Research On Pretreatment, Component Fractionation And Enzymatic Hydrolysis Of Lignocellulosic Materials

In this study, the pretreatment process for second-generation bioethanol production from lignocellulosic materials was mainly discussed. The research focused on the optimal conditions of steam explosion pretreatment, main components and high-value utilization of lignocellulosic materials, mechanism and effect of cellulose-dissolving pretreatment, and recycle and fractionation of crude lignin for further application. Lespedeza crytobotrya, as an excellent source for bioethanol production, was employed for bioconversion to produce sugars. As an environmentally friendly pretreatment method, the efficiency of steam explosion, mainly steam pressure and incubation time, was successfully reflected by the physicochemical characteristics, microscopic structure and following enzymatic hydrolysis. A new fractionation process was proposed, based on the synergistic effect of steam-explosion pretreatment and alkaline solution post-treatment. The structural characteristics of the main fractions were completely investigated, aiming to obtain some evidence for the further high-value application. The structure-activity relationship among cellulose solvent, crystal structure and enzymatic conversion was established. Besides, organic solvents were used to fractionate the crude Kraft-AQ lignin. We expected to get more results and data for the utilization of lignocellulosic materials, and provide some theoretic basis for its production in industrial scale. The results of this study were summarized as follows:1. The main components of Lespedeza crytobotrya stalks are cellulose 44.6%, hemicelluloses 29.3% and lignin 17.0%. Steam explosion significantly decreased the size of the materials, loosed the compact structure of lignocellulosic material, and degraded the components in the cell wall. Under the given conditions (1.5-2.5 MPa steam pressure, 2～10 min incubation time), the content of cellulose kept the same level (43.6%-47.1%) regardless of the severity of steam explosion, and the relative crystallinity of the pretreated samples were slightly increased from 10.55% to 16.30%, probably due to the removal of hemicelluloses, degradation of the amorphous cellulose and rearrangement of amorphous and para-crystalline cellulose to crystalline form. Hemicelluloses were significantly degraded during the pretreatment process and reached lowest content of 3.7%; however, the content of lignin was correspondingly increased to 17.7%-23.3%.2. Steam explosion is an efficient pretreatment method, which significantly increased the accessibility of cellulose and further increased the enzymatic hydrolysis efficiency. Based on the optimal enzymatic condition derived from the single-factor and orthogonal experiment, the average enzymatic conversion rates of the steam-pretreated sample at 2.25 MPa were 78%,93% and 97%, respectively, corresponding to the commercial cellulase products cellulaseⅠ,ⅡandⅢ,2.8 times higher than the raw materials. For the pretreated samples incubated for 4 min, elevating steam pressure obviously increased the enzymatic hydrolysis. At 1.5 MPa, the enzymatic conversion was slightly increased by about 20%. In comparison, reducing sugars analysis demonstrated a factor of two improvements in the bioconversion rate after steam exploded at 1.75 MPa for 4 min.3. A two-step process based on steam explosion pretreatment followed by alkaline ethanol solution post-treatment was used to fractionate Lespedeza cyrtobotrya stalks. The residues were mainl consisted of cellulosic components and the highest content of glucose was up to 94.3%. The significant effect of defibrillation of microfibers and isolation of non-cellulosic components led to the increasing reactive area. The degree of polymerization was obviously decreased as the steam pressure was higher than 2.0 MPa and incubation time was longer than 4 min, and reached the lowest level of 207. Both were benefit for the following enzymatic bioconversion process. Hemicelluloses, as the most thermally instable component, were extensively degraded during steam explosion process, evidenced by the average weight molecular weights decreased from 47960,63300 and 102400 g/mol to 6380,8100 and 16200 g/mol, respectively. The comprehensive debranching reaction cleaved the linkages between the mainchain of xylan and arabinose, lead to the obvious decrease of Ara/Xyl ratio from 0.08 to 0. At 2.5 MPa for 4 min,86.6% glucose was determined in the isolated hemicellulosic fraction, indicating the partial degradation of cellulose and co-precipitation with non-cellulosic polysaccharides. Due to the remarkable selectivity with respect to lignin, up to 73.4% and 93.7% lignin component was extracted from 1M NaOH solution and 60% ethanol solution containing 1% NaOH post-treatment, respectively. The significant cleave of the linkages between carbohydrate and lignin induced the extremely lower content of sugar (0.80%) in the lignin fraction. Based on the quantitative analysis of 1H NMR spectrum, the content of/β-O-4 ether bonds were decreased from 11% to 7% as prolonging the incubation time from 2 min to 10 min at 2.25 MPa. The depolymerization reactions were accompanied with the comprehensive repolymerization reactions during the steam explosion process, leading to the formation of new carbon-carbon bonds, which were chemically and thermally stable. Due to the steric hindrance of methoxyl group at C5 position in syringyl units, more guaiacyl units were participated in the condensation reactions, and S/G ratio was correspondingly increased. The results indicated the existence of the restricting interrelation between efficient fractionation of cell wall components and maintainance of structural characteristics of hemicelluloses and lignin. Taking Lespedeza stalks as the starting material, steam explosion pretreatment at middle or lower severity and alkaline solution post-treatment was proposed as a new environmentally friendly fractionation process, which is benefit for the further high-value utilization of the main components.4. Cellulose dissolving and following enzymatic hydrolysis aims to establish an efficient pretreatment process using cellulose-dissolution solvents to enhance the enzymatic saccharifi cation. LiOH/Urea, concentrated phosphoric acid, N-methyl-morpholine-N-oxide (NMMO) and ionic liquid (1-butyl-3-methylimidazolium chloride [BMIM]C1) were selected as the efficient agents, and the regenerated samples exhibited the significant crystal transformation from cellulose I to cellulose II or amorphous region. Consequently, the relative crystallinity and degree of polymerization (DP) were reduced to the lowest level of 8% and 60, respectively. Comparing to the untreated sample (33%), obvious enhancement on the bioconversion efficiency from dissolving process was observed not only on glucose yield, also hydrolysis rate. After 72 h enzymatic hydrolysis, the conversion of cellulose reached to 81%,92%,83% and 80%, respectively. Meanwhile, the recovery of xylose was also increased from 18.5% to 39.4%-49.6%.5. Kraft-AQ pulping lignin was sequentially fractionated by organic solvent extractions (hexane, diethylether, methylene chloride, methanol and dioxane) and the molecular properties of each fraction were characterized in detail. The average molecular weight and polydispersity of each lignin fraction increased with its hydrogen-bonding capacity (Hildebrand solubility parameter), from 493 g/mol to 2468 g/mol, and to 13651 g/mol. Based on 1H and 13C NMR spectra,β-0-4 ether bonds, the main subunit in lignin macromolecule, were relatively packed and protected, and rich in the fractions with higher molecular weight.