Enhancement of enzymatic saccharification of cellulose by cellulose dissolution pretreatments

Attempts were made to enhance cellulose saccharification by cellulase using cellulose dissolution as a pretreatment step. Four cellulose dissolution agents, NaOH/Urea solution, N-methylmorpholine-N-oxide (NMMO), ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and 85% phosphoric acid were employed to dissolve cotton cellulose. In comparison with conventional cellulose pretreatment processes, the dissolution pretreatments were operated under a milder condition with temperature <130 °C and ambient pressure. The dissolved cellulose was easily regenerated in water. The regenerated celluloses exhibited a significant improvement (about 2.7- to 4.6-fold enhancement) on saccharification rate during 1st h reaction. After 72 h, the saccharification yield ranged from 87% to 96% for the regenerated celluloses while only around 23% could be achieved for the untreated cellulose. Even with high crystallinity, cellulose regenerated from phosphoric acid dissolution achieved the highest saccharification rates and yield probably due to its highest specific surface area and lowest degree of polymerization (DP).     

Saccharification of native and degraded cotton cellulose and commercial microcrystalline cellulose by Trichoderma viride cellobiohydrolase I

The degree of polymerization of samples of acid degraded cotton cellulose has no appreciable influence on the saccharification by cellobiohydrolase I from Trichoderma viride. The increase in the number of cellulose molecule ends, achieved by a 30-fold decrease in molecular weight, does not produce the effect which could be expected for a pure end-wise mode of action of this exoglucanase. Microcrystalline celluloses saccharified by the same enzyme yield considerably more reducing sugars than cotton cellulose, either with a similar degree of polymerization or one of about 7000. It appears, therefore, that the difference in the susceptibility of the commercial substrates is not a consequence of their low degree of polymerization.     

Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis

The efficient conversion of lignocellulosic materials into fuel ethanol has become a research priority in producing affordable and renewable energy. The pretreatment of lignocelluloses is known to be key to the fast enzymatic hydrolysis of cellulose. Recently, certain ionic liquids (ILs) were found capable of dissolving more than 10wt% cellulose. Preliminary investigations [Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95, 904-910; Liu, L., Chen, H., 2006. Enzymatic hydrolysis of cellulose materials treated with ionic liquid [BMIM]Cl. Chin. Sci. Bull. 51, 2432-2436; Dadi, A.P., Schall, C.A., Varanasi, S., 2007. Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment. Appl. Biochem. Biotechnol. 137-140, 407-421] suggest that celluloses regenerated from IL solutions are subject to faster saccharification than untreated substrates. These encouraging results offer the possibility of using ILs as alternative and non-volatile solvents for cellulose pretreatment. However, these studies are limited to two chloride-based ILs: (a) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), which is a corrosive, toxic and extremely hygroscopic solid (m.p. approximately 70 degrees C), and (b) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), which is viscous and has a reactive side-chain. Therefore, more in-depth research involving other ILs is much needed to explore this promising pretreatment route. For this reason, we studied a number of chloride- and acetate-based ILs for cellulose regeneration, including several ILs newly developed in our laboratory. This will enable us to select inexpensive, efficient and environmentally benign solvents for processing cellulosic biomass. Our data confirm that all regenerated celluloses are less crystalline (58-75% lower) and more accessible to cellulase (>2 times) than untreated substrates. As a result, regenerated Avicel((R)) cellulose, filter paper and cotton were hydrolyzed 2-10 times faster than the respective untreated celluloses. A complete hydrolysis of Avicel((R)) cellulose could be achieved in 6h given the Trichoderma reesei cellulase/substrate ratio (w/w) of 3:20 at 50 degrees C. In addition, we observed that cellulase is more thermally stable (up to 60 degrees C) in the presence of regenerated cellulose. Furthermore, our systematic studies suggest that the presence of various ILs during the hydrolysis induced different degrees of cellulase inactivation. Therefore, a thorough removal of IL residues after cellulose regeneration is highly recommended, and a systematic investigation on this subject is much needed.     

Preparation of cellouronic acids and partially acetylated cellouronic acids by TEMPO/NaClO oxidation of water-soluble cellulose acetate

Water-soluble cellulose acetates with a degree of substitution (DS) of 0.5, prepared by partial deacetylation of cellulose acetate of DS=2.5, were oxidized with catalytic amount of 2,2,6,6,-tetramethyl-1-piperidinyloxy radical (TEMPO), sodium hypochlorite, and sodium bromide to provide useful cellouronic acids. The oxidation was conducted at a constant pH of 10 and at 2 degrees C to avoid the occurrence of side products. Whereas only the primary hydroxyl groups of cellulose acetate were oxidized, a variable degree of oxidation (DO) resulted in a range of 0.33 to 1.0, depending on the concentration in sodium hypochlorite. Thus, polyglucuronic acid as well as partially acetylated cellouronic acid, having a range of DO were obtained.     

Acetone-soluble cellulose acetates prepared by one-step homogeneous acetylation of cornhusk cellulose in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl)

Cellulose samples extracted from cornhusk have been successfully acetylated in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl). Without using any catalyst, cornhusk cellulose acetates (CCAs) with the degree of substitution (DS) in a range from 2.16 to 2.63 were prepared in one-step. Under the homogeneous state, the DS value of CCAs was easily controlled by the acetylation time. The obtained CCAs were characterized by means of FT-IR, ¹³C NMR, DSC, TGA, and a mechanical test. The NMR results showed that the distribution of the acetyl moiety among the three OH groups of the anhydroglucose unit shows a preference at the C₆ position. The CCAs exhibited good solubility in some organic solvents, such as acetone and DMSO. The cast CCA films from their acetone solutions had good mechanical properties. At the end of each acetylation of cornhusk cellulose, the ionic liquid AmimCl could be effectively recovered. Therefore, this study presents a promising approach and “green process” to make use of crop by-products.     

Cellulose acetates from linters and sisal: correlation between synthesis conditions in DMAc/LiCl and product properties

We report the acetylation of celluloses from sisal (untreated and alkali treated) and cotton linters (alkali treated), under homogeneous solution conditions, using DMAc/LiCl as solvent system. Our target was to evaluate the effects of cellulose dissolution and reactions conditions on the product properties. The products were characterized in terms of degree of substitution (DS) by 1H NMR, and molar weight distribution (MWD) by size exclusion chromatography. Changes in the DS of the products were correlated with reaction conditions and solution properties. It was found that the dissolution of celluloses and degree of substitution of cellulose derivatives depends on a fine adjustment of the dissolution/derivatization conditions, as well as on the origin (sisal or linters) of celluloses.     

Enhanced enzymatic hydrolysis of sugarcane bagasse by N-methylmorpholine-N-oxide pretreatment

The cellulose dissolution solvent used in Lyocell process for cellulose fiber preparation, N-methylmorpholine-N-oxide (NMMO) monohydrate, was demonstrated to be an effective agent for sugarcane bagasse pretreatment. Bagasse of 20wt% was readily dissolved in NMMO monohydrate at 130 degrees C within 1h. After dissolution, bagasse could be regenerated by rapid precipitation with water as a porous and amorphous mixture of its original components. The regenerated bagasse exhibited a significant enhancement on enzymatic hydrolysis kinetic. Not only the reducing sugars releasing rate but also hydrolysis yield was enhanced at least twofold as compared with that of untreated bagasse. The cellulose fraction of regenerated bagasse was nearly hydrolyzed to glucose after 72h hydrolysis with Cellulase AP3. The recycled NMMO demonstrated the same performance as the fresh one on bagasse pretreatment for hydrolysis enhancement. The regenerated bagasse was directly used in simultaneous saccharification and fermentation (SSF) for ethanol production by Zymomonas mobilis. No negative effect on ethanol fermentation was observed and ethanol yield approximately 0.15 g ethanol/g baggasse was achieved.     

Structural comparison and enhanced enzymatic hydrolysis of the cellulosic preparation from Populus tomentosa Carr., by different cellulose-soluble solvent systems

This study aims to establish an efficient pretreatment process using cellulose-dissolution solvents to enhance the enzymatic saccharification. LiOH/urea, LiCl/DMAc, concentrated phosphoric acid, ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and N-methyl-morpholine-N-oxide (NMMO) were selected as the cellulose dissolution agents. Except the cellulosic sample regenerated from LiCl/DMAc system, all the other treated samples exhibited lower cellulose crystallinity and degree of polymerization (DP), and consequently, exhibited a significant enhancement on enzymatic hydrolysis kinetic. Ionic liquid pretreatment offered unique advantages in the hydrolysis rate in the first 10 h, probably due to the extensively structural transformation of cellulose from the crystalline to the amorphous region. Meanwhile, the regenerated cellulose from concentrated phosphoric acid almost completely consisted of cellulose II, and achieved the highest saccharification yield.     

Beta-glucosidase of Trichoderma: its biosynthesis and role in saccharification of cellulose.

The extracellular beta-glucosidase of Trichoderma viride generally is present in low levels when the organism is cultured on cellulose because it is inactivated under the acid conditions which develop in the medium while the other enzymes of the cellulase complex are more stable. With the appropriate pH control, inactivation of beta-glucosidase is prevented and the activity of this enzyme increases during growth. In the saccharification of crystalline cellulose, or of cellulose at low concentrations, much of the glucose produced is the result of the cleavage of cellobiose by beta-glucosidase. However when high concentrations (10%) of pretreated cellulose are saccharified, significant quantities of glucose are produced by action of enzymes other than beta-glucosidase.     

Beta-glucosidase of Trichoderma: its biosynthesis and role in saccharification of cellulose.

The extracellular beta-glucosidase of Trichoderma viride generally is present in low levels when the organism is cultured on cellulose because it is inactivated under the acid conditions which develop in the medium while the other enzymes of the cellulase complex are more stable. With the appropriate pH control, inactivation of beta-glucosidase is prevented and the activity of this enzyme increases during growth. In the saccharification of crystalline cellulose, or of cellulose at low concentrations, much of the glucose produced is the result of the cleavage of cellobiose by beta-glucosidase. However when high concentrations (10%) of pretreated cellulose are saccharified, significant quantities of glucose are produced by action of enzymes other than beta-glucosidase.     

Hydration and saccharification of cellulose Iβ, II and IIII at increasing dry solids loadings

Crystalline cellulose Iβ (Avicel) was chemically transformed into cellulose II and III_I producing allomorphs with similar crystallinity indices (ATR-IR and XRD derived). Saccharifications by commercial cellulases at arrayed solids loadings showed cellulose III_I was more readily hydrolysable and less susceptible to increased dry solids levels than cellulose Iβ and II. Analysis by dynamic vapor sorption revealed cellulose II has a distinctively higher absorptive capacity than cellulose I and III_I. When equally hydrated (g water/g cellulose), low-field nuclear magnetic resonance (LF-NMR) relaxometry showed that cellulose II, on average, most constrained water while cellulase III_I left the most free water. LF-NMR spin–spin relaxation time distribution profiles representing distinct water pools suggest cellulose III_I had the most restricted pool and changes in water distribution during enzymatic saccharification were most dramatic with respect to cellulose III_I compared to celluloses Iβ and II.     

Factors influencing the regeneration of cellulose I from phosphoric acid

The regeneration of cellulose I from phosphoric acid solution was studied, with emphasis both on the conditions required for the regeneration of cellulose I and characterization of the resulting cellulose by X-ray diffraction. Raman spectroscopy and SS¹³C n.m.r. As the conditions of regeneration were varied with respect to temperature and time a variety of polymorphs were produced. As previously reported, the cellulose I polymorph dominated at high temperature and long regeneration time. The question of the authenticity of the regenerated cellulose I was addressed, with several tests confirming that it was not an insoluble residue. Further analysis of the various regenerated celluloses revealed that they all had a small cellulose I component that could be isolated by acid hydrolysis. It is suggested that during regeneration, nuclei of different cellulose polymorphs are formed simultaneously, the proportion of each dependent upon the relative rates of nucleation. Under degradative regeneration conditions the polymorphs more susceptible to hydrolysis are attacked preferentially, leaving behind resistant cellulose I     

TEMPO-mediated oxidation of native cellulose: Microscopic analysis of fibrous fractions in the oxidized products

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous suspensions of cotton linters, ramie and spruce holocellulose at pH 10.5, and water-insoluble fractions of the TEMPO-oxidized celluloses collected by filtration with water were analyzed by optical and transmission electron microscopy and others. The results showed that both fibrous forms and microfibrillar nature of the original native celluloses were maintained after the TEMPO-mediated oxidation, even though carboxylate and aldehyde groups of 0.67–1.16 and 0.09–0.21 mmol/g, respectively, were introduced into the water-insoluble fractions. Neither crystallinity nor crystal size of cellulose I of the original native celluloses was changed under the conditions adopted in this study. Carboxylate groups in the TEMPO-oxidized ramie were mapped by labeling with lead ions as their counter ions. The transmission electron micrographs indicated that some heterogeneous distribution of carboxylate groups along each cellulose microfibril or each bundle of cellulose microfibrils seemed to be present in the TEMPO-oxidized celluloses.     

Efficient approach to design stable water-dispersible nanoparticles of hydrophobic cellulose esters

Commercially prepared cellulose acetate, cellulose acetate propionate, -butyrate, and -phthalate as well as cellulose acetates prepared in the laboratory scale with varying degree of substitution (DS) self-assemble into regular nanoparticles, ranging in size from 86 to 368 nm, by using two different techniques of nanoprecipitation. Dialysis of polymers dissolved in N,N-dimethylacetamide results in the formation of regular nanospheres whereas the preparation in acetone by successive adding of water leads to bean-shaped particles in the nanoscale. One criterion for nanoprecipitation is the existence of dilute polymer solutions. Furthermore, the formation of nanoparticles strongly depends on DS and distribution of the substituents. Concerning this issue, quantitative (13)C NMR spectroscopy was applied for detailed structure characterization of selected cellulose acetates. The stability of the nanoparticle suspensions in the physiological pH range was observed by zeta potential measurements.     

Alternative pathway for glucose production from cellulose using Trichoderma reesei QM9414 cellulase

Summary Evidence is presented which supports the view that two routes exist for the formation of glucose when cellulosic material is saccharified using T. reesei enzyme preparations. The first is via cellobiose and for the second, glucose appears to be formed by a route not involving cellobiose. The second route becomes more apparent when dealing with less crystalline cellulose. This should be considered when constructing strains to produce enzyme preparations for saccharification of less crystalline cellulose.     

Synthesis and characterization of highly substituted deoxyfluorocellulose acetate.

Deoxyfluorocellulose acetates were prepared from cellulose acetate (CA, degree of substitution by acetyl groups: 2.2 and 1.7) by using diethylaminosulfur trifluoride (DAST) in 1,4-dioxane or diglyme. The maximum degree of substitution of fluorine of the products was approximately 0.60, and depolymerization was not significant during fluorination. The replacement of hydroxyl groups by fluorine atoms occurred exclusively at C-6, as confirmed by carbon-13 NMR spectroscopy. In the presence of pyridine, an N-pyridinium derivative of CA was obtained instead of a deoxyfluoro derivative of cellulose.     

木薯纤维素乙醇发酵的纤维素酶成本评价

木薯中的纤维素成分约占木薯干重的10％(W/W).文中以木薯燃料乙醇生产的木薯纤维素酒渣为原料,从纤维素酶成本角度评估了三种利用木薯纤维素组分发酵生产乙醇的方法,包括木薯纤维素酒渣的直接糖化和乙醇发酵、木薯纤维素酒渣预处理后的糖化与乙醇发酵、木薯乙醇发酵中同步淀粉与纤维素糖化以及乙醇发酵.结果表明,前两种方法的纤维素利用效率不高,酶成本分别达到13602、11659元/吨乙醇.第三种方法,即在木薯乙醇发酵过程同时加入糖化酶和纤维素酶,进行同步淀粉与纤维素糖化,进而进行乙醇发酵,木薯纤维素乙醇的收益最高.发酵结束时的乙醇浓度从101.5g/L提高到107.0g/L,纤维素酶成本为3 589元/吨乙醇.此方法利用木薯纤维素与木薯淀粉同时进行,不会带来额外的设备及操作投入,酶成本低于产品乙醇价格,可实现盈利,因此第三种方法为木薯纤维用于乙醇发酵的最适方法,本研究结果将为木薯乙醇产业深度利用木薯纤维提供依据.     

Homogeneous quaternization of cellulose in NaOH/urea aqueous solutions as gene carriers

Quaternized celluloses (QCs) were homogeneously synthesized by reacting cellulose with 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) in NaOH/urea aqueous solutions. The structure and solution properties of the QCs were characterized by using elemental analysis, FTIR, (13)C NMR, SEC-LLS, viscometer, and zeta-potential measurement. The results revealed that water-soluble QCs, with a degree of substitution (DS) value of 0.20-0.63, could be obtained by adjusting the molar ratio of CHPTAC to anhydroglucose unit (AGU) of cellulose and the reaction time. The QC solutions in water displayed a typical polyelectrolyte behavior, and the intrinsic viscosity ([eta]) value determined from the Fuoss-Strauss method increased with increasing DS value. Moreover, two QC samples (DS = 0.46 and 0.63) were selected and studied as gene carriers. The results of gel retardation assay suggested that QCs could condense DNA efficiently. QCs displayed relatively lower cytotoxicity as compared with PEI, and QC/DNA complexes exhibited effective transfection compared to the naked DNA in 293T cells. The quaternized cellulose derivatives prepared in NaOH/urea aqueous solutions could be considered as promising nonviral gene carriers.     

Far ultraviolet optical activity of saccharide derivatives. I. Xylan and cellulose acetates

Optial rotatory dispersion (ORD) and Circular dichroism (CD) spectra of several β-1,4 xylan and cellulose acetates and some of their oligomers were investigated. The CD spectra proved considerably superior in terms of information content and interpretability. Comparison of the xylan and cellulose acetate series which also included partially substited cellulose acetates showed that the sign of the CD of the C(6) acetyl was negative. Likewise, the combined contribution of C(1) and C(4) to their equatorially substituted acetyls was positive (with C(1) definitely Positive) and the combined contribution of C(2)_eq and C(3)_eq negative. The solution conformations of both polysaccharide acetates appeared to be random. The CD of White birch xylan acetate which is substituted on the average at every tenth residue with a 4-O-methyl glucuronic acid substituent, was sensitive to it. The xylan oligomer series also illustrated that CD may be used to determine the degree of polymerization of these oligomers up to about 20.