Kinetics Of Cellulose Hydrolysis Research Articles

Model of Processive Catalysis with Site Clustering and Blocking and Its Application to Cellulose Hydrolysis.

Interactions between particles moving on a linear track and their possible blocking by obstacles can lead to crowding, impeding the particles' transport kinetics. When the particles are enzymes processively catalyzing a reaction along a linear polymeric substrate, these crowding and blocking effects may substantially reduce the overall catalytic rate. Cellulose hydrolysis by exocellulases processively moving along cellulose chains assembled into insoluble cellulose particles is an example of such a catalytic transport process. The details of the kinetics of cellulose hydrolysis and the causes of the often observed reduction of hydrolysis rate over time are not yet fully understood. Crowding and blocking of enzyme particles are thought to be one of the important factors affecting the cellulose hydrolysis, but its exact role and mechanism are not clear. Here, we introduce a simple model based on an elementary transport process that incorporates the crowding and blocking effects in a straightforward way. This is achieved by making a distinction between binding and non-binding sites on the chain. The model reproduces a range of experimental results, mainly related to the early phase of cellulose hydrolysis. Our results indicate that the combined effects of clustering of binding sites together with the occupancy pattern of these sites by the enzyme molecules play a decisive role in the overall kinetics of cellulose hydrolysis. It is suggested that periodic desorption and rebinding of enzyme molecules could be a basis of a strategy to partially counter the clustering of and blocking by the binding sites and so enhance the rate of cellulose hydrolysis. The general nature of the model means that it could be applicable also to other transport processes that make a distinction between binding and non-binding sites, where crowding and blocking are expected to be relevant.

3D Printing of Cellulase-Laden Cellulose Nanofiber/Chitosan Hydrogel Composites: Towards Tissue Engineering Functional Biomaterials with Enzyme-Mediated Biodegradation.

The 3D printing of a multifunctional hydrogel biomaterial with bioactivity for tissue engineering, good mechanical properties and a biodegradability mediated by free and encapsulated cellulase was proposed. Bioinks of cellulase-laden and cellulose nanofiber filled chitosan viscous suspensions were used to 3D print enzymatic biodegradable and biocompatible cellulose nanofiber (CNF) reinforced chitosan (CHI) hydrogels. The study of the kinetics of CNF enzymatic degradation was studied in situ in fibroblast cell culture. To preserve enzyme stability as well as to guarantee its sustained release, the cellulase was preliminarily encapsulated in chitosan–caseinate nanoparticles, which were further incorporated in the CNF/CHI viscous suspension before the 3D printing of the ink. The incorporation of the enzyme within the CHI/CNF hydrogel contributed to control the decrease of the CNF mechanical reinforcement in the long term while keeping the cell growth-promoting property of chitosan. The hydrolysis kinetics of cellulose in the 3D printed scaffolds showed a slow but sustained degradation of the CNFs with enzyme, with approximately 65% and 55% relative activities still obtained after 14 days of incubation for the encapsulated and free enzyme, respectively. The 3D printed composite hydrogels showed excellent cytocompatibility supporting fibroblast cell attachment, proliferation and growth. Ultimately, the concomitant cell growth and biodegradation of CNFs within the 3D printed CHI/CNF scaffolds highlights the remarkable potential of CHI/CNF composites in the design of tissue models for the development of 3D constructs with tailored in vitro/in vivo degradability for biomedical applications.

Enzyme-assisted extraction of fucoidan from Kjellmaniella crassifolia based on kinetic study of enzymatic hydrolysis of algal cellulose

Fucoidan extraction from Kjellmaniella crassifolia was enhanced through β-glucosidase fortified cellulase treatment. Kinetic study of the hydrolysis of isolated seaweed cellulose by commercial enzymes was carried out, and cellulolytic enzyme cocktail was optimized according to the degradation kinetics using response surface methodology for fucoidan extraction. Cellulose (9.88 wt%, d.b.) found in the seaweed was highly crystalline (CrI: 83.24 %). β-glucosidase apparently improved the affinity of the cellulolytic enzymes for the cellulose by eliminating cellobiose feedback inhibition. Treating the seaweed with 30 FPU cellulase supplemented with 286.64 CBU β-glucosidase per gram cellulose, followed by conventional ethanol precipitation, the fucoidan extraction yield based on seaweed feedstock dry matter reached 4.74 %. The total sugar content and sulfate content of the product reached 76.68 % and 22.83 %, respectively, with fucose accounting for 62.46 % of the total sugar. The product extracted under optimal conditions exhibited apparently concentration-dependent antioxidant activity, with IC50 values for DPPH, Hydroxyl and ABTS radical scavenging reaching 2.43 mg/mL, 1.02 mg/mL and 1.12 mg/mL, respectively. The results showed that the proposed enzymatic treatment based on cellulose hydrolysis kinetics effectively disrupted the cell wall and facilitated the release and extraction of fucoidan from the seaweed.

A novel approach to study cellulose digestion kinetics in biogas fermentation applying feed-stop method and artificial medium to investigate effects of saccharides

Anaerobic cellulose digestion kinetics was studied using a novel artificial medium, which allowed cellulose mono-fermentation with a complex biogas biocenosis. Semi-continuous cellulose mono-fermentation was successfully performed with this medium for more than 18 months achieving high organic loading rates of 8.3 gl−1d−1 and short hydraulic residence times of 9.6 days. Cellulose hydrolysis kinetics was studied by monitoring the substrate depletion after short feed-stops using the data obtained to establish a 1st order reaction model. This method was used to assess the cellulose hydrolysis kinetics under the influence of selected carbohydrates, of which a mixture of cellooligosaccharides showed the highest potential. A qPCR-Method was established for quantitative monitoring of selected cellulolytic clostridia based on the extraction of metagenomic DNA and comparison to standards from single cultures. The monitoring indicated consistent growth of these clostridia in the novel cultivation approach.

High-temperature tolerant enzyme based extraction of lignocellulose derived C5 and C6 sugars for the integrated production of a promising supplement for a diesel/jet-engine fuel

Development of a novel, integrated production of C8 ester butyl butyrate (BuB), a promising supplement for diesel/jet-engine fuels, from lignocellulose derived C5 and C6 sugars is attempted by means of smart and innovative process design. In the pathway, enzymatic hydrolysis of biomass with high solid loading is an important step which requires reduced amount of water supply and energy input. However, increased substrate loading lead to diffusion limitations of enzymes, on top of nonproductive absorption of enzymes. Addition of ß-glucosidase over cellulase is another reason to increase the cost of enzymes. Viscozyme can be considered as a troubleshooter in these cases where it can offset high substrate loading and increased monomeric sugar generation. In the present study, 17.61 g/L of glucose, along with 7.43 g/L of xylose, have been recovered with a viscozyme concentration of 54.4 g/L. Additionally, thermal deactivation of viscozyme has been investigated. The deactivation constant is found to be gradually increasing with escalated enzyme loading from 24.2 g/L to 54.4 g/L. In this study, it has been found that hydrolysis of biomass follows a first order pseudo-kinetics and the same is eventually used to determine the fractal kinetics of cellulose hydrolysis. Fractal kinetics showed that increment of enzyme concentration is associated with lower fractal dimensions and increased binding capacity of substrate and enzyme. Diffusion limitation of the present enzyme-substrate system has also been investigated. It is observed that faster diffusion, along with increased resistances, can be achieved with increased enzyme concentration.

The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics

Interfacial enzyme reactions require formation of an enzyme-substrate complex at the surface of a heterogeneous substrate, but often multiple modes of enzyme binding and types of binding sites comp...

Effect of lignin-based amphiphilic polymers on the cellulase adsorption and enzymatic hydrolysis kinetics of cellulose

The origin, amount, hydrophilicity, charge, molecular weight and its distribution of lignin have significant influences on the enzymatic hydrolysis of lignocellulose. The enzymatic hydrolysis of lignocellulose was essentially enhanced by lignin-based polyoxyethylene ether (EHL-PEG), whereafter followed by PEG4600 and lignosulfonate (LS). The effect of LS, EHL-PEG and PEG4600 on the adsorption and enzymatic hydrolysis kinetics of cellulase on the gold surface and cellulose film was investigated by Quartz Crystal Microbalance with dissipation monitoring (QCM-D). Results showed that the interaction of LS or EHL-PEG with cellulase was electrostatic attractive and hydrophobic effect, respectively, and formed hydrophilic cellulase aggregates. LS-Cellulase peeled off the cellulose film layer by layer, while the hydrophobic phenylpropane structure of EHL-PEG-Cellulase acted as a cellulose binding domain to hydrolysis cellulose through “Hollow” effect and made cellulose become more loose and swollen. At last, a strategy to enhance the enzymatic hydrolysis of lignocellulose by lignin-based amphiphilic polymers was proposed as well.

Enhanced hydrolysis of mechanically pretreated cellulose in water/CO2 system

The aim of this work was to study promotion of ball milling and CO2 assistance on cellulose hydrolysis kinetics in water medium. Kinetic behaviors were analyzed based on first-order and shrinking core models. The results showed that cellulose hydrolysis is enhanced by ball milling and CO2 assistance. Ball milling reduced crystallinity and particle size of cellulose, resulting in high cellulose conversion, while hydrolysis promoted by CO2 assistance was weaker. Double-layer hydrolysis was observed for ball-milled cellulose, and rate constant in active layer is higher. Based on double-layer shrinking core model (DL-SCM), activation energy of cellulose conversion decreased from 73.6 to 39.8 kJ/mol when ball milling and CO2 assistance were applied. Hydrolysis active layer was about 0.9 μm, representing activated thickness of ball-milled cellulose. Hydrolysis promotion by crystallinity and particle size reduction was distinguished via DL-SCM, and crystal evolution possesses greater improvement than particle size decrease on hydrolysis of ball-milled cellulose.

Analysis of the Long Time Behavior of Enzymatic Cellulose Hydrolysis Kinetics

AbstractEnzymatic hydrolysis of biomass to produce sugars that can be converted to fuels and other valuable chemicals, is viewed as the prime technology for utilization of this renewable resource. To accelerate technology development, models are needed that are able to accurately predict the hydrolysis rate so that reactors can be tailored to the multitude of processing conditions and substrates that can be used. Of particular interest is the ability to predict the long time conversion in the hydrolysis reaction which dictates the maximum possible sugar concentration. It is our aim in this article to develop a simple model which is able to predict the long-term conversion of cellulose to soluble sugars. Drawing on the analogy from the theory of reactions in continuous mixtures, it is shown that analysis of the long time kinetics of hydrolysis by examining the behavior of the “lump” of the reacting material results in a simple expression which is capable of predicting the kinetics. Many features of actual enzyme systems can be included in the development of the hydrolysis model, such as the large size of the enzyme molecules, adsorption onto substrate, inhibition by different factors (solvent, glucose etc.), but, when the analysis is carried out to calculate the total sugar concentration, it is shown that the equations reduce to a simple expression. Analysis of this model is given with comparison to other models and experimental data available in the literature. In addition to predicting the long-term kinetics, it is shown that the model does a surprising job of predicting the initial hydrolysis rates as well.

Enzyme kinetics of cellulose hydrolysis of Miscanthus and oat hulls.

Experiments were done to model enzymatic hydrolysis of Miscanthus and oat hulls treated with dilute solutions of nitric acid and sodium hydroxide in direct and reverse sequences. The enzymatic hydrolysis kinetics of the substrates was studied at an initial solid loading from 30 to 120g/L. The effects of feedstock type and its pretreatment method on the initial hydrolysis rate and reducing sugar yield were evaluated. The fitting results by the developed models showed good agreement with the experimental data. These models designed for developing the production technology of concentrated glucose solutions can also be applied for glucose fermentation into ethanol. The initial solid loading of 60-90g/L provides the reducing sugar concentration of 40-80g/L necessary for ethanol synthesis. The kinetic model can also be applied to investigate enzymatic hydrolysis of other substrates (feedstock type, pretreatment method) under the similar conditions used herein, with adjusted empirical coefficient values.

Kinetics of cellulose hydrolysis by halostable cellulase from a marine Aspergillus niger at different salinities

Abstract Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, K m (15.6260 g/L) and the rate constant of deactivation, K de (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, V max (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.

Wheat straw hydrolytic pretreatment using maleic acid to enhance ethanol production from sugars

The acid hydrolysis of wheat straw was studied using a batch reactor. The hydrolysis was catalyzed by 0.01-0.09 M maleic acid for 0-50 min at 140°C-180°C. The hydrolysis kinetics of cellulose and hemicelluloses were simulated by a new model incorporating the acidity of the liquid phase. Moreover, the pH and the temperature profile during the process were included using a new modified severity factor R0*. The model parameters were calculated using nonlinear regression analysis. Process parameters investigated herein include variation in temperature, acid concentration and pH. The optimum reaction conditions resulting to maximum sugars yield were estimated according to the proposed model. For log R0* = 1.971 (0.05 M maleic acid, 180°C, 25 min) up to 18.2% w/w total glucose on cellulose basis, 91.0% w/w total xylose on xylan basis and 44.4% w/w total sugars on polysaccharides basis were obtained. The monosaccharides and oligosaccharides produced can be fermented to bioethanol. [Received: January 14, 2016; Accepted: September 26, 2016]

Wheat straw hydrolytic pretreatment using maleic acid to enhance ethanol production from sugars

The acid hydrolysis of wheat straw was studied using a batch reactor. The hydrolysis was catalyzed by 0.01-0.09 M maleic acid for 0-50 min at 140°C-180°C. The hydrolysis kinetics of cellulose and hemicelluloses were simulated by a new model incorporating the acidity of the liquid phase. Moreover, the pH and the temperature profile during the process were included using a new modified severity factor R0*. The model parameters were calculated using nonlinear regression analysis. Process parameters investigated herein include variation in temperature, acid concentration and pH. The optimum reaction conditions resulting to maximum sugars yield were estimated according to the proposed model. For log R0* = 1.971 (0.05 M maleic acid, 180°C, 25 min) up to 18.2% w/w total glucose on cellulose basis, 91.0% w/w total xylose on xylan basis and 44.4% w/w total sugars on polysaccharides basis were obtained. The monosaccharides and oligosaccharides produced can be fermented to bioethanol. [Received: January 14, 2016; Accepted: September 26, 2016]

Kinetics of Cellulose Hydrolysis in an Oscillatory Baffled Reactor

Enzymatic hydrolysis of microcrystalline cellulose in an oscillatory baffled reactor (OBR) and a parallel stirred tank (STR) were studied. In the proposed mechanistic model, saccharification of the intractable, crystalline portion of cellulose, E*Sx is important. Its rate, k3 (0.002), although much lower than k2 (9.05), the rate of product formation from the active E * Sc intermediate, makes appreciable contribution to the final glucose yield in the saccharification of cellulose. Shear deactivation of the cellulase is also considered in relation to average shear rate (ASR) in the reactor. At 200 Wm-3 ASR in the STR was twice that in the OBR (45:22.5 s -1 respectively). Correspondingly, 74% saccharification of cellulose was observed in the STR and 91% in the OBR after 168 h. The OBR has a more uniform shear field than the STR, hence, lower ASR even at the same power density as the STR. The OBR achieved the highest conversion at ~1/20th the power input of

Effect of the molecular structure of lignin-based polyoxyethylene ether on enzymatic hydrolysis efficiency and kinetics of lignocelluloses

Effect of the molecular structure of lignin-based polyoxyethylene ether (EHL–PEG) on enzymatic hydrolysis of Avicel and corn stover was investigated. With the increase of PEG contents and molecular weight of EHL–PEG, glucose yield of corn stover increased. EHL–PEG enhanced enzymatic hydrolysis of corn stover significantly at buffer pH 4.8–5.5. Glucose yield of corn stover at 20% solid content increased from 32.8% to 63.8% by adding EHL–PEG, while that with PEG4600 was 54.2%. Effect of EHL–PEG on enzymatic hydrolysis kinetics of cellulose film was studied by quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). An enhancing mechanism of EHL–PEG on enzymatic hydrolysis kinetics of cellulose was proposed. Cellulase aggregates dispersed by EHL–PEG excavated extensive cavities into the surface of cellulose film, making the film become more loose and exposed. After the maximum enzymatic hydrolysis rate, the film was mainly peeled off layer by layer until equilibrium.

A kinetics modeling study on the inhibition of glucose on cellulosome of Clostridium thermocellum

A simplified kinetics model was built to study the inhibition of glucose on cellulosome of Clostridium thermocellum. Suitable reaction conditions were adopted to evaluate the model. The model was evaluated at different temperatures and further with various activated carbon additions as adsorbent for glucose. Investigation results revealed that the model could describe the hydrolysis kinetics of cellulose by cellulosome quite well. Glucose was found to be an inhibitor for cellulosome based on the kinetics analysis. Inhibition increased with the increase in temperature. Activated carbon as adsorbent could lower the inhibition. Parameters in the model were further discussed based on the experiment. The model might also be used to describe the strong inhibition of cellobiose on cellulosome. Saccharification of cellulose by both cellulosome and C. thermocellum could be enhanced efficiently by activated carbon addition.

The effect of hemicelluloses and lignin on acid hydrolysis of cellulose

In acid hydrolysis of plant biomass, polysaccharides are converted to monosaccharides, which is basic raw material for biorefinery for fermentation based process. These monosaccharides, however, are not stable in acidic reaction medium, and are converted to organic acids via furans. Impact of hemicelluloses and lignin on acid hydrolysis of cellulose was investigated to focus on monosaccharide production with different degrees of cellulose purity. Two-step concentrated sulphuric acid process was applied to biomass for monosaccharide conversion. Kinetics of cellulose hydrolysis was analysed using 1H NMR spectroscopy. Higher reaction temperature in secondary hydrolysis caused severe degradation of the monosaccharides. In pure and holocellulose, further reaction of glucose in acidic reaction medium produced formic acid and levulinic acid. However, lignocellulosic biomass generated much less formic acid and levulinic acid under the same reaction condition. Humin (or pseudo-lignin) was also formed by the condensation of lignin and furans from monosaccharides in acidic reaction condition. Thus, the fermentation inhibitors, furans and formic acid, were generated in low quantities by lignocellulosic biomass than by delignified biomass such as pure cellulose or holocellulose.

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s(-1) at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

Kinetics of Glucose Production from Cellulose by Hydrolysis in Sub-Critical Water

Biomass is an important renewable and sustainable source of energy. Cellulose is the primary component of plant-derived biomass. Glucose, which can be further transformed to fuel alcohol in a fermentation process, is a biomass energy precursor. This study investigated the production of glucose from cellulose by hydrolysis in sub-critical water and the hydrolysis kinetics of cellulose. Kinetics was conducted in a temperature range of 240-260°C using a 200 ml stainless steel batch reactor. A simplified kinetic model base on parallel and consecutive reaction was proposed. The differential equations resulting from the model were fit to experimental data to obtain kinetic rate constants. The activation energy and the pre-exponential factor were determined. A good agreement between the simplified model and the experimental data was obtained. The experimental results show that the best hydrolysis technology is: reaction temperature 260°C, reaction time 120s. Under this condition, the glucose yield reaches 46.05%.

Transition of Cellulose Crystalline Structure and Surface Morphology of Biomass as a Function of Ionic Liquid Pretreatment and Its Relation to Enzymatic Hydrolysis

Cellulose is inherently resistant to breakdown, and the native crystalline structure (cellulose I) of cellulose is considered to be one of the major factors limiting its potential in terms of cost-competitive lignocellulosic biofuel production. Here we report the impact of ionic liquid pretreatment on the cellulose crystalline structure in different feedstocks, including microcrystalline cellulose (Avicel), switchgrass (Panicum virgatum), pine ( Pinus radiata ), and eucalyptus ( Eucalyptus globulus ), and its influence on cellulose hydrolysis kinetics of the resultant biomass. These feedstocks were pretreated using 1-ethyl-3-methyl imidazolium acetate ([C2mim][OAc]) at 120 and 160 °C for 1, 3, 6, and 12 h. The influence of the pretreatment conditions on the cellulose crystalline structure was analyzed by X-ray diffraction (XRD). On a larger length scale, the impact of ionic liquid pretreatment on the surface roughness of the biomass was determined by small-angle neutron scattering (SANS). Pretreatment resulted in a loss of native cellulose crystalline structure. However, the transformation processes were distinctly different for Avicel and for the biomass samples. For Avicel, a transformation to cellulose II occurred for all processing conditions. For the biomass samples, the data suggest that pretreatment for most conditions resulted in an expanded cellulose I lattice. For switchgrass, first evidence of cellulose II only occurred after 12 h of pretreatment at 120 °C. For eucalyptus, first evidence of cellulose II required more intense pretreatment (3 h at 160 °C). For pine, no clear evidence of cellulose II content was detected for the most intense pretreatment conditions of this study (12 h at 160 °C). Interestingly, the rate of enzymatic hydrolysis of Avicel was slightly lower for pretreatment at 160 °C compared with pretreatment at 120 °C. For the biomass samples, the hydrolysis rate was much greater for pretreatment at 160 °C compared with pretreatment at 120 °C. The result for Avicel can be explained by more complete conversion to cellulose II upon precipitation after pretreatment at 160 °C. By comparison, the result for the biomass samples suggests that another factor, likely lignin-carbohydrate complexes, also impacts the rate of cellulose hydrolysis in addition to cellulose crystallinity.