Surface character of pulp fibres studied using endoglucanases

The endoglucanase Cel5A from Trichoderma reesei and an endoglucanase from Aspergillus sp. (Novozym 476 from Novozyme A/S) were evaluated as probes for the surface properties of soft- and hardwood chemical pulp fibres. The hydrolysis time curves were in accordance with a two-phase degradation model described by a biexponential function. The kinetic parameters corresponding to the amount of fast and slow degraded parts of the substrate correlated to tensile index, relative bonded area and z-strength of the paper. All paper properties showing a correlation with enzyme kinetic parameters were related to fibre-fibre interactions. Fluorescence labelling of the reducing end groups in pulp fibres followed by enzyme treatment indicated that the fast substrate class corresponds to the population of "loose" cellulose chain ends not tightly associated with the bulk cellulose. The correlation between the parameters of enzyme kinetics and mechanical properties of the paper produced from the corresponding pulp found in this study should allow a rapid evaluation of the raw fibre material used in paper making process.     

The effect of crystallinity of cellulose on the rate of reducing sugars production by heterogeneous enzymatic hydrolysis

A kinetic model is devised, from the reaction mechanism steps, to predict the rate of reducing sugar production by hydrolysis of two types of cellulose, namely, amorphous carboxymethylcellulose (CMC) and highly crystalline wood shavings, using Aspergillus niger cellulase. Experimental results in a stirred batch reactor at 40 degrees C show that the production of reducing sugar reduced at much shorter times for wood shavings in comparison to CMC at the same initial substrate concentration. The experimental results are used to determine the kinetic parameters of the model equations. The significance of crystallinity was determined using inert fraction coefficient, which is assumed to be constant and equals 0.05 and 0.98 for CMC and wood shavings, respectively. It is shown there is a good agreement between the experimental results and proposed kinetic model predictions. The effect of the inert fraction coefficient on the production of reducing sugar by the enzymatic hydrolysis of cellulose is also determined. It is found that the cellulase used extracted from A. niger is much more sensitive towards the substrate structure in comparison to that extracted from Trichoderma reesei.     

Equivalent models of indanol isomers adsorption on cellulose tribenzoate

The adsorption isotherm data of (R)- and (S)-1-indanol and of their racemic mixture on cellulose tribenzoate were measured by frontal analysis. The experimental data for each enantiomers were fitted to the single-component bilangmuir isotherm model. The competitive experimental data were fitted to the ideal adsorption solution model (IAS), the real adsorption solution model (RAS), and the bilangmuir thermodynamically consistent model (BTC). The mass transfer kinetic parameters were estimated from systematic comparisons between the experimental single-component band profiles and profiles calculated using the general rate model (GR) of chromatography coupled with the generalized Maxwell-Stefan equation (GMS). The validation of the isotherm model and of the mass transfer kinetic model was made by comparing the experimental band profiles obtained for solutions of the two enantiomers and those calculated with the competitive GR-GMS model. The excellent agreement observed proves that a combination of the BTC isotherm model and the GMS kinetic model, using the best values of the BTC and GMS parameters estimated from single component experiments, allows an excellent prediction of the binary isotherm and the binary mass transfer kinetics.     

Use of thermogravimetric analysis to monitor the effects of natural laccase mediators on flax pulp

The effects of 1-hydroxybenzotriazole (HBT) and the natural laccase mediators gallic acid, caffeic acid and p-hydroxybenzoic acid, on the enzymatic bleaching of flax pulp were compared. The treatment was performed under atmospheric air and oxygen pressure, and, for the first time, monitored by thermogravimetric analysis (TGA) for comparison with chemical analysis, FTIR and X-ray diffraction (XRD) spectroscopies. Thermogravimetric data were simulated by applying a nucleation kinetic equation to a combustion model based on four pseudo-components (hemicellulose, amorphous and crystalline cellulose, and lignin). The results thus obtained show that the effects of the natural mediators are similar to those of HBT and lead to increased order in cellulose microfibril surfaces. An increase in pulp crystallinity was additionally exposed by the XRD and FTIR techniques, and a reduction in microfibril equatorial size by the XRD patterns. Simulated DTG curves were used to determine the kinetic parameters for thermal degradation.     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Kinetic studies on insoluble cellulose-cellulase system.

Enzymatic hydrolysis of insoluble amorphous cellulose by Trichoderma viride cellulase was investigated in a batch reactor at several substrate concentrations and three enzyme levels. The reactions were carried out at 50 degrees C and pH 4.8. Enzyme was rapidly adsorbed onto solids on contact, then gradually returned to the liquid phase as the reaction proceeded. A kinetic model that considered the fast adsorption which was followed by the slow reaction, and subsequent product inhibition was developed to interpret the experimental observations. The resulting equation successfully correlated the data for up to 70% conversion. The methods for determining the kinetic parameters are discussed.     

Kinetic modeling of rapid enzymatic hydrolysis of crystalline cellulose after pretreatment by NMMO

Pretreatment of cellulose with an industrial cellulosic solvent, N-methylmorpholine-N-oxide, showed promising results in increasing the rate of subsequent enzymatic hydrolysis. Cotton linter was used as high crystalline cellulose. After the pretreatment, the cellulose was almost completely hydrolyzed in less than 12 h, using low enzyme loading (15 FPU/g cellulose). The pretreatment significantly decreased the total crystallinity of cellulose from 7.1 to 3.3, and drastically increased the enzyme adsorption capacity of cellulose by approximately 42 times. A semi-mechanistic model was used to describe the relationship between the cellulose concentration and the enzyme loading. In this model, two reactions for heterogeneous reaction of cellulose to glucose and cellobiose, and a homogenous reaction for cellobiose conversion to glucose was incorporated. The Langmuir model was applied to model the adsorption of cellulase onto the treated cellulose. The competitive inhibition was also considered for the effects of sugar inhibition on the rate of enzymatic hydrolysis. The kinetic parameters of the model were estimated by experimental results and evaluated.     

Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass

A semimechanistic multi‐reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose‐to‐glucose and two heterogeneous reactions of cellulose‐to‐cellobiose and cellulose‐to‐glucose. Adsorption of cellulase onto pretreated CWR during enzymatic hydrolysis was modeled via a Langmuir adsorption isotherm. This is the first kinetic model which incorporated the negative role of lignin (nonproductive adsorption) using a Langmuir‐type isotherm adsorption of cellulase onto lignin. The model also reflected the competitive inhibitions of cellulase by glucose and cellobiose. The Matlab optimization function of “lsqnonlin” was used to fit the model and estimate kinetic parameters based on experimental data generated under typical conditions (8% solid loading and 15 FPU/g‐cellulose enzyme concentration without the addition of background sugars). The model showed high fidelity for predicting cellulose hydrolysis behavior over a broad range of solid loading (4–12%, w/w, dry basis), enzyme concentration (15–150 FPU/ g‐cellulose), sugar inhibition (glucose of 30 and 60 mg/mL and cellobiose of 10 mg/mL). In addition, sensitivity analysis showed that the incorporation of the nonproductive adsorption of cellulase onto lignin significantly improved the predictability of the kinetic model. Our model can serve as a robust tool for developing kinetic models for system optimization of enzymatic hydrolysis, hydrolysis reactor design, and/or other hydrolysis systems with different type of enzymes and substrates. Biotechnol. Bioeng. 2009;102: 1558–1569. © 2008 Wiley Periodicals, Inc.     

Comparison of two-phase lipase-catalyzed esterification on micro and bench scale

Lipase type B from Candida antarctica was used to catalyze the esterification of propionic acid and 1-butanol in a water/n-decane two-phase system on micro and on bench scale. The reaction was described by a Ping Pong Bi Bi mechanism with alcohol inhibition. The kinetic parameters on micro and bench scale were compared; no significant differences were found. Furthermore, effects of temperature on activation and inactivation of the enzyme were found to be similar on micro and bench scale. Therefore, parameters found on either scale can be used for the other scale. Enzyme kinetic parameters can be determined on a micro scale, with very low consumption of reagents and catalyst, and then be applied to bench scale. This can reduce the cost of optimizing enzyme processes by downscaling.     

Biodegradation of 4-nitrophenol in a two-phase sequencing batch reactor: concept demonstration,kinetics and modelling

The objectives of this work were to demonstrate the potential of a two-phase sequencing batch reactor in degrading xenobiotics and to evaluate the kinetic parameters leading to a mathematical model of the system. 4-Nitrophenol (4NP), a typical representative of substituted phenols, was selected as the target xenobiotic; this compound has never been remediated in a two-phase bioreactor before. Partition tests were conducted to determine the most appropriate partitioning solvent, and among the three investigated solvents (1-undecanol, 2-undecanone and oleyl alcohol), 2-undecanone was chosen because of its favourable partition coefficient and its negligible emulsion-forming tendencies. Moreover, the selected solvent showed satisfactory biocompatibility characteristics with respect to the biomass, with only minor effects on the intrinsic microbial kinetics. Kinetic tests were then performed in a sequencing batch reactor (2-l volume) operated in both conventional one- and two-phase configurations, with the two-phase system showing a significant improvement in the process kinetics in terms of reduced inhibition and increased maximum removal rate. The obtained kinetic parameters suggest that the two-phase sequencing batch system may find full-scale application, as the maximum removal rate k _max (~3 mg 4NP mgVSS⁻¹ day⁻¹) is of the same order of magnitude of heterotrophic bacteria operating in wastewater treatment plants.     

Modeling intrinsic kinetics of enzymatic cellulose hydrolysis

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.     

Synergism of endoenzyme and exoenzyme on hydrolysis of soluble cellulose derivatives

A kinetic model that represents the reaction of hydrolysis of water-soluble cellulose derivatives by a mixed endo- and exoenzyme system is proposed with the following assumptions: at an early stage of the reaction, endoenzymes split the substrate molecule in order to supply the newly formed nonreducing ends to exoenzymes until the molecular weight of the substrates reaches a low value; after that point, the reaction kinetics obeys only the rate equation of the reaction of the exoenzymes in which the reaction parameters change linearly with decrease of the molecular weight of the substrates. Hydrolysis experiments of soluble cellulose derivatives, carboxymethyl cellulose and hydroxyethyl cellulose, were carried out with endo-and exoenzymes separated from Trichoderma Koningii cellulase. The critical molecular weight of the substrate, from that point the action of endoenzyme can be neglected, was determined from the experimental data. That was ca. 4000 D. With that value, the model fits well the experimental data. Synergism of both enzymes appears as enhancement of the rate of the reaction at the early stage of the reaction.     

Evaluating kinetics of enzymatic saccharification of lignocellulose by fractal kinetic analysis

The effects of impacting factors, including cellulase loading, operation temperature, product glucose inhibition, and high solid pretreated biomass loading were examined systemically on the enzymatic saccharification of lignocellulose (dilute acid pretreated corn stover) in the presence and absence of tri-block copolymer L64 (also referred to polymeric nonionic surfactant). The complex kinetics of enzymatic saccharification of cellulose were subjected to fractal kinetic analysis based on a fractal kinetic model, which is described with fractal kinetic parameters of the rate constant and fractal exponent. The results indicate that glucose inhibition including high lignocellulose loading is indexed by decreasing rate constant while lignin inhibition and high operation temperature is indexed by increasing fractal exponent. The effect of a nonionic surfactant on the enzymatic saccharification of lignocellulose mainly contributed to the elimination of lignin inhibition by decreasing the corresponding fractal exponent. However, the effect of the nonionic surfactant on cellulase activity and stability was very limited.     

Pulp properties and their influence on enzymatic degradability

Endoglucanase treatment of pulp for the adjustment of viscosity and the increase in pulp reactivity is a promising step in the concept for the beneficial production of dissolving pulps from paper grade pulps. To promote the commercial applicability of these enzymes, the influence of pulp properties such as carbohydrate composition, pulp type and cellulose morphology on the enzymatic degradability of a pulp was examined. High contents of hemicelluloses and lignin were shown to impair the accessibility of the cellulose to the enzymes. Due to the elevated swelling capacity of cellulose II, conversion of the cellulose morphology from I to II upon alkaline treatments showed a large increasing effect on the cellulose accessibility, and enzymatic degradability. Reactivity measurements of softwood sulfite pulps after enzymatic degradation and acid-catalyzed hydrolysis, respectively, revealed elevated reactivity for the pulp after acid treatment. This is in contrast to effects of enzyme treatments reported for CCE treated kraft pulps.     

The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility

The influence of cellulose accessibility and protein loading on the efficiency of enzymatic hydrolysis of steam pretreated Douglas-fir was assessed. It was apparent that the lignin component significantly influences the swelling/accessibility of cellulose as at low protein loadings (5 FPU/g cellulose), only 16% of the cellulose present in the steam pretreated softwood was hydrolyzed while almost complete hydrolysis was achieved with the delignified substrate. When lignin (isolated from steam pretreated Douglas-fir) was added back in the same proportions it was originally found to the highly accessible and swollen, delignified steam pretreated softwood and to a cellulose control such as Avicel, the hydrolysis yields decreased by 9 and 46%, respectively. However, when higher enzyme loadings were employed, the greater availability of the enzyme could overcome the limitations imposed by both the lignin’s restrictions on cellulose accessibility and direct binding of the enzymes, resulting in a near complete hydrolysis of the cellulose.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: Derivation of a mechanistic kinetic model

A comprehensive mechanistic kinetic model for enzymatic hydrolysis of insoluble cellulose has been synthesized by combining models for several key aspects which have been derived independent of each other. The model takes into account the major contributing factors: the nature of the enzyme system, the structure of cellulose, and the mode of interaction between the enzyme and cellulose molecules. It consists of a set of simultaneously occurring ordinary differential equations with ten kinetic constants. All of the kinetic constants have been determined independently by carrying out critically designed experiments, and they appear in the comprehensive model without any arbitrary manipulations. The governing equations of the model have been numerically simulated by means of the computer subroutine CSMP III. The model predicts the progress of hydrolysis of cellulose over a wide range of experimental conditions and hydrolysis times reasonably well. The model can even be applied to predict the progress of hydrolysis for intensively pretreated cellulose with a minor adjustment. The applicability of the model for the actual process development is also discussed.     

A functionally based model for hydrolysis of cellulose by fungal cellulase

A new functionally based kinetic model for enzymatic hydrolysis of pure cellulose by the Trichoderma cellulase system is presented. The model represents the actions of cellobiohydrolases I, cellobiohydrolase II, and endoglucanase I; and incorporates two measurable and physically interpretable substrate parameters: the degree of polymerization (DP) and the fraction of beta-glucosidic bonds accessible to cellulase, F(a) (Zhang and Lynd, 2004). Initial enzyme-limited reaction rates simulated by the model are consistent with several important behaviors reported in the literature, including the effects of substrate characteristics on exoglucanase and endoglucanase activities; the degree of endo/exoglucanase synergy; the endoglucanase partition coefficient on hydrolysis rates; and enzyme loading on relative reaction rates for different substrates. This is the first cellulase kinetic model involving a single set of kinetic parameters that is successfully applied to a variety of cellulosic substrates, and the first that describes more than one behavior associated with enzymatic hydrolysis. The model has potential utility for data accommodation and design of industrial processes, structuring, testing, and extending understanding of cellulase enzyme systems when experimental date are available, and providing guidance for functional design of cellulase systems at a molecular scale. Opportunities to further refine cellulase kinetic models are discussed, including parameters that would benefit from further study.     

Parameter determination and validation for a mechanistic model of the enzymatic saccharification of cellulose‐Iβ

Cost‐effective production of fuels and chemicals from lignocellulosic biomass often involves enzymatic saccharification, which has been the subject of intense research and development. Recently, a mechanistic model for the enzymatic saccharification of cellulose has been developed that accounts for distribution of cellulose chain lengths, the accessibility of insoluble cellulose to enzymes, and the distinct modes of action of the component cellulases [Griggs et al. (2012) Biotechnol. Bioeng., 109(3):665–675; Griggs et al. (2012) Biotechnol. Bioeng., 109(3):676–685]. However, determining appropriate values for the adsorption, inhibition, and rate parameters required further experimental investigation. In this work, we performed several sets of experiments to aid in parameter estimation and to quantitatively validate the model. Cellulosic materials differing in degrees of polymerization and crystallinity (α‐cellulose‐I_β and highly crystalline cellulose‐I_β) were digested by component enzymes (EG_I/CBH_I/ ) and by mixtures of these enzymes. Based on information from the literature and the results from these experiments, a single set of model parameters was determined, and the model simulation results using this set of parameters were compared with the experimental data of total glucan conversion, chain‐length distribution, and crystallinity. Model simulations show significant agreement with the experimentally derived glucan conversion and chain‐length distribution curves and provide interesting insights into multiple complex and interacting physico‐chemical phenomena involved in enzymatic hydrolysis, including enzyme synergism, substrate accessibility, cellulose chain length distribution and crystallinity, and inhibition of cellulases by soluble sugars. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1237–1248, 2015     

Effects of drying-induced fiber hornification on enzymatic saccharification of lignocelluloses

This study investigated the effect of fiber hornification during drying on lignocellulosic substrate enzymatic saccharification. Two chemically pretreated wood substrates and one commercial bleached kraft hardwood pulp were used. Heat drying at 105 and 150°C and air drying at 50% RH and 23.8°C for different durations were applied to produce substrate with various degrees of hornification. It was found that substrate enzymatic digestibilities (SEDs) of hornified substrates made from the same never-dried sample correlate very well to an easily measurable parameter, water retention value (WRV), and can be fitted by a Boltzmann function. The hornification-produced SED reduction at a given degree of hornification as the percentage of the total SED reduction when the substrate is completely hornified depends on two parameters. The first is WRVˉ, which is primarily a function of the effective enzyme molecule size, and Δ, which is related to the substrate pore size distribution shape. The low values of SED(CH), SED of a completely hornified substrate, obtained from curve fittings for the three sets of samples studied, suggest that enzyme accessibility to cellulose is mainly through the pores in the cell wall rather than substrate external surface. The SEDs of hornified substrates were found to correlate to Simons' staining measurements well. A new parameter was proposed to better correlate enzyme accessibility to cellulose using the two-color Simons' staining technique.     

Kinetic modeling of cellulosic biomass to ethanol via simultaneous saccharification and fermentation: Part II. Experimental validation using waste paper sludge and anticipation of CFD analysis

A kinetic model of cellulosic biomass conversion to ethanol via simultaneous saccharification and fermentation (SSF) developed previously was validated experimentally using paper sludge as the substrate. Adsorption parameters were evaluated based on the data obtained at various values for fractional cellulose conversion. The adsorption model was then combined with batch SSF data to evaluate the cellulose hydrolysis parameters. With the parameters evaluated for the specific substrate, the discrete model was able to predict SSF successfully both with discrete addition of cellulase only and with discrete feeding of substrate, cellulase, and media. The model tested in this study extends the capability of previous SSF models to semi-continuous feeding configurations, and invites a mechanistic interpretation of the recently observed trend of increasing conversion with decreasing feeding frequency [Fan et al. (2007a) Bioprocess Biosyst Eng 30(1):27-34]. Our results also support the feasibility and utility of determining adsorption parameters based on data obtained at several conversions, particularly when the model is to be applied to extended reaction times rather than only initial hydrolysis rates. The revised model is considerably more computationally efficient than earlier models, and appears for many conditions to be within the capability of simulation using computational fluid dynamics.