Enzymatic Saccharification Yield Research Articles

A lignin-first biorefinery by integrated deep eutectic solvents towards formaldehyde-free plywood adhesive and monomeric sugars

A profitable and sustainable biorefining process will facilitate its scale-up production. In this study, we combined the advantages of both organic acid-based DES and polyol-based DES into a new ternary DES basing on the lignin-first biorefinery to fractionate bamboo, directly obtained a superstrong lignin adhesive and improved enzymatic saccharification. This integrated DES could enhance the enzymatic saccharification yield to 100 % by substantial removals of lignin (76.25 %) and xylan (79.72 %). Remarkably, the obtained lignin was modified and stabilized by polyols during the fractionation, possessing a well-stabilized β-O-4 structure (as high as 42.66/100Ar), which showed excellent adhesion performance of dry and wet bonding strength (1.87/1.62 MPa), outperforming the commercial phenolic resin and urea resin for plywood production. The formation mechanism of the lignin adhesive was comprehensively studied and demonstrated to be the graft reactions induced by the diol hydroxyl tails onto the α position of lignin’s side chain via etherification. This work proved that the combination of organic acid and polyol-based DES is an ideal strategy to generate a formaldehyde-free plywood adhesive and digestible carbohydrates as coproducts.

In situ pretreatment of wood samples with deep eutectic solvents for enhanced lignin removal and enzymatic Saccharification efficiency optimization

Deep eutectic solvents (DESs) are commonly utilized as an effective pretreatment method for biomass materials, particularly for enhancing the value of lignocellulosic biomass. However, to the best of our knowledge, the utilization of DESs for in situ pretreatment of wood samples has been seldom reported. In this study, the ability of five DESs systems to in-situ pretreatment of balsa wood samples was investigated. Among of them, the Choline Chloride-Ethylene Glycol-P-Toluenesulfonic Acid (ChCl-EG-PTSA) system showing an excellent lignin removal rate (the lignin removal of 87.34 % and cellulose retention of 86.32 %), which is significantly better than the delignification effect of the traditional acid chlorite method (lignin removal of 72,13 % and cellulose retention of 64.57 %). In addition, in the subsequent enzymatic saccharification experiments, after the wood samples were pretreated in situ by the ChCl-EG-PTSA system, the enzymatic saccharification yield of cellulose reached 75.46 %, and the enzymatic saccharification yield of xylose reached 92 %. At the same time, this study also used SEM, FTIR, XPS, TGA and other testing technologies to conduct detailed analysis and characterization of the pretreated samples. This comprehensive analysis unequivocally demonstrated the viability of the ChCl-EG-PTSA system as an environmentally sustainable and efficient approach for pretreating forest biomass. Significantly, this in-situ wood pretreatment strategy significantly lays a robust research groundwork for the development of multifunctional wood fiber composites in subsequent stages.

Elucidating the Correlation of Lignocellulosic Compositions and Physicochemical Alterations in Oil Palm (Elaeis guineensis) Biomass on Enzymatic Saccharification Yield

Elucidating the Correlation of Lignocellulosic Compositions and Physicochemical Alterations in Oil Palm (Elaeis guineensis) Biomass on Enzymatic Saccharification Yield

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities

BackgroundThe polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear.ResultsAn LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid).ConclusionsThis study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

Integrated multi-objective optimization of sodium bicarbonate pretreatment for the outer anatomical portion of corncob using central composite design, artificial neural networks, and metaheuristic algorithms

Effective pretreatment of raw biomass is crucial in establishing a successful biorefinery process, and response surface methodology (RSM), a statistical optimization method, is routinely employed for optimizing pretreatment methodologies. However, relying solely on statistical methods fails to capture the nonlinear relationships between pretreatment methodology and lignocellulose recalcitrance. Machine learning (ML) approaches excel at understanding these relationships. Therefore, combining statistical and ML optimization can establish better pretreatment parameters. This study presents a multi-stage approach to identify and optimize a chemical pretreatment for the outer anatomical portion of corncobs (CO). Various chemicals were screened using fixed factor screening and regular two-level factorial design (2FI), and among them NaHCO3 was chosen for further optimization, using central composite design (CCD). Moderate delignification (33.96%), minimum sugar loss (5.26 mg/g of CO), and a high enzymatic saccharification yield of up to 82% were achieved with the design. Three distinct metaheuristic algorithms, namely Teaching-Learning-Based Optimization (TLBO), Particle Swarm Optimization (PSO), and Genetic Algorithm (GA) were employed to optimize three specific hyperparameters of the artificial neural network (ANN), to create hybrid metaheuristic-optimized ANN models, which were subsequently used to validate the results obtained from the CCD. Among the different models, The TLBO-optimized ANN model provided the most accurate predictions than the CCD.

Integrated understanding of enhancing enzymatic hydrolysis of bulrush through the metal chloride-assisting choline chloride: Glycerol pretreatment

To improve the saccharification efficiency of bulrush, the supplementary of metal chlorides in pretreatment of lignocellulose had been researched. The effect of metal chloride type and dosage on the ability of deep eutectic solvent (DES ChCl: Gly) pretreatment was evaluated by composition analysis of biomass, characterization analysis [scanning electron microscope, Fourier transform infra spectra, X-ray diffraction, accessibility of cellulose (Direct red dye adsorption experiment), surface area of lignin (Azure B dye adsorption experiment)], and enzymatic saccharification. One-pot pretreatment of bulrush using the AlCl3/ChCl:Gly (molar ratio, 1:2:0.06) with the solid liquid ratio of 1:10 (w:w), lignin (70.8%) and hemicellulose (87.7%) could be effectively removed, and the enzymatic saccharification yield could reach 90.1%, much higher than that of raw bulrush (21.4%). AlCl3/ChCl:Gly pretreated bulrush showed an apparent reduction in lignin surface area due to lignin removal. The correlations between the Azure B dye adsorption data and delignification [Y(delignification) = 0.29 × X(surface area of lignin) + 226.9, R2 = 0.90] and glucose yield [Y(glucose yield) = −0.31 × X(surface area of lignin) + 247.9, R2 = 0.93] were observed during the AlCl3/ChCl:Gly pretreatment. This constructed pretreatment system could improve the bioconversion efficiency of bulrush, which had potential application in biorefinery of lignocellulose.

An innovative and efficient hydrogen peroxide-citric acid pretreatment of bamboo residues to enhance enzymatic hydrolysis and ethanol production

Organic peracids has attracted widespread attention from researchers in biomass pretreatment. As a weak acid with high production, low price and toxicity, citric acid (CA) was mixed with hydrogen peroxide at the room temperature to generate peroxy-citric acid with strong oxidative functions. An innovative and efficient pretreatment method using peroxy-citric acid (HPCA) was proposed to enhance enzymatic hydrolysis and bioethanol production of bamboo residues. After D. giganteus (DG) was pretreated with HPCA at 80 °C for 3 h, lignin of 95.36% and xylan of 55.41% was effectively removed, and the enzymatic saccharification yield of HPCA-treated DG enhanced by about 8–9 times compared with CA pretreated DG. The ethanol recovery of 17.18 g/L was achieved. This work provided a reference for mild biomass pretreatment, which will promote the large-scale application of organic peracids system in biorefinery processes.

Follow-up of solid-state fungal wood pretreatment by a novel near-infrared spectroscopy-based lignin calibration model

Lignin removal plays a crucial role in the efficient bioconversion of lignocellulose to fermentable sugars. As a delignification process, fungal pretreatment has gained great interest due to its environmental friendliness and low energy consumption. In our previous study, a positive linear correlation between acid-insoluble lignin degradation and the achievable enzymatic saccharification yield has been found, hereby highlighting the importance of the close follow-up of lignin degradation during the solid-state fungal pretreatment process.However, the standard quantification of lignin, which relies on the two-step acid hydrolysis of the biomass, is highly laborious and time-consuming. Vibrational spectroscopy has been proven as a fast and easy alternative; however, it has not been extensively researched on lignocellulose subjected to solid-state fungal pretreatment. Therefore, the present study examined the suitability of near-infrared spectroscopy (NIR) for the rapid and easy assessment of lignin content in poplar wood pretreated with Phanerochaete chrysosporium. Furthermore, the predictive power of the obtained calibration model and the recently published ATR-FTIR spectroscopy-based model were compared for the first time using the same fungus-treated wood data set.PLSR was used to correlate the NIR spectra to the acid-insoluble lignin contents (19.9%–27.1%) of pretreated wood. After normalization and second derivation, a PLSR model with a good coefficient of determination (RCV2 = 0.89) and a low root mean square error (RMSECV = 0.55%) were obtained despite the heterogeneous nature of the fungal solid-state fermentation. The performance of this PLSR model was comparably good to the one obtained by ATR-FTIR (RCV2 = 0.87) while it required more extensive spectral pre-processing. In conclusion, both methods will be highly useful for the high-throughput and user-friendly monitoring of lignin degradation in a solid-state fungal pretreatment-based biorefinery concept.

Alkali-facilitated deep eutectic solvent for effective bamboo saccharification

Herein, a Na2S promoted deep eutectic solvent (DES) was established to reduce the natural recalcitrance of moso bamboo (MB) and improve the subsequent enzymatic saccharification. It was found that the addition of Na2S (Choline chloride/Ethylene glycol/Na2S) dramatically promoted the deconstructions of lignin with highest removal of 74.67 %, but at the same time preserved glucan and hemicellulose to the maximum extent. With the fractionation, the enzymatic saccharification yield of pretreated MB can reach 100 % under the pretreatment condition of 140 °C, and lignin could be readily recovered with a high yield of 81.47 %. The proposed DES is superior to normal alkaline DES in terms of the higher lignin removal and recovery yield, carbohydrate preservation and enzymatic digestibility, which indicated Na2S as a novel and powerful reinforcer enhancing the DES fractionation efficiency.

Rapid lignin quantification for fungal wood pretreatment by ATR-FTIR spectroscopy

Lignin determination in lignocellulose with the conventional two-step acid hydrolysis method is highly laborious and time-consuming. However, its quantification is crucial to monitor fungal pretreatment of wood, as the increase of acid-insoluble lignin (AIL) degradation linearly correlates with the achievable enzymatic saccharification yield. Therefore, in this study, a new attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy method was developed to track fungal delignification in an easy and rapid manner.Partial least square regression (PLSR) with cross-validation (CV) was applied to correlate the ATR-FTIR spectra with the AIL content (19.9 %–27.1 %). After variable selection and normalization, a PLSR model with a high coefficient of determination (RCV2 = 0.87) and a low root mean square (RMSECV = 0.60 %) were obtained despite the heterogeneous nature of the fungal solid-state fermentation. These results show that ATR-FTIR can reliably predict the AIL content in fungus-treated wood while being a high-throughput method. This novel method can facilitate the transition to the wood-based economy.

Investigation of hydrogen peroxide-acetic acid pretreatment to enhance the enzymatic digestibility of bamboo residues

Bamboo biomass was widely considered as a promising substitute for lignocellulose to produce fermentable sugars and biofuels in the south of China. When P. amarus were treated using hydrogen peroxide and acetic Acid pretreatment in the presence of sulphuric acid at 60 ℃ for 2 h, 82.63% lignin was removed from the bamboo residue, and enzymatic saccharification yield of 79.3% and ethanol content of 13.31 g/L were obtained. Analysis indicated that HPAC pretreatment increased the hydrophilic and porous nature of substrate, which can improve the enzyme accessibility to cellulose. When HPAC-pretreated D. sinicus, B. lapidea, N. affinis, andD. giganteus were used as the substrates of enzymatic saccharification, glucose yields of 71–84% at 72 h were achieved. HPAC pretreatment was a highly efficient and environmentally friendly method for bamboo biorefinery in the south of China.

Rapid and efficient microwave-assisted guanidine hydrochloride deep eutectic solvent pretreatment for biological conversion of castor stalk

Microwave-assisted guanidine hydrochloride deep eutectic solvents (DESs) was developed for rapid and efficient pretreatment of castor stalk. The DES synthesized with guanidine hydrochloride and lactic acid showed a better delignification (92.02%) and enzymatic saccharification yield (96.3%) than choline chloride and lactic acid DES resulted. In addition, high-purity (up to 98%) lignin was recovered from the pretreatment liquor. The good recyclability of the guanidine hydrochloride-based DES was also proven with up to 90% cellulose hydrolysis with third-time recycled DES without post purification. The proposed microwave-assisted guanidine hydrochloride/lactic acid DES showed its great potentials as a highly effective and recyclable pretreatment solvent for future biorefinery strategies.

Fiber Cell-Specific Expression of the VP16-Fused Ethylene Response Factor 41 Protein Increases Biomass Yield and Alters Lignin Composition.

Arabidopsis thaliana transcription factors belonging to the ERFIIId and ERFIIIe subclade (ERFIIId/e) of the APETALA 2/ethylene response factor (AP2/ERF) family enhance primary cell wall (PCW) formation. These transcription factors activate expression of genes encoding PCW-type cellulose synthase (CESA) subunits and other genes for PCW biosynthesis. In this study, we show that fiber-specific expression of ERF035-VP16 and ERF041-VP16, which are VP16-fused proteins of ERFIIId/e members, promote cell wall thickening in a wild-type background with a concomitant increase of alcohol insoluble residues (cell wall content) per fresh weight (FW) and monosaccharides related to the PCW without affecting plant growth. Furthermore, in the ERF041-VP16 lines, the total amount of lignin and the syringyl (S)/guaiacyl (G) ratio decreased, and the enzymatic saccharification yield of glucose from cellulose per fresh weight improved. In these lines, PCW-type CESA genes were upregulated and ferulate 5-hydropxylase1 (F5H1), which is necessary for production of the S unit lignin, was downregulated. In addition, various changes in the expression levels of transcription factors regulating secondary cell wall (SCW) formation were observed. In conclusion, fiber cell-specific ERF041-VP16 improves biomass yield, increases PCW components, and alters lignin composition and deposition and may be suitable for use in future molecular breeding programs of biomass crops.

Pilot Scale Elimination of Phenolic Cellulase Inhibitors From Alkali Pretreated Wheat Straw for Improved Cellulolytic Digestibility to Fermentable Saccharides.

Depleting supplies of fossil fuel, regular price hikes of gasoline and environmental deterioration have necessitated the search for economic and eco-benign alternatives of gasoline like lignocellulosic biomass. However, pre-treatment of such biomass results in development of some phenolic compounds which later hinder the depolymerisation of biomass by cellulases and seriously affect the cost effectiveness of the process. Dephenolification of biomass hydrolysate is well cited in literature. However, elimination of phenolic compounds from pretreated solid biomass is not well studied. The present study was aimed to optimize dephenoliphication of wheat straw using various alkalis i.e., Ca(OH)2 and NH3; acids i.e., H2O2, H2SO4, and H3PO4; combinations of NH3+ H3PO4 and H3PO4+ H2O2 at pilot scale to increase enzymatic saccharification yield. Among all the pretreatment strategies used, maximum reduction in phenolic content was observed as 66 mg Gallic Acid Equivalent/gram Dry Weight (GAE/g DW), compared to control having 210 mg GAE/g DW using 5% (v/v) combination of NH3+H3PO4. Upon subsequent saccharification of dephenoliphied substrate, the hydrolysis yield was recorded as 46.88%. Optimized conditions such as using 1%+5% concentration of NH3+ H3PO4, for 30 min at 110°C temperature reduced total phenolic content (TPC) to 48 mg GAE/g DW. This reduction in phenolic content helped cellulases to act more proficiently on the substrate and saccharification yield of 55.06% was obtained. The findings will result in less utilization of cellulases to get increased yield of saccharides by hydrolyzing wheat straw, thus, making the process economical. Furthermore, pilot scale investigations of current study will help in upgrading the novel process to industrial scale.

Water retention value predicts biomass recalcitrance for pretreated biomass: biomass water interactions vary based on pretreatment chemistry and reflect composition

Processing of lignocellulosic biomass is complex due to the heterogeneity of the substrate, but also due to lengthy unit operations, which complicates process control including for enzymatic saccharification. Methods for predicting enzymatic saccharification yield based on the properties of the pretreated biomass would be advantageous to process optimization and control. Biomass-water interaction measurements provide a method for quickly predicting biomass recalcitrance. Correlating water retention value (WRV) and enzymatic saccharification yield (ESY) on pretreated biomass has shown promise, especially when assessing only single biomass types pretreated with one specific chemistry. However, with comparisons between different types of biomasses, predictive powers have been low. We investigate the effect of pretreatment chemistry on the predictive power of WRV, when keeping the biomass static. Wheat straw was pretreated with dilute acid, hydrothermal, or alkaline chemistries at five different temperatures. Furthermore, low field nuclear magnetic resonance was used to measure water constraint in the pretreated materials, to better understand how biomass-water interactions change with pretreatment severity and chemistry. We show that the correlation of WRV and ESY is highly pretreatment dependent, while WRV strongly predicts ESY within each pretreatment chemistry. While ESY and WRV correlated under all chemistries, the direction of the correlations were divergent, suggesting a more complex interplay between recalcitrance and biomass-water interactions. Using T2 relaxation profiles, reductions in hemicellulose composition was related to the decrease in size of the most constrained water population present in the pretreated biomasses for all chemistries, suggesting a new identification of this population of constrained water.

Effect of hydrogen bond donor on the choline chloride-based deep eutectic solvent-mediated extraction of lignin from pine wood

In this work, twenty-five kinds of choline chloride (ChCl)-based deep eutectic solvents (DESs) containing acid, hydroxyl, amide, and binary hydrogen bond donors (HBDs) were prepared and successfully used to pretreat pine wood powder. As a result of the pretreatment, the glucan content in the pretreated biomass was increased, whereas the contents of hemicellulose and lignin were significantly decreased. The biomass pretreatment efficiency of the DESs had improved with increasing the polarity and hydrogen bond acidity (α) of the DESs. Among the studied DESs, ChCl:lactic acid:formic acid (1:1:1) with the highest α value was the most efficient DES in extracting lignin from biomass. The pretreated biomass also showed an enhanced enzymatic saccharification yield owing to the decreased particle size of the biomass and reduced content of hemicellulose and lignin. During the pretreatment process of biomass using DESs, the extracted lignin could be recovered successfully, with a yield of up to 60% and purity of over 90%. The molecular weight of the extracted lignin was much lower than that of the native cellulolytic enzyme lignin. The DES used for pretreatment process could be also successfully reused with high recovery yield of DES and high retention of delignification capacity.

Effects of Additional Xylanase on Saccharification and Ethanol Fermentation of Ammonia-Pretreated Corn Stover and Rice Straw

Synergistic effect of cellulase and hemicellulase (xylanase) was evaluated because lignocellulosic material is a heterogeneous complex of cellulose and hemicellulose. Various effects of HTec2 addition on enzymatic saccharification and fermentation were evaluated using two different substrates such as corn stover and rice straw. Corn stover and rice straw were pretreated by the LMAA (low-moisture anhydrous ammonia) method at the preselected same conditions (90 °C, 120 h, moisture content = 50%, NH3 loading = 0.1 g NH3/g). It was observed that the enzymatic saccharification yield of pretreated corn stover (76.4% for glucan digestibility) was higher than that of pretreated rice straw (70.9% for glucan) using CTec2 cellulase without HTec2 addition. Glucan digestibility of pretreated corn stover was significantly increased from 76.4% to 91.1% when the HTec2/CTec2 (v/v) increased from 0 to 10. However, it was interesting that the ethanol production was decreased from 89.9% to 76.3% for SSF and 118.0% to 87.9% for SSCF at higher HTec2/CTec2. As the glucan loading increased from 2.0% to 7.0%, the ethanol yields of both SSF and SSCF were decreased from 96.3% to 88.9% and from 116.6% to 92.4%, respectively. In addition, the smallest inoculum size (optical density of 0.25) resulted in the highest ethanol production (20.5 g/L).

Novel sterilization method combining food preservative use and low temperature steaming for treatment of lignocellulosic biomass with white rot fungi

In the bioconversion of lignocellulosic biomass to useful chemicals, pretreatment of the material prior to hydrolysis is necessary. The aim of the present study was to develop a method to reduce the energy costs of lignocellulosic biomass treatment with white rot fungi. We developed a novel contaminant mold suppressing method combining the food preservative Vitagen AS5N (thiamine dilauryl sulfate) and low temperature steam sterilization (80 °C for 15 min). First, heat-resistant contaminant molds were isolated from sugarcane bagasse (Saccharum officinarum L.) sterilized at 80 °C, and a difference in sensitivity to Vitagen AS5N (200, 300, 500, and 1000 μg/mL, at pH 5.2 and 9.0) was observed between the isolated contaminant molds and white rot fungi. The sugarcane bagasse was subsequently impregnated with Vitagen AS5N solution (500 or 1000 μg/mL at pH 9.0), which did not affect the white rot fungi but suppressed the contaminant molds, and autoclaved at 80 °C. The sterilized sugarcane bagasse medium was inoculated with three types of white rot fungi, and the enzymatic saccharification yield of fungal bagasse was determined after 8 weeks. High enzymatic saccharification yields were attained with each fungal bagasse: 79.5 and 76.2 % for Ceriporiopsis subvermispora (500 and 1000 μg/mL Vitagen AS5N), 80.1 and 79.5 % for Lentinus edodes, and 74.5 and 78.1 % for Coriolus versicolor. These enzymatic saccharification yields were similar to that of normal fungal bagasse. The results indicated that the proposed combination method may potentially reduce the energy required for the treatment of lignocellulosic biomass with white rot fungi.

Exploring the Effect of Water Content and Anion on the Pretreatment of Poplar with Three 1-Ethyl-3-methylimidazolium Ionic Liquids.

We report on the pretreatment of poplar wood with three different 1-ethyl-3-methylimidazolium ionic liquids, [EMim][OAc], [EMim][MeSO3], and [EMim][HSO4], at varying water contents from 0–40 wt% at 100 °C. The performance was evaluated by observing the lignin and hemicellulose removal, as well as enzymatic saccharification and lignin yield. The mechanism of pretreatment varied between the ionic liquids studied, with the hydrogen sulfate ionic liquid performing delignification and hemicellulose hydrolysis more effectively than the other solvents across the investigated water content range. The acetate ionic liquid produced superior glucose yield at low water contents, while the hydrogen sulfate ionic liquid performed better at higher water contents and produced a recoverable lignin. The methanesulfonate ionic liquid did not introduce significant fractionation or enhancement of saccharification yield under the conditions used. These findings help distinguish the roles of anion hydrogen bonding, solvent acidity, and water content on ionic liquid pretreatment and can aid with anion and water content selections for different applications.

Effect of [EMIM]Ac Recycling on Salix gracilistyla Miq. Pretreatment for Enzymatic Saccharification

Recycling of ionic liquid (1-ethyl-3-methylimidazolium acetate, [EMIM]Ac) after the pretreatment of Salix gracilistyla Miq. was conducted and the effect of the recycling number on the enzymatic saccharification yield was investigated. Enzymatic saccharification was performed using an enzyme cocktail (Acremonium cellulase and Optimash BG) at 50 °C for 72 h. All recycled [EMIM]Ac samples showed a lower amount of water soluble fraction than pure [EMIM]Ac. On increasing the recycling number from 1 to 4, the amount of water soluble fraction decreased from 18% to 15%. The X-ray diffraction pattern of the products pretreated with recycled [EMIM]Ac showed cellulose I crystalline polymorph. The crystallinity of the product pretreated with recycled [EMIM]Ac was 47-49%, which was lower than 33% of that with pure [EMIM]Ac. The yields of glucose and xylose decreased in the pretreatment with recycled [EMIM]Ac compared to that with pure [EMIM]Ac.