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Cellulose was undoubtedly man's first fuel, being the major component of wood for his fires. Therefore, when we consider cellulose as a “novel” energy source we have more indirect use in mind, such as its conversion to synthetic fuels (Huge, 1968) and to food (Meller, 1969).

This article sets forth a simple cellulase assay procedure. Cellulose is variable in nature, insoluble and resistant to enzymatic attack. As a result there have been a bevy of bewildering cellulase assays published that yielded irrational results. Certain protocols focused on the rapidity of the assay while ignoring that only the most readily susceptible cellulose regions were being hydrolyzed. Other assays simplified the system by using modified soluble substrates and yielded results that bore no relationship to the real world hydrolysis of insoluble cellulose. In this study Mandels, Andreotti and Roche utilized a common substrate, Whatman filter paper. Hydrolysis of a 50 mg sample of the paper was followed to roughly 4% degradation, which circumvented the problems of attack of only the most susceptible zones. This common hydrolysis target range also resulted in some balance with regard to the interaction of the several cellulase components. The method was subsequently widely adopted. Douglas E Eveleigh

Strains of Trichoderma, particularly T. reesei and its mutants, are good sources of extracellular cellulase suitable for practical saccharification. They secrete a complete cellulase complex containing endo- and exo-glucanases plus β-glucosidase (cellobiase) which act syngergistically to degrade totally even highly resistant crystalline cellulose to soluble sugars. All strains investigated to date are inducible by cellulose, lactose, or sophorose, and all are repressible by glucose. Induction, synthesis and secretion of the β-glucanase enzymes appear to be closely associated. The composition and properties of the enzyme complex are similar regardless of the strain or inducing substrate although quantities of the enzyme secreted by the mutants are higher. Enzyme yields are proportional to initial cellulose concentration. Up to 15 filter paper cellulase units (20 mg of cellulase protein) per ml and productivities up to 80 cellulase units (130 mg cellulase protein) per litre per hour have been attained on 6% cellulose. The economics of glucose production are not yet competitive due to the low specific activity of cellulase (0.6 filter paper cellulase units/mg protein) and poor efficiency (about 15% of predicted sugar levels in 24 h hydrolyses of 10–25% substrate). As hydrolysis proceeds, the enzyme reaction slows due to increasing resistance of the residue, product inhibition, and enzyme inactivation. These problems are being attacked by use of pretreatments to increase the quantity of amorphous cellulose, addition of β-glucosidase to reduce cellobiose inhibition, and studies of means to overcome instability and increase efficiency of the cellulases. Indications are that carbon compounds derived from enzymatic hydrolysis of cellulose will be used as fermentation and chemical feedstocks as soon as the process economics are favourable for such application .

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.

Enzyme stability studies have been reinvestigated under the conditions used for cellulose hydrolysis (pH 4.8, 50 degrees C, 24 hr). The cellobiohydrolase (CBH) component as measured on Avicel is less stable than other enzymes of the cellulase complex, and is 60% inactivated by merthiolate (and other Hg compounds) under the above conditions. Endo-beta-1,4-glucanase is much more stable, and more resistant to merthiolate and other compounds. Under unshaken conditions the Avicelase of the Rutgers strain C 30 shows greater stability to heat than that of other available strains. Biocides must be selected not only for their ability to prevent contamination, but also for their compatibility with cellulases. Tetracycline and chlortetracycline are inexpensive, effective in very low concentrations, have no harmful effect on the enzymes, and are compatible with the yeasts that subsequently grow on the sugar solutions to produce alcohol. Attempts have been made to stabilize the enzymes by chemical modification in such a way as to maintain their solubility. Glutaraldehyde treatment greatly increased the enzyme size, lowered the pI values, and gave a slight shift in the pH activity curve. There was, unfortunately, no increase in enzyme stability, and the activity of enzymes on solid celluloses was adversely affected. Shaking greatly reduced the hydrolysis of Avicel by Trichoderma reesei C 30 enzyme. The adverse effect was accompanied by a decrease in recoverable enzyme and protein.

Some studies on the adsorption of cellulase on cellulose revealed part of the mechanisms involved in the enzymatic hydrolysis of cellulose and provided some clues to the synergistic mechanism of cellulase complex. The adsorption of cellulase was significantly affected by the reaction conditions and physical chemical characteristics of cellulose. Endoglucanase consisted of adsorbable and nonadsorbable components. Cellobiohydrolase had the strongest adsorption affinity. Each cellulase component is postulated to have distinctly different adsorption sites on cellulose, corresponding to the active sites in the hydrolysis reaction. Competitive adsorption kinetics between cellulase components were also observed during the adsorption process. The degree of competitive adsorption was most remarkable when the composition of cellulase components was nearly the same as that in the crude cellulase complex. This seems to show the optimal relative composition of cellulase components. The synergism between cellobiohydrolase and endoglucananse could be elucidated more clearly by this competitive adsorption model of the reaction mechanism.

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, MO, and for carbon source, MC, are 0.85 mmol O2/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: YX/O = 32.3 g biomass/mol O2, YX/C = 1.1 g biomass/g C or YX/C = 0.44 g biomass/g hexose, YX/N = 12.5 g biomass/g nitrogen for the cell growth stage, and YX/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ∼ 0.028 hr−1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.

The aim of this review is to examine the literature relevent to the hydrolysis of cellulose in lignocellulosic materials. It is a complex process involving the nature of the cellulose itself and its association with other substances such as lignin and hemicellulose, as well as a multicomponent enzyme system which appears to be able to hydrolyze the cellulose in at least two ways. Any process to convert this cellulose to monomeric glucose requires both pretreatment and hydrolysis steps. The two steps are interdependent and it is not practical to study them in isolation. 350 references.

Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 hours. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given time was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration. (Refs. 28).

Waste cellulose was a suitable carbon source for cellulose production by Trichoderma viride. The enzyme can be produced in submerged fermentation using newspaper as a growth substrate. A variety of pure and complex cellulosic materials were hydrolyzed by culture filtrates. Saccharification of 5% slurries after 48 hr ranged from 2–92%. The rate and extent of hydrolysis was controlled by degree of crystallinity, particle size, and presence of impurities. Newspaper was used to evaluate methods for the pretreatment of substrate. The best pretreatment was ball milling which gave good size reduction, maximum bulk density, and maximum susceptibility. Hammer milling, fluid energy milling, colloid milling, or alkali treatments were less satisfactory. Dissolving cellulose in cuprammonium, or carbon disulfide (Viscose) and then reprecipitating gave a susceptible, but low bulk density product. However the susceptibility was lost if the substrate was dried. Because of costs, low bulk density, necessity of keep ing the substrate wet, and generation of chemical waste streams dissolving cellulose to increase reactivity does not seem a practical approach. Cellulose fractions separated from municipal trash or agricultural residues such as milled fibres from bovine manure are promising substrates for conversion.

Enzymes capable of hydrolyzng β-(1→3) glucan (laminarin) have been found in callus cultures of bean (Phaseolus), lettuce (Lactuca), carrot (Daucus) and pepper (Capsicum). Laminaribiose is the major product. Lichenin and oat glucan were not attacked by these laminarinases.

β-D-1,2-Glucans apparently occur rarely in nature, and yet many fungi have the ability to produce a β-1,3-glucanase capable of hydrolyzing them. The β-1,2-glucanases in fungi are adaptive, like the β-1,4-glucanases, and thus differ from the β-1.3- and the β-1,6-glucanases which are constitutive. Enzymatic hydrolysis of the β-1,2-glucan is random, a series of β-1,2-linked oligosaccharides, including sophorose, being formed.

A β-thioglucosidase capable of hydrolyzing mustard oil glucosides is of rare occurrence in fungi. Aspergillus sydowi QM 31c is a good source of the enzyme. The fungal enzyme resembles mustard seed myrosinase. Both enzymes degrade sinigrin producing glucose, KHSO4, and allyl isothiocyanate. They attack only the mustard oil thioglucosides, and are inactive on thioglucosides not having this configuration. The fungal enzyme is relatively more active on sinalbin and glucotropaeolin than is myrosinase. The fungal enzyme is less resistant to heat, acids, alkalies, and chemical inhibitors than is the mustard enzyme.

Mycodextranase is an α-glucanase which splits only the α-1,4 linkages in a substrate having alternating α-1,3 and α-1,4 links. It ïs a fungal enzyme of rather infrequent occurrence, adaptive in nature, and in its properties strongly resembling other fungal glucanases. The products of its action on mycodextran are a tetramer and nigerose. The hypothesis is advanced that this glucanase, and others, contain a site which binds a dimeric portion of the polymer; in mycodextranase, the bound dimer is nigerose, the α-1,3-disaccharide.

Mandels, Mary (Quartermaster Research and Engineering Center, Natick, Mass.), Fredrick W. Parrish, and Elwyn T. Reese . Sophorose as an inducer of cellulase in Trichoderma viride . J. Bacteriol. 83: 400–408. 1962.—The impurity in glucose responsible for cellulase induction in Trichoderma viride QM 6a has been isolated and characterized as sophorose (2-O-β- d -glucopyranosyl- d -glucose). It is present at 0.0058% in reagent grade glucose. Sophorose is a very powerful inducer of cellulase for Trichoderma viride , being 2500 times as active as cellobiose. Modifications of sophorose, such as reduction or glycoside formation, destroy its inducing ability. The high activity of sophorose as an inducer is specific for T. viride .

Cellulose strongly adsorbs cellulase at pH 4.0-5.0, 25-50°C, conditions which are optimum for the enzyme action. The adsorbed enzyme is sufficient to digest the cellulose with no replenishment of enzyme even though the liquid phase containing the sugar is continuously removed. As cellulose is digested, the released enzyme is readsorbed on excess or newly added cellulose with retention of activity. Sugars can be separated from the enzyme-cellulose complex by simple filtration or centrifugation or by retaining the enzyme-cellulose complex in a column from which the sugar solution is eluted. Adsorbed cellulase has been used to produce syrups containing 5-14% glucose continuously from stirred reactors containing 10-20% cellulose, or from cellulose columns.

β-D-1,6-Glucans apparently occur rarely in nature, yet many fungi are able to produce an enzyme capable of hydrolyzing them. The β-1,6-glucanases tend to be constitutive enzymes, similar in this respect to the β-1,3-glucanases. They act on β-1,6-glucans in a random fashion, producing a series of β-1,6-linked oligosaccharides, including gentiobiose. The β-1,6-glucanases can be separated from other β-glucanases by zone electrophoresis.

The yields of the extracellular sucrases produced by fungi and yeasts were markedly increased by using sucrose monopalmitate as carbon source. The sucrases of 10 fungi moved as single components in zone electrophoresis. The sucrases of six fungi were composed of two separable components. In one fungus, three sucrase components were observed.

A review on Trichoderma reese as the best available source of extracellular cellulase. (Refs. 56).

This symposium is good evidence of the current interest in utilization of renewable resources via fermentation. For substrates, various forms of plant biomass are available in large quantities or may be produced on energy farms. Solar energy is fixed by photosynthesis of green plants initially as small molecules, but these are rarely available in high concentrations or large quantities. Most of the excess over immediate metabolic requirements are rapidly converted into more complex molecules which are utilized by the plants as reserve foods or as structural materials. Most of these are polymers that are awkward to use for chemical or biological processes because they are chemically and physically complex and are attacked only by limited groups of specialized organisms that do not normally produce high levels of fermentations products of commercial interst. Therefore, we would like to convert these polymers back to the more usable monomers. An exception to the above is sucrose, a non-polymeric plant reserve food which is a simple soluble molecule, readily separated from plant juices in a high degree of purity. Not surprisingly, sucrose molasses is the basis for many industrial fermentations, notably the production of ethanol by yeasts.

Both batch culture and two-stage continuous culture systems were employed to study the growth characteristics, cellulase productivity, the specific rates of nutrient assimilation, and metabolic regulation related to the cellulase biosynthesis. Four mutant strains of Trichoderma reesei studied were designated as QM 6a, QM 9414, C 30 (7) and MCG 77 (3). These strains were then compared in terms of specific enzyme productivity, the nutrient requirements and economics, the nature of cellulase components, and the stability and utilization efficiency of cellulase.

Increased resistance of weathered cotton cellulose to microbial breakdown has been shown to be the result of development of resistance to the action of fungal cellulases. Photochemical activity during weathering exposure transforms the cellulose into an altered substrate that prevents access of the enzymes to susceptible sites of the cellulose molecule. It is postulated that the altered substrate consists of cellulose molecules of low degrees of polymerization, with some ring openings and altered chain ends. Weathered cellulose fails to adsorb cellulases.

One possible means of controlling microbial decomposition is through inactivation of the extracellular enzymes involved (Siu, 1951). This possibility was investigated for cellulose decomposition (Reese and Mandels, 1957). The conclusion reached Was that it requires more of the inhibitor to inactivate the degrading enzymes than it does to kill the organism.

Callus structures were isolated from a number of edible plants and maintained on simple defined media for extended periods. Growth rates are slow in comparison to other microbial systems, and increase tends to be linear. Static cultures on solid media double in 5-10 days, yield up to 0.26 mg dry weight per ml per day, and attain a maximum weight of about 12 mg dry weight per ml. Suspension cultures double in 2-5 days, yield up to 1.1 mg dry weight per ml per day, and attain a maximum weight of about 23 mg dry weight per ml. These growth rates are of the same order of magnitude as those for higher plants growing conventionally. Considerable improvements in these growth rates will be required before use of plant cell cultures as food can be realized economically.

β-D-1,3 Glucanases are of common occurrence in fungi, being detected in the culture nitrates of 96% of the organisms tested in shake flasks and in the sporophores of six basidiomycetcs. The enzyme is constitutive. Basidiomycete QM 806 and Sporotrichum pruinosum QM 826 are excellent sources of β-D-1,3 glucanase of the exo-type giving glucose as the sole reducing product of laminarin hydrolysis. Rhizopus arrhizus QM 1032 produces a β-D-1,3 glucanase of the endo-type giving laminaribiose and higher oligosaccharides as the products of hydrolysis of β-D-1,3 glucans. By controlling the conditions of growth β-D-1,3 glucanases can be produced free of β-1,4 glucanase (cellulase).

A mutant strain that secretes twice as much cellulase as its parent was obtained by irradiating conidia of Trichoderma viride QM 6a with a linear accelerator.