Kinetic modeling of the enzymatic hydrolysis of pretreated cellulose

Biotechnology and Bioengineering, 1987

The process of enzymatic cellulose saccharification has been widely investigated recently since cellulose is the most abundant renewable resource and glucose formed as a product of hydrolysis can be converted to ethanol, microbial products, and other chemicals. The efficiency of enzymatic cellulose hydrolysis is known to be affected by many factors related to the source of the cellulase preparation,14 composition and structure of cellulosic material, the method of its pretreatment,'-1° process conditions,"I2 and the reactor design.'",." Since these factors are closely interrelated, a complex study of hydrolysis and a quantitative estimation of the role of the individual factors affecting its efficiency are needed to optimize the process.

Process Biochemistry, 2018

A kinetic model based on Michaelis-Menten assumptions is developed here for enzymatic hydrolysis of cellulose using cellulase enzyme derived from Trichoderma reesei. Further, to assess the inhibition of enzyme and the nature of inhibition by different sugars (glucose, galactose, mannose, xylose and cellobiose) the primary kinetic model is modified with the inhibition kinetics. The enzymatic reaction was carried out at pH = 4.8, temperature = 50°C, solid loading of 1:20 and at 100 rpm stirrer speed. It was observed among all the sugars, only xylose (five carbon sugars) showed non-competitive inhibition with K i = 27.2 mM and V max = 0.19 g L −1 h −1. Glucose and cellobiose manifested competitive inhibition with almost comparable inhibition constants (K i) of 24.7 and 26.3 mM respectively, while V max = 0.26 g L −1 h −1. On the contrary, with galactose and mannose, the inhibition constants decrease to 10.9 and 11.1 mM respectively. Statistical analysis shows that cellobiose attributes to a maximum inhibition with 72% reduction in reducing sugar production. On the contrary, the minimum inhibitory effect with 11% reduction in sugar production was observed with xylose. At the end, ANOVA analysis manifests that the effect of different sugars are much significant in inhibiting the enzyme compared to the substrate concentration in case with the cellulase enzyme from Trichoderma reesei.

Energy and Sustainability V, 2014

Biofuel production such as ethanol from lignocellulosic biomass consists of three fundamental processes: pretreatment, enzymatic hydrolysis, and fermentation. Enzymatic hydrolysis uses two types of enzymes simultaneously: endoglucanase I (EG 1) and cellobiohydrolase I (CBH 1), to break the cellulose chains into sugar in the form of cellobiose or glucose. We studied a currently proposed kinetic model for enzymatic hydrolysis of cellulose that uses the population balance equation. The model describes the changes in the cellulose chain length distribution. The complexity of the model makes finding the analytical solution difficult. Therefore, we split the full model into two cases of individual enzyme hydrolysis action and perform mathematical analysis of a single pure enzyme of both cases. The approximate solutions for both cases were derived by employing the asymptotic analysis method. The integrodifferential equation in the first case is solved using Laplace transform. Some significant characteristics are captured. The higher the rate of exposure of cellulose substrates to enzymes, the higher the number of cellulose chains generated from the breakage process. And also, the rate coefficient for CBH1 to locate and thread a reducing end of a cellulose chain is a key factor in bioconversion.

Applied Biochemistry and Biotechnology, 2014

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.

Applied Biochemistry and Biotechnology, 2006

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K m = 21 mM, K ic = 0.035 mM; K icl = 1.5 × 10 15 mM; k cat = 12 h-1) or assuming variable substrate (K m = 16 mM, K ic = 0.037 mM; K icl = 5.8 × 10 14 mM; k cat = 9 h-1), showed a good similarity between these two alternative methodologies and pointed out that both ethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K icl. In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration. 28 Bezerra et al.

2019

Corn cob is one of lignocellulosic material used for bioethanol production due to its high content of cellulose which can be converted into glucose through enzymatic hydrolysis. Unfortunately, the hydrolysis process is time consuming and the yield is relatively low. It is necessary to develop a more efficient process by applying optimum conditions. The objective of this research is to study the effect of the substrate concentration and the stirring rate on the amount of the glucose produced, to optimize these parameters and to derive a kinetic model of the enzymatic hydrolysis process studied. Pre-treated corn cobs and multiple enzymes (Cellic® C-Tec2 and Cellic® H-Tec2 from Novozymes) are used. The results show that the higher substrate concentration and the higher stirring rate lead to a higher amount of glucose. However, if the stirring rate is greater than 150 rpm, the enzymes activity decreases as they are sensitive to a mechanical stress. The optimum values of the substrate co...

In this paper, the kinetics of enzymatic hydrolysis of cellulose samples with different structural characteristics has been studied using the equation of Avrami-Kolmogarov-Erofeev (AKE): ln(1-α) = -K tn, where α is conversion degree; K is effective rate constant; t is time, and n is effective order of the kinetic process. It was shown that AKE-equation adequately describes the experimental kinetic curves. In case of hydrolysis of highly crystalline microcrystalline cellulose, the coefficient n in the AKE-equation is 0.5, which is typical for diffusion mechanism of the process. With the decrease of crystallinity degree of cellulose, the coefficient n increases and reaches 1 for completely amorphous cellulose in a wet state that indicates on the reaction of first-order. The intermediate n-value from 0.5 to 1 shows that the enzymatic hydrolysis of the sample is limited by diffusion of the large enzyme molecules into the cellulose structure. Drying of cellulose samples causes a decrease of pore volume and amplifies the contribution of diffusion to integral hydrolysis process. Effective rate constant K of enzymatic hydrolysis also increases with decreasing of crystallinity of the cellulose sample. Furthermore, the K-value for the wet sample was higher than for the dry sample. The use of parameters of AKE-equation

Biotechnology and Bioengineering, 1987

A reactor's design is of great importance in the enzymatic hydrolysis of cellulose. Three types of reactors or variations of these are usually in use: a batchstirred which is the most often employed, a continuous-stirred tank reactor with an ultrafiltration and a continuous plug-flow column reactor.8-"

Metabolic Engineering, 2008

Microcrystalline cellulose (Avicel) was subjected to three different pretreatments (acid, alkaline, and organosolv) before exposure to a mixture of cellulases (Celluclast). Addition of beta-glucosidase, to avoid the well-known inhibition of cellulase by cellobiose, markedly accelerated cellulose hydrolysis up to a ratio of activity units (beta-glucosidase/cellulase) of 20. All pretreatment protocols of Avicel were found to slightly increase its degree of crystallinity in comparison with the untreated control. Adsorption of both cellulase and beta-glucosidase on cellulose is significant and also strongly depends on the wall material of the reactor. The conversion-time behavior of all four states of Avicel was found to be very similar. Jamming of adjacent cellulase enzymes when adsorbed on microcrystalline cellulose surface is evident at higher concentrations of enzyme, beyond 400 U/L cellulase/8 kU/L beta-glucosidase. Jamming explains the observed and well-known dramatically slowing rate of cellulose hydrolysis at high degrees of conversion. In contrast to the enzyme concentration, neither the method of pretreatment nor the presence or absence of presumed fractal kinetics has an effect on the calculated jamming parameter for cellulose hydrolysis.

Biocatalysis and Biotransformation, 2007

The kinetics of cellulose hydrolysis by commercially available Cellubrix were described mathematically, with Avicel and wheat straw as substrates. It was demonstrated that hydrolysis could be described by three reactions: direct glucose formation and indirect glucose formation via cellobiose. Hydrolysis did not involve any soluble oligomers apart from low amounts of cellobiose. Phenomena included in the mathematical model were substrate limitation, adsorption of enzyme onto substrate, glucose inhibition, temperature dependency of reaction rates, and thermal enzyme inactivation. In addition, substrate heterogeneity was described by a recalcitrance constant. Model parameters refer to both enzyme characteristics and substrate-specific characteristics. Quantitative model development was carried out on the basis of Avicel hydrolysis. In order to describe wheat straw hydrolysis, wheat straw specific parameter values were measured. Updating the pertinent parameters for wheat straw yielded a satisfactory description of wheat straw hydrolysis, thus underlining the generic potential of the model.

Applied Biochemistry and Biotechnology, 2000

Acids catalyze the hydrolysis of cellulose and hemicellulose to produce sugars that organisms can ferment to ethanol and other products. However, advanced low- and no-acid technologies are critical if we are to reduce bioethanol costs to be competitive as a pure fuel. We believe carbohy drate oligomers play a key role in explaining the performance of such hydrolysis processes and that kinetic models would help us understand their role. Various investigations have developed reaction rate expressions based on an Arrhenius temperature dependence that is first order in substrate concentration and close to first order in acid concentration. In this article, we evaluate these existing hydrolysis models with the goal of providing a foundation for a unified model that can predict performance of both current and novel pretreatment process configurations.

Biotechnology and Bioengineering, 1988

Cellulose materials can readily be degraded into cellobiose and glucose by hydrolysis of the enzymes cellulase and P-glucosidase in aqueous media. Product inhibition does, however, retard the reaction rate and reduce productivity. This may be avoided by carrying out the degradation of cellulose in an aqueous two-phase system, which permits the enzymes and the substrate to be partitioned to one phase and the products to be extracted into a second phase. In addition, two-phase systems also allow recycling of the enzymes. Here, three models previously developed for "one-phase" enzymic degradation are compared to data from enzymic degradation in an aqueous two-phase system. The models tested agreed relatively well with batch experiments during a period of 200 h. For one of the models tested, continuous degradation also gave accurate agreement.

Acid Hydrolysis of Cellulose. Part I. Experimental Kinetic Analysis, 1993

The main objective of this investigation is to obtain experimental data for the sulfuric acid hydrolysis of cotton and mechanically pretreated cotton fibres. These data indicate that some glycosidic bonds of cellulose have very high accessibility to catalytic ions. It was also shown that milling increases the accessibility of some glycosidic bonds of cellulose and decreases the volume of the crystalline regions of cotton. From the glucose yield versus time data, it was found that the effect of milling on the rate of cellulose depolymerization depends on the reactivity and accessibility of the glycon rings of cellulose. It was also found that at 1OO " C, the rate of cellulose depolymerization was not affected by the extraction of cotton wax and this was related to a rolling up process of cotton wax caused by melting. The kinetic constants of glucose degradation and cellobiose hydrolysis have been determined for the stochastic simulation of cellulose depolymerization which is the subject of the second part of this work. L'objectif principal de ce travail est l'obtention de donnCes exp6rimentales sur l'hydrolyse acide (sulfurique) des fibres du coton nature1 et du coton pretraitt mtcaniquement. Les rCsultats obtenus montrent que certaines liaisons glycosi-diques de la cellulose prksentent une grande accessibilitk aux ions catalytiques. Nous avons aussi montrC qu'un pretraitement mCcanique augmente I'accessibilite de certains liens glycosidiques de la cellulose et diminue le volume des regions cristallines du coton. Les donnCes exPCrimentales du glucose produit en fonction du temps, indiquent que les effets d'un pritraitement mtcanique sur la vitesse de dipolymerisation de la cellulose dCpendent de la rkactivitd et de l'acces-sibilitC des glycones de la cellulose. Nous avons aussi constat6 qu'il 1OO " C, la vitesse de d6polymirisation de la cellulose n'est pas affectee par l'extraction des cires du coton. Nous avons relit5 celil il une globulisation des cires causCe par leur fusion. Les constantes cinktiques de la degradation du glucose et de l'hydrolyse du cellobiose ont CtC deter-minees pour la simulation stochastique de la ddpolymerisation de la cellulose qui fera l'objet de la seconde partie de ce travail.

Kinetic aspects of enzyme hydrolysis of cellulose fiber material, 2015

The kinetic dependences characterizing enzyme hydrolysis of cellulose fiber material (bleached hardwood pulp) by cellulasic enzyme complexes are studied. It is found that the topochemical mechanism provides a good interpretation of the cellulase action. The kinetics of the process is described by the modified topochemical equation of Prout – Tompkins. It is applied to the processes starting on the easily accessible outer surface of the pulp to continue further through by a gradual penetration to the capillary system of the fiber. The structural features of the system pulp – enzyme control the rate of the process. The activation energy of the hydrolysis is found constant indicating that the energy characteristics of the cellulose are identical on the surface and in the bulk of the fiber matrix. The pre-exponential factor accounts for the accessibility and the varying dimensions of the reaction area, which is formed by the cellulose–enzyme complexes and change during the process. The rate decrease in the course of the process is determined not only by the pre-exponential factor decrease but by the enzyme inhibition as well.

Journal of Chemical Engineering of Japan, 1988