John A. Heitmann

Dependency of polyelectrolyte complex stoichiometry on the order of addition. 1. Effect of salt concentration during streaming current titrations with strong poly-acid and poly-base

Titrations were carried out between solutions of a strong poly-acid (polyvinylsulfate, potassium salt) and a strong poly-base (poly-diallyldimethylammonium chloride (poly-DADMAC)) over a range of salt concentrations. Streaming current (SC) analysis of the titration endpoints appeared to show increasing deviations from 1:1 stoichiometry of complexation with increased salt. The results depended on the direction of the titration, such that a stoichiometric excess of the titrant (second additive) was required to achieve a SC reading of zero. These symmetrical results, depending on the order of addition, were obtained despite the fact that the plastic surfaces of the SC device had a slight negative charge and differing adsorption tendencies for the two kinds of polymer. A qualitative model of molecular events, based on non-equilibrium entrapment of non-complexed polymer segments was found to be inconsistent with results of tests carried out over a range of initial polymer concentration. Results were better described by a qualitative model involving formation of polyelectrolyte complexes (PECs) in solution, in which near-stoichiometric core complexes are stabilized by an excess of the second additive on their surface. Implications of the latter model were compared with the results of turbidimetric tests, aqueous contact angles on polymer-treated plastic surfaces, and microelectrophoresis of PECs. Results of this study have consequences for interpretation of polyelectrolyte titrations, as well as for industrial operations that involve the mixing of oppositely charged polyelectrolytes.

Influence of lignin and its degradation products on enzymatic hydrolysis of xylan.

The influence of lignin, lignin model compounds, and black liquor from the kraft pulping process on the hydrolysis of xylan by xylanase was investigated. Addition of vanillic acid, acetovanillone, and protocatechuic acid increased the rate of hydrolysis of xylan by as much as 18-50% at low concentrations, but reached maxima at about 0.05% concentration. Addition of vanillin caused a 15% improvement in xylan hydrolysis, while addition of guaiacol more than doubled the hydrolysis rate. Increasing concentrations of either lignin or black liquor also increased the hydrolysis rate of xylan. Circular dichroism spectroscopy indicated a change in the structure of xylanase in the presence of black liquor.

In Situ Monitoring of Cellulase Activity by Microgravimetry with a Quartz Crystal Microbalance

Quartz crystal microgravimetry (QCM) was used to investigate the interactions between cellulase enzymes and model cellulose substrates. The substrates consisted of thin films of cellulose that were spin-coated onto polyvinylamine (PVAm) precoated quartz crystal sensors carrying conductive gold surfaces. In QCM the quartz crystals are piezoelectrically driven and the frequency and dissipation shifts allow monitoring of substrate hydrolysis at various temperatures and enzyme concentrations in situ and in real time. The changes in frequency of cellulose-coated quartz resonators during their incubation in cellulase solutions were related to contributions from the liquid phase properties, the adsorptions of cellulase enzymes, and the hydrolysis of the substrate. Cellulase adsorption was found to be nonspecific and irreversible on gold-, PVAm-, and cellulose-coated quartz crystal sensors. The contribution to frequency shifts due to the bulk fluid properties of the cellulase solutions (at concentrations lower than 0.5 mg/mL) was minimal compared to the frequency shifts produced by cellulase binding. The maximum frequency decreases were fitted to a Langmuir model. The adsorption constant and the maximum adsorption were estimated by the fitting parameters of this model. The hydrolysis process was modeled by using a dose-response model that was then used to estimate the maximum hydrolysis rate, to compare the relative effects of temperature on adsorption and hydrolysis rate, and to obtain the apparent activation energy of cellulose hydrolysis. The hydrolysis rate increased with incubation temperature while apparent adsorption decreased. The apparent activation energy for the hydrolysis of the cellulose films employed was calculated to be 37 kJ/mol.

Quantification of cellulase activity using the quartz crystal microbalance technique.

The development of more efficient utilization of biomass has received increased attention in recent years. Cellulases play an important role in processing biomass through advanced biotechnological approaches. Both the development and the application of cellulases require an understanding of the activities of these enzymes. A new method to determine the activity of cellulase has been developed using a quartz crystal microbalance (QCM) technique. We compare the results from this technique with those from the IUPAC (International Union of Pure and Applied Chemistry) dinitrosalicylic acid (DNS) standard method and also from biccinchoninic acid and ion chromatography methods. It is shown that the QCM technique provides results closer to those obtained by measuring the actual reducing sugars. The elimination of the use of color development in the standard redox methods makes the QCM platform easier to implement; it also allows more flexibility in terms of the nature of the substrate. Finally, validation of the proposed method was carried out by relating the crystallinity of different substrates to the cellulase activity. Numerical values of cellulase activities measured with the QCM method showed that celluloses with higher crystallinity indices were hydrolyzed slower and to a lower extent than those of lower crystallinity indices for the cellulase mixtures examined.

Adsorption of hemicellulases onto bleached kraft fibers

Abstract The presence of a cellulose binding domain was the main factor which influenced the adsorption of two Trichoderma reesei mannanases and a Trichoderma longibrachiatum xylanase onto bleached kraft fibers. The mannanase containing the binding domain adsorbed to a much greater extent than the mannanase and xylanase without binding domains. Once-dried fibers exhibited lower hemicellulase adsorption than the never-dried fibers. The adsorption onto once-dried fibers was only 30–50% the amount adsorbed onto virgin fibers. Adjustment of the ionic strength and pH of treatment was used to modify the charge on the fibers. Ionic strength had opposite effects on mannanase and mannanase core adsorption. Mannanase adsorption increased at high ionic strengths, while the mannanase core adsorption decreased. An increase in pH and fiber charge significantly enhanced the adsorption of xylanase.

Adsorption of a Trichoderma reesei endoglucanase and cellobiohydrolase onto bleached Kraft fibres

Cellulases can be used to modify pulp fibres. For the development of biotechnical applications, a better understanding of the adsorption of cellulases onto commercial wood fibres is needed. In this work, the adsorption behaviour of purified CBH I and EG II on bleached Kraft fibres was investigated. Three variables were studied with respect to their effect on adsorption: fibre type (hardwood or softwood), fibre history (never-dried or once-dried), and ionic strength. The results showed that fibre history had the largest influence on the extent of adsorption of each enzyme. The effect of ionic strength was shown to be dependent on the enzyme and fibre type. At high ionic strength, CBH I exhibited a higher affinity for both once-dried and never-dried fibres at low enzyme concentrations; however, salt was shown to decrease the extent of adsorption at higher enzyme dosages. In contrast, salt increased the maximum adsorption of EG II, most notably on the once-dried hardwood fibres. Fibre type was also shown to affect adsorption behaviour. CBH I had a higher affinity for softwood fibres than for hardwood fibres at low enzyme concentrations. The maximum adsorption of EG II onto once-dried softwood fibres increased by 80% compared to the once-dried hardwood fibres. Interestingly, this did not correlate to in creased fibre hydrolysis.

Enzyme activity recovery from secondary fiber treated with cellulase and xylanase

Abstract One of the major problems with implementing biotechnical processes in the recycled paper industry, such as enzyme-enhanced deinking and enzymatic enhancement of pulp drainage properties, is the cost of commercial enzyme preparations. Thus, several factorial studies were performed to determine if enzyme activity can be successfully removed from simulated recycled fiber (once-dried, bleached hardwood and softwood kraft fiber) treated with low concentrations (0.2% or 2.0% on oven-dry fiber) of cellulase or xylanase. Enzyme activity recovery was accomplished by washing treated fiber with dilute NaOH in combination with a low concentration of the nonionic surfactant Tween 80 under a variety of mild conditions. Various cellulase activities i.e., endoglucanase, exoglucanase, and filter paper, can be effectively recovered depending on the washing conditions, the cellulase charge, and the fiber type. Xylanase activity was effectively recovered from softwood, but not hardwood. The results suggest that enzyme activity recovery may be a possible means of decreasing the operating costs for biotechnical processes in the paper industry.

RHEOLOGY OF CARBOXYMETHYL CELLULOSE SOLUTIONS TREATED WITH CELLULASES

The effect of cellulase treatments on the rheology of carboxymethyl cellulose (CMC) solutions was studied using a rotational viscometer. The rheological behaviors of CMC solutions of different molecular mass and degrees of substitution where studied as a function of time after various treatments. These solutions were subjected to active and heat-denatured cellulase, a cationic polyelectrolyte (C-PAM), as well as different shear rates. A complex protein-polymer interaction was observed, leading to a potential error source in the measurement of enzymatic activity by changes in the intrinsic viscosity. The interaction was termed a polymeric effect and defined as a reduction in viscosity of the substrate solution without significant formation of reducing sugars from enzymatic hydrolysis. The cause of the reduction in viscosity appears to be related to the interaction between the enzymes as amphipathic particles and the soluble CMC. Thus, the polymeric effect may cause a considerable experimental error in the measurement of enzymatic activity by viscometric methods.

Dimensional and hygroexpansive behaviors of cellulose microfibrils (MFs) from kraft pulp-based fibers as a function of relative humidity

Abstract Cellulose aggregate fibrils (CAFs) with dimensions of 100,000×3000×300 nm from unbleached kraft pulp (KP), oxygen delignified KP (KPox.del), and fully bleached kraft pulp (BKP) were liberated by a series of high shear and fractionation operations. The CAFs served as microfibril model material to evaluate their dimensional and hygroexpansive behaviors when submitted to variable relative humidities (RHs). The atomic force microscopy images of CAFs from different sources of kraft fibers were obtained during a RH cycle from 50% RH to 78% RH and then to 21% RH while being kept at 23°C. The resulting images were analyzed to determine dimensional changes in length, its concurrent cross-sectional area, width, and height. The mean value of changes in length and width was in the range of 2.3–3.2% and 1.9–3.3%, respectively. The changes in area and height were in the range of 14.5–18.2% and 12.4–17.3%, respectively. The length of CAFs showed a negative correlation with RH, whereas cross-sectional area, width, and height changes correlated positively with RHs. In the out-of-plane direction, such as area and height, the hygroexpansivity was one order of magnitude larger than the in-plane hygroexpansivity, i.e., in terms of length and width.

Preventing Strength Loss of Unbleached Kraft Pulp

Kraft pulp fibers lose inter-fiber bonding ability when they are dried during the manufacture of paper. Adverse environmental consequences of this loss include (a) limitations on the number of times that kraft fibers can be recycled, (b) reduced paper strength, sometimes making it necessary to use heavier paper or paperboard to meet product strength requirements, increasing the usage of raw materials, (c) decreased rates of paper production in cases where the fiber furnish has been over-refined in an attempt to regain inter-fiber bonding ability. The present study is the first of its type to focus on unbleached kraft fibers, which are a main ingredient of linerboard for corrugated containers. About 90 million tons of unbleached kraft fiber are used worldwide every year for this purpose.