Transition of cellulose crystalline structure and surface morphology of biomass as a function of ionic liquid pretreatment and its relation to enzymatic hydrolysis

Cellulose is inherently resistant to breakdown, and the native crystalline structure (cellulose I) of cellulose is considered to be one of the major factors limiting its potential in terms of cost-competitive lignocellulosic biofuel production. Here we report the impact of ionic liquid pretreatment on the cellulose crystalline structure in different feedstocks, including microcrystalline cellulose (Avicel), switchgrass (Panicum virgatum), pine ( Pinus radiata ), and eucalyptus ( Eucalyptus globulus ), and its influence on cellulose hydrolysis kinetics of the resultant biomass. These feedstocks were pretreated using 1-ethyl-3-methyl imidazolium acetate ([C2mim][OAc]) at 120 and 160 °C for 1, 3, 6, and 12 h. The influence of the pretreatment conditions on the cellulose crystalline structure was analyzed by X-ray diffraction (XRD). On a larger length scale, the impact of ionic liquid pretreatment on the surface roughness of the biomass was determined by small-angle neutron scattering (SANS). Pretreatment resulted in a loss of native cellulose crystalline structure. However, the transformation processes were distinctly different for Avicel and for the biomass samples. For Avicel, a transformation to cellulose II occurred for all processing conditions. For the biomass samples, the data suggest that pretreatment for most conditions resulted in an expanded cellulose I lattice. For switchgrass, first evidence of cellulose II only occurred after 12 h of pretreatment at 120 °C. For eucalyptus, first evidence of cellulose II required more intense pretreatment (3 h at 160 °C). For pine, no clear evidence of cellulose II content was detected for the most intense pretreatment conditions of this study (12 h at 160 °C). Interestingly, the rate of enzymatic hydrolysis of Avicel was slightly lower for pretreatment at 160 °C compared with pretreatment at 120 °C. For the biomass samples, the hydrolysis rate was much greater for pretreatment at 160 °C compared with pretreatment at 120 °C. The result for Avicel can be explained by more complete conversion to cellulose II upon precipitation after pretreatment at 160 °C. By comparison, the result for the biomass samples suggests that another factor, likely lignin-carbohydrate complexes, also impacts the rate of cellulose hydrolysis in addition to cellulose crystallinity.     

Sulfonated hierarchical H-USY zeolite for efficient hydrolysis of hemicellulose/cellulose

Sulfonated hierarchical H-USY zeolite was prepared and characterized by X-ray diffraction, N₂ physisorption, Fourier transform infrared spectroscopy, inductively coupled plasma atomic emission spectroscopy, temperature-programmed desorption of ammonia, and acid–base titration. It was proved that sulfonic group was successfully anchored onto the hierarchical H-USY zeolite. The acidity of the hierarchical H-USY was remarkably improved. Sulfonated hierarchical H-USY zeolite was efficient for the hydrolysis of hemicellulose and cellulose. The yield of TRS for hydrolysis of hemicellulose reached 78.0% at 140 °C for 9 h. For hydrolysis of α-cellulose, 60.8% conversion with 22.4% yield of glucose was obtained. Even for microcrystalline cellulose, 43.7% conversion with 15.1% yield of glucose can be obtained. These results are much higher than those obtained over hierarchical H-USY zeolite, indicating that both the acidity and the pore structure determine the activity of zeolite as catalyst in the hydrolysis of biomass.     

Statistical correlation of spectroscopic analysis and enzymatic hydrolysis of poplar samples

Spectroscopic characterization of poplar wood samples with different crystallinity indices, lignin contents, and acetyl contents was performed to determine changes in the biomass spectra and the effects of these changes on the hydrolysis yield. The spectroscopic methods used were X-ray diffraction for determining cellulose crystallinity (CrI), diffuse reflectance infrared (DRIFT) for changes in C-C and C-O bonds, and fluorescence to determine lignin content. Raman spectroscopy was also used to determine its effectiveness in the determination of crystallinity and C-C and C-O bond changes in the biomass as a complement to better-known methods. Changes in spectral characteristics and crystallinity were statistically correlated with enzymatic hydrolysis results to identify and better understand the fundamental features of biomass that influence enzymatic conversion to monomeric sugars. In addition, the different spectroscopic methods were evaluated separately to determine the minimum amount of spectroscopic data needed to obtain accurate predictions. The principal component regression (PCR) model with only the DRIFT data gives the best correlation and prediction for both initial rate of hydrolysis and also the 72-h hydrolysis yield. The factor that most affects both the initial rate and the 72-h conversion is the O-H bond content of the sample, which directly relates to the breakage of structural carbohydrates into smaller molecules.     

Highly hydrophobic biopolymers prepared by the surface pentafluorobenzoylation of cellulose substrates

New highly hydrophobic/lipophobic biopolymers were prepared by the controlled heterogeneous pentafluorobenzoylation of cellulose substrates, i.e., plant and bacterial cellulose fibers. The characterization of the modified fibers was performed by elemental analysis, FTIR spectroscopy, X-ray diffraction, thermogravimetry, and surface analysis (XPS, ToF-SIMS, and contact angle measurements). The degree of substitution of the ensuing pentafluorobenzoylated fibers ranged from 0.014 to 0.39. The hydrolytic stability of these perfluorinated cellulose derivatives was also evaluated and showed that they were quite water stable, although of course the fluorinated moieties could readily be removed by hydrolysis in an aqueous alkaline medium.     

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     

Organosolvent pretreatment and enzymatic hydrolysis of rice straw for the production of bioethanol

The present study investigates the operational conditions for organosolvent pretreatment and hydrolysis of rice straw. Among the different organic acids and organic solvents tested, acetone was found to be most effective based on the fermentable sugar yield. Optimization of process parameters for acetone pretreatment were carried out. The structural changes before and after pretreatment were investigated by scanning electron microscopy, X-ray diffraction and Fourier transform infrared (FTIR) analysis. The X-ray diffraction profile showed that the degree of crystallinity was higher for acetone pretreated biomass than that of the native. FTIR spectrum also exhibited significant difference between the native and pretreated samples. Under optimum pretreatment conditions 0.458 g of reducing sugar was produced per gram of pretreated biomass with a fermentation efficiency of 39%. Optimization of process parameters for hydrolysis such as biomass loading, enzyme loading, surfactant concentration and incubation time was done using Box–Benhken design. The results indicate that acetone pretreated rice straw can be used as a good feed stock for bioethanol production.     

The study of factors affecting the enzymatic hydrolysis of cellulose after ionic liquid pretreatment

Cellulose resource has got much attention as a promising replacement of fossil fuel. The hydrolysis of cellulose is the key step to chemical product and liquid transportation fuel. In this paper a serials of chloride, acetate, and formate based ionic liquids were used as solvents to dissolve cellulose. The cellulose regenerated from ILs was characterized by FTIR and X-ray powder diffraction. From the characterization and analysis, it was found that the original close and compact structure has changed a lot. After enzymatic hydrolysis, different kinds of ionic liquids (ILs) have different yields of the reducing sugar (TRS). They are 100%, 90.72%, and 88.92% from 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), 1-butyl-3-methylimidazolium formate ([BMIM][HCOO]) respectively after enzymatic hydrolysis at 50 °C for 5 h. The results indicated that the yields and the hydrolysis rates were improved apparently after ILs pretreatment comparing with the untreated substrates.     

Effect of biomass concentration and inoculum source on the rate of anaerobic cellulose solubilization

This study determines cellulose solubilization kinetics from controlled batch digestions and shows the effect of inoculum biomass concentrations. Separate measurements and analyzes were performed for sessile biomass (biofilms) and planktonic biomass (free suspensions). Experiments were conducted using either leachate enriched on cellulose or rumen fluid as inoculum to assess if the effect of biomass concentration was consistent for microbial populations from different source environments. All batch digestions were fitted to a first-order kinetic model (R² ranging from 0.94 to 0.99). Regression analysis used to compare the first-order hydrolysis rate showed that the first-order hydrolysis rate was most strongly correlated with the concentration of sessile biomass rather than with the concentration of total or planktonic biomass. The correlation between solubilization rate and sessile biomass was statistically the same for the rumen and leachate inoculated reactors indicating that at low concentration ratios of inoculum to cellulose, the rate of cellulose solubilization is dependant primarily on sessile biomass concentration rather than the species profile of the cellulolytic community.     

X-Ray crystal structure of the multidomain endoglucanase Cel9G from Clostridium cellulolyticum complexed with natural and synthetic cello-oligosaccharides

Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the beta-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-A resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family III(c) cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 A, respectively.     

Effect of phosphoric acid pretreatment on enzymatic hydrolysis of microcrystalline cellulose

Microcrystalline cellulose (MCC) was pretreated with phosphoric acid at 323 K for 10 h. X-ray diffraction (XRD) and Atomic Force Microscope (AFM) analyses revealed that the fiber surface morphology of pretreated MCC (P-MCC) were uneven and rough with the crystalline diffraction peaks of P-MCC decreased to a distinct range. The X-ray Photoelectron Spectroscopy (XPS) analysis showed that the uneven and rough surface of P-MCC could enhance the adsorption of cellulose to the molecular surface of cellulose, which is one of the key factors affecting enzymatic hydrolysis of cellulose. A reversible first order kinetics was employed to describe the adsorption kinetics of cellulase to MCC and P-MCC, and the adsorption rate constants of MCC and P-MCC were found to be 0.016, 0.024, 0.041, and 0.095, 0.149, 0.218 min^− 1, respectively at 278 K, 293 K and 308 K. The activation energies of MCC and P-MCC hydrolysis reactions were found to be 22.257 and 19.721 kJ mol^− 1. The major hydrolysis products of MCC and P-MCC were cellobiose and glucose. Hydrolysis of MCC for 120 h resulted in yields of glucose (7.21%), cellobiose (13.16%) and total sugars (20.37%). However, after the pretreatment with phosphoric acid, the corresponding sugar yields resulted from enzymatic hydrolysis of P-MCC were increased to 24.10%, 41.42%, and 65.52%; respectively, which were 3.34, 3.15, and 3.22 times of the sugars yields from enzymatic hydrolysis of MCC.     

Elucidation of the effect of ionic liquid pretreatment on rice husk via structural analyses

ABSTRACT: BACKGROUND: In the present study, three ionic liquids, namely 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), and 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIM]DEP), were used to partially dissolve rice husk, after which the cellulose were regenerated by the addition of water. The aim of the investigation is to examine the implications of the ionic liquid pretreatments on rice husk composition and structure. RESULTS: From the attenuated total reflectance Fourier transform-infrared (ATR FT-IR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM) results, the regenerated cellulose were more amorphous, less crystalline, and possessed higher structural disruption compared with untreated rice husk. The major component of regenerated cellulose from [BMIM]Cl and [EMIM]DEP pretreatments was cellulose-rich material, while cellulose regenerated from [EMIM]OAc was a matrix of cellulose and lignin. Cellulose regenerated from ionic pretreatments could be saccharified via enzymatic hydrolysis, and resulted in relatively high reducing sugars yields, whereas enzymatic hydrolysis of untreated rice husk did not yield reducing sugars. Rice husk residues generated from the ionic liquid pretreatments had similar chemical composition and amorphousity to that of untreated rice husk, but with varying extent of surface disruption and swelling. CONCLUSIONS: The structural architecture of the regenerated cellulose and rice husk residues showed that they could be used for subsequent fermentation or derivation of cellulosic compounds. Therefore, ionic liquid pretreatment is an alternative in the pretreatment of lignocellulosic biomass in addition to the conventional chemical pretreatments.     

Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass

The switchgrass (SG) samples pretreated by cellulose solvent‐ and organic solvent‐based lignocellulose fractionation were characterized by enzymatic hydrolysis, substrate accessibility assay, scanning electron microscopy, X‐ray diffraction (XRD), cross polarization/magic angle spinning (CP/MAS) ¹³C nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). Glucan digestibility of the pretreated SG was 89% at hour 36 at one filter paper unit of cellulase per gram of glucan. Crystallinity index (CrI) of pure cellulosic materials and SG before and after cellulose solvent‐based pretreatment were determined by XRD and NMR. CrI values varied greatly depending on measurement techniques, calculation approaches, and sample drying conditions, suggesting that the effects of CrI data obtained from dried samples on enzymatic hydrolysis of hydrated cellulosic materials should be interpreted with caution. Fast hydrolysis rates and high glucan digestibilities for pretreated SG were mainly attributed to a 16.3‐fold increase in cellulose accessibility to cellulase from 0.49 to 8.0 m²/g biomass, because the highly ordered hydrogen‐bonding networks in cellulose fibers of biomass were broken through cellulose dissolution in a cellulose solvent, as evidenced by CP/MAS ¹³C‐NMR and FTIR. Biotechnol. Bioeng. 2011; 108:521–529. © 2010 Wiley Periodicals, Inc.     

Hydrolysis of lignocellulosic materials for ethanol production: a review

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.     

Chlorine-free extraction of cellulose from rice husk and whisker isolation

This work reports the isolation of cellulose whiskers from rice husk (RH) by means of an environmental friendly process for cellulose extraction and bleaching. The multistep process begins with the removal of pectin, cutin, waxes and other extractives from rice husk, then an alkaline treatment for the removal of hemicelluloses and lignin, and a two-step bleaching with hydrogen peroxide/tetra-acetylethylenediamine (TAED), followed by a mixture of acetic and nitric acids, for further delignification of the cellulose pulp. The techniques of infrared absorption spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), modulated differential scanning calorimetry (MDSC) and X-ray diffraction (XRD) showed that the overall process is adequate to obtain cellulose with high purity and crystallinity. This cellulose was submitted to sulfuric acid hydrolysis with the aim to isolate the whiskers. They showed the typical elongated rod-like aspect as revealed by transmission electron microscopy (TEM) and atomic force microscopy (AFM).     

Changes in the structural properties and rate of hydrolysis of cotton fibers during extended enzymatic hydrolysis

An extended enzymatic hydrolysis of cotton fibers by crude cellulase from Trichoderma pseudokoningii S-38 is described with characterization of both the enzyme changes of activities and cellulose structure. The hydrolysis rates declined drastically during the early stage and then slowly and steadily throughout the whole hydrolysis process the same trend could be seen during the following re-hydrolysis process. Morphological and structural changes to the fibers, such as swelling, frequent surface erosion, and variation in the packing and orientation of microfibrils, were investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Observation of X-ray diffraction and IR spectra suggests that the hydrolysis process results in a gradual increase in the relative intensity of the hydrogen bond network, and a gradual decrease in the apparent crystal size of cellulose. The I(alpha) crystal phase was hydrolyzed more easily than was the I(beta) crystal phase. Apart from the inactivation of CBHs activity, changes in the packing and arrangement of microfibrils and the structural heterogeneity of cellulose during hydrolysis could be responsible for the reduction in the rate of reaction, especially in its later stages. The results indicate that the enzymatic hydrolysis of cellulose occurs on the outer layer of the fiber surface and that, following this, the process continues in a sub-layer manner.     

Effects of chemical treatments and heating on the crystallinity of celluloses and their implications for evaluating the effect of crystallinity on cellulose biodegradation

Chemical treatments similar to those routinely used to extract cellulose from plant biomass caused significant increases in the relative crystallinity index (RCI) of Sig-macell 100 (a commercial cellulose of moderate crystallinity), as measured by x-ray powder diffraction in both the reflectance and transmittance modes. In general, the largest increases in RCI were observed following higher (rather than lower) temperature treatments. Substantial increases in crystalliity were also observed upon resuspension in water prior to drying, with higher temperatures again resulting in the greatest increases in RCI. Measurement of the RCIs of wetted Sigmacell 100 samples by acid hydrolysis kinetics revealed that most of the increased crystallinity occurred rapidly upon contact with water. In contrast to Sigmacell 100, a cellulose of higher initial crystallinity (the microcrystalline cellulose Sigmacell 50) showed little change in crystallinity following the above treatments. The results provide a partial explanation for the inconsistent relationships reported between cellulose crystallinity and cellulose biodegradation. (c) 1995 John Wiley & Sons, Inc.     

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

Background

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates.

Results

Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 °C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability, and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl₃-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose.

Conclusions

Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.

     

Preparation of cellulase concoction using differential adsorption phenomenon

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while β-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.     

Hemicelluloses negatively affect lignocellulose crystallinity for high biomass digestibility under NaOH and H2SO4 pretreatments in Miscanthus

ABSTRACT: BACKGROUND: Lignocellulose is the most abundant biomass on earth. However, biomass recalcitrance has become a major factor affecting biofuel production. Although cellulose crystallinity significantly influences biomass saccharification, little is known about the impact of three major wall polymers on cellulose crystallization. In this study, we selected six typical pairs of Miscanthus samples that presented different cell wall compositions, and then compared their cellulose crystallinity and biomass digestibility after various chemical pretreatments. RESULTS: A Miscanthus sample with a high hemicelluloses level was determined to have a relatively low cellulose crystallinity index (CrI) and enhanced biomass digestibility at similar rates after pretreatments of NaOH and H2SO4 with three concentrations. By contrast, a Miscanthus sample with a high cellulose or lignin level showed increased CrI and low biomass saccharification, particularly after H2SO4 pretreatment. Correlation analysis revealed that the cellulose CrI negatively affected biomass digestion. Increased hemicelluloses level by 25% or decreased cellulose and lignin contents by 31% and 37% were also found to result in increased hexose yields by 1.3-times to 2.2-times released from enzymatic hydrolysis after NaOH or H2SO4 pretreatments. The findings indicated that hemicelluloses were the dominant and positive factor, whereas cellulose and lignin had synergistic and negative effects on biomass digestibility. CONCLUSIONS: Using six pairs of Miscanthus samples with different cell wall compositions, hemicelluloses were revealed to be the dominant factor that positively determined biomass digestibility after pretreatments with NaOH or H2SO4 by negatively affecting cellulose crystallinity. The results suggested potential approaches to the genetic modifications of bioenergy crops.