All enzymatic processes consist of four major steps that may be combined in a variety of ways: pretreatment, enzyme production, hydrolysis, and fermentation
It has long been recognized that some form of treatment is necessary to achieve reasonable rates and yields in the enzymatic hydrolysis of biomass. Pretreatment has generally been practiced to reduce the crystallinity of cellulose, to lessen the average polymerization of the cellulose and the lignin防emicellulose sheath that surround the cellulose, and to increase available surface area for the enzymes to attack.
Mechanical pretreatments such as intensive ball milling and roll milling have been investigated as means of increasing the surface area, but they require exorbitant amounts of energy. The efficiency of a chemical process can be understood by considering the interaction between the enzymes and the substrate. The hydrolysis of cellulose into sugars and other oligomers is a solid-phase reaction in which the enzymes must bind to the surface to catalyze the reaction. Cellulase enzymes are large proteins, with molecular weights ranging from 30,000 to 60,000 and are thought to be ellipsoidal with major and minor dimensions of 30 to 200 ﾅ. The internal surface area of wood is very large, but only 20% of the pore volume is accessible to cellulase-sized molecules. By breaking down the hemicellulose僕ignin matrix, hemicellulose or lignin can be separated and the accessible volume greatly increased. This removal of material greatly enhances enzymatic digestibility.
The hemicellulose僕ignin sheath can be disrupted by either acidic or basic catalysts. Basic catalysts simultaneously remove both lignin and hemicellulose, but suffer large consumption of base through neutralization by ash and acid groups in the hemicellulose. In recent years attention has been focused on acidic catalysts. They can be mineral acids or organic acids generated in situ by autohydrolysis of hemicellulose.
Various types of pretreatments are used for biomass conversion. The pretreatments that have been studied in recent years are steam explosion autohydrolysis, wet oxidation, organosolv, and rapid steam hydrolysis (RASH). The major objective of most pretreatments is to increase the susceptibility of cellulose and lignocellulose material to acid and enzymatic hydrolysis. Enzymatic hydrolysis is a very sensitive indicator of lignin depolymerization and cellulose accessibility. Cellulose enzyme systems react very slowly with untreated material; however, if the lignin barrier around the plant cell is partially disrupted, then the rates of enzymatic hydrolysis are increased dramatically.
Most pretreatment approaches are not intended to actually hydrolyze cellulose to soluble sugars, but rather to generate a pretreated cellulosic residue that is more readily hydrolyzable by cellulase enzymes than native biomass. Dilute acid hydrolysis processes are currently being proposed for several near-term commercialization ventures until lower-cost commercial cellulase preparations become available. Such dilute acid hydrolysis processes typically result in no more than 60% yields of glucose from cellulose.
The enzyme of interest is the cellulase, which is needed for the hydrolysis of the cellulose. Cellulase is a multicomponent enzyme system consisting of: endo-ﾟ-1,4-glycanases; exo-ﾟ-1,4-glucan gluco hydrolases; and exo-ﾟ-1,4-glucan cellobiohydrolase. Cellobiose is the dominant product of this system but is highly inhibitory to the enzymes and is not usable by most organisms. Cellobiase hydrolyzes cellobiose to glucose, which is much less inhibitory and highly fermentable. Many of the fungi produce this, and most of the work that is presently going on is on Trichoderma reesei (viride). This cellulase is much less inhibited than other cellulases, which is a major advantage for industrial purposes.
The type of inhibition exhibited by cellulases is the subject of much confusion. Although most researchers favor competitive inhibition, some cellulases are noncompetitively inhibited. Trichoderma reesei enzyme on substrates like solka floc (purified cellulose), wheat straw, and bagasse (biomass remaining after sugarcane stalks are crushed to extract their juice) is competitively inhibited by glucose and cellobiose. On the other hand, some enzyme is noncompetitively inhibited by cellobiose, using other substrates like rice straw and avicel. Trichoderma viride is uncompetitively inhibited by glucose in a cotton waste substrate.
Many mutants have been produced following Trichoderma reesei. The most prominent among these is the Rut C-30, the first mutant with ﾟ-glucosidase production. 0ther advantages of the strain are that it is hyperproducing and is carbolite-repression resistant.
Cellulases from thermophilic bacteria have also been extensively examined. Among these, Clostridium thermocellum is perhaps the most extensively characterized organism; it is an anaerobic, thermophilic, cellulolytic, and ethanogenic bacterium capable of directly converting cellulosic substrate into ethanol. The enzymes isolated from thermophilic bacteria may have superior thermostability and hence will have longer half-lives at high temperatures. Although this is not always the case, cellulases isolated from Clostridium thermocellum have high specific activities, especially against crystalline form of cellulose that have proved to be resistant to other cellulase preparations.
Enzyme production with trichoderma reesei is difficult because cellulase production terminates in the presence of easily metabolizable substrates. Thus, most production work has been carried out on insoluble carbon sources such as steamexploded biomass or Solka-Flocｮ. In such systems, the rate of growth and cellulase production is limited because the fungi must secrete the cellulase and carry out slow enzymatic hydrolysis of the solid to obtain the necessary carbon. Average productivities have been approximately l00 IU/l/h (Hydrolytic activity of cellulose is generally in terms of international filter unit [IU]. This is a unit defined in terms of the amount of sugar produced per unit time from a strip of Whatman filter paper.) The filter paper unit is a measure of the combined activities of all three enzymes on the substrate. High productivities have been reported with Trichoderma reesei mutant in a fed-batch system using lactose as carbon source and steam-exploded aspen as an inducer. Although lactose is not available in quantities required to supply a large ethanol industry, this does suggest that it may be possible to develop strains that can produce cellulases with soluble carbon sources such as xylose and glucose.
Increases in productivities dramatically reduce the size and cost of the fermenters used to produce the enzyme. More rapid fermentations would also decrease the risk of contamination and might allow for less expensive construction. Alternatively, using a soluble substrate may allow simplification of fermenter design or allow the design of a continuous enzyme production system.
Low-cost but efficient enzymes for lignocellulosic ethanol technology must be developed to reduce the operational cost and improve the productivity of the process.
Cellulose hydrolysis and fermentation can be achieved by two different process schemes, depending on where the fermentation is carried out: