Novel Penicillium cellulases for total hydrolysis of lignocellulosics

The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35 °C while at 45 °C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.     

Enzyme adsorption on SO2 catalyzed steam-pretreated wheat and spruce material

Lignocellulose is widely recognized as a sustainable substrate for biofuels production, and the enzymatic hydrolysis is regarded as a critical step for the development of an effective process for the conversion of cellulose into ethanol. One key factor affecting the overall conversion rate is the adsorption capacity of the cellulase enzymes to the surface of the insoluble substrate. Pretreatment has a strong impact on hydrolysis, which could be related to both chemical changes and morphological changes of the material. In the current work, the accessibility of four differently pretreated wheat straw substrates, two differently pretreated spruce materials, and Avicel cellulose was investigated. Adsorption isotherms (at 4 °C and 30 °C) for a cellulase preparation were obtained, and the rates of hydrolysis were determined for the different materials. Furthermore, the surface area and pore size distribution of the various materials were measured and compared to adsorption and hydrolysis properties, and the structures of the pretreated materials were examined using scanning electron microscopy (SEM).The results demonstrated a positive correlation between enzyme adsorption and the substrate specific surface area within each feedstock. Overall, the amount of enzyme adsorbed was higher for pretreated spruce than for the pretreated wheat straw, but this was not accompanied by a higher initial rate of hydrolysis for spruce. Also, the difference in the measured endoglucanase adsorption and overall FPU adsorption suggests that a larger fraction of the enzyme adsorbed on spruce was unproductive binding. The SEM analysis of the material illustrated the structural effects of pretreatment harshness on the materials, and suggested that increased porosity explains the higher rate of hydrolysis of more severely pretreated biomass.     

Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris

The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265 U/L and 300 mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145 U/L and 200 mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100 U/L) and protein yield (2.0 g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.     

Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides

In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and β-glucosidase played an important role in mannan hydrolysis. Addition of 10 mg/ml mannan reduced the glucose yield of Avicel (at 20 g/l) from 40.1 to 24.3%. No inhibition of β-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on β-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.     

Effect of hydrothermolysis process conditions on pretreated switchgrass composition and ethanol yield by SSF with Kluyveromyces marxianus IMB4

Hot compressed liquid water was used to treat switchgrass in a method called hydrothermolysis to disrupt lignin, dissolve hemicellulose, and increase accessibility of cellulose to cellulase. Three temperatures (190, 200, and 210 °C) and hold times (10, 15, and 20 min) were tested. Switchgrass treated at 190 °C for 10 min had the greatest xylan recovery in the prehydrolyzate. Less than 0.65 g/L glucose were released into the prehydrolyzate for all pretreatment conditions, indicating most glucose was retained as cellulose in the solid substrate. 5-Hydroxymethylfurfural (HMF) and furfural formation in the prehydrolyzate were found to be less than 1 g/L for all treatments. The highest concentration of ethanol, 16.8 g/L (72% of theoretical), was produced from switchgrass pretreated at 210 °C and 15 min using simultaneous saccharification and fermentation (SSF) at 45 °C with the thermotolerant yeast Kluyveromyces marxianus IMB4 and 15 FPU cellulase/g glucan.     

Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Characterization of enzymatic saccharification for acid-pretreated lignocellulosic materials with different lignin composition

The enzymatic saccharification of three different feedstocks, rice straw, bagasse and silvergrass, which had been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharification was affected by the lignin composition of the raw materials. There was a quantitatively inverse correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4% sulfuric acid. The lignin accounted for about 18.8–21.8% of pretreated rice straw, which was less than the 23.1–26.5% of pretreated bagasse and the 21.5–24.1% of pretreated silvergrass. The maximum glucose yield achieved, under an enzyme loading 6.5 FPU g^?1 DM for 72 h, was close to 0.8 g glucose/g glucan from the enzymatic hydrolysis of the pretreated rice straw; this was twice that from bagasse and silvergrass. A decrease in initial rate of glucose production was observed in all cases when the raw materials underwent enzymatic saccharification with 4% sulfuric acid pretreatment. It is suggested that the higher acid concentration led to an inhibition of β-glucosidase activity. Fourier transform infrared (FTIR) spectroscopy further indicated the chemical properties of the rice straw and silvergrass become more hydrophilic after pretreatment using 2% of sulfuric acid, but the pretreated bagasse tended to become more hydrophobic. The hydrophilic nature of the pretreated solid residues may increase the inhibitive effects of lignin on the cellulase and this could become very important for raw materials such as silvergrass that contain more lignin.     

Ethanol and furfural production from corn stover using a hybrid fractionation process with zinc chloride and simultaneous saccharification and fermentation (SSF)

A two-stage hybrid fractionation process was investigated to produce cellulosic ethanol and furfural from corn stover. In the first stage, zinc chloride (ZnCl₂) was used to selectively solubilize hemicellulose. During the second stage, the remaining treated solids were converted into ethanol using commercial cellulase and Saccharomyces cerevisiae or recombinant Escherichia coli, KO11. This hybrid fractionation process recovered 93.8% of glucan, 89.7% of xylan, 71.1% of arabinan, and 74.9% of lignin under optimal reaction conditions (1st stage: 5% acidified ZnCl₂, 7.5 ml/min, 150 °C (10 min) and 170 °C (10 min); 2nd stage: simultaneous saccharification and fermentation (SSF) using S. cerevisiae). The furfural yield from the hemicellulose hydrolysates was 58%. The SSF of the treated solids resulted in 69–98% of the theoretical maximum ethanol yields based on the glucan content in the treated solids. After fermentation, the solid residues contained primarily lignin. Based on the total lignin in untreated corn stover, the lignin recovery yield was 74.9%.     

Recombinant expression,characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11

An open reading frame (XylX) with 1131 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in E. coli. It encodes a family 11 endoxylanase, designated as XylX, of 41 kDa. The homology of the amino acid sequence deduced from XylX is only 73% identical to the next closest sequence. XylX contains a family 11 catalytic domain of the glycoside hydrolase and a family 6 cellulose-binding module. The recombinant xylanase was fused to a His-tag for affinity purification. The XylX activity was 2392 IU/mg, with a K_m of 6.78 mg/ml and a V_max of 4953 mol/min/mg under optimal conditions (pH 7, 60 °C). At pH 11, 60 °C, the activity was still as high as 517 IU/mg. Xylanase activities at 60 °C under pH 5 to pH 9 remained at more than 69.4% of the initial activity level for 8 h. The addition of Hg²⁺ at 5 mM almost completely inhibited xylanase activity, whereas the addition of tris-(2-carboxyethyl)-phosphine (TCEP) and 2-mercaptoethanol stimulated xylanase activity. No relative activities for Avicel, CMC and d-(+)-cellobiose were found. Xylotriose constitutes the majority of the hydrolyzed products from oat spelt and birchwood xylan. Broad pH and temperature stability shows its application potentials for biomass conversion, food and pulp/paper industries.     

Directed bioconversion of Kraft lignin to polyhydroxyalkanoate by Cupriavidus basilensis B-8 without any pretreatment

This work presents here a new fundamental strategy for bio-converting Kraft lignin (KL) into useful products. Cupriavidus basilensis B-8 (here after B-8) was able to use KL as the sole carbon source. Fully 41.5% of lignin, 37.7% of total carbon (TC) and 43.0% of color were removed after 7 days of incubation. At the same time, lignin was depolymerized into small fragments, which was confirmed by scanning electron microscopy (SEM) and gel permeation chromatography (GPC). Bacterial biomass accumulated to 735.6 mg/L at the initial KL concentration of 5 g L⁻¹, and the corresponding volumetric productivity of polyhydroxyalkanoate (PHA) was 128 mg/L. PHA productivity was significantly improved through fed batch fermentation and reached to 319.4 mg/L. GC–MS analysis showed that PHA polymer was composed of three basic monomers: 98.3 mol% of (S)-3-hydroxy-butanoic acid (S3HB), 1.3 mol% of ^®-3-hydroxybutyric acid (R3HB) and 0.4 mol% of 3-hydroxy-butanoic acid (3HB).     

Enhanced ethanol and chitosan production from wheat straw by Mucor indicus with minimal nutrient consumption

Recently, Mucor indicus was introduced as a promising ethanol producing microorganism for fermentation of lignocellulosic hydrolysates, showing a number of advantages over Saccharomyces cerevisiae. However, high nutrient requirement is the main drawback of the fungus in efficient ethanol production from lignocelluloses. In this study, application of fungal extract as a potential nutrient source replacing all required nutrients in fermentation of wheat straw by M. indicus was investigated. Wheat straw was pretreated with N-methylmorpholine-N-oxide (NMMO) at 120 °C for 1–5 h prior to enzymatic hydrolysis. Hydrolysis yield was improved at least by 6-fold for 3 h pretreated straw compared with that of untreated one. A fungal extract was produced by autolysis of M. indicus biomass, an unavoidable byproduct of fermentation. Maximum free amino nitrogen (2.04 g/L), phosphorus (1.50 g/L), and total nitrogen (4.47 g/L) as well as potassium, magnesium, and calcium in the fungal extract were obtained by autolysis of the biomass at 50 °C and pH 5.0. The fungal extract as a nutrient-rich supplement substituted yeast extract and all other required minerals in fermentation and enhanced the ethanol yield up to 92.1% of the theoretical yield. Besides, appreciate amounts of chitosan were produced as another valuable product of the autolysis.     

Enhanced ethanol production via fermentation of rice straw with hydrolysate-adapted Candida tropicalis ATCC 13803

Neutralized hydrolysate and pretreated rice straw obtained from a 2% (w/v) sulfuric acid pretreatment were mixed at 10% (w/v) and subjected to simultaneous saccharification and co-fermentation (SSCF), with cellulase, β-glucosidase, and Candida tropicalis cells at 15 FPU/g-ds, 15 IU/g-ds and 1 × 10⁹ cells/ml, respectively. A 36-h SSCF with adapted cells resulted in YP/S and ethanol volumetric productivity of 0.36 g/g and 0.57 g/l/h, respectively. In addition to ethanol, insignificant amounts of glycerol and xylitol were also produced. Adapted C. tropicalis cells produced nearly 1.6 times more ethanol than non-adapted cells. Ethanol yield (Yp/s), ethanol volumetric productivity and a xylitol concentration of 0.48 g/g, 0.33 g/l/h and 0.89 g/l, respectively, were produced from fermentation of remaining hydrolysate with adapted C. tropicalis cells. The 0.20 g/g ethanol yield and 77% production efficiency from SSCF of pretreated rice straw indicate scale-up potential for the process. This study demonstrated that C. tropicalis produced ethanol and xylitol from a mixed-sugar stream, although cell adaptation affected ethanol and xylitol yields. Scanning electron microscopy indicated agglomeration of cellulose microfibrils and globular deposition of lignin in acid-pretreated rice straw.     

Horizontal transmission of the Picea glauca foliar endophyte Phialocephala scopiformis CBS 120377

We have studied the presence of the foliar endophtye of Picea glauca (white spruce) Phialocephala scopiformis CBS 120377 and its affect on the growth of Choristoneura fumiferana (spruce budworm). Here we examine the transmission of this fungus from 50 trees planted in a test field site to 250 P. glauca seedlings planted under the emerging canopies. After 3 y, the endophyte spread to 40 % of these trees (now 20–30 cm) with an average rugulosin (an anti-insect toxin) concentration of 1 μg g^?1. All woody plants within 2 m of the test trees were collected. These were all shown to be negative for P. scopiformis except for some spruce seedlings that arose from seeds (natural generation). This is positive evidence for the horizontal transmission of P. scopiformis and its apparent specificity to P. glauca under field conditions.     

Germination responses of Croton macrostachyus (Euphorbiaceae) to various physico-chemical pretreatment conditions

Croton macrostachyus Hochst. ex Del. (Euphorbiaceae) is a multipurpose, deciduous, and medium sized tree of pantropic occurrence. Because the species has numerous useful qualities (e.g., establishment and growth in disturbed sites, drought tolerance, fast growth rate, copious litter/necromass production, suitability for agroforestry, and ability to attract avian frugivores), its speedy restoration has become increasingly critical. Germination studies were therefore conducted on seeds pooled from five widely located provenances with a view to supporting efforts geared toward the speedy propagation and restoration of this valuable tree species. Seed pretreatments were achieved using various dilution levels of plant-derived smoke–water (1:1, 1:10, 1:100 and 1:1000), as well as gibberellic acid (GA₃) or potassium nitrate (KNO₃) ranging in concentration from 0.1 to 100 μmol. The control was to use distilled water for seed pretreatment. Seeds were germinated under either illuminated (ca 60 μmol m^− 2 s^− 1; cool-white fluorescent lamp) or non-illuminated conditions. Experiments on the impact of seed storage durations, as well as storage temperatures were also conducted. The study found that germination percentage (GP: ca 90%), and mean germination time (MGT: 14 days) were significantly (P < 0.001) better when seeds were pretreated with smoke–water and germinated under non-illuminated conditions, than when these were pretreated with various concentrations of GA₃ or KNO₃ (GP and MGT of ca 65% and 20 days, respectively). Germination percentage (GP) and germination vigor (GV) declined with increasing storage-time for all storage temperatures, but GV's decline was faster for seeds stored at 22 °C than for those stored at 5 and 15 °C. On the other hand, mean germination time (MGT) increased significantly (P < 0.01) with seed storage-time of up to 8 months at 5, 15, and 22 °C, but the increase was more marked for seeds stored at 22 °C than for those stored at 5 and 15 °C. From these investigations, it is concluded that germination of C. macrostachyus seeds through use of smoke–water is faster, cheaper, and technically less demanding, compared to that of either GA₃ or KNO₃. The study also concludes that C. macrostachyus is intermediate between orthodox and recalcitrant seeds, and that it is non-photoblastic.     

Effect of hydrolytic lignin on formation of protein–lignin complexes and protein degradation by rumen microbes

Several methods have been developed to protect feed protein from rumen microbial degradation. The current study aimed to evaluate the potential use of an industrial lignin, namely hydrolytic lignin, to protect protein from rumen microbial degradation. The hydrolytic lignins assessed in this study were extracted from wheat straw previously subjected to various steam treatment conditions (pressure: 15, 17 and 19 bar; reaction time: 0, 5 and 10 min; use of acidic catalyst: without and with 2% H₂SO₄ on DM basis). Results indicated that hydrolytic lignin can precipitate protein when measured by a standard bovine serum albumin assay. It was also observed that protein-precipitating capacity of lignin increased with increasing harshness of steam treatment until a point from which no further effect was observed. The effect of lignin upon protein degradation in vitro was clearly detected. Both ammonia nitrogen and iso-acid concentration in vitro were significantly decreased (P<0.01) when lignin was added to fermentation flask containing casein. Unlike tannins, hydrolytic lignins do not inhibit rumen microbial activity. Additionally, it was observed that lignin’s ability to bind and protect protein is a pH-dependent reaction. Protein binding to lignin is markedly reduced at pH<3.0.     

Extraction method for increasing antioxidant activity of raw garlic using steam explosion

Novel extraction method for increasing the antioxidant activity of raw garlic was proposed using steam explosion. Raw garlic was hydrolyzed by high temperature (183–258 °C) and pressure steam (10–45 atm), and then crushed by the rapid decompression. The antioxidant activity of raw garlic treated by steam explosion was higher than that of black garlic, i.e. aging garlic. The lowest EC₅₀ value, i.e. the highest antioxidant activity, of extract from raw garlic was obtained at a steam pressure of 45 atm for a steaming time of 5 min, but the highest amount of phenolic compounds, i.e. 93.7 mg-catechin equiv./g-dry raw garlic, was obtained at a steam pressure of 30 atm for a steaming time of 5 min.     

Seed and forage yield,and forage quality determinants of nine legume shrubs in a non-tropical dryland environment

The objective was to identify legume shrub species for development of agroforestry technologies based on seed and forage (leaves and twigs < 10 mm diameter) yield, and determinants of forage quality. Ten individual plants of Bituminaria bituminosa ‘Ecotypes 1’, B. bituminosa ‘Ecotypes 2’, Medicago citrina, and M. arborea from Spain; Colutea istria and Onobrychis aurantiaca from Syria; C. istria from Jordan; Chamaecytisus mollis from Morocco; and Coronilla glauca from France were randomly selected from plots established in a non-tropical dryland environment in northwest Syria in 2000. Five individual plants of each species were cut back to 0.5 m above ground in March 2004. Coppice regrowths were pruned in December 2004 and April 2005 to determine forage yield and proportion of forage in the total above ground biomass (PEFB). Forage samples were analyzed for concentrations of crude protein (CP), lignin(sa), acid detergent fibre (ADFom), neutral detergent fibre (aNDFom), in vitro organic matter (OM) digestibility (IVOMD), and in vitro 24 h gas production (IVGP24h). Matured seeds were hand harvested from the remaining five plants of each species to estimate seed yield. Forage (21–250 kg DM/ha) and seed (0–200 kg DM/ha) yields; PEFB (0.22–0.96); and concentrations of CP (85–115 g/kg DM), lignin(sa) (14–42 g/kg DM), ADFom (94–170 g/kg DM), aNDFom (122–217 g/kg DM), IVOMD (456–617 g/kg OM), and IVGP24h (27–42 ml 200 mg/DM) varied (P<0.05) among shrub species. The IVOMD and IVGP24h were positively correlated (r = 0.75, P<0.032), whereas IVOMD and IVGP24h were negatively correlated with ADFom, lignin(sa) and aNDFom. In terms of forage and seed yields and determinants of forage quality, C. istria from Jordan, M. arborea, B. bituminosa ‘Ecotype-2’, C. istria and O. aurantiaca have higher potential than C. mollis, C. glauca and B. Bituminosa ‘Ecotype-1’ for the development of agroforestry technologies in non-tropical dry areas.     

Sono-chemical preparation of cellulose nanocrystals from lignocellulose derived materials

This study examined the production of cellulose nanocrystals from microcrystalline wood cellulose, Avicel and recycled pulp of wood pulp using sono-chemical-assisted hydrolysis. Two hydrolysis systems: deionized water and maleic acid were evaluated. In deionized water, Avicel produced cellulose nanocrystals with average diameter of 21 ± 5 nm (minimum 15 nm and maximum 32 nm). Cellulose nanocrystals from recycled pulp were not distinctively spherical and had an average diameter of 23 ± 4 nm (minimum 14 nm and maximum 32 nm). Maleic acid (50 mM) sono-chemical assisted hydrolysis of Avicel at 15 °C and 90% power output for 9 min produced cellulose nanocrystals which were cylindrical in shape and were of dimensions, length 65 ± 19 nm and width 15 nm.