The pretreatment effect on wheat straw saccharification

Enzymatic hydrolysis of cellulose is potentially an attractive method for converting cellulose into glucose which can then be used as a chemical feed or as a growth substrate for a number of microorganisms to produce microbial products. An enzymatic hydrolysis of wheat straw with cellulase preparation “Trichocease” was made. The wheat straw used was pretreated mechanically and with NaOH. A procedure of pretreatment was investigated in 26 variants. The dynamics of enzymatic hydrolysis was studied. An assay of this dynamics based on the amount of reducing sugars formed during the cellulase reaction and depending upon enzyme and substrate concentration and time of action was carried out.     

Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step

Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay.     

Economic impact of total solids loading on enzymatic hydrolysis of dilute acid pretreated corn stover

In process integration studies of the biomass-to-ethanol conversion process, it is necessary to understand how cellulose conversion yields vary as a function of solids and enzyme loading and other key operating variables. The impact of solids loading on enzymatic cellulose hydrolysis of dilute acid pretreated corn stover slurry was determined using an experimental response surface design methodology. From the experimental work, an empirical correlation was obtained that expresses monomeric glucose yield from enzymatic cellulose hydrolysis as a function of solids loading, enzyme loading, and temperature. This correlation was used in a technoeconomic model to study the impact of solids loading on ethanol production economics. The empirical correlation was used to provide a more realistic assessment of process cost by accounting for changes in cellulose conversion yields at different solids and enzyme loadings as well as enzyme cost. As long as enzymatic cellulose conversion drops off at higher total solids loading (due to end-product inhibition or other factors), there is an optimum value for the total solids loading that minimizes the ethanol production cost. The optimum total solids loading shifts to higher values as enzyme cost decreases.     

Factors affecting cellulose and hemicellulose hydrolysis of alkali treated brewers spent grain by Fusarium oxysporum enzyme extract

The enzymatic degradation of polysaccharides to monosaccharides is an essential step in bioconversion processes of lignocellulosic materials. Alkali treated brewers spent grain was used as a model substrate for the study of cellulose and hemicellulose hydrolysis by Fusarium oxysporum enzyme extract. The results obtained showed that cellulose and hemicellulose conversions are not affected by the same factors, implementing different strategies for a successful bioconversion. Satisfactory cellulose conversion could be achieved by increasing the enzyme dosage in order to overcome the end-product inhibition, while the complexity of hemicellulose structure imposes the presence of specific enzyme activities in the enzyme mixture used. All the factors investigated were combined in a mathematical model describing and predicting alkali treated brewers spent grain conversion by F. oxysporum enzyme extract.     

A comparison of two empirical models for the enzymatic hydrolysis of pretreated poplar wood

Two empirical models for the enzymatic hydrolysis of cellulose are used to analyze the same set of experimental data to determine if one model is superior to the other. Both models adequately describe the data. The parameters in both models may be correlated to the hydrolysis conditions of enzyme, substrate, and product inhibitor concentrations. Both empirical models have features which are consistent with theoretical models of cellulose hydrolysis.     

Three-stage enzymatic hydrolysis of steam-exploded corn stover at high substrate concentration

The feasibility of three-stage hydrolysis of steam-exploded corn stover at high-substrate concentration was investigated. When substrate concentration was 30% and enzyme loading was 15-30 FPU/g cellulose, three-stage (9+9+12 h) hydrolysis could reach a hydrolysis yield of 59.9-81.4% in 30 h. Compared with one-stage hydrolysis for 72 h, an increase of 34-37% in hydrolysis yield could be achieved. When steam-exploded corn stover was used as the substrate for enzyme synthesis and hydrolysis was conducted at a substrate concentration of 25% with an enzyme loading of 20 FPU/g cellulose, a hydrolysis yield of 85.1% was obtained, 19% higher than that the commercial cellulase could reach under the same conditions. The removal of end products was suggested to improve the adsorption of cellulase on the substrate and enhance the productivity of enzymatic hydrolysis.     

Recycle of enzymes and substrate following enzymatic hydrolysis of steam-pretreated aspenwood

The commercial production of chemicals and fuels from lignocellulosic residues by enzymatic means still requires considerable research on both the technical and economic aspects. Two technical problems that have been identified as requiring further research are the recycle of the enzymes used in hydrolysis and the reuse of the re calcitrant cellulose remaining after incomplete hydrolysis. Enzyme recycle is required to lower the cost of the enzymes, while the reuse of the spent cellulose will lower the feedstock cost. The conversion process studied was a combined enzymatic hydrolysis and fermentation (CHF) procedure that utilized the cellulolytic enzymes derived from the fungus Trichoderma harzianum E58 and the yeast Saccharomyces cerevisiae. The rate and extent of hydrolysis and ethanol production was monitored as was the activity and hydrolytic potential of the enzymes remaining in the filtrate after the hydrolysis period. When a commercial cellulose was used as the substrate for a routine 2-day CHF process, 60% of the original treated, water-extracted aspenwood was used as the substrate, only 13% of the original filter paper activity was detected after a similar procedure. The combination of 60% spent enzymes with 40% fresh enzymes resulted in the production of 30% less reducing sugars than the original enzyme mixture. Since 100% hydrolysis of the cellulose portion is seldom accomplished in an enzymatic hydrolysis pro cess, the residual cellulose was used as a substrate for the growth of T. harzianum E58 and production of celulolytic enzymes. The residue remaining after the CHF process was used as a substrate for the production of the cellulolytic enzymes. The production of enzymes from the residue of the Solka Floc hydrolysis was greater than the production of enzymes from the original Solka Floc.     

Effect of particle size on the rate of enzymatic hydrolysis of cellulose

The effect of particle size on enzymatic hydrolysis of cellulose has been investigated. The average size of microcrystalline cotton cellulose has been reduced to submicron scale by using a media mill. The milled products were further subjected to hydrolysis using cellulase. High cellulose concentration (7%) appeared to retard the size reduction and resulted in greater particles and smaller specific surface areas than those at low concentration (3%) with the same milling time. Initial rate method was employed to explore the rate of enzymatic hydrolysis of cellulose. The production rate of cellobiose was increased at least 5-folds due to the size reduction. The yield of glucose was also significantly increased depending upon the ratio of enzyme to substrate. A high glucose yield (60%) was obtained in 10-h hydrolysis when the average particle size was in submicron scale.     

Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls

The study of biomass deconstruction by enzymatic hydrolysis has hitherto not focussed on the importance of supramolecular structures of cellulose. In lignocellulose fibres, regions with a different organisation of the microfibrils are present. These regions are called dislocations or slip planes and they are known to be more susceptible to various forms of degradation such as acid hydrolysis. Traditionally the cellulose within these regions has been assumed to be amorphous, but in this study it is shown by use of polarized light microscopy that dislocations are birefringent. This indicates that they have a crystalline organisation. Dislocations may be entry points for endoglucanases. Using a fluorescent labelled endoglucanase combined with confocal fluorescence microscopy, it is shown that the enzyme selectively binds to dislocations during the initial phase of the hydrolysis. Using a commercial cellulase mixture on hydrothermally treated wheat straw, it was found that the fibres were cut into segments corresponding to the sections between the dislocations initially present, as has previously been observed for acid hydrolysis of softwood pulps. The results indicate that dislocations are important during the initial part of enzymatic hydrolysis of cellulose. The implications of this phenomenon have not yet been recognized or explored within cellulosic biofuels.     

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems

Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.     

Fractionation of sugar beet pulp into pectin, cellulose, and arabinose by arabinases combined with ultrafiltration.

Incubation of beet pulp with two arabinases (alpha-L-arabinofuranosidase and endo-arabinase), used singularly or in combination at different units of activity per gram of beet pulp, caused the hydrolysis of arabinan, which produced a hydrolyzate consisting mainly of arabinose. Pectin and a residue enriched with cellulose were subsequently separated from the incubation mixture. The best enzymatic hydrolysis results were obtained when 100 U/g of beet pulp of each enzyme worked synergistically with yields of 100% arabinose and 91.7% pectin. These yields were higher than those obtained with traditional chemical hydrolysis. The pectin fraction showed a low content of neutral sugar content and the cellulose residue contained only a small amount of pentoses. Semicontinuous hydrolysis with enzyme recycling in an ultrafiltration unit was also carried out to separate arabinose, pectin, and cellulose from beet pulp in 7 cycles of hydrolysis followed by ultrafiltration. The yields of separation were similar to those obtained in batch experiments, with an enzyme consumption reduced by 3.5 times and some significant advantages over batch processes.     

Changes in the enzymatic hydrolysis rate of Avicel cellulose with conversion

The slow down in enzymatic hydrolysis of cellulose with conversion has often been attributed to declining reactivity of the substrate as the more easily reacted material is thought to be consumed preferentially. To better understand the cause of this phenomenon, the enzymatic reaction of the nearly pure cellulose in Avicel was interrupted over the course of nearly complete hydrolysis. Then, the solids were treated with proteinase to degrade the cellulase enzymes remaining on the solid surface, followed by proteinase inhibitors to inactive the proteinase and successive washing with water, 1.0 M NaCl solution, and water. Next, fresh cellulase and buffer were added to the solids to restart hydrolysis. The rate of cellulose hydrolysis, expressed as a percent of substrate remaining at that time, was approximately constant over a wide range of conversions for restart experiments but declined continually with conversion for uninterrupted hydrolysis. Furthermore, the cellulose hydrolysis rate per adsorbed enzyme was approximately constant for the restart procedure but declined with conversion when enzymes were left to react. Thus, the drop off in reaction rate for uninterrupted cellulose digestion by enzymes could not be attributed to changes in substrate reactivity, suggesting that other effects such as enzymes getting "stuck" or otherwise slowing down may be responsible.     

CP/MAS 13C NMR analysis of cellulase treated bleached softwood kraft pulp

Fully bleached softwood kraft pulps were hydrolyzed with cellulase (1,4-(1,3:1,4)-beta-D-glucan 4-glucano-hydrolase, EC 3.2.1.4) from Trichoderma reesei. Supra-molecular structural features of cellulose during enzymatic hydrolysis were examined by using CP/MAS 13C NMR spectra in combination with line-fitting analysis. Different types of cellulose allomorphs (cellulose I(alpha), cellulose I(beta), para-crystalline) and amorphous regions were hydrolyzed to a different extent by the enzyme used. Also observed was a rapid initial phase for hydrolysis of regions followed by a slow hydrolysis phase. Cellulose I(alpha), para-crystalline, and non-crystalline regions of cellulose are more susceptible to enzymatic hydrolysis than cellulose I(beta) during the initial phase. After the initial phase, all the regions are then similarly susceptible to enzymatic hydrolysis.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Effect of surfactants on cellulose hydrolysis

The effect of surfactants on the heterogeneous enzymatic hydrolysis of Sigmacell 100 cellulose and of steam-exploded wood was studied. Certain biosurfactants (sophorolipid, rhamnolipid, bacitracin) and Tween 80 increased the rate of hydrolysis of Sigmacell 100, as measured by the amount of reducing sugar produced, by as much as seven times. The hydrolysis of steam-exploded wood was increased by 67% in the presence of sophorolipid. At the same time, sophorolipid was found to decrease the amount of enzyme adsorbed onto the cellulose at equilibrium. Sophorolipid had the greatest effect on cellulose hydrolysis when it was present from the beginning of the experiment and when the enzyme/cellulose ratio was low. (c) 1993 John Wiley & Sons, Inc.     

Ionic liquid pretreatment of cellulosic biomass: enzymatic hydrolysis and ionic liquid recycle

Ionic liquids (ILs) are promising solvents for the pretreatment of biomass as certain ILs are able to completely solubilize lignocellulose. The cellulose can readily be precipitated with an anti-solvent for further hydrolysis to glucose, but the anti-solvent must be removed for the IL to be recovered and recycled. We describe the use of aqueous kosmotropic salt solutions to form a three-phase system that precipitates the biomass, forming IL-rich and salt-rich phases. The phase behavior of [Emim][Ac] and aqueous phosphate salt systems is presented, together with a process for recycling the [Emim][Ac] and enzymatically hydrolyzing the cellulose. This process reduces the amount of water to be evaporated from recycled IL, permitting efficient recycle of the IL. Material balances on the process, with multiple recycles of the [Emim][Ac], quantify the major components from a Miscanthus feedstock through the pretreatment, separation, and enzymatic hydrolysis steps. A more rapid and higher yielding conversion of cellulose to glucose is obtained by use of the three-phase system as compared to the cellulose obtained from biomass pretreated with IL and precipitated with water. The addition of a kosmotropic salt during the precipitation results in partial delignification of the biomass, which makes the substrate more accessible, enhancing the enzymatic hydrolysis.     

Comparison of the 3,5-dinitrosalicylic acid and Nelson-Somogyi methods of assaying for reducing sugars and determining cellulase activity

A comparison has been made between the 3,5-dinitrosalicylic acid (DNS) and alkaline copper methods of assaying for reducing sugars released during the enzymatic hydrolysis of cellulose by culture filtrates from Trichoderma harzianum E58. The DNS method was shown to be more readily influenced by the incubation conditions and by components derived from lignocellulosic substrates. The endo-1,4-β-d-glucanase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] values obtained with the DNS assay were always considerably higher than those obtained with the alkaline copper method and did not give reducing values that were proportional to the actual number of hemiacetal reducing groups. The alkaline copper assay was not affected by the degree of polymerization of the substrate. Although this latter method appeared to be superior to the DNS assay it was still affected by the incubation conditions, nature of the substrate and the influence of other cellulase components on each of the specific enzyme assays.     

Enhancement of enzymatic hydrolysis by simultaneous attrition of cellulosic substrates

It has been shown that simultaneous attrition of cellulose in an attritor containing stainlesssteel beads results in a substantial enhancement of the enzymatic hydrolysis. The attrition exerts two opposing effects, continuous delamination and comminution of the substrate with formation of new reactive sites and a gradual denaturation and inactivation of the enzyme. Consequently, the hydrolysis proceeds very rapidly at first and levels off at about 70% saccharification of the substrate. Accumulation of hydrolysis products is also responsible for inhibition of the enzyme. The attrition method is effective for the saccharification of cottonwood in which the cellulosic microfibrils are embedded in a matrix of lignin and hemicelluloses. A comparison between the saccharification of wood, lignocellulose, holocellulose, and cellulose with simultaneous attrition showed that the lignin component provided more hindrance toward the saccharification process than hemicelluloses, which are themselves subject to enzymatic hydrolysis.     

Reconstitution of cellulose and lignin after [C2mim][OAc] pretreatment and its relation to enzymatic hydrolysis

Although the effects of cellulose crystallinity and lignin content as two major structural features on enzymatic hydrolysis have been extensively studied, debates regarding their effects still exist. In this study, reconstitution of cellulose and lignin after 1‐ethyl‐3‐methylimidazolium acetate ([C₂mim][OAc]) pretreatment was proposed as a new method to study their effects on enzymatic digestibility. Different mechanisms of lignin content for reduction of cellulose hydrolysis were found between the proposed method and the traditional method (mixing of cellulose and lignin). The results indicated that a slight change of the crystallinity of the reconstituted materials may play a minor role in the change of enzyme efficiency. In addition, the present study suggested that the lignin content does not significantly affect the digestibility of cellulose, whereas the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase plays an important role when an ionic liquid pretreatment of biomass was conducted. Biotechnol. Bioeng. 2013; 110: 729–736. © 2012 Wiley Periodicals, Inc.     

Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process

The biochemical conversion of cellulosic biomass to ethanol, a promising alternative fuel, can be carried out efficiently and economically using the simultaneous saccharification and fermentation (SSF) process. The SSF integrates the enzymatic hydrolysis of cellulose to glucose, catalyzed by the synergistic action of cellulase and beta-glucosidase, with the fermentative synthesis of ethanol. Because the enzymatic step determines the ethanol. Because the enzymatic step determines the availability of glucose to the ethanologenic fermentation, the kinetic of cellulose hydrolysis by cellulase and beta-glucosidase and the susceptibility of the two enzymes to inhibition by hydrolysis and fermentation products are of significant importance to the SSF performance and were investigated under realistic SSF conditions. A previously developed SSF mathematical model was used to conceptualize the depolymerization of cellulose. The model was regressed to the collected data to determine the values of the enzyme parameters and was found to satisfactorily predict the kinetics of cellulose hydrolysis. Cellobiose and glucose were identified as the strongest inhibitors of cellulase and beta-glucosidase, respectively. Experimental and modeling results are presented in light of the impact of enzymatic hydrolysis on fuel ethanol production. (c) 1993 Wiley & Sons, Inc.