Investigation Of Cellulase Production By Immobilized Cell Fermentation And Application For Cellulosic Ethanol Production

Lignocellulose is the most abundant renewable resource and its application in biorefineryis potential. Lignocellulases can convert lignocellulose to fermentable sugars for variousbiochemicals, including bioethanol. Thus, lignocellulases play an important role inbiorefinery. The objectives in this study were mainly to obtain a novel cellulase productionwith high activity and a high titer ethanol production. Based on this, two strains producingcellulases with high activity were first screened and identified, and then, coculture andimmobilized cell fermentations were conducted. In order to explore the advantages ofimmobilized cell fermentation, cell morphology and secretory proteins were also investigated.Subsequently, the practical application of cellulases in cellulosic ethanol production wasfurther studied to obtain a high titer ethanol, which involved the main three processes ofproducing cellulosic ethanol, i.e. pretreatment, hydrolysis and fermentation. The detailedcontents are as follows:(1) In this chapter, forty mycelial isolates were selected and purified from naturallyfermented SCB with the medium in which SCB was the only carbon source. Based on theactivities of CMCase, FPase, cellobiase, and xylanase and the saccharification yield ofsugarcane bagasse, two cellulase-overproducing isolates, SCUT-18and SCUT-20werescreened. By determining18S rDNA sequences and examining mycelial morphologies, thesetwo isolates were identified as Aspergillus awamori and Aspergillus fumigatus, respectively.(2) In this chapter, the submerged coculture of Aspergillus awamori and Aspergillusfumigatus was studied (culture conditions:28oC,90rpm, initial pH4.8), and the resultsshowed that the saccharification yield of SCB by the coculture broth was~30%higher thanthose by the individual culture broths. Adding2%wheat bran in the cocultured fermentationincreased the saccharification yield of the produced broth from32.2%to42.6%. When usingthe broth produced by the coculture with cells immobilized on a polypropylene cloth, thesaccharification yield further increased to48.3%. A higher saccharification yield of68.6%was obtained when the immobilized-cell fermentation broth was concentrated5-fold toincrease its protein content to a comparable level to that of the commercial lignocellulases,Genencor ACCELLERASE1500, which gave a saccharification yield of62.1%(hydrolysisconditions:50oC,140rpm,48h).(3) On the one hand, coculture fermentation of A. awamori and A. fumigatus was morecomplex than individual fermentation, which made further analysis on coculture fermentationmore difficult; on the other hand, the information on gene sequences and protein analysis of a model strain is richer than that of an ordinary one. Thus, the individual fermentation of themodel strain is more helpful to the further study on cell morphology and secretory proteins. Inthis chapter, the submerged fermentations with free cells in a7L stirred-tank reactor (STR)and immobilized cells in a7L rotating fibrous-bed bioreactor (RFBB) by the cellulase-overproducing model strain Trichoderma viride were conducted by using sugarcane bagasseand wheat bran as the carbon source. Through the analysis of enzyme activities,saccharification yield of SCB and cell morphology, it was found that immobilized-cellfermentation gave35.5%higher FPase activity and69.7%higher saccharification yield ofSCB compared to free-cell fermentation and the difference in cell morphology between thetwo fermentations demonstrated the advantages of oxygen and nutrients transfer to cells inimmobilized-cell fermentation.(4) In this chapter, the difference in secretory proteins between the broths of free-cell andimmobilized-cell fermentations by T. viride was analyzed with two-dimensional gelelectrophoresis (2-DE) and MALDI-TOF-TOF mass spectrometry. The results showed thatthe secretory proteins had significant differences between the broths of the two fermentationsand24protein spots with significantly differential expression levels were successfullyidentified, including CBHI, CBHII, EGII, EGIV, α-amylase and swollenin. Among them,cellobiohydrolase CBHIΙ was highly expressed and secreted in the immobilized-cellfermentation, while the free-cell fermentation produced more CBH, and the activity ofCBHII is2-fold higher than that of CBHI, which probably was responsible for explaining thedifference of saccharification of sugarcane bagasse between free-cell fermentation andimmobilized-cell fermentation.(5) In this chapter, to further (1) learn the practical application of cellulases in cellulosicethanol production,(2) the whole process of producing cellulosic ethanol,(3) the correlationsof factors in production process, and (4) produce a high titer ethanol, a series of studies weredone, including the relationships between lignocellulose pretreatment method and enzymatichydrolysis efficiency, improvement of enzymatic hydrolysis and elimination of inhibitors inhydrolysis and fermentation processes. The results showed that enzymatic saccharificationresponse curves of lignocellulose substrates by various pretreatments (dilute acid, alkaline,and SPORL) were very different based on pH. The maximal saccharification of the lodgepolepine pretreated by SPORL occurred at the substrate suspension of pH6.2which wassignificantly different from pH4.8-5.2reported in literature. Then, the lodgepole pinepretreated by SPORL was used as the substrate, a fed-batch simultaneous saccharification andfermentation with the liquor derived from pretreatment was conducted at up to19.5%total solids loading at the optimal pH6.2. A maximum ethanol titer of47.4g/L was achieved,resulting in a calculated yield of285L/ton of dry substrate. This result is potential incommerce.