Low Cellulase Loading Research Articles

Biobased lignin-blockers enable efficient production of glucose from lignocelluloses

Enzymatic hydrolysis of cellulose in lignocelluloses exhibits low efficiency due to low cellulose accessibility and adverse lignin effects. By using phenylsulfonic acid pretreatment to improve cellulose accessibility, we have developed several new plant proteins such as corn germ and green rapeseed proteins that extracted from inexpensive defatted meals as lignin-blockers. For pretreated lignocellulosic substrates with high cellulose accessibility, such plant proteins could effectively overcome negative lignin effects and achieve robust cellulose enzymatic conversion (88–97 %) at a low cellulase loading of 5 FPU/g glucan. To reach the same level of cellulose enzymatic conversion, adding corn germ protein could save cellulase loading by 3.6 times as compared to those without lignin-blockers. But these plant proteins showed insignificant promotion effects on substrates with low cellulose accessibility. Overall, this study coupled high cellulose-accessible substrates and efficient lignin-blockers to synergistically improve the profitability of lignocellulosic biorefinery.

Comparison of organosolv pretreatment of masson pine with different solvents in promoting delignification and enzymatic hydrolysis efficiency

Organosolv pretreatment has been verified to facilitate the enzymatic hydrolysis by efficient delignification. Recently, novel solvents derived from biomass have been applied in lignocellulose pretreatment. However, few studies on the pretreatment of softwood with novel solvents have been reported. In this study, organosolv pretreatments with three green solvents, including γ-valerolacton (GVL), tetrahydrofuran (THF), and 2-methyltetrahydrofuran (2-MTHF), were performed on masson pine, and compared with conventional ethanol (EtOH) pretreatment. The results showed that GVL pretreatment exhibited the best performance for removing lignins from masson pine, achieving a pretty high lignin removal ratio of 87.2 % at 150 °C with 75 mM H2SO4. Correspondingly, the highest glucose yield of 76.8 % was observed in the enzymatic hydrolysis of GVL pretreated masson pine with a relatively low cellulase loading (20 FPU/g glucan). The relations between relative energy difference of the solvent-lignin interactions and both of the lignin removal and enzymatic digestibility of organosolv pretreated substrates were evaluated. Furthermore, the structural features of four organosolv lignins were characterized, including molecular weights, functional groups, inter-unit linkages and aromatic information. The results provide a better understanding of organosolv pretreatment effects on enzymatic digestibility of softwood biomass, and give comprehensive lignin properties for valorization of organosolv lignins from softwood.

Enhancing ethylene glycol and ferric chloride pretreatment of rice straw by low-pressure carbon dioxide to improve enzymatic saccharification

Ethylene glycol and ferric chloride pretreatment assisted by low-pressure carbon dioxide (1 MPa CO2) realized the targeted deconstruction of lignocelluloses at 170 °C for 5 min, achieving 98 % cellulose recovery with removal of 92 % lignin and 90 % hemicellulose. After the pretreatment, the formation of stable platform mono-phenol components would be with the destruction of the lignin-carbohydrate complexes structure, and the surface of rice straw became rough, with a less negative charge and higher specific surface area, while the enzyme adsorption rate increased by 8.1 times. Furthermore, the glucose yield of pretreated straw was remarkably increased by 5.6 times that of the untreated straw, reaching 91 % after hydrolyzed for 48 h. With Tween 80 added in concentrated solid (12 %) hydrolysis at low cellulase loading (3 FPU/g dry substrate), half of the hydrolysis time was shortened than that without Tween 80, with 45 % higher glucose yield.

Extracting high β-O-4 content lignin and by-producing substrate susceptible to enzymatic hydrolysis by a green flow through process

Maleic acid (MA), a solid dicarboxylic acid can be obtained from renewable lignocellulose, was used to extract lignin through flow through (FT) process for the first time. Due to the high boiling point and flash point of MA, the FT process was carried out at atmospheric pressure without VOCs release and explosion risk. 50 wt% MA aqueous solution could extract 60 % lignin from birch wood, and the dilution factor for precipitating lignin was only 3. MA-extracted lignin preserved more than 80 % of βO4 bonds and a monophenol yield (＞30 %) comparable to that of milled wood lignin was obtained by catalytic hydrogenolysis. The recyclability of MA was demonstrated through three rounds of lignin extraction by recovered MA. And the cellulose-rich residue could be efficiently converted into glucose (glucose yield＞90 %) at a low cellulase loading of 10 FPU/g glucan due to the rich carboxyl groups of lignin. In addition, the low corrosiveness of MA and the mild operating conditions make the process have strong industrial application prospects.

Enhanced enzymatic hydrolysis of poplar cellulosic residue fractionated by a magnetic carbon-based solid-acid catalyst in the γ-valerolactone–water system

The conventional pretreatment method of poplar comprises multiple steps, including different procedures for fractionating hemicellulose and lignin separately. In our study, hemicellulose and lignin were removed simultaneously by a one-step method. In the γ-valerolactone (GVL)–water environment, the cellulose retention, hemicellulose removal, and lignin removal rates of 84.94%, 89.08%, and 72.28%, respectively, were achieved over a magnetic carbon-based solid acid (MMCSA) catalyst, under best conditions (160 °C, 30 min, 2 g of poplar, 2 g of MMCSA, 35 mL of GVL, and 15 mL of water). The pretreatment of fresh poplar in the reused MMCSA–GVL–water environment showed similar fractionation results as the first time. Scanning electron microscopy characterization of the cellulosic residue revealed the presence of noticeable structural fragmentation. Brunauer–Emmett–Teller characterization showed that the total pore volume of the residue was 2.13 times that of the raw material. The above features of the residue confirmed the high enzymatic hydrolysis potential of the pretreated residue. The enzymatic hydrolysis efficiency of the poplar residue was 67% at a cellulase loading of 20 FPU/g cellulose in dry matter, and it increased to 77.02% at 40 FPU/g cellulose in dry matter. Interestingly, the addition of Tween 80 did not improve the enzymatic hydrolysis efficiency at high cellulase loadings (30 and 40 FPU/g cellulose in dry matter) compared to the case at low cellulase loadings. The relative mechanisms were also analyzed. In this study, a one-step pretreatment method comprising the MMCSA–GVL system for the catalytic depolymerization of poplar wood was developed. The system was verified to be very effective for the subsequent enzymatic hydrolysis of the residues.

Efficiency enhancement of a new cellulase cocktail at low enzyme loading for high solid digestion of alkali catalyzed atmospheric glycerol organosolvent pre-treated sugarcane bagasse

The acquisition during biomass saccharification of elevated levels of fermentable sugars with lower cellulase concentration is central to ensuring an economically viable and industrially relevant hydrolytic process. Thus, using a new cellulase preparation (LT4) at low cellulase loading (2 mg protein/g dried substrate), this study assessed the possible boosting effect of integrating accessory enzymes and additives on high-solids hydrolysis of sugarcane bagasse via fed-batch feeding. Hydrolysis which commenced with initial 8% solids loading and subsequent substrate feeding of 4% solids at 6 h, 18 h, and 24 h respectively, proved optimal for the 20% high-solids saccharification producing 158 g/L total sugars and 83% glucose yield after 72 h with the combined optimized additives and accessory enzymes. The results obtained indicate that the integration of accessory enzymes and additives offers a benignant approach to minimizing the enzyme load and cost of high solids saccharification of lignocellulosic heteropolymers while also boosting enzyme hydrolytic performance.

Understanding the promoting effect of non-catalytic protein on enzymatic hydrolysis efficiency of lignocelluloses

Lignin deposits formed on the surface of pretreated lignocellulosic substrates during acidic pretreatments can non-productively adsorb costly enzymes and thereby influence the enzymatic hydrolysis efficiency of cellulose. In this article, peanut protein (PP), a biocompatible non-catalytic protein, was separated from defatted peanut flour (DPF) as a lignin blocking additive to overcome this adverse effect. With the addition of 2.5 g/L PP in enzymatic hydrolysis medium, the glucose yield of the bamboo substrate pretreated by phenylsulfonic acid (PSA) significantly increased from 38 to 94% at a low cellulase loading of 5 FPU/g glucan while achieving a similar glucose yield required a cellulase loading of 17.5 FPU/g glucan without PP addition. Similar promotion effects were also observed on the n-pentanol-pretreated bamboo and PSA-pretreated eucalyptus substrates. The promoting effect of PP on enzymatic hydrolysis was ascribed to blocking lignin deposits via hydrophobic and/or hydrogen-bonding interactions, which significantly reduced the non-productive adsorption of cellulase onto PSA lignin. Meanwhile, PP extraction also facilitated the utilization of residual DPF as the adhesive for producing plywood as compared to that without protein pre-extraction. This scheme provides a sustainable and viable way to improve the value of woody and agriculture biomass.Peanut protein, a biocompatible non-catalytic protein, can block lignin, improve enzymatic hydrolysis efficiency and thereby facilitate the economics of biorefinery.Graphical abstract

Comparison of Two Acid Hydrotropes for Sustainable Fractionation of Birch Wood.

This study reports on a comparative study of acid hydrotropic fractionation (AHF) of birch wood using maleic acid (MA) and p-toluenesulfonic acid (p-TsOH). Under the same level of delignification, lignin dissolved by MA is much less condensed with a higher content of ether aryl β-O-4 linkages. Lignin depolymerization dominated in MA hydrotropic fractionation (MAHF) and resulted in a single lower molecular weight peak, in contrast to the competitive depolymerization and repolymerization in p-TsOH AHF with a bimodal distribution. The less condensed MA-dissolved lignin facilitated catalytic conversion to monophenols. Carboxylation of residual lignin in fractionated cellulosic water-insoluble solids (WISs) enhanced enzymatic saccharification by decreasing nonproductive cellulase binding to lignin. At a low cellulase loading of 10 FPU g-1 glucan, saccharification of WIS-MT120 from MAHF at 120 °C was 95 % compared with 48 % for WIS-PT85 from p-TsOH AHF at 85 °C under the same level of delignification of 63 %. Residual lignin carboxylation also facilitated nanofibrillation of WIS for producing lignin-containing cellulose nanofibrils (LCNFs) through an enhanced lignin lubrication effect, which substantially decreases fibrillation energy. LCNFs from only one pass of microfluidization of WIS-MT120 have the same morphology as those from WIS-PT85 after three passes. MA also has a lower solubility and higher minimal hydrotropic concentration, which facilitated acid recovery. MA is U.S. Food and Drug Administration (FDA)-approved as an indirect food additive, affording significant advantages compared with p-TsOH for biorefinery applications.

Fed-batch high-solids enzymatic saccharification of lignocellulosic substrates with a combination of additives and accessory enzymes

The operation of high-solids enzymatic saccharification at low cellulase loading is required for the commercialization of the current cellulase-mediated lignocellulosic bioethanol. In this study, high-solids (30 %) fed-batch enzymatic saccharification at low cellulase loading (3 FPU/g) was attempted by fed-batch mode with a mixture of additives and accessory enzymes. The saccharification process began with 10 % (w/v) solids content and 6 %, 6 %, and 8 % of substrates were fed at 6, 12, and 24 h to achieve 30 % (w/v) of the total solids content. With the optimization of additives, namely 40 mg/g Tween 80, 30 mg/g tea saponin, 8 mg/g BSA, and 2.4 mg/g xylanase and 0.4 mg/g AA9 as accessory enzymes, the saccharification process released 180 g/L of fermentable sugars with 70 % glucose yield after 72 h. The fed-batch mode along with the mixtures of some additives and accessory enzymes has been found to be a beneficial approach to run a high-solids enzymatic saccharification at low enzyme loading.

Integrating Benzenesulfonic Acid Pretreatment and Bio-Based Lignin-Shielding Agent for Robust Enzymatic Conversion of Cellulose in Bamboo.

A hydrotrope-based pretreatment, benzenesulfonic acid (BA) pretreatment, was used to fractionate bamboo in this work. With optimized content (80 wt %) of BA in pretreatment liquor, about 90% of lignin and hemicellulose could be removed from bamboo under mild conditions (95 °C, 30 min or 80 °C, 60 min). The potential accessibility of BA pretreated substrate to cellulase was thus significantly improved and was also found to be much higher than those of acidic ethanol and dilute acid pretreatments. But the deposition of lignin on the surface of solid substrates, especially the BA pretreated substrate, was also observed, which showed a negative effect on the enzymatic hydrolysis efficiency. The addition of inexpensive soy protein, a bio-based lignin-shielding agent, could readily overcome this negative effect, leading the increase of enzymatic conversion of cellulose in BA pretreated substrate from 37% to 92% at a low cellulase loading of 4 FPU/g glucan. As compared to acidic ethanol and dilute acid pretreatments, the combination of BA pretreatment and soy protein could not only stably improve the efficiency of non-cellulose components removal, but also could significantly reduce the loading of cellulase.

Fractionation of corn stover for efficient enzymatic hydrolysis and producing platform chemical using p-toluenesulfonic acid/water pretreatment

Aromatic sulfonic acid, p-toluenesulfonic acid (p-TsOH), was employed to pretreat the corn stover in aqueous for improving the enzymatic hydrolysis of cellulose-enriched residue, and was compared to ethanol/water pretreatment. The glucose yield of corn stover from p-TsOH/water pretreatment (97.6 %) was higher than that from ethanol/water pretreatment (89.5 %) after 96 h hydrolysis with high cellulase loading of 15 FPU/g-cellulose. More than 80 % of glucose yield was still obtained with low cellulase loading (7.5 FPU/g-cellulose) after p-TsOH/water pretreatment, while only 65.2 % was obtained after ethanol/water pretreatment. Composition and structure analysis showed that p-TsOH/water pretreatment severely break the fibre, removed more lignin (especially of surface lignin), well increased cellulose accessibility, and resulted in more hydroxyl exposed on the surface. Moreover, more monomeric sugars (xylose and glucose) derived from hemicellulose were released after p-TsOH/water pretreatment, and they could be converted to valuable platform chemical of levulinic acid by simply heating the prehydrolysate.

High-concentrated substrate enzymatic hydrolysis of pretreated rice straw with glycerol and aluminum chloride at low cellulase loadings

Rice straw was pretreated with glycerol and AlCl3 for enzymatic hydrolysis at low cellulase loadings. Based on a central composite design, 83% delignification, 94% hemicellulose removal, and 92% cellulose recovery (or 76% cellulose in solid residue) were achieved under the optimized pretreatment conditions (0.08 mol/L AlCl3 as catalyst at 146.8 °C for 20 min with 90% glycerol). During glycerol-AlCl3 pretreatment, the lignin-carbohydrate complex was depolymerized, resulting in the complex and recalcitrant construction of straw effectively being destroyed. The enzyme adsorption ability of pretreated straw was 16.5 times that for the original sample. After pretreatment, glucose yield was increased by 2.4 times to 74% for 48 h. Moreover, concentrated solid (15%) with low cellulase loading (3.3 FPU/g dry substrate) achieved 58.6% glucose yield, and further increased by 12% to 65.7% by adding Tween 80. Glycerol-AlCl3 pretreatment was a promising approach to realize high-concentrated solid hydrolysis for sugars at low cellulase loadings.

Near-complete removal of non-cellulosic components from bamboo by 1-pentanol induced organosolv pretreatment under mild conditions for robust cellulose enzymatic hydrolysis

Based on Hildebrand solubility parameter (δH) theory, a novel organosolv pretreatment medium (OPM), 1-pentanol (80%, v/v)–water mixture with a δH value of 27.4 (J/cm3)−1/2, was discovered. This OPM achieved near-complete removal of non-cellulosic components (hemicellulose 96.2% and lignin 87.8%) from bamboo under mild conditions [130 °C, 20 min, 4% (w/w) H2SO4]. For the OPMs with similar δH values [DMSO (80%, v/v)- and 1-pentanol (80%, v/v)-water, 30.9 vs. 27.4 (J/cm3)−1/2] to that of bamboo lignin [28.3 (J/cm3)−1/2] used in this study, the effect of the δh value (contribution of hydrogen-binding force to the δH) on lignin removal was also discussed. After overcoming the negative effect of lignin deposits on enzymes with biocompatible soy protein (SP), a decent cellulose enzymatic conversion (EC) of 92.6% was achieved by 1-pentanol (80%, v/v)-water induced organosolv pretreatment at a low cellulase loading (5 FPU/g glucan). This cellulose EC was much higher than those of other OPMs (< 63%) with the δH values very different from that of bamboo lignin. Integration of organosolv pretreatment with an ideal OPM and enzymatic hydrolysis using SP as an additive can be thereby viewed as a viable way to achieve the robust cellulose EC at low enzyme loading, which shows great potential for improving the economics of organosolv pretreatment-based lignocellulose biorefinery.

Enhanced High-Solids Fed-Batch Enzymatic Hydrolysis of Sugar Cane Bagasse with Accessory Enzymes and Additives at Low Cellulase Loading

High cellulase loading is still a major impediment in the production of fermentative sugars from high-solids enzymatic hydrolysis of lignocellulosic substrates in the enzyme-based “biorefinery” industry. This study attempted a high-solids (20%) enzymatic hydrolysis of lignocellulosic substrate at a very low cellulase loading with mixed use of additives and accessory enzymes by fed-batch mode. To avoid the high initial biomass viscosity, the high-solids enzymatic hydrolysis of lignocellulosic substrates was initiated with a solids content of 8%. Thereafter, 4% of the additional substrates were consecutively fed into the hydrolysis system after 6, 12, and 18 h to reach a final solids content of 20%. Some additive mixtures (40 mg/g substrateTween 80 + 10 mg/g substrate tea saponin +20 mg/g substrate BSA) were observed to enable this fed-batch hydrolysis to increase 30% of the glucose yield after the 48 h. The combination of these additives and accessory enzymes (2.4 mg/g substrate xylanase and 1 mg/g substra...

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches

BackgroundNew biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis.ResultsSugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O-methyl-glucuronic acid and p-coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio.ConclusionsHemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

Mild pretreatment of yellow poplar biomass using sequential dilute acid and enzymatically-generated peracetic acid to enhance cellulase accessibility

Biomass contains cellulose, xylan and lignin in a complex interwoven structure that hinders enzymatic hydrolysis of the cellulose. To separate these components in yellow poplar biomass, we sequentially pretreated with dilute sulfuric acid and enzymatically-generated peracetic acid. In the first step, the dilute acid with microwave heating (140°C, 5 min) hydrolyzed 90% of xylan. The xylose yield in hydrolysate after dilute acid pretreatment was 83.1%. In the second step, peracetic acid (60°C, 6 h) removed up to 80% of lignin. This sequential pretreatment fractionated biomass into xylan and lignin, leaving a solid residue enriched in cellulose (~80%). The sequential pretreatment enhanced enzymatic digestibility of the cellulase by removal of the other components in biomass. The glucose yield after enzymatic hydrolysis was 90.5% at a low cellulase loading (5 FPU/g of glucan), which is 1.6 and 18 times higher than for dilute acid-pretreated biomass and raw biomass, respectively. This novel sequential pretreatment with dilute acid and peracetic acid efficiently separates the three major components of yellow poplar biomass, and reduces the amount of cellulase needed.

Bioprocessing of tea oil fruit hull with acetic acid organosolv pretreatment in combination with alkaline H2O2

BackgroundAs a natural renewable biomass, the tea oil fruit hull (TOFH) mainly consists of lignocellulose, together with some bioactive substances. Our earlier work constructed a two-stage solvent-based process, including one aqueous ethanol organosolv extraction and an atmospheric glycerol organosolv (AGO) pretreatment, for bioprocessing of the TOFH into diverse bioproducts. However, the AGO pretreatment is not as selective as expected in removing the lignin from TOFH, resulting in the limited delignification and simultaneously high cellulose loss.ResultsIn this study, acetic acid organosolv (AAO) pretreatment was optimized with experimental design to fractionate the TOFH selectively. Alkaline hydrogen peroxide (AHP) pretreatment was used for further delignification. Results indicate that the AAO–AHP pretreatment had an extremely good selectivity at component fractionation, resulting in 92% delignification and 88% hemicellulose removal, with 87% cellulose retention. The pretreated substrate presented a remarkable enzymatic hydrolysis of 85% for 48 h at a low cellulase loading of 3 FPU/g dry mass. The hydrolyzability was correlated with the composition and structure of substrates by using scanning electron microscopy, confocal laser scanning microscopy, and X-ray diffraction.ConclusionThe mild AAO–AHP pretreatment is an environmentally benign and advantageous scheme for biorefinery of the agroforestry biomass into value-added bioproducts.

Solvent-based delignification and decrystallization of wheat straw for efficient enzymatic hydrolysis of cellulose and ethanol production with low cellulase loadings

Several solvents and ionic liquids, namely formic acid (Formiline process), concentrated phosphoric acid (CPA), N-methylmorpholine-N-oxide (NMMO) and 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), were used to pretreat wheat straw in order to increase cellulose digestibility for ethanol production. When being directly used to pretreat the raw wheat straw under corresponding optimized conditions, the improvement of cellulose hydrolyzability followed the order of CPA > Formiline > NMMO > [AMIM]Cl. However, when Formiline pretreated (delignified) substrates were further post-treated by the above cellulose solvents, the cellulose digestibility was significantly improved particularly with low cellulase loadings. Cellulose solvent post-treatment resulted in deconstruction of hydrogen-bond networking, alteration of cellulose polymorphs, decrease in crystallinity, depolymerization of cellulose chain with dramatic reduction in particle size, thus greatly increasing cellulose accessible surface area. CPA post-treatment showed the best efficacy. Semi-simultaneous scarification and fermentation (sSSF) of a CPA post-treated substrate obtained an ethanol concentration of 41.6 g L−1 with 91.2% of yield at a relatively low cellulase loading (5 FPU per g solid) within 24 h incubation. A biorefining process was proposed based on Formiline pretreatment coupled with CPA post-treatment to achieve a co-production of ethanol, furfural and high-purity lignin, which greatly increases the potential revenue of the process.

Saccharification of Acacia auriculiformis Benth. biomass for the production of glucose using phosphoric acid pretreatment

Acacia auriculiformis is an exotic plant species luxuriantly grown in different parts of India. Because of its drought tolerance potential and wood quality, it is widely grown as timber crops in some part of the country. In this study we have addressed the possible use of A.auriculiformis for production of glucose, an intermediate for the production of ethanol. Chemical composition of this species was carried out and it was found that this species contains about 34 % glucan. Using Cellulose –Solvent and Organic-solvent based Lignocellulose Fractionation pretreatment the biomass of the species was hydrolyzed at two different dosages of cellulase. It was found that even at low cellulase loadings [1 Filter Paper Unit (FPU)] about 10 International Units (IU) of β-glucosidase about 88 % glucan conversion took place. This study suggests the use of this species as a biofuel feedstock for next generation fuels.

Addition of expansin to cellulase enhanced bioethanol production

Abstract A metagenomic clone (pUC-189) that exhibited clear zone on CMC agar plate showed homology with glycosyl hydrolase (GH) family 12 endoglucanase. Purified recombinant cellulase (C18) exhibited the highest activity towards CMC followed by Avicel PH101 and waste paper which was confirmed by chromatography. Role of expansin (E11CB) was investigated to address the poor activity of C18 towards waste paper. The Langmuir isotherm for binding of the purified E11CB to waste paper showed a good fit (R 2 = 0.97). Recombinant E11CB protein bound to waste paper was detected in situ by UV fluorescence of [Ln(DPA) 3 ] 3− complex. Scanning electron microscopy confirmed that binding of E11CB transformed smooth microfibrils into rough and amorphic surface. Improvement in the enzymatic hydrolysis of pure cellulose and lignocelluloses was observed after treatment with purified E11CB at low cellulase loading. The enzymatic hydrolysate of waste cellulose paper was then used as the substrate for bioethanol production by Saccharomyces cerevisiae.