| Ramie is the traditional fiber crops in China. Its fibers are considered as the longest, strongest, and silkiest in plant fibers and have excellent properties as natural textile material, such as preeminent absorption, quick drying, easy dyeing, shrinkage resistance, good bacteria, mildew, and insect resistance. However, decorticated ramie fibers contain20-35%gum, which mainly consists of pectin and hemicellulose. This gum should be removed as much as possible for most applications. Conventional degumming using NaOH solution consumes large amounts of chemicals and energy, and causes serious environmental pollution. Enzymatic degumming is a gentle reaction, with less damage to fibers and flexible operation, as well as easy processing and quality control, and so on. Hence, it is considered a potential alternative to chemical degumming and has attracted wide attention. The research on efficient degumming enzyme, high-efficient degumming enzyme components and degumming mechanism can promote the understanding and development of the enzymatic degumming. Up to now, the ramie stalk and kenaf stalk have not been used effectively. The stalk contains a lot of cellulose and therefore can be used for the production of bioethanol. The research on the pretreatment of the stalks and fermentation for bioethanol production could provide reference for bioethanol production using this kind of raw materials. Lignin has been always deemed to have a negative impact on the saccharification of lignocellulosic feedstocks by physically barring and unproductively adsorbing hydrolytic enzymes. The lignin sources and structural features of lignin affect the inhibition on the saccharification. The characteristics and compositions of lignin are significantly different in diverse types of biomass. The native difference in lignin is the reason for the differences in their adsorption capacity. However, the mechanism on how it affects the inhibitory hydrolysis was not explained. Based on above, the research contents and the main results of the thesis are as follows: 1. Screening of strains capable of producing effective degumming enzyme, enzyme characterization and optimization of enzyme productionIt was found that the crude enzyme from wild-type Bacillus sp. Y1had a powerful and fast degumming ability. Its polygalacturonate lyase (PGL) activity was the highest at pH9.6-10.0and60℃, and stable at pH7.0-10.5and30-50℃. The PGL have a wide scope of pH and temperature, and high H2O2tolerance. The gum loss and brightness of fibers could be significantly improved when H2O2was added during enzymatic degumming with the PGL, showed a synergistic action between the PGL and H2O2on the degumming and bleaching of ramie fibers. It was very suitable for a joint process of enzymatic degumming and H2O2bleaching.The medium components and fermentation conditions for the pectinase production were optimized by some statistical methods, and the activities of polygalacturonate lyase (PGL) and polygalacturonase (PG) were increased to2.00-and3.44-fold respectively, and the fermentation time shortened12hours (from72hrs to60hrs) after optimization. The final optimal medium components and fermentation conditions were as follows:carbon source,10.5%,(NH4)2SO4,0.37%; MgSO4,0.3%; Tween-80,0.1%(w/v); initial pH,8.2; and inoculum number of1.3mL (with the OD600of the seed medium about5.77) per50mL of fermentation medium on a swing shaker (100rpm) at34℃for60h.2. Degumming mechanism with crude enzyme from Bacillus sp. Y1The mechanism about effective degumming with crude enzyme from Bacillus sp. Yl was studied by SDS-PAGE, MS/MS, and the adding experments of enzymes and purified protein. It was found that the crude enzyme of wild-type Bacillus sp. Y1contains more proteins compared with the control pectinase, and protease component in it was further substantiated to play an important role in the degumming process. There was a synergistic action between protease and pectinase during degumming.Chemical composition and degumming performance of ramie from different growth period were analyzed. Few differences were found in chemical composition and degumming performance between the first ramie and second ramie, and the degumming performance of the third ramie is the worst. The chemical composition analysis showed that the third ramie contains more hemicellulose. It was found from comparative study on the ramie degumming in different time that the degumming process was finished after1h, and the removal of pectin and hemicellulose in the third ramie was obviously lower than that in the first ramie. SEM observation showed that the gum adhesions on the surface of the third ramie was significant more than that on the first ramie, and more difficult to be removed.3. Pretreatment of ramie stalk and kenaf stalk and Q-SSF for bioethanol productionThe effects of different chemical pretreatment on enzymatic digestibility of ramie stalk and kenaf stalk were studied. Ramie and kenaf stalks pretreated with alkali were chosen to produce ethanol using quasi-simultaneous saccharification and fermentation (Q-SSF) process. The results show that for the stalk pretreated with4%NaOH and0.02%anthraquinone-2-sulfonic acid sodium salt (AQSS) as catalyzer at170℃for1h, the ethanol concentration could reach51g/L after fermentation for168h at18%of solid substrate concentration. By fed-batch to20%of solid substrate concentration, the ethanol concentration could reach63g/L, and the cellulose conversion was77%and79%for ramie stalk and kenaf stalk, respectively. For kenaf stalk pretreated with5.2%NaHSO3and0.2%H2SO4at170℃for1h, the ethanol concentration and cellulose conversion could reach to65g/L and72%, respectively.4. Differences in the adsorption of enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanismIt was found that lignin sources affected enzyme adsorption using structural features, such as functional groups and lignin composition. G lignin had a higher adsorption capacity on enzymes than S lignin. The adsorption capacity of cellulase onto lignin decreased in the order:pine lignin (PL)> corn stover lignin (CL)> aspen lignin (AL)> kenaf lignin (KL). The lignin with low S/G ratio has higher adsorption capacity on enzyme. A higher content of phenolic hydroxyl groups and a lower content of carboxylic acid groups resulted in stronger adsorption affinity for CL than for KL and AL. The lower amount of aliphatic hydroxyls that increased hydrophobic interactions could explain the higher adsorption capacity of PL than CL. For different mono-component enzymes, the adsorption ability decreased in the order: cellobiohydrolase (CBH), xylanase> endoglucanase (EG)> β-glucosidase (BG), indicating the important role of carbohydrate-binding module (CBM) in protein adsorption. |
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