Pretreatment And Saccharification Fermentation Of Oil Palm Empty Fruit Bunch(EFB) For Ethanol Production And Mechanism Of Pretreatment

Most supplies of oil palm are currently produced in South East Asia. Oil palm was introduced to South East Asia in the 19th century. Malaysia and Indonesia are the two largest producers of oil palm, collectively accounting for approximately 85% of the world oil palm production. Lignocellulosic fibers can be extracted from the trunk, frond, fruit mesocarp, and empty fruit bunch (EFB) of an oil palm tree. EFB contains high content of glucan (35-50%) and xylan (10 to 20% ). EFB has been usually burned in incinerators of palm oil mills, causing biomass resources waste and environmental pollution. Lignocellulosic EFB has recently been considered as a potential low-cost material, and an alternative renewable bioresource for the production of bioethanol. Pretreatment is necessary for reducing the hemicellulose and lignin contents in EFB for effective bioethanol fermentation. Several studies have evaluated the effects of some pretreatment processes, such as alkaline pretreatment, aqueous ammonia soaking pretreatment, sequential pretreatment with dilute acid and then alkali, on enzymatic digestibility of EFB. However, it was shown that the pretreatment processes were little effective for improvement of digestibility of EFB, and not resulted in high sugar yield. Based on the above background, different pretreatment processes were explored to obtain an effective method for increasing the enzymatic digestibility of EFB and ethanol production. Moreover, possible mechanism of the ideal pretreatment for improvement of enzymatic hydrolysis of EFB from oil palm was studied, and biorefinery process of EFB was also investigated. The main contents of this thesis are as follows:1. Pretreatment of empty fruit bunch from oil palm for fuel ethanol production and proposed biorefinery processWet-oxidation, oxygen-alkali (OA) and bisulfite pretreatments were selected to treat EFB prior to enzymatic hydrolysis stage, and experimental results showed that the bisulfite pretreatment was practical for EFB pretreatment. Moreover, the optimum pretreatment conditions of the bisulfite pretreatment (180 ℃,30 min,8% NaHSO3, 1% H2SO4) were identified. After undergoing bisulfite pretreatment with 8% NaHSO3 and 1% H2SO4, the glucose yield in subsequent enzyme hydrolysis was 0.318 g/g EFB after 72 h. Then, the bisulfite-pretreated EFB sample under optimum pretreatment condition was chosen as substrate for the quasi-simultaneous saccharification and fermentation (Q-SSF), and found that the final ethanol titers were 52 g/L for the pretreated EFB after 160 h of fermentation time, reaching 95% of the theoretical maximum yield of ethanol. The ethanol productivity from the Q-SSF for pretreated EFB was 0.325 g/L/h until 160 h.A biorefinery process was proposed for production of bioethanol and high-value products from the EFB material. The pretreatment liquor of bisulfite process was used to produce furfural. Moreover, the optimum conditions were identified:180 ℃,25 min,0.75% H2SO4. On the optimum conditions, the furfural yield was 18.8 g/L, and the conversation rate of xylan to furfural was reached up to 82.5%. Then, fraction of waste liquor from bisulfite pretreatment process was studied for its comprehensive utilization. Through different methods to deal with the pretreatment liquor, we found that the macroporous resin DM130 could be used to effectively separate the sugar and lignin sulfonate component from the pretreatment waste liquor.2. Changes in the structure and properties of EFB after bisulfite treatmentThe surface structure and properties of unpretreated and bisulfite-pretreated EFB were analyzed by scanning electron microscopy (SEM), X-ray diffraction, fiber-specific surface area, and Fourier-transform infrared spectrometry (FT-IR). In order to better explore bisulfite pretreatment mechanism, several pretreated smaples from dilute acid process, OA process with alkaline impregnation process and bisulfite process, which have roughly same total removal of hemicellulose and lignin, were selected.The crystallinity of cellulose in the EFB samples changed from 62.3% to 74.4% after bisulfite pretreatment. The HBI of bisulfite-pretreated EFB was reduced from 4.75 to 3.24. SEM observation showed that the EFB external surface was damaged and became rough after bisulfite pretreatment. The volume of pores with diameters of 50 nm or higher increased after bisulfite pretreatment, which implied the increased internal specific area of EFB. The increased pore volume with bigger pore sizes was also beneficial because it improved the penetration of enzymes into the substrate. Bisulfite pretreatment could clearly and effectively increase the specific surface area of the EFB (from 0.99 to 1.18).3. The changes of microstructure of EFB by bisulfite pretreatment and observation of cell wall structure change during enzymatic hydrolysis with cellulase.Optical microscope, scanning electron microscope (SEM), fluorescence microscope (FM), confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) was used for analyzing the microstructure. From the results of the observation, EFB mostly consists of parenchyma cells, scleroid cells, vessels and epidermal cells, etc. The overall structure of EFB was destroyed by bisulfite process. Scleroid cell separated, part of parenchyma cell was also destroyed because of bisulfite process. The fiber was shortened, the width decreased, and the content of fine increased after bisulfite pretreatment.We also observed the change in cell wall structure of bisulfite pretreated EFB during enzymatic hydrolysis. The results showed that most of cell walls began to degrade after 1 h of cellulase hydrolysis. With the extension of time, the degradation degree of cell wall is aggravating, and reached to maximum after 5 h cellulase hydrolysis.4. Changes in structural and properties of hemicellulose and lignin fractions in EFB by bisulfite pretreatmentFractions of hemicellulose and lignin were isolated from the bisulfite-pretreated EFB, and their structural characteristics were investigated by spectroscopic techniques, such as FT-IR, gel-permeation chromatography (GPC),1H-nuclear magnetic resonance (NMR),13C-NMR, and 1H-13C two-dimensional (2D) heteronuclear single-quantum correlation (HSQC) spectroscopy. The results implied that the hemicellulose fractions consisted of the main chain of xylan, which contained hydroxyl and methyl groups in untreated and bisulfite-pretreated EFBs. The esterification of the aromatic and alcohol groups of the propane chain (Ca and Cy) in lignin fractions occurred during delignification using bisulfite process, other reaction included the degradation and dissolution of syringyl, condensed guaiacyl and guaiacyl units, and partial removal of methoxyl groups during bisulfite pretreatment.Lignin physically hinders the accessibility of enzymes to cellulose, and soluble lignin-derived compounds may cause enzyme inhibition. Therefore, milled wood lignin (MWL) was isolated from unpretreated and bisulfite-pretreated EFB, respectively, and the differences in the adsorption of cellulase on the MWLs were investigated.