable and environmentally friendly,which are significant for sustainable socio-economic development.A general scheme for the biorefinery includes biomass pretreatment,cellulase production,enzymatic hydrolysis of the cellulose component,microbial femientation and unit integration and process optimization,but high cost of cellulase production is one of the bottlenecks.Tirich.odeirma ireesei is the best cellulase-producer,but its celulase production needs reduction.Cellulose is a natural inducer for cellulase biosynthesis by the species,in particular microcrystalline cellulose,although it is expensive.However,cellulose is insoluble,which needs to be hydrolyzed slowly by the basal cellulase to release true inducers for cellulase production at large quantity,which substantially prolongs the fermentation process,and consequently compromises cellulase productivity.Since cellulase fermentation is aerobic and characterized by Non-Newtonian fluid properties with high viscosity,low productivity makes cellulase production extremely energy-intensive.Pre-treated lignocellulosic biomass?corn stover,wheat and rice straw and bagasse?and paper sludge have been explored to replace microcrystalline cellulose,but they are not effective for hydrolysis by the basal cellulase to relase true inducers,making cellulase production even more energy-intensive.Soluble inducers such as lactose can be uptaken directly by the species to address the challenge with insoluble inducers,but they are expensive and less effective in cellulase induction.The aim of this work is to develop low cost and more efficient soluble inducers for robust production of cellulase by T.reesei Rut C30,and in the meantime optimize the cellulase cocktail for more efficient enzymatic hydrolysis of the pre-treated biomass.Disaccharides were synthesized from glucose through the transglycosylation reaction catalyzed by p-glucosidase.Since large amount of glucose was left when the reaction was terminated,the mixture of glucose-disaccharides?MGD?was used as substrate for mycelial growth and inducer for cellulase production,which was tested through flask culture.In addition,another soluble inducer ?-1,3-glucan was also selected and studied for cellulase induction.The experimental results indicated that MGD and ?-1,3-glucan were more assimilable and effective in cellulase induction,and cellulase production was 1.24 and 1.16 times,respectively,of that induced by lactose,which was confirmed by the transcription of genes encoding cellulolytic enzymes.No significant defference was observed for cellulase induction between MGD and p₁,3-glucan,but MGD would be more cost-effective,and thus suitable for cellulase induction.Sugars in MGD wrere further analyzed by ion chromatography,and gentiobiose,cellobiose and sophorose were identified as the major disaccharides,but sophorose was experimentally validated to be the key inducer.Therefore,another route for sophorose production through stevioside hydrolysis by acid was explored,but the process was not reliable,and sophorose production might not be economically feasible.Cellulase overproduction by T.reesei Rut C30 was carried out in 7 L fermentor with MGD as substrate and inducer.In order to eliminate its inhibition in cellulase production,glucose was controlled between 0.05 and 0.30 g/L through the batch-feeding of MGD.As a result,cellulase titer as high as 90.3 FPU/mL was achieved at 144 h,10 folds higher than that achieved with lactose as inducer,and consequently cellulase productivity was increased to 627.1 FPU/L/li,at least 3-5 folds higher than previously reported for cellulase production by the fungal species using different inducers and fermentation systems.Proteomics analysis was performed for the crude enzymes Cel-MGD and Cel-APCS induced by MGD and alkali pretreatment corm stover?APCS?,respectively,and the results indicated that specific cellulase and ?-glucosidase activities in Cel-MGD were 1.74 and 2.42 times higher than that decteced in Cel-APCS,but much higher xylanase activity was observed in Cel-APCS.Activity of ?-glucosidase was lower in both Cel-MGD and Cel-APCS.The cassette containing heterologous ?-glucosidase gene aabgl1 under the control of the pdcl promoter was constructed and transformed into T.reesei Rut C30 by Agrobacterium tunmefaciens-mtdiated transformation,and the recombinant T.reesei PB-3 with the highest ?-glucosidase activity of 310.12 CBU/mL,about 70 times of that produced by the host strain,was selected.Under batch-feeding conditions,cellulase activity of 50 FPU/mL was achieved at 156 h by T.reesei PB-3,and the crude enzyme?Cel-MGD2?was tested by hydrolyzing 15%APCS.No significant difference in the hydrolysis with sugar yield of 92%was observed for Cel-MGD2 and Cel-MGD formulated with commercial ?-glucosidase,and 44.8 g/L ethanol was produced from the sugar by Saccharomyces cerevisiae.Fed-batch strategy was applied for the hydrolysis with solid uploading increased to 20%,and separate hydrolysis and fermentation?SHF?and simultaneous saccharification and fermentation?SSF?were performed,respectively.As a result,119.9 g/L glucose was produced at 108 h for the SHF process,which was consumed within 12 h with 54.2 g/L ethanol produced by the yeast.For the SSF process,52.1 g/L ethanol was produced at 96 h.Although ethanol yield was slightly lower for the SSF process,much higher ethanol productivity was obtained.These results illustrated that the crude cellulase Cel-MGD2 was effective for hydrolyzing APCS.A recombinant strain T.reesei YH18 overexpressing cbh2 was developed to improve cellulase production,and a mixture of MGD and APCS was optimized for cellulase production with improved xylanase activity to hydrolyze hemicelluloses.Fed-batch fermentation was performed with T.reesei YH18 using the mixture with the optimized ratio of MGD/APCS,and cellulase activity of 7.17 FPU/mL and xylanase activity of 577.55 FPU/mL were achieved at 60 h,which were significantly higher than those reported using cellulosic biomass as inducer.After cellulase fermentation,APCS was supplemented directly at 20%solid uploading,and 122.5 g/L glucose and 40.2 g/L xylose were released,the highest reported so far using on-site produced cellulase for biomass hydrolysis.The progress with this work provides alternative strategies for robust cellulase production and its on-site use for the biorefinery of lignocellulosic biomass.