Optimization of ethanol production by Saccharomyces cerevisiae UFPEDA 1238 in simultaneous saccharification and fermentation of delignified sugarcane bagasse

Ethanol production by Saccharomyces cerevisiae UFPEDA1238 was performed in simultaneous saccharification and fermentation of delignified sugarcane bagasse. Temperature (32 °C, 37 °C), agitation (80; 100 rpm), enzymatic load (20 FPU/g cellulose and 10%, v/v β-glucosidase or 10 FPU/g cellulose and 5% β-glucosidase) and composition of culture medium were evaluated. Ethanol concentration, enzymatic convertibility of cellulose and volumetric productivity were higher than 25 g/L, 72% and 0.70 g/L h, respectively, after 30 h, when the culture medium 1 and 20 FPU/g cellulose/10%, v/v β-glucosidase or the culture medium 2 and 10 FPU/g cellulose/5% β-glucosidase were used in SSF at 37 °C and 80 rpm. In the SSF with culture medium 2 (supplemented with ammonium, phosphate, potassium and magnesium), 150 L ethanol/t bagasse was achieved, with minimum enzyme loading (10 FPU/g cellulose and 5%, v/v β-glucosidase) for 8%, w/v of solids, which is often an important requirement to provide cost-efficient second generation ethanol processes.     

Fractionation of flax shives with pressurized aqueous ethanol

Fractionation of flax shives into major biopolymer constituents, such as cellulose, hemicelluloses, and lignin, was carried out with pressurized aqueous ethanol in a pressurized low-polarity water extractor. The effect of processing parameters such as temperature, ethanol concentration, flow rate, sample size and solvent/feed ratio on the simultaneous extraction of hemicelluloses and lignin was determined. More than 80% of total hemicelluloses and ∼78% of total lignin were removed simultaneously in a single step under the following conditions: 180 °C, 30% (v/v) ethanol concentration, 3 mL/min flow rate, and 45 mL/g solvent/feed ratio. Under these extraction conditions, cellulose degradation was negligible. Further, the separation of lignin from hemicelluloses was carried out using two simple alternative methodologies based on precipitation. Since no acidic or alkali catalysts were used, the degradation of biopolymers was negligible and the oligomer/monomer ratio of sugars was 825:1. Characterization of fractionated biopolymers was carried out with scanning electron microscopy and a Fourier-transform infrared spectrometer (FT-IR). FT-IR spectra of isolated lignin and hemicelluloses showed that both polymers were comparable to commercially available products.     

Process optimization for ethanol production from photoperiod-sensitive sorghum: Focus on cellulose conversion

Photoperiod-sensitive sorghum, as a competitive biomass for ethanol production, was investigated to develop an integrated process for improving ethanol yield. Response surface methodology was employed to study the relationship between pretreatment variables (including temperature, sulfuric acid concentration, and reaction time) and cellulose recovery, as well as efficiency of enzymatic hydrolysis (EEH) in the solid part. Recovery yield decreased and EEH increased as the pretreatment temperature, acidic concentration, and reaction time increased. A model was successfully developed to predict total glucose yield with a maximum value of 82.2%. Conditions of co-fermentation were also optimized, and the optimal ethanol yield was obtained with constant-temperature simultaneous saccharification and fermentation at 38 °C. Acetate buffer at a concentration of 50 mM was found helpful for increasing efficiency of enzymatic hydrolysis, as well as ethanol yield. The maximum ethanol yield was 0.21 g ethanol per dry mass at the conditions of 38 °C, 0.05 g yeast/L, and 50 mM acetate buffer. A complete cellulose balance was provided for the whole process.     

Preparation of regenerated cellulose fiber via carbonation. I. Carbonation and dissolution in an aqueous NaOH solution

Cellulose carbonate was prepared by the reaction of cellulose pulp and CO₂ with treatment reagents, such as aqueous ZnCl₂ (20–40 wt%) solution, acetone or ethyl acetate, at −5–0°C and 30–40 bar (CO₂) for 2 hr. Among the treatment reagents, ethyl acetate was the most effective. Cellulose carbonate was dissolved in 10% sodium hydroxide solution containing zinc oxide up to 3 wt% at −5–0°C. Intrinsic viscosities of raw cellulose and cellulose carbonate were measured with an Ubbelohde viscometer using 0.5 M cupriethylenediamine hydroxide (cuen) as a solvent at 20°C according to ASTM D1795 method. The molecular weight of cellulose was rarely changed by carbonation. Solubility of cellulose carbonate was tested by optical microscopic observation, UV absorbance and viscosity measurement. Phase diagram of cellulose carbonate was obtained by combining the results of solubility evaluation. Maximum concentration of cellulose carbonate for soluble zone was increased with increasing zinc oxide content. Cellulose carbonate solution in good soluble zone was transparent and showed the lowest absorbance and the highest viscosity. The cellulose carbonate and its solution were stable in refrigerator (−5°C and atmospheric pressure).     

Optimisation of a solvent for the complete extraction of prolamins from heated foods

Wheat bread was lyophilised, ground, extracted and centrifuged. The supernatants were analysed for gluten content by RP-HPLC and a commercial sandwich ELISA. Prolamin extraction solvents contained tris(2-carboxyethyl)phosphine (TCEP; 1, 2, 5, 10, 20, 50 mmol/L), guanidine hydrochloride (GUA; 0 or 2 mol/L) and a buffered salt solution. A commercial cocktail solution (250 mmol/L mercaptoethanol (ME), 2 mol/L GUA, buffered salt solution) as well as 60% (v/v) ethanol were used as control solvents. Wheat flour was the control for the extractability of the native prolamins. 60% ethanol only extracted 37% of the prolamins from wheat bread (cocktail = 100%). When ME was replaced by TCEP the protein yield increased from 35% at the lowest TCEP-level to 95% when 20–50 mmol/L TCEP were used. The use of GUA was essential to extract prolamins quantitatively. Comparative protein analysis using RP-HPLC and ELISA showed that both methods provided comparable prolamin (gliadin) concentrations of the wheat flour (40.3–45.7 mg/g), when 60% ethanol was used as extraction solvent. The extraction yields from bread were considerably lower (16.7–24.7 mg/g). Cocktail and TCEP extracted almost the same amount of protein from flour and bread with TCEP showing slightly lower yields. Total extractable protein (gliadin + glutenin) as determined by RP-HPLC was 70.5–75.3 mg/g, and total gliadin as determined by ELISA was 42.7–44.2 mg/g. Thus, the study has shown that TCEP in combination with GUA extracts proteins from heated, gluten-containing foods as effective as the commercial cocktail solution.     

Improving the barrier and mechanical properties of corn starch-based edible films: Effect of citric acid and carboxymethyl cellulose

The films produced from pure starch are brittle and difficult to handle. Chemical modifications (e.g. cross-linking) and using a second biopolymer in the starch based composite have been studied as strategies to produce low water sensitive and relatively high strength starch based materials. A series of corn starch films with varying concentrations (0-20%, W/W) of citric acid (CA) and carboxymethyl cellulose (CMC) were produced by casting method. The effects of CA and CMC on the water vapor permeability (WVP), moisture absorption, solubility and tensile properties were investigated. The water vapor barrier property and the ultimate tensile strength (UTS) were improved significantly (p < 0.05) as the CA percentage increased from 0 to 10% (W/W). At the level of 15% (W/W) CMC, the starch films showed the lowest WVP values (2.34 × 10⁻⁷ g Pa⁻¹ h⁻¹ m⁻¹) and UTS increased from 6.57 MPa for the film without CMC to 16.11 MPa for that containing 20% CMC.     

Antioxidant activity of phenolic compounds obtained by autohydrolysis of corn residues

This study aimed to evaluate the effect of autohydrolysis temperature of corn residues in the antioxidant activity of the phenolic compounds extracted from the liquid phase. The treatments were carried out at 160, 180, 190 and 200 °C for 30 min in a pressurized batch reactor. Two different methods for phenolic compounds extraction from the autohydrolysis liquors were investigated. For that purpose, solvent extraction with ethyl acetate and acidic precipitation were performed for phenolic compounds recovery. These methods have been compared in terms of extraction yield, physicochemical properties of obtained polyphenols (characterization by Fourier Transform Infrared Spectroscopy, Thermogravimetric Analysis and Gel Permeation Chromatography), total phenolic content, total antioxidant capacity and Trolox equivalent antioxidant capacity (TEAC) values, measured in DPPH (2,2-diphenyl-1-picrylhydrazyl) test system. The maximum phenolic contents ranged from 6.04 mg GAE/100 mg extract in acidic precipitated samples to 16.45 mg GAE/100 mg extract in ethyl acetate soluble fractions. The results indicated that the ethyl acetate fractions possessed the highest antioxidant activity, reaching after 30-60 min the same capacity reported for the reference synthetic antioxidants (Trolox).     

Characterization of depolymerized residues from extremely low acid hydrolysis (ELA) of sugarcane bagasse cellulose: Effects of degree of polymerization, crystallinity and crystallite size on thermal decomposition

Sugarcane bagasse cellulose was subjected to the extremely low acid (ELA) hydrolysis in 0.07% H₂SO₄ at 190, 210 and 225 °C for various times. The cellulose residues from this process were characterized by TGA, XRD, GPC, FTIR and SEM. A kinetic study of thermal decomposition of the residues was also carried out, using the ASTM and Kissinger methods. The thermal studies revealed that residues of cellulose hydrolyzed at 190, 210 and 225 °C for 80, 40 and 8 min have initial decomposition temperature and activation energy for the main decomposition step similar to those of Avicel PH-101. XRD studies confirmed this finding by showing that these cellulose residues are similar to Avicel in crystallinity index and crystallite size in relation to the 110 and 200 planes. FTIR spectra revealed no significant changes in the cellulose chemical structure and analysis of SEM micrographs demonstrated that the particle size of the cellulose residues hydrolyzed at 190 and 210 °C were similar to that of Avicel.     

Blends of castor meal and castor husks for optimized use as organic fertilizer

Production of oil from castor (Ricinus communis) generates two main by-products: husks and meal. For each ton of castor oil, 1.31 ton of husks and 1.13 ton of meal are produced. Castor meal is the most important by-product due to its high nitrogen content, and presently it is predominantly used as an organic fertilizer. This greenhouse study aimed to find optimized blends of castor husks and meal as organic fertilizer for growth of castor plants. The by-products were mixed to sandy soil in the doses (v:v) of 0 + 10%, 2.5 + 7.5%, 5.0 + 5.0%, 7.5 + 2.5% and 10.0 + 0% of castor meal and castor husks, respectively. At 50 days after sowing, data was taken on plant growth and shoot nutrients content. Blends of castor meal and castor husks used as fertilizer promoted substantial plant growth up to the dose of 4.5% (in volume) of meal. Doses higher than 4.5% caused reduction in plant growth and even plant death. It seems that rapidly mineralizing nitrogen from the castor meal is the reason for both the intensive plant growth in low doses, and the reduction of plant growth in high doses due to excessive mineral N. Increments in castor meal dose resulted in increased N and Ca shoot contents. Other macronutrients were not influenced. Castor meal is a good organic fertilizer, due to its high N and P contents, but blending with castor husks is not necessary.     

Development of transparent bacterial cellulose nanocomposite film as substrate for flexible organic light emitting diode (OLED) display

Nanocomposite film composed of bacterial cellulose (10-50 wt.%) and poly-urethane (PU) based resin was fabricated and utilized as a substrate for flexible organic light emitting diode (OLED) display. The performance of the nanocomposite satisfied the criteria for the substrate of OLED with an additional feature of flexibility. The visible light transmittance of the nanocomposite film was as high as 80%. Its thermal stability was stable up to 150 °C while its dimensional stability in terms of coefficient of thermal expansion (CTE) was less than 20 ppm/K. After OLED was fabricated on the substrate through thermal evaporation technique, the OLED performed highest current efficiency of 0.085 cd/A and power efficiency of 0.021 lm/W at 200 cd/m² while retained its flexible feature, suggesting that bacterial cellulose nanocomposite is a promising material for the development of substrate for flexible OLED display.     

Comparison of lignin and cellulose solubilities in ionic liquids by COSMO-RS analysis and experimental validation

In this work, the solubilities of lignin and cellulose in ionic liquids (ILs) have been analyzed and compared by COSMO-RS simulation. The excess enthalpy of the IL + lignin/cellulose mixtures is evaluated as reference property to describe the affinity of lignin and cellulose for different ionic liquids, in terms of the intermolecular interactions contributing to their mixing properties. Thus, the anion plays the main role in the dissolution process of both lignin and cellulose, in which the hydrogen bonding forces are the major interactions. The most promising anions for the cellulose and lignin dissolution are acetate, formate and chloride, whereas different cations can be employed. Computational results indicated that a rational combination of the anions and cations allows designing ILs able to dissolve with different selectivity lignin and cellulose. Some of these solutions are tested in the laboratory to validate the model employed and the regenerated solids are analyzed by FTIR spectroscopy.     

Optimization of formic/acetic acid delignification of Miscanthus × giganteus for enzymatic hydrolysis using response surface methodology

A Box-Behnken experimental design and response surface methodology were employed to optimize the pretreatment parameters of a formic/acetic acid delignification treatment of Miscanthus × giganteus for enzymatic hydrolysis. The effects of three independent variables, namely cooking time (1, 2 and 3 h), formic acid/acetic acid/water ratio (20/60/20, 30/50/20 and 40/40/20) and temperature (80, 90 and 107 °C) on pulp yield, residual Klason lignin content, concentration of degradation products (furfural and hydroxymethylfurfural) in the black liquor, and enzymatic digestibility of the pulps were investigated. The major parameter influencing was the temperature for pulp yield, delignification degree, furfural production and enzymatic digestibility. According to the response surface analysis the optimum conditions predicted for a maximum enzymatic digestibility of the glucan (75.3%) would be obtained using a cooking time of 3 h, at 107 °C and with a formic acid/acetic acid/water ratio of 40/40/20%. Glucan digestibility was highly dependent on the delignification degree.     

Comparative study of organosolv lignins from wheat straw

Dewaxed wheat straw was treated with acetic acid–H₂O (65/35, v/v), acetic acid–H₂O (80/20, v/v), acetic acid–H₂O (90/10, v/v), formic acid–acetic acid–H₂O (20/60/20, v/v/v), formic acid–acetic acid–H₂O (30/60/10, v/v/v), methanol–H₂O (60/40, v/v) and ethanol–H₂O (60/40, v/v) using 0.1% HCl as a catalyst at 85 °C for 4 h, in which 78.2, 80.0, 88.2, 89.4, 94.1, 23.5 and 37.4% of the original lignin, and 42.4, 58.7, 70.0, 65.1, 76.5, 14.2 and 22.2% of the original hemicelluloses was released, respectively. Lignins obtained were characterized by their content of hemicelluloses, composition of phenolic acids and aldehydes, molecular weight, thermal stability and by UV, Fourier transform infrared (FT-IR), ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The results showed that aqueous organic acid was more effective than aqueous organic alcohol for extensive delignification and selective fractionation of cellulose, lignin and hemicelluloses from the straw. In particular, the addition of formic acid gave a significant effect on the dissolution of lignin. All the acid-insoluble lignin fractions contained small amounts of contaminated hemicelluloses as shown by their content of neutral sugars, 0.9–4.3%, and had weight-average molecular weight between 3960 and 4340 g mol⁻¹. An increase in concentration of acetic acid or formic acid in organosolv resulted in an increment in release of guaiacyl units and in lignin condensation. However, the lignin preparations released during the treatment with aqueous organic alcohol without organic acid contained almost equal amounts of non-condensed guaiacyl and syringyl units with fewer p-hydroxyphenyl units. The β-O-4 ether bonds together with β-β, β-5 and 5-5′ carbon–carbon linkages were identified to be present in lignin substructures.     

Effect of pre-extraction on soda-anthraquinone (AQ) pulping of rice straw

The effect of hot-water and alkaline pre-extraction of rice straw on soda-anthraquinone pulping was carried out. The pre-extraction with hot water at 150 °C for 1 h dissolved 34.7% biomass and the pre-extracted liquor comprised of 16.6% sugars, 6.7% lignin, 6.6% acetic acid and other unknown products. But the pre-extraction with 1% NaOH at 100 °C for 1 h dissolved 10.2% sugars, 5.1% lignin and 10% acetic acid from rice straw. Pre-extracted rice straw was cooked by soda-anthraquinone process with varying alkali charges. The pulp from pre-extracted rice straw was low in kappa number with reduced pulp yield. The drainage resistance (°SR) improved obviously on pre-extraction of rice straw. Pulp strength properties such as the tensile index and the burst index were found to be lower, but the tear index was higher both with hot-water and alkaline pre-extraction. After bleaching, the gaps of the overall pulp yield and strength properties between pre-extracted and non-extracted rice straw became narrower. The alkaline pre-extraction showed improved yield and properties compared with hot-water pre-extracted rice straw.     

Porous Laponite/Poly(L-lactic acid) Membrane with Controlled Release of TCH and Efficient Antibacterial Performance

Fabrication of porous polymer membrane with controlled drug release and efficient antibacterial performances is of great interest in biomedical fields. In this study, Laponite (LAP) nanodisks were first used to encapsulate a model antibiotic drug, tetracycline hydrochloride (TCH). Then, drug-loaded LAP nanodisks with an optimized loading efficiency (85.3 %) were mixed with poly(L-lactic acid) (PLLA) polymer to form drug-loaded composite porous membrane via solvent coasting. The structure, morphology and swelling property of the porous membranes formed with varied solvent ratio of methylene dichloride (DCM) and dimethyl formamide (DMF) in the mixture solvent were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy and swelling test. In vitro drug release behavior, the cytotoxicity and the antibacterial activity of drug-loaded composite membranes were evaluated. Results showed that the TCH release was dependent on the physical structure of PLLA membrane and the presence of LAP nanodisks effectively weakened the initial burst release of TCH, and improved the sustained release property of porous PLLA membrane. The released TCH of TCH/LAP/PLLA_3:1 and TCH/LAP/PLLA_4:1 was 10.0 % and 5.3 % within initial 1 h, respectively. More importantly, the porous TCH/LAP/PLLA membrane was cytocompatible and displayed considerable antibacterial activity, solely associated with the loaded TCH drug, confirming its potential utility in wound dressings and tissue engineering.     

Microwave pretreatment: A clean and dry method for palm oil production

The present study shows that microwave pretreatment coupled with solvent extraction is a clean and dry process for the production of crude palm oil (CPO). The results indicate that palm fruits microwave heated for 3 min achieved oil yield comparable to the conventional and commercial palm oil milling process with an average of 20%. Fatty acid composition (FAC) of the CPO produced shows an increment in lauric acid (C12:0) as the duration of microwave exposure increases. Due to the absence of wet-heat treatment or sterilization process, the resultant palm oil also exhibits desirable and very low free fatty acid (FFA) (0.26%) and moisture content (0.05%). The current approach has also provided surprisingly high vitamin E and carotenes content. The highest concentration of vitamin E (2345 ppm) and carotene content (1585 ppm) was recorded for palm fruits subjected to microwave for 2 min. It shows that under the investigated conditions in the present study, the quality of CPO produced using microwave treatment coupled with hexane extraction possesses superior qualities compared to the conventional palm oil milling process.     

Optimization of ultrasonic-assisted extraction for herbicidal activity of chicory root extracts

The present study evaluated the herbicidal potential of extracts from chicory (Cichorium intybus L.) roots on the germination of Echinochloa crusgalli L. Beauv and Amaranthus retroflexus L. Eight ultrasonic assisted-extraction (UAE) conditions were optimized, using an orthogonal matrix design. The extract concentrations that would yield the largest allelopathic effects on the plant species were estimated by a modeling analysis. Our results showed that an alcohol solvent extract of chicory root had significant herbicidal activity which depended on the extract concentrations and the target species. The half-inhibitory concentrations of crude extract of chicory root ranged from 0.5 g l⁻¹ to 40.5 g l⁻¹. At a frequency of 40 kHz, the optimum UAE conditions to produce an extract for use as herbicide against E. crusgalli L. included an ethanol content (Ec) of 50% (v/v), a solvent-to-solid ratio (SR) of 16:1, an ultrasound temperature (UT) of 35 °C, an impregnation time (Imt) of 24 h with two rounds of impregnation (Imr), a sonication period (St) of 120 min with two rounds of sonication (Sr) and an ultrasound input power (P) of 200 W. The optimum conditions to produce an extract for use against A. retroflexus L. included an Ec of 100% (v/v), a SR of 16:1, an UT of 20 °C, an Imt of 48 h with two Imr, a St of 30 min with one Sr and a P of 400 W. The extract had the largest inhibitory effects on the germination index and root growth of both E. crusgalli L. and A. retroflexus L. at concentrations ranging from 30.8 to 33.7 g l⁻¹. At a concentration of 4.2 g l⁻¹, the extract significantly enhanced the shoot growth of A. retroflexus L. Overall, chicory root extract has potential for use as a main ingredient in natural herbicides or for development as a novel plant-derived herbicide.     

Genetic structure of natural Tunisian Hypericum humifusum L. (Hypericacae) populations as assessed by allozymes and RAPDs

The genetic diversity within and among seven Tunisian natural populations of Hypericum humifusum L., from different geographic regions and bioclimates, was assessed using 11 isozymic polymorphic loci, and 166 RAPD markers amplified by 8 primers. The genetic diversity within populations based on allozymes was higher (P = 72.46%, Ap = 2.01 and H_e = 0.29), than that revealed by RAPDs (29.52 < P < 39.16% and 0.150 < H < 0.200). Both markers yielded high estimates of genetic differentiation and low gene flow (Nm = 0.257 and 0.508 for RAPD and allozymes, respectively) among populations at all space scales. However, the level of differentiation revealed by RAPDs (Φ_ST = 0.494; G_ST = 0.561) was higher than that based on allozymes (F_ST = 0.117). No correlation (Mantel test) among genetic (F_ST and Φ_ST matrices) and geographical distance matrices was observed indicating no isolation by distance. Cluster analyses from allozyme and RAPD loci did not completely agree. The dendrogram based on allozymes yielded higher separation among most populations, while that from RAPDs separated populations into three distinct subclusters. Groupings of populations, in both dendrograms, did not reflect spatial geographic or bioclimatic patterns, indicating specific adaptation of populations to local environments. The correlation between matrices of allozyme and RAPD band frequencies was not significant (Mantel test). The dendrogram obtained from combined data yielded similar population groupings to that probed by RAPDs suggesting higher accuracy of these markers. Given the high differentiation among all populations even at a low geographic distance, ex situ conservation should involve extensive seed collection from all populations from all bioclimatic zones. The populations from the upper semi-arid bioclimate exhibiting relatively high level of genetic diversity should be first protected.     

Composite materials of thermoplastic starch and fibers from the ethanol-water fractionation of bagasse

The aim of this study was to evaluate the potential of the fibrous material obtained from ethanol-water fractionation of bagasse as reinforcement of thermoplastic starches in order to improve their mechanical properties. The composites were elaborated using matrices of corn and cassava starches plasticized with 30 wt% glycerin. The mixtures (0, 5, 10 and 15 wt% bagasse fiber) were elaborated in a rheometer at 150 °C. The mixtures obtained were pressed on a hot plate press at 155 °C. The test specimens were obtained according to ASTM D638. Tensile tests, moisture absorption tests for 24 days (20-23 °C and 53% RH, ASTM E104), and dynamic-mechanical analyses (DMA) in tensile mode were carried out. Images by scanning electron microscopy (SEM) and X-ray diffraction were obtained. Fibers (10 wt% bagasse fiber) increased tensile strength by 44% and 47% compared to corn and cassava starches, respectively. The reinforcement (15 wt% bagasse fiber) increased more than fourfold the elastic modulus on starch matrices. The storage modulus at 30 °C (E_30 °C′) increased as the bagasse fiber content increased, following the trend of tensile elastic modulus. The results indicate that these fibers have potential applications in the development of biodegradable composite materials.     

Sugarcane bagasse whiskers: Extraction and characterizations

This work evaluates the use of sugarcane bagasse (SCB) as a source of cellulose to obtain whiskers. These fibers were extracted after SCB underwent alkaline peroxide pre-treatment followed by acid hydrolysis at 45 °C. The influence of extraction time (30 and 75 min) on the properties of the nanofibers was investigated. Sugarcane bagasse whiskers (SCBW) were analyzed by transmission electron microscopy (TEM), X-ray diffraction (XRD) and thermogravimetric analysis (TGA) in air atmosphere. The results showed that SCB could be used as source to obtain cellulose whiskers and they had needle-like structures with an average length (L) of 255 ± 55 nm and diameter (D) of 4 ± 2 nm, giving an aspect ratio (L/D) around 64. More drastic hydrolysis conditions (75 min) resulted in less thermally stable whiskers and caused some damage on the crystal structure of the cellulose as observed by XRD analysis.