Deficiency of Cellulase Activity Measurements for Enzyme Evaluation

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial β-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.     

Effects of Substrate Loading on Enzymatic Hydrolysis and Viscosity of Pretreated Barley Straw

In this study, the applicability of a “fed-batch” strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a β-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily.     

Potential of Agricultural Residues and Hay for Bioethanol Production

Production of bioethanol from agricultural residues and hays (wheat, barley, and triticale straws, and barley, triticale, pearl millet, and sweet sorghum hays) through a series of chemical pretreatment, enzymatic hydrolysis, and fermentation processes was investigated in this study. Composition analysis suggested that the agricultural straws and hays studied contained approximately 28.62-38.58% glucan, 11.19-20.78% xylan, and 22.01-27.57% lignin, making them good candidates for bioethanol production. Chemical pretreatment with sulfuric acid or sodium hydroxide at concentrations of 0.5, 1.0, and 2.0% indicated that concentration and treatment agent play a significant role during pretreatment. After 2.0% sulfuric acid pretreatment at 121 degrees C/15 psi for 60 min, 78.10-81.27% of the xylan in untreated feedstocks was solubilized, while 75.09-84.52% of the lignin was reduced after 2.0% sodium hydroxide pretreatment under similar conditions. Enzymatic hydrolysis of chemically pretreated (2.0% NaOH or H2SO4) solids with Celluclast 1.5 L-Novozym 188 (cellobiase) enzyme combination resulted in equal or higher glucan and xylan conversion than with Spezyme(R) CP- xylanase combination. The glucan and xylan conversions during hydrolysis with Celluclast 1.5 L-cellobiase at 40 FPU/g glucan were 78.09 to 100.36% and 74.03 to 84.89%, respectively. Increasing the enzyme loading from 40 to 60 FPU/g glucan did not significantly increase sugar yield. The ethanol yield after fermentation of the hydrolyzate from different feedstocks with Saccharomyces cerevisiae ranged from 0.27 to 0.34 g/g glucose or 52.00-65.82% of the theoretical maximum ethanol yield.     

The enzymatic hydrolysis of pretreated agricultural residues and the fermentation of the liberated sugars to ethanol

Agricultural residues were pretreated by steam explosion and the cellulosic component of these substrates were converted to ethanol using a combined enzymatic hydrolysis and fermentation (CHF) process. The enzymatic hydrolysis was carried out using culture filtrates ofTrichoderma harzianum E58 while the liberated sugars were fermented to ethanol byS. cerevisiae. Initially, pretreatment conditions were optimized to ensure that the substrates were readily hydrolyzed and fermented. The agricultural residues were steamed for various times between 30 and 120 s at approximately 240‡C prior to rapid decompression (explosion) in a small masonite-type gun. The various substrates were selectively extracted by water and alkali to see whether the enzymatic hydrolysis and fermentability of the substrates were enhanced. A comparison between the overall conversion of wheat and barley straw was made since these are the two most readily available agricultural residues in Canada. Steam explosion did not affect the hexosan content of the residues, although the pentosan content of the substrates decreased with increasing duration of steaming. The hexosan (cellulose) content of wheat straw was 50.7% of the total substrate while a slightly higher 52.9% cellulose content was detected in the barley straw. Wheat straw was more efficiently hydrolyzed after it had been steamed for 90 s while optimum hydrolysis of the barley straw was detected after 60 s. Steam exploded wheat and barley straw that was subsequently extracted with water was readily hydrolyzed to their component sugars.S. cerevisiae could almost quantitatively convert these sugars to ethanol. This indicated that water washing not only enhanced the enzymatic hydrolysis of the steam exploded substrates, it also removed inhibitory material that restricted the growth of S.cerevisiae. Maximum hydrolysis (78.5%) and ethanol yields (10 mg/mL) were obtained when wheat straw was steamed for 90 s. Slightly lower hydrolysis (76.0%) and ethanol yields (9.5 mg/mL) were obtained with barley straw that had been steamed for 120 s.     

Ensiling Agricultural Residues for Bioethanol Production

The potential of using ensiling, with and without supplemental enzymes, as a cost-effective pretreatment for bioethanol production from agricultural residues was investigated. Ensiling did not significantly affect the lignin content of barley straw, cotton stalk, and triticale hay ensiled without enzyme, but slightly increased the lignin content in triticale straw, wheat straw, and triticale hay ensiled with enzyme. The holocellulose (cellulose plus hemicellulose) losses in the feedstocks, as a result of ensiling, ranged from 1.31 to 9.93%. The percent holocellulose loss in hays during ensiling was lower than in straws and stalks. Ensiling of barley, triticale, wheat straws, and cotton stalk significantly increased the conversion of holocellulose to sugars during subsequent hydrolysis with two enzyme combinations. Enzymatic hydrolysis of ensiled and untreated feedstocks by Celluclast 1.5 L-Novozyme 188 enzyme combination resulted in equal or higher saccharification than with Spezyme CP-xylanase combination. Enzyme loadings of 40 and 60 FPU/g reducing sugars gave similar sugar yields. The percent saccharification with Celluclast 1.5 L-Novozyme 188 at 40 FPU/g reducing sugars was 17.1 to 43.6%, 22.4 to 46.9%, and 23.2 to 32.2% for untreated feedstocks, feedstocks ensiled with, and without enzymes, respectively. Fermentation of the hydrolysates from ensiled feedstocks resulted in ethanol yields ranging from 0.21 to 0.28 g/g reducing sugars.     

Steam pretreatment of acid-sprayed and acid-soaked barley straw for production of ethanol

Barley is an abundant crop in Europe, which makes its straw residues an interesting cellulose source for ethanol production. Steam pretreatment of the straw followed by enzymatic hydrolysis converts the cellulose to fermentable sugars. Prior to pretreatment the material is impregnated with a catalyst, for example, H₂SO₄, to enhance enzymatic digestibility of the pretreated straw. Different impregnation techniques can be applied. In this study, soaking and spraying were investigated and compared at the same pretreatment condition in terms of overall yield of glucose and xylose. The overall yield includes the soluble sugars in the liquid from pretreatment, including soluble oligomers, and monomer sugars obtained in the enzymatic hydrolysis. The yields obtained differed for the impregnation techniques. Acid-soaked barley straw gave the highest overall yield of glucose, regardless of impregnation time (10 or 30 min) or acid concentration (0.2 or 1.0 wt%). For xylose, soaking gave the highest overall yield at 0.2 wt% H₂SO₄. An increase in acid concentration resulted in a decrease in xylose yield for both acid-soaked and acid-sprayed barley straw. Optimization of the pretreatment conditions for acid-sprayed barley straw was performed to obtain yields using spraying that were as high as those with soaking. For acid-sprayed barley straw the optimum pretreatment condition for glucose, 1.0 wt% H₂SO₄ and 220°C for 5 min, gave an overall glucose yield of 92% of theoretical based on the composition of the raw material. Pretreatment with 0.2wt% H₂SO₄ at 190°C for 5 min resulted in the highest overall xylose yield, 67% of theoretical based on the composition of the raw material.     

Combined steam pretreatment and enzymatic hydrolysis of starch-free wheat fibers

Steam treatment of an industrial process stream, denoted starch-free wheat fiber, was investigated to improve the formation of monomeric sugars in subsequent enzymatic hydrolysis for further bioconversion into ethanol. The solid fraction in the process stream, derived from a combined starch and ethanol factory, was rich in arabinose (21.1%), xylose (30.1%), and glucose (18.6%), in the form of polysaccharides. Various conditions of steam pretreatment (170–220°C for 5–30 min) were evaluated, and their effect was assessed by enzymatic hydrolysis with 2 g of Celluclast + Ultraflo mixture/ 100 g of starch-free fiber (SFF) slurry at 5% dry matter (DM). The highest overall sugar yield for the combined steam pretreatment and enzymatic hydrolysis, 52g/100 g of DM of SFF, corresponding to 74% of the theoretical, was achieved with pretreatment at 190°C for 10 min followed by enzymatic hydrolysis.     

A Dynamic Model for Cellulosic Biomass Hydrolysis: a Comprehensive Analysis and Validation of Hydrolysis and Product Inhibition Mechanisms

The objective of this study is to perform a comprehensive enzyme kinetics analysis in view of validating and consolidating a semimechanistic kinetic model consisting of homogeneous and heterogeneous reactions for enzymatic hydrolysis of lignocellulosic biomass proposed by the U.S. National Renewable Energy Laboratory (Kadam et al., Biotechnol Prog 20(3):698–705, 2004) and its variations proposed in this work. A number of dedicated experiments were carried out under a range of initial conditions (Avicel® versus pretreated barley straw as substrate, different enzyme loadings and different product inhibitors such as glucose, cellobiose and xylose) to test the hydrolysis and product inhibition mechanisms of the model. A nonlinear least squares method was used to identify the model and estimate kinetic parameters based on the experimental data. The suitable mathematical model for industrial application was selected among the proposed models based on statistical information (weighted sum of square errors). The analysis showed that transglycosylation plays a key role at high glucose levels. It also showed that the values of parameters depend on the selected experimental data used for parameter estimation. Therefore, the parameter values are not universal and should be used with caution. The model proposed by Kadam et al. (Biotechnol Prog 20(3):698–705, 2004) failed to predict the hydrolysis phenomena at high glucose levels, but when combined with transglycosylation reaction(s), the prediction of cellulose hydrolysis behaviour over a broad range of substrate concentrations (50–150 g/L) and enzyme loadings (15.8–31.6 and 1–5.9 mg protein/g cellulose for Celluclast and Novozyme 188, respectively) was possible. This is the first study introducing transglycosylation into the semimechanistic model. As long as these type of models are used within the boundary of their validity (substrate type, enzyme source and substrate concentration), they can support process design and technology improvement efforts at pilot and full-scale studies.     

Fermentation of Biologically Pretreated Wheat Straw for Ethanol Production: Comparison of Fermentative Microorganisms and Process Configurations

The pretreatment of lignocellulosic biomass with white-rot fungi to produce bioethanol is an environmentally friendly alternative to the commonly used physico-chemical processes. After biological pretreatment, a solid substrate composed of cellulose, hemicellulose and lignin, the two latter with a composition lower than that of the initial substrate, is obtained. In this study, six microorganisms and four process configurations were utilised to ferment a hydrolysate obtained from wheat straw pretreated with the white-rot fungus Irpex lacteus. To enhance total sugars utilisation, five of these microorganisms are able to metabolise, in addition to glucose, most of the pentoses obtained after the hydrolysis of wheat straw by the application of a mixture of hemicellulolytic and cellulolytic enzymes. The highest overall ethanol yield was obtained with the yeast Pachysolen tannophilus. Its application in combination with the best process configuration yielded 163 mg ethanol per gram of raw wheat straw, which was between 23 and 35 % greater than the yields typically obtained with a conventional bioethanol process, in which wheat straw is pretreated using steam explosion and fermented with the yeast Saccharomyces cerevisiae.     

Two-stage dilute-acid pretreatment of softwoods

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.     

A technical and economic analysis of acid-catalyzed steam explosion and dilute sulfuric acid pretreatments using wheat straw or aspen wood chips

Lignocellulosic biomass is one of the most plentiful and potentially cheapest feedstocks for ethanol production. The cellulose component can be broken down into glucose by enzymes and then converted to ethanol by yeast. However, hydrolysis of cellulose to glucose is difficult, and some form of pretreatment is necessary to increase the susceptibility of cellulose to enzymatic attack. An analysis has been completed of two pretreatment options, dilute sulfuric acid hydrolysis and sulfur dioxide impregnated steam explosion, for two feedstocks, wheat straw and aspen wood chips. Detailed process flow sheets and material and energy balances were used to generate equipment cost information. A technical and economic analysis compared the two feedstocks for each of the two pretreatments. For the same pretreatment, sugars produced from aspen wood hydrolysis were cheaper because of the higher carbohydrate content of aspen, whereas dilute acid pretreatment is favored over acid-catalyzed steam explosion.     

High solids simultaneous saccharification and fermentation of pretreated wheat straw to ethanol

Wheat straw was pretreated with dilute (0.5%) sulfuric acid at 140°C for 1 h. Pretreated straw solids were washed with deionized water to neutrality and then stored frozen at –20°C. The approximate composition of the pretreated straw solids was 64% cellulose, 33% lignin, and 2% xylan. The cellulose in the pretreated wheat straw solids was converted to ethanol in batch simultaneous saccharification and fermentation experiments at 37°C using cellulase enzyme fromTrichoderma reesei (Genencor 150 L) with or without supplementation with β–glucosidase fromAspergillus niger (Novozyme 188) to produce glucose sugar and the yeastSaccharomyces cerevisiae to ferment the glucose into ethanol. The initial cellulose concentrations were adjusted to 7.5, 10, 12.5, 15, 17.5, and 20% (w/w). Since wheat straw particles do not form slurries at these concentrations and cannot be mixed with conventional impeller mixers used in laboratory fermenters, a simple rotary fermenter was designed and fabricated for these experiments. The results of the simultaneous saccharification and fermentation (SSF) experiments indicate that the cellulose in pretreated wheat straw can be efficiently fermented into ethanol for up to a 15% cellulose concentration (24.4% straw concentration).     

Dilute Acid Pretreatment of Black Spruce Using Continuous Steam Explosion System

The pretreatment of lignocellulosic materials prior to the enzymatic hydrolysis is essential to the sugar yield and bioethanol production. Dilute acid hydrolysis of black spruce softwood chip was performed in a continuous high temperature reactor followed with steam explosion and mechanical refining. The acid-soaked wood chips were pretreated under different feeding rates (60 and 92 kg/h), cooking screw rotation speeds (7.2 and 14.4 rpm), and steam pressures (12 and 15 bar). The enzymatic hydrolysis was carried out on the acid-insoluble fraction of pretreated material. At lower feeding rate, the pretreatment at low steam pressure and short retention time favored the recovery of hemicellulose. The pretreatment at high steam pressure and longer retention time recovered less hemicellulose but improved the enzymatic accessibility. As a result, the overall sugar yields became similar no matter what levels of the retention time or steam pressure. Comparing with lower feeding rate, higher feeding rate resulted in consistently higher glucose yield in both liquid fraction after pretreatment and that released after enzymatic hydrolysis.     

Effect of Double-Step Steam Explosion Pretreatment in Bioethanol Production from Softwood

The study investigated the production of bioethanol from softwood, in particular pine wood chip. The steam explosion pretreatment was largely investigated, evaluating also the potential use of a double-step process to increase ethanol production through the use of both solid and liquid fraction after the pretreatment. The pretreatment tests were carried out at different conditions, determining the composition of solid and liquid fraction and steam explosion efficiency. The enzymatic hydrolysis was carried out with Ctec2 enzyme while the fermentation was carried out using Saccharomyces Cerevisiae yeast “red ethanol”. It was found that the best experimental result was obtained for a single-step pretreated sample (10.6 g of ethanol/100 g of initial biomass dry basis) for a 4.53 severity. The best double-step overall performance was equal to 8.89 g ethanol/100 g of initial biomass dry basis for a 4.27 severity. The enzymatic hydrolysis strongly depended on the severity of the pretreatment while the fermentation efficiency was mainly influenced by the concentration of the inhibitors. The ethanol enhancing potential of a double-step steam explosion could slightly increase the ethanol production compared to single-step potential.     

Enzymatic Hydrolysis and Ethanol Fermentation of High Dry Matter Wet-Exploded Wheat Straw at Low Enzyme Loading

Wheat straw was pretreated by wet explosion using three different oxidizing agents (H₂O₂, O₂, and air). The effect of the pretreatment was evaluated based on glucose and xylose liberated during enzymatic hydrolysis. The results showed that pretreatment with the use of O₂ as oxidizing agent was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15–20% will be necessary. However, high DM hydrolysis and fermentation are limited by high viscosity of the material, higher inhibition of the enzymes, and fermenting microorganism. The wet-explosion pretreatment method enabled relatively high yields from both enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) to be obtained when performed on unwashed slurry with 14% DM and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%.     

Effects of Liquid Hot Water Pretreatment on Enzymatic Hydrolysis and Physicochemical Changes of Corncobs

Liquid hot water (LHW) pretreatment is an efficient chemical-free strategy for enhancing enzymatic digestibility of lignocellulosic biomass for conversion to fuels and chemicals in biorefinery. In this study, effects of LHW on removals of hemicelluloses and lignin from corncobs were studied under varying reaction conditions. LHW pretreatment at 160 °C for 10 min promoted the highest levels of hemicellulose solubilization into the liquid phase, resulting into the maximized pentose yield of 58.8% in the liquid and more than 60% removal of lignin from the solid, with 73.1% glucose recovery from enzymatic hydrolysis of the pretreated biomass using 10 FPU/g Celluclast?. This led to the maximal glucose and pentose recoveries of 81.9 and 71.2%, respectively, when combining sugars from the liquid phase from LHW and hydrolysis of the solid. Scanning electron microscopy revealed disruption of the intact biomass structure allowing increasing enzyme’s accessibility to the cellulose microfibers which showed higher crystallinity index compared to the native biomass as shown by x-ray diffraction with a marked increase in surface area as revealed by BET measurement. The work provides an insight into effects of LHW on modification of physicochemical properties of corncobs and an efficient approach for its processing in biorefinery industry.     

Bioconversion of mixed solids waste to ethanol

A mixed solids waste (MSW) feedstock, comprising construction lumber waste (35% oven-dry basis), alm ond treeprunings (20%), wheat straw (20%), office waste paper (12.5%), and newsprint (12.5%), was converted to ethanol via dilute-acid pretreatment followed by enzymatic hydrolysis and yeast fermentation. The MSW was pretreated with dilute sulfuricacid (0.4% w/w) at 210°C for 3 min in a 4-L stea mexplosion reactor, then washed with water to recover the solubilized hemicellulose. The digestibility of water-washed, pretreated MSW was 90% in batch enzymatic hydrolysis at 66 FPU/g cellulose. Using an enzyme-recycle bioreactor system, greater than 90% cellulose hydrolysis was achieved at a net enzyme loading of about 10 FPU/g cellulose. Enzyme recycling using mebrane filtration and a fed-batch fermentation technique is a promising option for significantly reducing the cost of enzyme in cellulose hydrolysis. The hexosesugars were readily fermentable using a Saccharomyces cerevisiae yeast strain that was adapted to the hydrolysate. Solid residue after enzyme digestion was subjected to various furnace experiments designed to assess the fouling and slagging characteristics. Results of these analyses suggest the residue to be of a low to moderate slagging and fouling type if burned by itself.     

Evaluation of Selected White-Rot Fungal Isolates for Improving the Sugar Yield from Wheat Straw

Biological pretreatment of lignocellulosic biomass by fungi can represent a low-cost and eco-friendly alternative to physicochemical methods to facilitate enzymatic hydrolysis. However, fungal metabolism can cause cellulose loss and it is therefore necessary to use the appropriate fungal strain-biomass type combination. In this work, the effects of biological pretreatments carried out by five different fungi on enzymatic hydrolysis of wheat straw were investigated. The best results were obtained with a Ceriporiopsis subvermispora strain, which minimized weight and cellulose losses and gave the highest net sugar yield (calculated with respect to the holocellulose content of the untreated straw), up to 44 % after a 10-week pretreatment, more than doubling the yields obtained with the other isolates. Moreover, prolonging the pretreatment from 4 up to 10 weeks produced a 2-fold increase, up to 60 %, in digestibility (sugar yield, calculated considering the holocellulose content of the pretreated material). The hemicellulose content of the pretreated material resulted inversely correlated with digestibility, and it could thus be utilized as an index of the pretreatment efficacy. Finally, a correlation was also found between digestibility and the difference between the absorbance values at 290 and 320 nm of pretreated wheat straw extracts.     

Lime pretreatment of crop residues bagasse and wheat straw

Lime (calcium hydroxide) was used as a pretreatment agent to enhance the enzymatic digestibility of two common crop residues: bagasse and wheat straw. A systematic study of pretreatment conditions suggested that for short pretreatment times (1–3 h), high temperatures (85-135°C) were required to achieve high sugar yields, whereas for long pretreatment times (e.g., 24 h), low temperatures (50–65°C) were effective. The recommended lime loading is 0.1 g Ca(OH)₂/g dry biomass. Water loading had little effect on the digestibility. Under the recommended conditions, the 3-d reducing sugar yield of the pretreated bagasse increased from 153 to 659 mg Eq glucose/g dry biomass, and that of the pretreated wheat straw increased from 65 to 650 mg Eq glucose/g dry biomass. A material balance study on bagasse showed that the biomass yield after lime pretreatment is 93.6%. No glucan or xylan was removed from bagasse by the pretreatment, whereas 14% of lignin became solubilized. A lime recovery study showed that 86% of added calcium was removed from the pretreated bagasse by ten washings and could be recovered by carbonating the wash water with CO₂ at pH 9.5.     

Effect and Modeling of Glucose Inhibition and In Situ Glucose Removal During Enzymatic Hydrolysis of Pretreated Wheat Straw

The enzymatic hydrolysis of lignocellulosic biomass is known to be product-inhibited by glucose. In this study, the effects on cellulolytic glucose yields of glucose inhibition and in situ glucose removal were examined and modeled during extended treatment of heat-pretreated wheat straw with the cellulolytic enzyme system, Celluclast? 1.5 L, from Trichoderma reesei, supplemented with a β-glucosidase, Novozym? 188, from Aspergillus niger. Addition of glucose (0–40 g/L) significantly decreased the enzyme-catalyzed glucose formation rates and final glucose yields, in a dose-dependent manner, during 96 h of reaction. When glucose was removed by dialysis during the enzymatic hydrolysis, the cellulose conversion rates and glucose yields increased. In fact, with dialytic in situ glucose removal, the rate of enzyme-catalyzed glucose release during 48–72 h of reaction recovered from 20–40% to become ≈70% of the rate recorded during 6–24 h of reaction. Although Michaelis–Menten kinetics do not suffice to model the kinetics of the complex multi-enzymatic degradation of cellulose, the data for the glucose inhibition were surprisingly well described by simple Michaelis–Menten inhibition models without great significance of the inhibition mechanism. Moreover, the experimental in situ removal of glucose could be simulated by a Michaelis–Menten inhibition model. The data provide an important base for design of novel reactors and operating regimes which include continuous product removal during enzymatic hydrolysis of lignocellulose.