Cellulase kinetics as a function of cellulose pretreatment

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.     

Automated colorimetric determination of cellulase activity in fermentation samples using a ferricyanide-molybdoarsenic acid reagent

An automated method for the determination of cellulase activity has been developed. The enzyme incubation with the cellulose substrate is stopped at alkaline pH, after which the reaction mixture is dialyzed. The concentration of reducing sugar in the dialyzer recipient is determined by means of the colorimetric ferricyanide-molybdoarsenic acid reaction. In this reaction the ferrocyanide formed by the ferricyanide reaction is coupled with molybdoarsenic acid during formation of a molybdenum-blue color. An optimization of the analytical conditions and the reagent concentrations has been carried out. The result of the automation is an analysis with high precision (mean = 36.4; SD = 0.7%) and accuracy as well as high rate of analysis. The sensitivity is 5 mg of glucose/liter.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.     

Investigation of biomass concentration,lipid production,and cellulose content in Chlorella vulgaris cultures using response surface methodology

The microalgae Chlorella vulgaris produce lipids that after extraction from cells can be converted into biodiesel. However, these lipids cannot be efficiently extracted from cells due to the presence of the microalgae cell wall, which acts as a barrier for lipid removal when traditional extraction methods are employed. Therefore, a microalgae system with high lipid productivity and thinner cell walls could be more suitable for lipid production from microalgae. This study addresses the effect of culture conditions, specifically carbon dioxide and sodium nitrate concentrations, on biomass concentration and the ratio of lipid productivity/cellulose content. Optimization of culture conditions was done by response surface methodology. The empirical model for biomass concentration (R² = 96.0%) led to a predicted maximum of 1123.2 mg dw L^?1 when carbon dioxide and sodium nitrate concentrations were 2.33% (v/v) and 5.77 mM, respectively. For lipid productivity/cellulose content ratio (R² = 95.2%) the maximum predicted value was 0.46 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 when carbon dioxide concentration was 4.02% (v/v) and sodium nitrate concentration was 3.21 mM. A common optimum point for both variables (biomass concentration and lipid productivity/cellulose content ratio) was also found, predicting a biomass concentration of 1119.7 mg dw L^?1 and lipid productivity/cellulose content ratio of 0.44 (mg lipid L^?1 day^?1)(mg cellulose mg biomass^?1)^?1 for culture conditions of 3.77% (v/v) carbon dioxide and 4.01 mM sodium nitrate. The models were experimentally validated and results supported their accuracy. This study shows that it is possible to improve lipid productivity/cellulose content by manipulation of culture conditions, which may be applicable to any scale of bioreactors. Biotechnol. Bioeng. 2013; 110: 2114–2122. © 2013 Wiley Periodicals, Inc.     

Cellulase production and ammonia metabolism in Trichoderma reesei on high levels of cellulose.

Trichoderma can be cultured in stirred-tank fermentors on high (8%) cellulose concentrations without increasing the salt concentration of the medium when NH4OH is used to control pH and as a nitrogen source. Approximately 90% of the ammonia consumed by the organism can be added as NH4OH. The advantage of using high concentrations of cellulose is that culture filtrates with greater cellulase activity are obtained. The advantage of a low salts medium is that unwanted solutes in the final enzyme preparation are reduced. The appearance of cellulase in the medium occurs later than net ammonia uptake so that only 20% of the final amount of cellulase has appeared when 80% of the maximum amount of ammonia has been consumed.     

Influence of external concentration fluctuations on leukocyte chemotactic orientation

The quantitative dependence of leukocyte chemotactic orientation on imprecision in the measurement of chemoattractant concentrations from thermal fluctuations is analyzed. First, a mathematical model relating orientation to differences in receptor occupancy across cell dimensions is developed. This is then coupled with an extension of Berg and Purcell's analysis (1) of the precision of attractant concentration measurements by means of receptor occupancy. Our results show that thermal fluctuations in external concentrations can limit the accuracy of orientation, unless the measurement noise is reduced by averaging the measurements over a period of time. Comparison of our model predictions to experimental orientation data suggests that leukocytes do overcome this limitation, and allows estimation of the time-averaging period necessary to do so. For the orientation observed in a visual bridge assay by Zigmond (2) using the attractant peptide FNLLP, we estimate that receptor occupancy measurements for spatial comparison across cell dimensions must be averaged for a few minutes. Otherwise, the fluctuations in the attractant concentration near the cell will be too great to allow the observed degree of orientation. Our analysis also suggests that the ratio of signal-to-signal noise does not adequately characterize orientation accuracy. Accurate orientation can, in some situations, occur when this ratio is substantially less than unity; in other situations, a ratio much greater than unity is required for accurate orientation.     

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.     

Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.     

Column cellulose hydrolysis reactor: the effect of retention time,temperature, cellulase concentration and exogenously added cellobiase on the overall process

Summary A novel column cellulose hydrolysis reactor with constant enzyme recycling was operated under various conditions to determine the effects of retention time, temperature, cellulase concentration and exogenously added cellobiase on the concentration of the product stream and the productivities of the reactor. Short term (7 days) hydrolysis was best at 42°C while longer term (14 days) hydrolysis was better at 37°C. A retention time of 11 h and reactor cellulase concentration of 30 filter paper units per gram of cellulose gave the best compromise for efficient operation by minimizing product inhibition, maximizing product concentration and minimizing enzyme consumption. The addition of cellobiase to the reactor increased cellulose hydrolysis and raised the proportion of monomeric sugars in the hydrolysate. Continuous cellulose hydrolyses were maintained for 7 and 14 days at 42°C and 37°C, respectively, resulting in volumetric productivities of 6.82 and 4.84 g/l/h and average sugar concentrations of 7.3% and 6.0% (w/v), respectively. Greater than 95% (w/w) of the sugars produced were in the monomeric state. Average cellulase used for the two runs were 8.4 and 5.3 filter paper units per gram of sugar produced, respectively.     

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.     

Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover

Biological processing of cellulosic biomass to fuels and chemicals would open up major new agricultural markets and provide powerful societal benefits, but pretreatment operations essential to economically viable yields have a major impact on costs and performance of the entire system. However, little comparative data is available on promising pretreatments. To aid in selecting appropriate systems, leading pretreatments based on ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime were evaluated in a coordinated laboratory program using a single source of corn stover, the same cellulase enzyme, shared analytical methods, and common data interpretation approaches to make meaningful comparisons possible for the first time. Each pretreatment made it possible to subsequently achieve high yields of glucose from cellulose by cellulase enzymes, and the cellulase formulations used were effective in solubilizing residual xylan left in the solids after each pretreatment. Thus, overall sugar yields from hemicellulose and cellulose in the coupled pretreatment and enzymatic hydrolysis operations were high for all of the pretreatments with corn stover. In addition, high-pH methods were found to offer promise in reducing cellulase use provided hemicellulase activity can be enhanced. However, the substantial differences in sugar release patterns in the pretreatment and enzymatic hydrolysis operations have important implications for the choice of process, enzymes, and fermentative organisms.     

The first evidence that a single cellulase can be essential for cellulose degradation in a cellulolytic microorganism

Cellulose is the most abundant carbon source in nature but it is very difficult to degrade because of its insolubility, quasi‐crystalline structure and its presence in plant cell walls in a matrix with other polymers that limit access to the cellulose surface. Most cellulose in soils is degraded by cellulolytic microorganisms that use a number of different approaches to overcome the recalcitrance of cellulose in plant cell walls. All of these approaches involve multiple cellulases and, since cellulose is insoluble and microorganisms cannot ingest particles, the cellulases are present outside of the cell although they can be attached to its outer surface. An impressive article by Tolonen et al. in this issue of Molecular Microbiology shows that deletion of the single family 9 cellulase gene in Clostridium phytofermentans prevents growth on cellulose although the mutant strain grows perfectly well on glucose and its other cellulase genes are transcribed normally. These results show for the first time that a single cellulase can be essential for cellulose degradation by an organism despite the presence of several other cellulases. It will be interesting to learn the detailed mechanism that C. phytofermentans uses to degrade cellulose.     

The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft

BACKGROUND: DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. METHODS: Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. RESULTS: PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. DISCUSSION: We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.     

Adsorption of cellulase on cellulose: Effect of physicochemical properties of cellulose on adsorption and rate of hydrolysis

In the cellulase-cellulose reaction system, the adsorption of cellulase on the solid cellulose substrate was found to be one of the important parameters that govern the enzymatic hydrolysis rate of cellulose. The adsorption of cellulase usually parallels the rate of hydrolysis of cellulose. The affinity for cellulase varies depending on the structural properties of cellulose. Adsorption parameters such as the half-saturation constant, the maximum adsorption constant, and the distribution coefficient for both the cellulase and cellulsoe have been experimentally determined for several substrates. These adsorption parameters vary with the source of cellulose and the pretreatment methods and are correlated with the crystallinity and the specific surface area of cellulose substrates. The changing pattern of adsorption profile of cellulase during the hydrolysis reaction has also been elucidated. For practical utilization of cellulosic materials, the cellulose structural properties and their effects on cellulase adsorption, and the rate of hydrolysis must be taken into consideration.     

Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

Localization of the cellulase activity of Bacteroides cellulosolvens

The major portion of cellulase activity of Bacteroides cellulosolvens was cell-associated. Cells grown on cellulose had a distinctive morphology, characterized by an amorphous outer cell wall layer with irregular, 'fluffy' projections. These cells had greater cell-associated cellulase activity than the cellobiose grown cells which exhibited a smooth, distinct outer layer. It is suggested that the outer layer characteristic of cellulose grown cells is the location of cellulase activity and the site for close cellulose-cell contact.     

Acetic acid formation in Escherichia coli fermentation

Theoretical analysis of cellulase product inhibition (by cellobiose and glucose) has been performed in terms of the mathematical model for enzymatic cellulose hydrolysis. The analysis showed that even in those cases when consideration of multienzyme cellulase system as one enzyme (cellulase) or two enzymes (cellulase and beta-glucosidase) is valid, double-reciprocal plots, usually used in a product inhibition study, may be nonlinear, and different inhibition patterns (noncompetitive, competitive, or mixed type) may be observed. Inhibition pattern depends on the cellulase binding constant, enzyme concentration, maximum adsorption of the enzyme (cellulose surface area accessible to the enzyme), the range in which substrate concentration is varied, and beta-glucosidase activity. A limitation of cellulase adsorption by cellulose surface area that may occur at high enzyme/substrate ratio is the main reason for nonlinearity of double-reciprocal plots. Also, the results of calculations showed that material balance by substrate, which is usually neglected by researchers studying cellulase product inhibition, must be taken into account in kinetic analysis even in those cases when the enzyme concentration is rather low. (c) 1992 John Wiley & Sons, Inc.     

The Presence of Cellulose in Heterocyst Envelopes of Blue-Green Algae and its Role in Relation to Nitrogen Fixation

The present study gives evidence for the presence of cellulose in the heterocyst envelope of blue-green algae by means of electron microscopy, cellulase treatments and specific staining and demonstrates the role of this cellulose for the protection of the heterocyst nitrogenase during acetylene reduction. Experiments with lysozyme and cellulase suggest that nitrogen fixation in heterocystous blue-green algae under aerobic conditions is functionally effective only when an intimate relationship exists between vegetative cells and heterocysts and both cell types have intact wall structures.     

Carbon mass balance evaluation of cellulase production on soluble and insoluble substrates

A methodology is described and applied for performing carbon mass balances across cellulase enzyme production processes using both soluble sugar and insoluble cellulose substrates. The fungus Trichoderma reesei was grown on either glucose, lactose, or cellulose in aerobic batch mode, and the evolution of the main carbonaceous components (cell mass, cellulose, soluble protein, adsorbed protein, sugars, and carbon dioxide) was followed. A variety of analytical techniques were utilized to measure these components, including (i) gravimetric analysis, (ii) near-infrared spectroscopy, (iii) bicinchoninic acid based soluble protein measurement, (iv) gas mass spectrometry and flow rate, (v) CHNS/O elemental analyses, and (vi) high-performance liquid chromatography. The combined set of measurements allowed carbon mass balances across the cellulase production process to be assessed to determine the consistency of the underlying kinetic data. Results demonstrate the capability to determine the levels and distribution of all major carbonaceous components during the cellulase production process on both soluble and insoluble substrates. Average carbon mass balance closures were near 100% during early stages (<72 h) of the cultivations using glucose, lactose, or cellulose as the substrates, but carbon mass closures trended high later in the cultivation. Analysis of carbon allocation results suggests that an error in the gas mass flow rate measurement was the primary cause for carbon mass balance closures to exceed 110% late in the process.