Cellulase Adsorption The enzymatic hydrolysis of cellulose proceeds by adsorption of cellulase enzyme on the lignacious residue as well as the cellulose fraction. The adsorption on the lignacious residue is an attractive factor from the viewpoint of the recovery of enzyme after the reaction and recycling it for use on the fresh substrate. Obviously, the recovery is reduced by the adsorption of enzyme on lignacious residue, an important consideration, because a large fraction of the total operating cost is due to the production of enzyme. As the capacity of lignacious residue to adsorb the enzyme is influenced by the pretreatment conditions, the pretreatment should be evaluated, in part, by how much enzyme adsorbs on the lignacious residue at the end of hydrolysis, as well as its effect on the rate and extent of the hydrolysis reaction.
The adsorption of cellulase on cellulose and lignacious residue has been investigated by Ooshima et al.33 using cellulase from Trichoderma reesei and hardwood pretreated by dilute sulfuric acid with explosive decomposition. The cellulase was found to adsorb on the lignacious residue as well as on the cellulose during hydrolysis of the pretreated wood. A decrease in the enzyme recovery in the liquid phase with an increase in the substrate concentration has been reported owing to the adsorption on the lignacious residue. The enzyme adsorption capacity of the lignacious residue decreases as the pretreatment temperature is increased, whereas the capacity of the cellulose increases. The reduction of the enzyme adsorbed on the lignacious residue as the pretreatment temperature increases is important in increasing the ultimate recovery of the enzyme, as well as enhancing the enzyme hydrolysis rate and extent.
An enzymatic hydrolysis process involving solid lignocellulosic materials can be designed in many ways. The common features are that the substrates and the enzyme are fed into the process, and the product stream (sugar solution) along with a solid residue leaves it at various points. The residue contains adsorbed enzymes that are lost when the residue is removed from the system.
To ensure that the enzymatic hydrolysis process is economically efficient, a certain degree of enzyme recovery is essential. Both the soluble enzymes and the enzyme adsorbed onto the substrate residue must be reutilized. It is expected that the loss of enzyme is influenced by the selection of the stages at which the enzymes in the solution and adsorbed enzymes are recirculated and the point at which the residue is removed from the system. Vallander and Erikkson defined an enzyme loss function, L, assuming that no loss occurs through filtration:
The amount of enzyme lost through removal of residue
_________________________________________________
The amount of enzyme at the start of hydrolysis
They developed a number of theoretical models, finally concluding that increased enzyme adsorption leads to increased enzyme loss. The enzyme loss decreases if the solid residue is removed late in the process. Both the adsorbed and dissolved enzymes should be reintroduced at the starting point of the process. This is particularly important for the dissolved enzymes. Washing of the entire residue is likely to result in significantly lower recovery of adsorbed enzymes than if a major part (60% or more) of the residue with adsorbed enzymes is recirculated. Uninterrupted hydrolysis over a given time period leads to a lower degree of saccharification than when hydrolysate is withdrawn several times. Saccharification is also favored if the residue is removed at a late stage. Experimental investigations of the theoretical hydrolysis models have recovered more than 70% of the enzymes.
Mechanism of Hydrolysis The overall hydrolysis is based on the synergistic action of three distinct cellulase enzymes, depending on the concentration ratio and the adsorption ratio of the component enzymes: endo-ﾟ-gluconases, exo-beta-gluconases, and beta-glucosidases. Endo-beta-gluconases attack the interior of the cellulose polymer in a random fashion, exposing new chain ends. Because this enzyme catalyzes a solid-phase reaction, it adsorbs strongly but reversibly to the microcrystalline cellulose (also known as avicel). The strength of the adsorption is greater at lower temperatures. This enzyme is necessary for the hydrolysis of crystalline substrates. The hydrolysis of cellulose results in a considerable accumulation of reducing sugars, mainly cellobiose, because the extracellular cellulase complex does not possess cellobiose activity. Sugars that contain aldehyde groups that are oxidized to carboxylic acids are classified as reducing sugars.
Exo-beta-gluconases remove cellobiose units (two glucose units) from the nonreducing ends of cellulose chains. This is also a solid-phase reaction, and the exogluconases adsorb strongly on both crystalline and amorphous substrates. The mechanism of the reaction is complicated because there are two distinct forms of both endo- and exo-enzymes, each with a different type of synergism with the other members of the complex. As these enzymes continue to split off cellobiose units, the concentration of cellobiose in solution may increase. The action of exo-gluconases may be severely inhibited or even stopped by the accumulation of cellobiose in the solution.
The cellobiose is hydrolyzed to glucose by action of beta-glucosidase. Glucosidase is any enzyme that catalyzes hydrolysis of glucoside. beta-Glucosidase catalyzes the hydrolysis of terminal, nonreducing beta-D-glucose residues with release of beta-D-glucose. The effect of beta-glucosidase on the ability of the cellulase complex to degrade avicel has been investigated by Kadam and Demain.
They determined the substrate specificity of the ﾟ-glucosidase and demonstrated that its addition to the cellulase complex enhances the hydrolysis of avicel specifically by removing the accumulated cellobiose. A thermostable ﾟ-glucosidase form, Clostridium thermocellum, which is expressed in Escherichia coli, was used to determine the substrate specificity of the enzyme. The hydrolysis of cellobiose to glucose is a liquid-phase reaction, and ﾟ-glucosidase absorbs either quickly or not at all on cellulosic substrates. The action can be slow or halted by the inhibitive action of glucose accumulated in the solution. The accumulation may also induce the entire hydrolysis to a halt as inhibition of the ﾟ-glucosidase results in a buildup of cellobiose, which in turn inhibits the action of exo-gluconases. The hydrolysis of the cellulosic materials depends on the presence of all three enzymes in proper amounts. If any one of these enzymes is present in less than the required amount, the others will be inhibited or will lack the necessary substrates to act upon.
The hydrolysis rate increases with increasing temperature. However, because the catalytic activity of an enzyme is related to its shape, the deformation of the enzyme at high temperature can inactivate or destroy the enzyme. To strike a balance between increased activity and increased deactivation, it is preferable to run fungal enzymatic hydrolysis at approximately 40 to 50ｰC.
Researchers at the National Renewable Energy Laboratory reported results for a dilute acid hydrolysis of softwoods in which the conditions of the reactors were as follows:
Their bench-scale tests also confirmed the potential of achieving yields of 89% for mannose, 82% for galactose, and 50% for glucose, respectively. Fermentation with Saccharomyces cerevisiae achieved ethanol conversion of 90% of the theoretical yield.
Cellulose hydrolysis and fermentation can be achieved by two different process schemes, depending on where the fermentation is carried out:
separate hydrolysis and fermentation (SHF) In SHF, the hydrolysis is carried out in one vessel and the hydrolysate is then fermented in a second reactor. The most expensive items in the overall process costare the cost of feedstock, enzyme production, hydrolysis, and utilities. The feedstock and utility costs are high because only about 73% of the cellulose is converted to ethanol in 48 h, whereas the remainders of the cellulose, hemicellulose and lignin, are burned. Enzyme production is a costly step because of the large amount of the enzyme used in an attempt to overcome the end product inhibition, as well as its slow rate of production. The hydrolysis step is also expensive, owing to the large capital and operating costs associated with large-size tanks and agitators. The most important parameters are the hydrolysis section yield, product quality, and the required enzyme loading, which are all interrelated. Yields are typically higher in more dilute systems, where inhibition of enzymes by glucose and cellobiose is minimized. Increasing the amount of enzyme loading can help overcome inhibition and increase yield and concentration. Increased reaction time also leads to higher yields and concentrations. Cellulase enzymes from different organisms can result in markedly different performances. Figure 11.10 shows the effect of yield at constant solid and enzyme loading and the performance of different enzyme loadings. Increase in enzyme loading beyond a particular point is of no value. It would be economical to operate at a minimum enzyme loading level, or the enzyme could be recycled by appropriate methods. As the cellulose is hydrolyzed, the endo- and exogluconase components are released back into the solution. Because of their affinity for cellulose, these enzymes can be recovered and reused by contacting the hydrolysate with fresh feed. The amount of recovery is limited because of ﾟ-glucosidase, which does not adsorb on the feed. Some of the enzyme remains attached to the lignin, and unreacted cellulose and enzymes are thermally denatured during hydrolysis. A major difficulty in this type of process is maintaining the sterility, which would otherwise be contaminated. The power consumed in agitation is also significant and does affect the economics of this process
simultaneous saccharification and fermentation (SSF). The operating cost of this process is generally lower than that of SHF. As the name implies, both hydrolysis and fermentation are carried out in the same vessel. In this process, yeast ferments the glucose to ethanol as soon as the glucose is produced, preventing the sugars from accumulating and inhibiting the end product. Using the yeast, Candida brassicae and the Genencor enzyme (by Genencor International), the yield is increased to 79% and the ethanol concentration produced is 3.7%.
Even in SSF, cellobiose (the soluble sugar) inhibition occurs to an appreciable extent. The enzyme loading for SSF is only 7 IU/g of cellulose, compared to 33 IU/g in SHF. The cost of energy and feedstock is somewhat reduced because of the improved yield, and the increased ethanol concentration significantly reduces the cost of distillation and utilities. The cost of SSF process is slightly less than the combined cost of hydrolysis and fermentation in the SHF process. The decreasing factor of the reactor volume due to the higher concentration of ethanol offsets the increasing factor in the reactor size caused by the longer reaction times (7 d for SSF vs. 2 d for hydrolysis and 2 d for fermentation). Experiments show that fermentation is the rate-controlling step and not the enzymatic hydrolysis process. The hydrolysisis carried out at 37ｰC and increasing the temperature increases the reaction rate; however, the ceiling temperature is limited by yeast cell viability. The concentration of ethanol is also a limiting factor. (This was tested by connecting a flash unit to the SSF reactor and removing the ethanol periodically. This technique showed higher productivities up to 44%.) Recycling the residual solids may also increase process yield. However, the most important limitation in enzyme recycling comes from the presence of lignin, which is inert to the enzyme. High recycling rates increase the fraction of lignin in the reactor and cause handling difficulties.
Two major types of enzyme-recycling schemes have been proposed, one in which enzymes are recovered in the liquid phase and the other in which enzymes are recoveredby recycling unreacted solids.45 Systems of the first type have been recommended for SHF processes, which operate at 50ｰC. These systems are favored at such a high temperature because increasing temperature increases the proportion of enzyme that remains in the liquid phase. Conversely, as the temperature is decreased, the amount of enzyme adsorbed on the solid increases. Therefore, at the lower temperatures encountered in SSF processes, solid recycling appears to be more effective.
Please contact us for a Comparison between SSF and SHF Processes