| Lignocellulosic biomass is the most abundant renewable resource in the world. It has been considered that bioconversion of lignocellulose to fuel ethanol is of great benefits to reduction of the greenhouse effect, mitigation of the energy crisis, improvement of environment qualities and sustaining development of our economy and society. Kenaf (Hibiscus cannabinus L.) is a traditional quick-growing fiber crop with high biomass yields and has been regarded as a quality plant for papermaking and good substitute for the wood. It has been reported that the highest yield of kenaf biomass is up to 30 t·h-2, 3-4 times as much as the wood , and the CO2-assimilation rate of kenaf is 4-5 times more than the trees. Kenaf is usually cultivated on coastal salt lick and inland arid hilly area because of its drought and salt tolerance. Moreover, cultivation of kenaf is helpful to improvement of cultivated fields and not to contend with food for farmland. Thus, production of kenaf is beneficial to full use of marginal land throughout the country and land-use ratio to be improved. Kenaf core contains abundant cellulose and hemicellulose, and it is of great socioeconomic significance for bioconversion of kenaf lignocellulose to fuel ethanol. Pretreatment process of raw materials, costs of cellulase and xylose fermentation are the main bottlenecks to restrict the development of kenaf lignocellulosic ethanol. In view of these factors, key technologies in fuel ethanol production from kenaf lignocellulosic biomass were investigated in this dissertation, based on efficient pretreatment of raw materials and screening of kenaf lignocellulosic degrading fungi and xylose fermentation yeast. The main results are as follows:1. Content of the lignocellulosic components of kenaf was determined, which contained 42.31% of cellulose, 22.58% of hemicellulose and 23.79% of lignin in the stalk. Three different pretreatment methods for kenaf core were studied. Hot water (121℃, 60 min), dilute H2SO4 (3% v/v, 121℃, 60 min) and aqueous sodium hydroxide (1.5 % w/v, 121℃, 60 min) were employed in this study to determine how each method affected the digestibility of kenaf core during enzymatic hydrolysis. The average conversion rate of kenaf cellulose to fermentable reducing sugar was 12.23%, 25.62% and 85.34%, respectively. The result showed that alkaline pretreatment of kenaf core was a more suitable method compared to the other two. The application of simultaneous saccharification and fermentation (SSF) with alkali-pretreated kenaf core as substrate was performed and an ethanol yields of 76.71% were achieved after 168 h-SSF, giving ethanol concentration of 26.06 g·L-1.2. Kenaf core was pretreated by the white-rot fungus Pleurotus sajor-caju in solid-state cultivation with kenaf core medium. Delignification and subsequent enzymatic saccharification and fermentation of microbial-pretreatment kenaf core were investigated in order to evaluate effects of microbial pretreatment on bioconversion of kenaf lignocellulose to fuel ethanol. The highest delignification efficiency of 50.20% was obtained after 2535-day cultivation by P. sajor-caju, which degraded lignin of kenaf lignocellulose efficiently and improved subsequent enzymatic hydrolysis (The saccharification efficiency of pretreated kenaf core was 69.3378.64%). However, untreated sample could not be pretreated efficiently with enzyme extraction on kenaf culture by P. sajor-caju. Simultaneous saccharification and fermentation (SSF) with microbial-pretreatment kenaf core as substrate was performed. A highest overall ethanol yields of 68.31% was achieved after 72 h-SSF, giving ethanol concentration of 18.3518.90 g·L-1.3. A strain of fungus, named Trichoderma sp. HC-35, was screened to degrade kenaf lignocellulose efficiently. Then it was mutated by ultraviolet and NTG and DES, and a mutant strain of Trichoderma sp. HC-35 was selected, which is named as MHC-18. Filter paper activity (FPA) and CMCase activity of MHC-18's cellulase production were up to 29 and 260 U·mL-1, respectively, after solid state fermentation in suitable conditions. Compared to initial strain Trichoderma sp. HC-35, the cellulase enzymatic activities of MHC-18 were raised to 2 times. Enzymatic activities of cellulase production of successive five generations were relatively stable and it showed that the mutant strain, MHC-18, had a good genetic stability.4. Solid-state fermentation medium for cellulase production of MHC-18 was initially got by analyzing medium ingredients and fermentation conditions. The humidity of a mixture of kenaf core and wheat bran (7∶3,w/w) was adjusted to 60%75% using mixed solution of (NH4)2SO4 and MgSO4 (25 g·L-1 of (NH4)2SO4 and 0.5 g·L-1 of MgSO4 , pH5.06.0). Proper quantities of conidia of MHC-18 were inoculated in the media and cultivated in the conditions of 2530℃for 5-7 d. Then, the maximum enzymatic activities of cellulase production of Trichoderma sp. MHC-18 were reached, up to 239.67279.31 U·mL-1 of CMCase and 24.3339.29 U·mL-1 of FPA (95% of confidence level). Better hydrolysis catalyzed by MHC-18 cellulase occurred in the conditions of 45℃60℃of reaction temperature and 4.56.0 of pH, and optimum temperature and pH were 50℃and 5.0, respectively. However, less stable cellulase solution could only be kept for 72 h in 4℃. Moreover, the incomplete system of cellulase production of MHC-18 had showed low activity ofβ-glucosidase.5. Solid-state mixed fermentation of A. niger and MHC-18 was investigated, due to higherβ-glucosidase activity of cellulase production from A. niger and less different conditions of enzymatic hydrolysis between cellulase productions from the two strains. MHC-18 was the first to be inoculated in the fermentation media, 24 h earlier than A. niger. Higher activities of cellulase production from A. niger and MHC-18 by solid-state mixed fermentation, including FPA and CMCase andβ-glucosidase, were achieved in the conditions of 28℃and 120 h of total fermentation time.6. Solid-state mixed fermentation medium of MHC-18 and A. niger was optimized for cellulase production using L16(45) orthogonal test. Kenaf ligno- cellulose was used as the primary carbon source in the optimized medium, and the humidity of a mixture of kenaf core and wheat bran (7∶3,w/w) was adjusted to 60%75% using mixed solution of (NH4)2SO4 and MgSO4 and KH2PO4 (25 g·L-1 of (NH4)2SO4 and 3.5 g·L-1 of MgSO4 and 7 g·L-1 of KH2PO4, pH5.06.0). Activities of FPA and CMCase andβ-glucosidase of the cellulase production were up to 35.7246.44 U·mL-1, 320.22384.67 U·mL-1 and 197.44229.86 U·mL-1 (95% of confidence level), respectively, after 5-d mixed fermentation.7. A strain of yeast, named as RX-8, was screened to utilize xylose for ethanol fermentation, and the fermentation was a kind of limited oxygen fermentation. The fermentation medium contained 50 g·L-1 of glucose and 5.0 g·L-1 of peptone and 3.0 g·L-1 of yeast extract, and an overall ethanol yield of 33.37% was achieved after 3-d fermentation in conditions of 28℃, 120 r/min and 10% of inoculation concentration, giving ethanol concentration of 10.4611.70μL·mL-1 (95% of confidence level) in fermentation liquor.8. Fermentation of glucose for ethanol by RX-8 was also investigated, while the fermentation medium contained 120 g·L-1 of glucose and 5 g·L-1 of peptone and 3 g·L-1 of yeast extract. An higher overall ethanol yield of 71.67% was achieved after 3-d fermentation in conditions of 28℃, 120 r/min and 10% of inoculation concentration, giving ethanol concentration of 10.4611.70μL·mL-1 (95% of confidence level ) in fermentation liquor. The results showed good qualities of glucose fermentation and higher glucose tolerance of RX-8. Thus, co-fermentation of glucose and xylose for ethanol by RX-8 was possible and the strain of yeast RX-8 would be potentially applied to lignocellulosic ethanol industry.
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