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Accellerase 1000 cellulase, Spezyme CP cellulase, β-glucosidase, Multifect xylanase, and beta-xylosidase were evaluated for hydrolysis of pure cellulose, pure xylan, and switchgrass solids from leading pretreatments of dilute sulfuric acid, sulfur dioxide, liquid hot water, lime, soaking in aqueous ammonia, and ammonia fiber expansion. Distinctive sugar release patterns were observed from Avicel, phosphoric acid swollen cellulose (PASC), xylan, and pretreated switchgrass solids, with accumulation of significant amounts of xylooligomers during xylan hydrolysis. The strong inhibition of cellulose hydrolysis by xylooligomers could be partially attributed to the negative impact of xylooligomers on cellulase adsorption. The digestibility of pretreated switchgrass varied with pretreatment but could not be consistently correlated to xylan, lignin, or acetyl removal. Initial hydrolysis rates did correlate well with cellulase adsorption capacities for all pretreatments except lime, but more investigation is needed to relate this behavior to physical and compositional properties of pretreated switchgrass., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Current technology for conversion of biomass to ethanol is an enzyme-based biochemical process. In bioethanol production, achieving high sugar yield at high solid loading in enzymatic hydrolysis step is important from both technical and economic viewpoints. Enzymatic hydrolysis of cellulosic substrates is affected by many parameters, including an unexplained behavior that the glucan digestibility of substrates by cellulase decreased under high solid loadings. A comprehensive study was conducted to investigate this phenomenon by using Spezyme CP and Avicel as model cellulase and cellulose substrate, respectively. The hydrolytic properties of the cellulase under different substrate concentrations at a fixed enzyme-to-substrate ratio were characterized. The results indicate that decreased sugar yield is neither due to the loss of enzyme activity at a high substrate concentration nor due to the higher end-product inhibition. The cellulase adsorption kinetics and isotherm studies indicated that a decline in the binding capacity of cellulase may explain the long-observed but little-understood phenomenon of a lower substrate digestibility with increased substrate concentration. The mechanism how the enzyme adsorption properties changed at high substrate concentration was also discussed in the context of exploring the improvement of the cellulase-binding capacity at high substrate loading.

Microcrystalline cellulose (MCC) was pretreated with subcritical water in a continuous flow reactor for enhancing its enzymatic reactivity with cellulase enzyme. Cellulose/water suspension was mixed with subcritical (i.e., pressurized and heated) water and then fed into the reactor maintained at a constant temperature and pressure. After the reaction, product was immediately cooled in a double-pipe heat exchanger. The solid portion of the product (i.e., treated MCC) was separated and tested for molecular structure and enzymatic reactivity. Experiments were conducted at temperatures ranging from 200 to 315 degrees C, at 27.6 MPa, and for 3.4-6.2 s reaction times. The treated MCC was characterized for degree of polymerization (DP(v)) by viscosimetry, and crystallinity by X-ray diffraction (XRD). In addition, differential scanning calorimetry and scanning electron microscopy (SEM) analyses were carried out to study any transformation in the cellulose structure. As expected, DP(v) of cellulose steadily decreased with increase in the pretreatment temperature, with a rapid drop occurring above 300 degrees C. On the other hand, XRD analysis did not show any decrease in crystallinity upon pretreatment but, partial transformation of celluloses I-II structure was noticed in the MCC treated at 300 degrees C. Development of surface cracks and trenches were observed in the SEM images for all the treated samples. Enzymatic reactivity was increased after the treatment at > or = 300 degrees C.

Cellulase reaction mechanism was investigated with the use of following pure cellulosic substrates: Microcrystalline cellulose (Avicel), alpha-cellulose (Sigma), filter paper, cotton, and non-crystalline cellulose (NCC). NCC is amorphous cellulose prepared in our laboratory by treatment with concentrated sulfuric acid. When hydrolyzed with cellulase, NCC produces significant amount of cello-oligosaccharides (COS) as reaction intermediates along with glucose and cellobiose. The COS produced by cellulase were categorized into two different moieties based upon their degree of polymerization (DP): low DP (less than 7) COS (LD-COS) and high DP COS (HD-COS). Endo-glucanase (Endo-G) reacts rapidly on the NCC reducing its DP to 30-60, after which the Endo-G reaction with NCC ceases. HD-COS is produced from NCC by the action of Endo-G, whereas LD-COS is produced by exo-glucanase (Exo-G). beta-Glucosidase (beta-G) hydrolyzes LD-COS to produce cellobiose, but it does not hydrolyze HD-COS. DP of NCC affects the action of Exo-G in such a way that the overall yield is high for high DP NCC. This is in line with previous findings that substrate-recognition by Exo-G requires binding on beta-glucan chain with DP of 10 for the hydrolysis to take place. The individual cellulose chain residues within solid having DP less than 10 therefore remain unreacted. The percentage of the unreacted portion would be lower for high DP NCC, which results high overall conversion. The surface area and the number of reactive sites on the substrate facilitate adsorption of enzyme therefore the initial rate of the hydrolysis. The overall extent of conversion of cellulose, however, is controlled primarily by its inherent characteristics such as DP and crystallinity., (2008 Wiley Periodicals, Inc.)

A multistep approach was taken to investigate the intrinsic kinetics of the cellulase enzyme complex as observed with hydrolysis of noncrystalline cellulose (NCC). In the first stage, published initial rate mechanistic models were built and critically evaluated for their performance in predicting time-course kinetics, using the data obtained from enzymatic hydrolysis experiments performed on two substrates: NCC and alpha-cellulose. In the second stage, assessment of the effect of reaction intermediates and products on intrinsic kinetics of enzymatic hydrolysis was performed using NCC hydrolysis experiments, isolating external factors such as mass transfer effects, physical properties of substrate, etc. In the final stage, a comprehensive intrinsic kinetics mechanism was proposed. From batch experiments using NCC, the time-course data on cellulose, cello-oligosaccharides (COS), cellobiose, and glucose were taken and used to estimate the parameters in the kinetic model. The model predictions of NCC, COS, cellobiose, and glucose profiles show a good agreement with experimental data generated from hydrolysis of different initial compositions of substrate (NCC supplemented with COS, cellobiose, and glucose). Finally, sensitivity analysis was performed on each model parameter; this analysis provides some insights into the yield of glucose in the enzymatic hydrolysis. The proposed intrinsic kinetic model parametrized for dilute cellulose systems forms a basis for modeling the complex enzymatic kinetics of cellulose hydrolysis in the presence of limiting factors offered by substrate and enzyme characteristics.

Biological processing of cellulosic biomass to fuels and chemicals would open up major new agricultural markets and provide powerful societal benefits, but pretreatment operations essential to economically viable yields have a major impact on costs and performance of the entire system. However, little comparative data is available on promising pretreatments. To aid in selecting appropriate systems, leading pretreatments based on ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime were evaluated in a coordinated laboratory program using a single source of corn stover, the same cellulase enzyme, shared analytical methods, and common data interpretation approaches to make meaningful comparisons possible for the first time. Each pretreatment made it possible to subsequently achieve high yields of glucose from cellulose by cellulase enzymes, and the cellulase formulations used were effective in solubilizing residual xylan left in the solids after each pretreatment. Thus, overall sugar yields from hemicellulose and cellulose in the coupled pretreatment and enzymatic hydrolysis operations were high for all of the pretreatments with corn stover. In addition, high-pH methods were found to offer promise in reducing cellulase use provided hemicellulase activity can be enhanced. However, the substantial differences in sugar release patterns in the pretreatment and enzymatic hydrolysis operations have important implications for the choice of process, enzymes, and fermentative organisms.

For the first time, a single source of cellulosic biomass was pretreated by leading technologies using identical analytical methods to provide comparative performance data. In particular, ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime approaches were applied to prepare corn stover for subsequent biological conversion to sugars through a Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, and Texas A&M University. An Agricultural and Industrial Advisory Board provided guidance to the project. Pretreatment conditions were selected based on the extensive experience of the team with each of the technologies, and the resulting fluid and solid streams were characterized using standard methods. The data were used to close material balances, and energy balances were estimated for all processes. The digestibilities of the solids by a controlled supply of cellulase enzyme and the fermentability of the liquids were also assessed and used to guide selection of optimum pretreatment conditions. Economic assessments were applied based on the performance data to estimate each pretreatment cost on a consistent basis. Through this approach, comparative data were developed on sugar recovery from hemicellulose and cellulose by the combined pretreatment and enzymatic hydrolysis operations when applied to corn stover. This paper introduces the project and summarizes the shared methods for papers reporting results of this research in this special edition of Bioresource Technology.

Cellulosic plant material represents an as-of-yet untapped source of fermentable sugars for significant industrial use. Many physio-chemical structural and compositional factors hinder the enzymatic digestibility of cellulose present in lignocellulosic biomass. The goal of any pretreatment technology is to alter or remove structural and compositional impediments to hydrolysis in order to improve the rate of enzyme hydrolysis and increase yields of fermentable sugars from cellulose or hemicellulose. These methods cause physical and/or chemical changes in the plant biomass in order to achieve this result. Experimental investigation of physical changes and chemical reactions that occur during pretreatment is required for the development of effective and mechanistic models that can be used for the rational design of pretreatment processes. Furthermore, pretreatment processing conditions must be tailored to the specific chemical and structural composition of the various, and variable, sources of lignocellulosic biomass. This paper reviews process parameters and their fundamental modes of action for promising pretreatment methods.

Hydrolysis of alpha-cellulose by H2SO4 is a heterogeneous reaction. As such the reaction is influenced by physical factors. The hydrolysis reaction is therefore controlled not only by the reaction conditions (acid concentration and temperature) but also by the physical state of the cellulose. As evidence of this, the reaction rates measured at the high-temperature region (above 200 C) exhibited a sudden change in apparent activation energy at a certain temperature, deviating from Arrhenius law. Furthermore, alpha-cellulose, once it was dissolved into concentrated H2SO4 and reprecipitated, showed a reaction rate two orders of magnitude higher than that of untreated cellulose, about the same magnitude as cornstarch. The alpha-cellulose when treated with a varying level of H2SO4 underwent an abrupt change in physical structure (fibrous form to gelatinous form) at about 65% H2SO4. The sudden shift of physical structure and reaction pattern in response to acid concentration and temperature indicates that the main factor causing the change in cellulose structure is disruption of hydrogen bonding. Finding effective means of disrupting hydrogen bonding before or during the hydrolysis reaction may lead to a novel biomass saccharification process.

The classic kinetic model for cellulose hydrolysis is often referred to as pseudo-homogeneous, a term revealing the insight that the process is actually heterogeneous. During the past 10-15 yr, the shortcomings of this model have been demonstrated in various studies and the interest in the heterogeneous aspects has increased. The present work presents a simplistic model in which the intrinsic, heterogeneous hydrolysis and transport rates are coupled by the assumption of a constant glucosidic surface concentration. The mechanisms affecting these two rates are largely unknown, but the model serves as a guideline for further exploration of the process.

Acid-catalyzed hydrolysis is controlled not only by temperature and acid concentration but also by the physical state of the cellulose. Under low temperature and acid condition the cellulose structure stays in stable crystalline form. Therefore, the prevailing reaction mode is endwise hydrolysis. Glucose then becomes the main sugar product. However, when temperature and/or acid concentration is raised to a certain level, the cellulose structure becomes unstable by breakage of hydrogen bonding, the primary force that holds the cellulose chains. Once the crystalline structure of the cellulose is disrupted, acid molecules can penetrate into the inner layers of the cellulose chains. In support of this hypothesis, we have experimentally verified that a substantial amount of oligomers is formed as reaction intermediates under extremely low-acid and high-temperature conditions. We also found that the breakage of hydrogen bonds occurs rather abruptly in response to temperature. One such condition is 210 degrees C, 0.07% H2SO4. Glucose, once it is formed in the hydrolysate, interacts with acid-soluble lignin, forming a lignin-carbohydrate complex. This occurs concurrently with other reactions involving glucose such as decomposition and reversion. On the basis of these findings, a comprehensive kinetic model is proposed. This model is in full compliance with our recent experimental data obtained under a broad range of reaction conditions.

Intra-particle diffusion of sulfuric acid into sugarcane bagasse, corn stover, rice straw and yellow poplar was investigated to determine the effective diffusivity of sulfuric acid within the porous biomass structure. Diffusion experiments were conducted over 25-75 degrees C for two different biomass sizes using dynamic diffusion test cells. Diffusivities of sulfuric acid in agricultural residues were significantly higher than those of hard wood. Diffusivity data for each biomass were fitted into the Arrhenius equation for extrapolation to higher temperatures. The diffusivity data were subsequently incorporated into a theoretical model to determine acid profile within the biomass matrix. The modeling results indicate that intra-particle diffusion of acid influences the rate of dilute-acid pretreatment if unground biomass feedstock is used under normal pretreatment conditions. A criterion was set up to determine the critical biomass size at which the intra-particle acid diffusion becomes a rate-influencing factor for a given pretreatment condition.

The kinetics of cellulose hydrolysis under extremely low acid (ELA) conditions (0.07 wt%) and at temperatures >200 degrees C was investigated using batch reactors and bed-shrinking flow-through (BSFT) reactors. The maximum yield of glucose obtained from batch reactor experiments was about 60% for alpha-cellulose, which occurred at 205 and 220 degrees C. The maximum glucose yields from yellow poplar feedstocks were substantially lower, falling in the range of 26-50%. With yellow poplar feedstocks, a large amount of glucose was unaccounted for at the latter phase of the batch reactions. It appears that a substantial amount of released glucose condenses with nonglucosidic substances in liquid. The rate of glucan hydrolysis under ELA was relatively insensitive to temperature in batch experiments for all three substrates. This contradicts the traditional concept of cellulose hydrolysis and implies that additional factors influence the hydrolysis of glucan under ELA. In experiments using BSFT reactors, the glucose yields of 87.5, 90.3, and 90.8% were obtained for yellow poplar feedstocks at 205, 220, and 235 degrees C, respectively. The hydrolysis rate for glucan was about three times higher with the BSFT than with the batch reactors. The difference of observed kinetics and performance data between the BSFT and the batch reactors was far above that predicted by the reactor theory.

Pretreatment of two different softwood-based lignocellulosic wastes (newsprint and Kraft pulp mill sludge) was investigated. Pretreatment was done by aqueous ammonia and hydrogen peroxide (H2O2), two delignifying reagents that are environmentally benign. Three different treatment schemes were employed: aqueous ammonia alone (ammonia recycled percolation [ARP]), mixed stream of aqueous ammonia and H2O2, and successive treatment with H2O2 and aqueous ammonia. In all cases there was a substantial degree of delignification ranging from 30 to 50%. About half of the hemicellulose sugars were dissolved into the process effluent. Retention of cellulose after pretreatment varied from 85 to 100% for newspaper feedstock and from 77 to 85% for the pulp mill sludge. After treatment with aqueous ammonia alone (ARP), the digestibility of newspaper and the pulp mill sludge was improved only by 5% (from 40 to 45% for the former and from 68 to 73% for the latter), despite a substantial degree of delignification occurring after the ARP process. The lignin content thus did not correlate with the digestibility for these substrates. Simultaneous treatment with H2O2 and aqueous ammonia did not bring about any significant improvement in the digestibility over that of the ARP. A successive treatment by H2O2 and ARP showed the most promise because it improved the digestibility of the newspaper from 41 to 75%, a level comparable to that of alpha-cellulose.

A strain of Clostridium thermoaceticum (ATCC 49707) was evaluated for its homoacetate potential. This thermophilic anaerobe best produces acetate from glucose at pH 6.0 and 59 degrees C with a yield of 83% of theoretical. Enzyme hydrolysis of two substrates, a-cellulose and a pulp mill sludge, yielded 68% and 70% digestion, respectively. The optimum conditions for the simultaneous saccharification and fermentation (SSF) were substrate dependent: 55 degrees C, pH 6.0 for alpha-cellulose, and 55 degrees C, pH 5.5 for the pulp mill sludge. In the SSF with alpha-cellulose, the overall yield of acetate was strongly influenced by the enzyme loading. In a fed-batch operation of SSF with alpha-cellulose, an overall acetic acid yield of 60 wt% was obtained. Among the factors limiting the yields were incomplete digestion by the enzyme and the end-product inhibition. In the SSF of pulp mill sludge, inhibitors present in the sludge severely limited bacterial action. A large accumulation of glucose developed over the entire process, changing the intended SSF operation into a separate hydrolysis and fermentation operation. Despite a long lag phase of microbial growth, a terminal yield of 85% was obtained with this substrate.

The enzymatic reaction in the simultaneous saccharification and fermentation (SSF) is operated at a temperature much lower than its optimum level. This forces the enzyme activity to be far below its potential, consequently raising the enzyme requirement. To alleviate this problem, a nonisothermal simultaneous saccharification and fermentation process (NSSF) was investigated. The NSSF is devised so that saccharification and fermentation occur simultaneously, yet in two separate reactors that are maintained at different temperatures. Lignocellulosic biomass is retained inside a column reactor and hydrolyzed at the optimum temperature for the enzymatic reaction (50 degrees C). The effluent from the column reactor is recirculated through a fermenter, which runs at its optimum temperature (20-30 degrees C). The cellulase enzyme activity is increased by a factor of 2-3 when the hydrolysis temperature is raised from 30 to 50 degrees C. The NSSF process has improved the enzymatic reaction in the SSF to the extent that it reduces the overall enzyme requirement by 30-40%. The effect of temperature on beta-glucosidase activity was the most significant among the individual cellulase compounds. Both ethanol yield and productivity in the NSSF are substantially higher than those in the SSF at the enzyme loading of 5 IFPU/g glucan. With 10 IFPU/g glucan, improvement in productivity was more discernible for the NSSF. The terminal yield attainable in 4 d with the SSF was reachable in 40 h with the NSSF.