The production of lactic acid from chemi-thermomechanical pulps using a chemo-catalytic approach.

Previous work has shown that sulfonation and oxidation of chemi-thermomechanical pulps (CTMPs) significantly enhanced enzyme accessibility to cellulose while recovering the majority of carbohydrates in the water-insoluble component. In the work reported here, modified (sulfonated and oxidized) CTMPs derived from hard-and-softwoods were used to produce a DL-mix of lactic acid via a chemo-catalytic approach using lanthanide triflate (Ln (OTf)3) catalysts (Ln = La, Nd, Er, and Yb). It was apparent that sulfonation and oxidation of chemi-thermomechanical pulps (CTMPs) also enhanced Ln(OTf)3 catalyst accessibility to the carbohydrate components of the pulps, with the Er(OTf)3 catalysts resulting in significant lactic acid production. Under optimum conditions (250 °C, 60 min, 0.5 mmol catalyst g-1 biomass), 72% and 67% of the respective total carbohydrate present in the hard-and-softwood CTMPs could be converted to lactic acid compared to the respective 59% and 51% yields obtained after energy-intensive ball milling.

Enhancing Enzyme-Mediated Cellulose Hydrolysis by Incorporating Acid Groups Onto the Lignin During Biomass Pretreatment.

Lignin is known to limit the enzyme-mediated hydrolysis of biomass by both restricting substrate swelling and binding to the enzymes. Pretreated mechanical pulp (MP) made from Aspen wood chips was incubated with either 16% sodium sulfite or 32% sodium percarbonate to incorporate similar amounts of sulfonic and carboxylic acid groups onto the lignin (60 mmol/kg substrate) present in the pulp without resulting in significant delignification. When Simon's stain was used to assess potential enzyme accessibility to the cellulose, it was apparent that both post-treatments enhanced accessibility and cellulose hydrolysis. To further elucidate how acid group addition might influence potential enzyme binding to lignin, Protease Treated Lignin (PTL) was isolated from the original and modified mechanical pulps and added to a cellulose rich, delignified Kraft pulp. As anticipated, the PTLs from both the oxidized and sulfonated substrates proved less inhibitory and adsorbed less enzymes than did the PTL derived from the original pulp. Subsequent analyses indicated that both the sulfonated and oxidized lignin samples contained less phenolic hydroxyl groups, resulting in enhanced hydrophilicity and a more negative charge which decreased the non-productive binding of the cellulase enzymes to the lignin.

Non-productive celluase binding onto deep eutectic solvent (DES) extracted lignin from willow and corn stover with inhibitory effects on enzymatic hydrolysis of cellulose.

In this work, deep eutectic solvent (DES) was prepared by mixing choline chloride (ChCl) with lactic acid (LA), and effects of cellulase non-productive binding onto DES-extracted lignin from willow and corn stover on enzymatic hydrolysis of cellulose was investigated. The correlation between hydrolysis yield of cellulose and chemical features of lignin was evaluated, and a potential inhibitory mechanism was proposed. Condensation of lignin was observed during DES treatment, and these condensed aromatic structures had an increased tendency to adsorb enzymes through hydrophobic interactions. As well as hydrophobic interactions mediated by lignin condensation, an increase in phenolic hydroxyl groups resulted in a greater amount of hydrogen bonds between cellulases and lignin that appeared to inhibit enzymatic hydrolysis yields of cellulose (39.96-42.86 % to 31.96-32.68 %). Although large amounts of COOHs were generated, the elevated electrostatic repulsion as a result of ionic groups was insufficient to decrease non-productive adsorption.

Enzyme-Mediated Lignocellulose Liquefaction Is Highly Substrate-Specific and Influenced by the Substrate Concentration or Rheological Regime.

The high viscosities/yield stresses of lignocellulose slurries makes their industrial processing a significant challenge. However, little is known regarding the degree to which liquefaction and its enzymatic requirements are specific to a substrate's physicochemical and rheological properties. In the work reported here, the substrate- and rheological regime-specificities of liquefaction of various substrates were assessed using real-time in-rheometer viscometry and offline oscillatory rheometry when hydrolyzed by combinations of cellobiohydrolase (Trichoderma reesei Cel7A), endoglucanase (Humicola insolens Cel45A), glycoside hydrolase (GH) family 10 xylanase, and GH family 11 xylanase. In contrast to previous work that has suggested that endoglucanase activity dominates enzymatic liquefaction, all of the enzymes were shown to have at least some liquefaction capacity depending on the substrate and reaction conditions. The contribution of individual enzymes was found to be influenced by the rheological regime; in the concentrated regime, the cellobiohydrolase outperformed the endoglucanase, achieving 2.4-fold higher yield stress reduction over the same timeframe, whereas the endoglucanase performed best in the semi-dilute regime. It was apparent that the significant differences in rheology and liquefaction mechanisms made it difficult to predict the liquefaction capacity of an enzyme or enzyme cocktail at different substrate concentrations.

Laccase-mediated hydrophilization of lignin decreases unproductive enzyme binding but limits subsequent enzymatic hydrolysis at high substrate concentrations.

One of the predominant mechanisms by which lignin restricts effective enzymatic deconstruction of lignocellulosic materials is the unproductive adsorption of enzymes. Although this inhibition can be partially mitigated through hydrophilization of lignin during thermochemical pretreatment, these types of treatments could potentially worsen slurry rheology, consequently making it more difficult to process the material at high substrate concentrations. In the work reported here, laccases were used to specifically modify lignin hydrophilicity within steam-pretreated substrate via in situ phenolic compound grafting. While lignin hydrophilization reduced unproductive enzyme adsorption, high-solids hydrolysis efficiency decreased significantly due to mass transfer limitations. It was apparent that low-solids hydrolysis experiments were a poor predictor of substrate digestibility at high-solids conditions and that substrate-water interactions impacted both substrate digestibility and slurry rheology.

The influence of lignin migration and relocation during steam pretreatment on the enzymatic hydrolysis of softwood and corn stover biomass substrates.

To be effective, steam pretreatment is typically carried out at temperatures/pressures above the glass transition point (Tg) of biomass lignin so that it can partly fluidize and relocate. The relocation of Douglas-fir and corn stover derived lignin was compared with the expectation that, with the corn stover lignin's lower hydrophobicity and molecular weight, it would be more readily fluidized. It was apparent that the Tg of lignin decreased as the moisture increased, with the easier access of steam to the corn stover lignin promoting its plasticization. Although the softwood lignin was more recalcitrant, when it was incorporated onto filter paper, it too could be plasticized, with its relocation enhancing enzymatic hydrolysis. When lignin recondensation was minimized, the increased hydrophobicity suppressed lignin relocation. It was apparent that differences in the accessibility of the lignin present in Douglas-fir and corn stover to steam significantly impacted lignin fluidization, relocation, and subsequent cellulose hydrolysis.

Lignin valorization: lignin nanoparticles as high-value bio-additive for multifunctional nanocomposites.

BACKGROUND: Although conversion of low value but high-volume lignin by-product to its usable form is one of the determinant factors for building an economically feasible integrated lignocellulose biorefinery, it has been challenged by its structural complexity and inhomogeneity. We and others have shown that uniform lignin nanoparticles can be produced from a wide range of technical lignins, despite the varied lignocellulosic biomass and the pretreatment methods/conditions applied. This value-added nanostructure lignin enriched with multifunctional groups can be a promising versatile material platform for various downstream utilizations especially in the emerging nanocomposite fields. RESULTS: Inspired by the story of successful production and application of nanocellulose biopolymer, two types of uniform lignin nanoparticles (LNPs) were prepared through self-assembling of deep eutectic solvent (DES) and ethanol-organosolv extracted technical lignins derived from a two-stage fractionation pretreatment approach, respectively. Both LPNs exhibited sphere morphology with unique core-shell nanostructure, where the DES-LNPs showed a more uniform particle size distribution. When incorporated into the traditional polymeric matrix such as poly(vinyl alcohol), these LPN products displayed great potential to formulate a transparent nanocomposite film with additional UV-shielding efficacy (reached ~80% at 400 nm with 4 wt% of LNPs) and antioxidant functionalities (reached ~160 µm mol Trolox g-1 with 4 wt% of LNPs). At the same time, the abundant phenolic hydroxyl groups on the shell of LNPs also provided good interfacial adhesion with PVA matrix through the formation of hydrogen bonding network, which further improved the mechanical and thermal performances of the fabricated LNPs/PVA nanocomposite films. CONCLUSIONS: Both LNPs are excellent candidates for producing multifunctional polymer nanocomposites using facile technical route. The prepared transparent and flexible LNPs/PVA composite films with high UV-shielding efficacy, antioxidant activity, and biocompatibility are promising in the advanced packaging field, which potentially provides an additional high-value lignin product stream to the lignocellulose biorefinery. This study could open the door for the production and application of novel LNPs in the nascent bioeconomy.Graphical abstractLignin nanoparticle for transparent nanocomposite film with UV-shielding efficacy.

A comparison of various lignin-extraction methods to enhance the accessibility and ease of enzymatic hydrolysis of the cellulosic component of steam-pretreated poplar.

BACKGROUND: Current single-stage delignification-pretreatment technologies to overcome lignocellulosic biomass recalcitrance are usually achieved at the expense of compromising the recovery of the polysaccharide components, particularly the hemicellulose fraction. One way to enhance overall sugar recovery is to tailor an efficient two-stage pretreatment that can pre-extract the more labile hemicellulose component before subjecting the cellulose-rich residual material to a second-stage delignification process. Previous work had shown that a mild steam pretreatment could recover >65% of the hemicellulose from poplar while limiting the acid-catalysed condensation of lignin. This potentially allowed for subsequent lignin extraction using various lignin solvents to produce a more accessible cellulosic substrate. RESULTS: A two-stage approach using steam and/or solvent pretreatment was assessed for its ability to separate hemicellulose and lignin from poplar wood chips while providing a cellulose-rich fraction that could be readily hydrolysed by cellulase enzymes. An initial steam-pretreatment stage was performed over a range of temperatures (160-200 °C) using an equivalent severity factor of 3.6. A higher steam temperature of 190 °C applied over a shorter residence time of 10 min effectively solubilized and recovered 75% of the hemicellulose while enhancing the ability of various solvents [deep eutectic solvent (DES), ethanol organosolv, soda/anthraquinone (soda/AQ) or a hydrotrope] to extract lignin in a second stage. When the second-stage treatments were compared, the mild DES treatment (lactic acid and betaine) at 130 °C, removed comparable amounts of lignin with higher selectivity than did the soda/AQ and organosolv pretreatments at 170 °C. However, the cellulose-rich substrates obtained after the second-stage organosolv and soda/AQ pretreatments showed the highest cellulose accessibility, as measured by the Simon's staining technique. They were also the most susceptible to subsequent enzymatic hydrolysis. CONCLUSIONS: The second-stage pretreatments varied in their ability to solubilize and extract the lignin component of steam-pretreated poplar while enhancing the enzymatic hydrolysis of the resulting cellulose-rich residual fractions. Although DES extraction was more selective in extracting lignin from the steam-pretreated substrates, the organosolv and soda/AQ post treatments disrupted the cellulose structure to a greater extent while enhancing the ease of enzymatic hydrolysis. Graphical abstractEffective hemicellulose removal via steam pretreatment followed by subsequent lignin extraction under acidic, alkaline or solvolytic conditions results in a highly accessible, more readily hydrolysed cellulose fraction.

The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis.

With the goal of enhancing overall carbohydrate recovery and reducing enzyme loading refiner mechanical pulping and steam pretreatment (210°C, 5 min) were used to pretreat poplar wood chips. Neutral sulphonation post-treatment indicated that, although the lignin present in the steam pretreated substrate was less reactive, the cellulose-rich, water insoluble component was more accessible to cellulases and Simons stain. This was likely due to lignin relocation as the relative surface lignin measured by X-ray photoelectron spectroscopy increased from 0.4 to 0.8. The integration of sulphite directly into steam pretreatment resulted in the solubilisation of 60% of the lignin while more than 80% of the carbohydrate present in the original substrate was recovered in the water insoluble fraction after Na2CO3 addition. More than 80% of the sugars present in the original cellulose and xylan could be recovered after 48 h using an enzyme loading of 20 mg protein/g cellulose at a 10% substrate concentration.

Steam pretreatment of agricultural residues facilitates hemicellulose recovery while enhancing enzyme accessibility to cellulose.

The origins of lignocellulosic biomass and the pretreatment used to enhance enzyme accessibility to the cellulosic component are known to be strongly influenced by various substrate characteristics. To assess the impact that fibre properties might have on enzymatic hydrolysis, seven agricultural residues were characterised before and after steam pretreatment using a single pretreatment condition (190°C, 5min, 3% SO2) previously shown to enhance fractionation and hydrolysis of the cellulosic component of corn stover. When the fibre length, width and coarseness, viscosity, water retention value and cellulose crystallinity were monitored, no clear correlation was observed between any single substrate characteristic and the substrate's ease of enzymatic hydrolysis. However, the amount of hemicellulose that was solubilised during pretreatment correlated (r(2)=0.98) with the effectiveness of enzyme hydrolysis of each pretreated substrate. Simons's staining, to measure the cellulose accessibility, showed good correlation (r(2)=0.83) with hemicellulose removal and the extent of enzymatic hydrolysis.

Fibre size does not appear to influence the ease of enzymatic hydrolysis of organosolv-pretreated softwoods.

To determine the effect of fibre size on enzymatic hydrolysis, organosolv-pretreated lodgepole pine was size-fractionated into six substrates ranging in average size from 0.20 to 3.4mm. Other than the fines fraction (<0.2mm) which contained most of the lignin, the fractionated substrates were more readily hydrolyzed than the original substrate with nearly complete hydrolysis after 72 h at 5 FPU g(-1) cellulose. Surprisingly, fibre size was found to have little influence on enzymatic hydrolysis likely due to similarities in the substrates' chemical composition, accessible surface area, cellulose crystallinity and degree of polymerization. To determine the influence of the fines on enzymatic hydrolysis, their content was artificially increased (from 8.9% to 55.4%) however; this did not have a noticeable effect. These results show that within the range of fibre sizes tested, other substrate characteristics likely play a more significant role in the ease of hydrolysis of pretreated substrates.

The influence of pretreatment and enzyme loading on the effectiveness of batch and fed-batch hydrolysis of corn stover.

To try to improve hydrolysis yields at elevated solids loadings, a comparison was made between batch and fed-batch addition of fresh substrate at the initial and later phases of hydrolysis. Both ethanol (EPCS) and steam-pretreated corn stover (SPCS) substrates were tested at low (5 FPU) and high (60 FPU) loadings of cellulase per gram of cellulose. The fed-batch addition of fresh substrate resulted in a slight decrease in hydrolysis yields when compared with the corresponding batch reactions. A 72-h hydrolysis of the SPCS substrate resulted in a hydrolysis yield of 66% compared with 51% for the EPCS substrate. When the enzyme adsorption and substrate characteristics were assessed during batch and fed-batch hydrolysis, it appeared that the irreversible binding of cellulases to the more recalcitrant original substrate limited their access to the freshly added substrate. After 72-h hydrolysis of the SPCS substrate at low enzyme loadings, ∼40-50% of the added cellulases were desorbed into solution, whereas only 20% of the added enzyme was released from the EPCS substrate. Both simultaneous and sequential treatments with xylanases and cellulases resulted in an up to a 20% increase in hydrolysis yields for both substrates at low enzyme loading. Simons' stain measurements indicated that xylanase treatment increased cellulose access, thus facilitating cellulose hydrolysis.

The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings.

Organosolv-pretreated Lodgepole pine substrates were physically and chemically treated to increase their hydrophilicity and swelling as these are two substrate attributes which have been shown to improve cellulolytic hydrolysis. Surprisingly, mechanical treatment of the organosolv-treated substrates by PFI-mill refining did not significantly increase hydrolysis yields despite decreases in particle size and crystallinity and increases in swelling. However, sulfonation of the substrate did, significantly, increase enzymatic hydrolysis at loadings of both 5 and 2.5 FPU g(-1) cellulose (from 80% to 95% and from 35% to 80%, respectively). In addition, sulfonation resulted in an increase in the amount of free enzymes detected during the course of hydrolysis to a maximum of 80% after 72 h. This suggested that the beneficial effects of sulfonation were primarily due to a decrease in the non-specific binding of the cellulases to the lignin.

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose.

Douglas-fir was SO(2)-steam pretreated at different severities (190, 200, and 210°C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and (13)C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins.

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin.

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel). An increase in the carboxylic content of the lignin preparation resulted in an increased hydrolysis yield. These results suggested that the carboxylic acids within the lignin partially alleviate non-productive binding of cellulases to lignin. To try to confirm this possible mechanism, dehydrogenative polymers (DHP) of monolignols were synthesized from coniferyl alcohol (CA) and ferulic acid (FA), and these model compounds were added to a typical enzymatic hydrolysis of Avicel. The DHP from FA, which was enriched in carboxylic acid groups compared with the DHP from CA, adsorbed a lower mount of cellulases and did not decrease hydrolysis yields when compared to the DHP from CA, which decreased the hydrolysis of Avicel by 8.4%. Thus, increasing the carboxylic acid content of the lignin seemed to significantly decrease the non-productive binding of cellulases and consequently increased the enzymatic hydrolysis of the cellulose.

The effect of varying organosolv pretreatment chemicals on the physicochemical properties and cellulolytic hydrolysis of mountain pine beetle-killed lodgepole pine.

Mountain pine beetle-killed lodgepole pine (Pinus contorta) chips were pretreated using the organosolv process, and their ease of subsequent enzymatic hydrolysis was assessed. The effect of varying pretreatment chemicals and solvents on the substrate's physicochemical characteristics was also investigated. The chemicals employed were MgCl2, H2SO4, SO2, and NaOH, and the solvents were ethanol and butanol. It was apparent that the different pretreatments resulted in variations in both the chemical composition of the solid and liquid fractions as well in the extent of cellulolytic hydrolysis (ranging from 21% to 82% hydrolysis after 12 h). Pretreatment under acidic conditions resulted in substrates that were readily hydrolyzed despite the apparent contradiction that pretreatment under alkaline conditions resulted in increased delignification (approximately 7% and 10% residual lignin for alkaline conditions versus 17% to 19% for acidic conditions). Acidic pretreatments also resulted in lower cellulose degree of polymerization, shorter fiber lengths, and increased substrate porosity. The substrates generated when butanol/water mixtures were used as the pretreatment solvent were also hydrolyzed more readily than those generated with ethanol/water. This was likely due to the limited miscibility of the solvents resulting in an increased concentration of pretreatment chemicals in the aqueous layer and thus a higher pretreatment severity.

The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates, on enzymatic hydrolysis.

The influence of the residual lignin remaining in the cellulosic rich component of pretreated lignocellulosic substrates on subsequent enzymatic hydrolysis was assessed. Twelve lignin preparations were isolated by two isolation methods (protease treated lignin (PTL) and cellulolytic enzymatic lignin (CEL)) from three types of biomass (corn stover, poplar, and lodgepole pine) that had been pretreated by two processes (steam and organosolv pretreatments). Comparative analysis of the isolated lignin showed that the CEL contained lower amounts of carbohydrates and protein than did the PTL and that the isolated lignin from corn stover contained more carbohydrates than did the lignin derived from the poplar and lodgepole pine. The lower yields of acid insoluble lignin (AIL) obtained from the corn stover when using the PTL method indicated that the lignin from the corn stover had a higher hydrophilicity than did the lignin from the poplar and lodgepole pine. The isolated lignin preparations were added to the reaction mixture containing crystalline cellulose (Avicel) and their possible effects on enzymatic hydrolysis were assessed. It was apparent that the lignin isolated from lodgepole pine and steam pretreated poplar decreased the hydrolysis yields of Avicel, whereas the other isolated lignins did not appear to decrease the hydrolysis yields significantly. The hydrolysis yields of the pretreated lignocellulose and those of Avicel containing the PTL showed good correlation, indicating that the nature of the residual lignin obtained after pretreatment significantly influenced hydrolysis.

Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates.

Fiber size analysis, water retention value, and Simons' stain measurements were assessed for their potential to predict the susceptibility of a given substrate to enzymatic hydrolysis. Slight modifications were made to the fiber size analysis and water retention protocols to adapt these measurements to evaluate substrates for cellulolytic hydrolysis rather than pulps for papermaking. Lodgepole pine was pretreated by the steam and ethanol-organosolv processes under varying conditions. The Simons' stain procedure proved to be an effective method for indicating the potential ease of enzymatic hydrolysis of substrates pretreated by either process or when the pretreatment conditions were altered.

Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated Lodgepole pine.

Recycling of cellulases is one way of reducing the high cost of enzymes during the bioconversion process. The effects of surfactant addition on enzymatic hydrolysis and the potential recycling of cellulases were studied during the hydrolysis of steam exploded Lodgepole pine (SELP) and ethanol pretreated Lodgepole pine (EPLP). Three cellulase preparations (Celluclast, Spezyme CP, and MSUBC) were evaluated to determine their hydrolysis efficiencies over multiple rounds of recycling. The surfactant, Tween 80, significantly increased the yield from 63% to 86% during the hydrolysis of the SELP substrate. The addition of surfactant to the hydrolysis of the EPLP substrate increased the free enzymes in the supernatant from 71% of the initial protein to 96%. Based on the Langmuir adsorption constants, cellulases (Celluclast and Spezyme CP) from Trichoderma reesei showed a higher affinity (3.48 mL/mg and 3.17 mL/mg) for the EPLP substrate than did the Penicillium enzyme (0.62 mg/mg). The Trichoderma reesei enzyme was used in four successive rounds of enzyme recycling using surfactant addition and readsorption onto fresh substrates during the hydrolysis of EPLP. In contrast, the Penicillium-derived enzyme preparation (MSUBC) could only be recycled once. When the same recycling strategy was carried out using the SELP substrate, the hydrolysis yield declined during each enzyme recycling round. These results suggested that the higher lignin content of the SELP substrate, and the low affinity of cellulases for the SELP substrate limited enzyme recycling by readsorption onto fresh substrates.

Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates.

The recycling of cellulase enzymes is one potential strategy for reducing the cost of the enzymatic hydrolysis step during the bioconversion of lignocellulosics to ethanol. To determine the influence of lignin on the post-hydrolysis distribution of cellulase enzymes between the liquid and solid phases, the hydrolysis of Avicel was compared to an organosolv-pretreated Douglas fir substrate with a lignin content of 3.0%. After a 12 h hydrolysis reaction on Avicel, 90% of the added cellulases (including beta-glucosidases) remained "free" in the liquid phase compared to only 65% in the case of the hydrolysis of the organosolv-pretreated Douglas fir substrate. The readsorption of free cellulases by supplementing the hydrolysis reaction with fresh substrate was explored as a potential means of recovering the free cellulases that remain in the liquid phase after hydrolysis. The Langmuir adsorption isotherm was used to develop a model predicting that 82% of the free cellulases could be recovered via readsorption onto fresh substrates during the hydrolysis of an ethanol-pretreated mixed softwood substrate with a lignin content of 6%. Recoverable free cellulase values of 85% and 88% based on cellulase activity and protein content, respectively, were obtained after experimental verification of the model. The readsorption of free cellulases onto fresh lignocellulosic substrates was shown to be an effective method for free enzyme recovery.