This treatise deals with a novel, ecologically benign, technically effective and economically practical cellulose-processing applied science (wood refinery) and its associated biotechnology; as well as the production of sugars and alcohols, particularly as derived from cellulosic materials; and industrial enzyme engineering processes.

It is clear that a large number of cellulose-, starch- and sugar-containing plants can be processed to produce sugars and alcohols. Sugar-containing plants such as sugarcane, sweet sorghum and nipa palm are the best candidates for the high-yield production of alcohol fuels. Similarly, the starch-containing crops such as cassava, sweet potatoes, yams, taro and tannia are also good candidates but require an additional step to break down starch to sugar. However, the emphasis of this report is on the major cellulose-containing biomass, which requires special treatment before it can be used to produce glucose and alcohols. To utilize cellulosic raw materials the following steps are necessary:

(1) Growth, harvest and delivery of raw materials to processing plants; or, alternatively, the collection and delivery of cellulosic "waste" products.

(2) Pre-treatment or conversion of the raw material by mechanical, physical, chemical or enzymatic methods to break down cellulose to sugars and to modify or remove unwanted side-products, usually lignin and hemicellulose.

(3) Recovery and purification of sugars from reaction mixture.

(4) Fermentation of sugars to alcohol and purification by distillation.

(5) Treatment of process residues to reduce pollution and to recover potentially valuable by-products.

From the considerable R&D work carried out in all areas of pre-treatment it appears that acid and enzymatic hydrolytic processes hold the most promise for developing countries. Though acid hydrolysis technology is more advanced, greater ultimate potential is expected for enzymatic hydrolysis which is, therefore, recommended.

The economics of producing alcohol from cellulosic materials is generally considered at this time to be unfavorable, although the data upon which this conclusion is based are limited. Therefore, it is recommended that further R&D be undertaken in this area in order to evaluate one or more commercially attractive processes for producing alcohol from fermenting cellulose.

The treatise consists of three major parts in six volumes: the first part presents the biochemical background of the enzymatic hydrolysis of cellulose and describes principles and approaches to the utilization of cellulosic raw materials (biomass) and principles of biotechnological processes; the second describes case studies of processes realized on a pilot scale (no industrial processes for enzymatic hydrolysis of cellulose exist as yet); the third describes the state-of-the art in enzyme engineering processes at the industrial level, some of which could be coupled with the novel cellulose-producing technology and associated biotechnology into an integrated industrial process(es), and some of which could be considered as case studies for courses in research and training.

Volume I - Biomass Conversion with Cellulases - deals with applied principles of the process, starting with some of the potential biodegradable agricultural and agro-industrial cellulosic residues and their availability, and provides a thorough review of various methods for pre-treatment of raw materials (including mechanical, chemical, physical, biological, and combinations of the above, that make cellulosics more reactive for enzymatic conversion to valuable products): cellulase enzymes, their natural sources, and cellulase preparations that are commercially available in several countries; composition and activity of available enzyme preparations and a description of their important properties that affect their potential industrial applications. This volume also describes the technology of glucose production in enzyme reactors, inhibition of the hydrolytic reaction by its products, mainly glucose and cellobiose, and principal ways to overcome this negative feature of the biotechnological process. The principles of practice of enzyme recycling are discussed in this section, as well as state-of-the-art production technologies, the economics of extant processes, and summary implications of the foregoing for developing countries

Volume II - Principles of the Enzymatic Degradation of Cellulose - covers the following principal biochemical areas of the enzymatic degradation of cellulose: adsorption of cellulases on cellulose; behavior of adsorbed cellulases on the cellulose surface; synergism between components of the cellulase system; mechanism(s) of degradation of cellulose by cellulases of varied origin; comparative consideration of cellulases of fungal and bacterial origin and design of cellulase systems with improved characteristics by protoplast fusion, enzyme fusion, protein engineering and genetic engineering in general. The overall aim of this consideration is to provide a contemporary basis for developing a strategy for industrial application of enzymatic conversion of cellulose to glucose (and then to ethanol, among other useful products). This section shows, in particular, how knowledge of some properties of cellulolytic enzymes, such as specific activity, adsorption capacity, thermostability, product inhibition, etc., can be translated into concrete requirements for cellulases for their potential industrial applications. Volume II also describes recombinant DNA technology that is essential for the generation of optimized enzymes for industrial utilization. Procedures which increase thermal stability and enhance catalytic efficiency are detailed. The text includes techniques which have been utilized on cellulases in particular as well as those which are applicable to the stabilization of enzymes in general. The protocols currently used for the generation of the engineered proteins include the polymerase chain reaction, site specific mutagenesis, random mutagenesis, cloning and expression of the individually altered proteins. Other genetic engineering techniques required for the achievement of the desired goals are outlined.

Volume III - Economics of Enzymatic Cellulose Hydrolysis - indicates that, as yet, no country has developed an industrial process for the enzymatic conversion of cellulose into sugars or alcohol. Several countries have designed and built pilot or pre-pilot scale solid waste treatment plants. The advantages and disadvantages of the major specific technologies being developed in this area can only be evaluated by comparing them with each other. This volume describes processes being developed to a certain degree in the USA, Canada, Russia, India, Finland, Sweden, Italy, Japan, and South Africa.

Eventually, an integrated set of commercially attractive industrial biotechnology processes based on biomass conversion and wood refinery will benefit developing countries, particularly:
  • Value-added exports derived from these technologies will promote a better balance of trade.
  • Employment, especially in rural areas, will increase.
  • Biotechnology and related areas will become competitive in global markets.
  • Chemical and biotechnological industries will expand.
  • An improved agricultural base utilizing alternative crops and what was previously termed agricultural waste will stabilize agricultural production.

Volumes IV, V and VI - Industrial Production with Immobilized Enzymes - describe the state-of-the-art of contemporary technological processes using immobilized enzymes and cells on the industrial level. Some of them could be combined with the enzymatic production of glucose from cellulose, described in Volumes IV (High Fructose Corn Syrups and Amino Acids for Food and Feed), V (Antibiotics, Food Additives and Dairy Products), and VI (Saccharose and Starch Hydrolysis), and/or with the subsequent microbiological conversion of that glucose into nutritional or other useful products. Others can serve as examples of modern industrial or semi-industrial processes based on practical application of principles of enzymology and biochemical engineering. Also, these enzyme processes can be considered as case studies for the course of research and training.

Enzyme engineering is a relatively new field of science and technology that deals with the development of useful products or processes based on the catalytic action of isolated enzymes and/or non-growing cells, converted into heterogeneous (insoluble) catalysts. Immobilized enzymes/cells are readily separable from the product solution and can be reused, are often more operationally stable in fixed form than in solution, and typically are much less expensive considering their longer period of use without replenishment. Volumes VI, V and VI describe in detail eight processes that use immobilized enzymes or microbial cells and have found industrial application:

   - Production of high-fructose syrups by immobilized glucose isomerase,

   - Optical resolution of amino acids by immobilized aminoacylase,

   - Production of optically active D-amino acids by immobilized hydantoinase,

   - Production of L-aspartic acid by immobilized aspartase,

   - Production of L-malic acid by immobilized fumarase,

   - Deacylation of penicillin G and cephalosporin derivatives by immobilized penicillin amidase,

   - Hydrolysis of lactose in dairy processing (milk and cheese whey) by immobilized lactase,

   - Production of glucose-fructose syrups from saccharose by immobilized invertase.

Each of these processes is considered in detail in terms of its background, available commercial preparations of respective immobilized enzymes, technological characteristics of the processes, economic estimations, and current (or previously reported) scale.



1. An interest in the enzymatic hydrolysis of cellulose began during the Second World War. Hordes of microscopic fungi inundated the American units stationed in the South Pacific. All cotton gear, tents, uniforms, knapsacks and cartridge belts, were ruined after a brief period in the humid tropical climate. Much cargo space was wasted just replenishing items that had become non-functional.

2. Since the situation was getting out of hand, solutions were called for. As a result, several laboratories within the Armed Services were established to investigate the nature of "rotting," to establish the causes, identify the invisible culprit and how it operated, and to find the ways and means to halt the damage.

3. The laboratories charged with these tasks carried out studies that included the investigation of more than 10,000 microorganisms. This enormous task included growing each one on textile strips and testing tens of thousands of strips for loss of tensile strength. It took several years to establish that the agent responsible was a special type of cellulose-destroying fungus. Therefore, it attacked cotton fabrics, since cellulose is the main component of cotton.

4. The chemical structure of cellulose is central to an understanding of these events. It is almost completely composed of "sugar", or more precisely, of glucose units. These are chemically bonded "head-to-tail" and form very long chains, containing hundreds and thousands of glucose units fixed into a rigidly ordered pattern rather like a crystalline lattice. The result is a stable structure because the chains are cross-linked by hydrogen bonds. In and of themselves the bonds are not very strong, but when blended with thousands of others they form a veritable monolith. As a result cellulose is not only insoluble in water, but its crystalline regions are virtually inaccessible to most chemical agents; neither water nor even strong acids easily penetrate the "crystallites".

5. However, the cellulose-destroying fungi achieved this by attaching themselves to cellulose-containing material and secreting a number of cellulose degrading enzymes. Several thousand different enzymes exist in nature and each is a catalyst for a specific chemical reaction. In the case under examination evolution has generated fungi which produce enzymes that degrade cellulose. Termed "cellulases", they act on cellulose in the immediate vicinity of the fungus, systematically cleaving the chemical bonds that link glucose molecules into polymer chains, until only glucose, the end product, remains.

6. Glucose, the main nutrient, enables the fungi to multiply, grow and spread over new areas, secreting more enzymes until the available substrate, cellulose, is exhausted.

7. In nature this usually takes a very long time. Years elapse before a tree stump decays. In some cases the process can be accelerated greatly, as in the South Pacific example. However, the reaction can be sped up even further. If the enzymes - cellulases - are isolated from the microorganisms, pooled and then added to the cellulose, the resulting glucose will not be consumed by the fungus, but will accumulate in the reaction mixture. There is another advantage; if cellulose-containing waste, not pure cellulose, were to be used as substrate, the waste problem would also be solved. Glucose can be fermented to form ethanol and used as fuel instead of other chemicals, and additionally, the dehydration of ethanol produces ethylene - a major compound used in much of the modern chemical industry. Depending on its purity and the economic efficiency of the process, glucose thus obtained could be used widely by the pharmaceutical, chemical, food and bioscience-based industries.

8. Cellulose heads the list of the world's renewable resources. The worldwide production of cellulose by nature runs into billions of tons annually. Mankind's use of these resources results in the production of very large quantities of cellulose-containing waste. If even a fraction of this waste were to be turned into useful products by enzymatic treatment, renewable supplies of edible carbohydrate and gasoline substitutes would become a major share of the world market. Given the enormity of the potential rewards, it is not surprising to find that research in these areas in recent years has been assiduous.

9. Generally speaking, cellulolytic enzymes are not the only agents capable of breaking down cellulose to sugars. This can also be done by subjecting cellulosics to a variety of physical and/or chemical practices. Physical methods include the steaming of cellulose or its g-irradiation, though they are so far not technologically attractive (unless the "steaming" is performed in a very specific manner, see below). The best known chemical methods involve the treatment of cellulose with strong mineral acids, e.g., sulfuric and hydrochloric acids.

10. The use of acids in industrial application followed by fermentation of the resulting glucose to alcohol began 80 years ago. The first commercial application was in 1913 at Georgetown, South Carolina, where a plant was built to hydrolyze southern pine mill waste with 2% sulfuric acid at 175°C in rotary steam-heated digesters. The dilute sugar solution, produced at a 25% yield, was fermented to yield 5,000 gallons of ethanol per day. A second plant was built subsequently in Fullerton, Louisiana. Both plants produced alcohol profitably until the middle 1920s, benefiting from extremely low-cost sawdust waste and the lack of environmental controls. Eventually, a fall in the price of blackstrap molasses led to cheap supplies of sugar from this source and the two plants became unprofitable.

11. During the 1930s Scholler in Germany developed a process by which wood in a percolator was subjected to hydrolysis with successive batches of dilute acid. Wood was pretreated with 1% HCl and then digested with 0.5% sulfuric acid at 130-190°C for 18-24 hours in stationary digesters. By removing sugar as it formed yields amounted to roughly 50%, about twice the typical yield of earlier processes. Three such plants were built in Germany and used during the Second World War. Additionally, one was built in Korea, and one at Ems, Switzerland. In the USSR, a percolation plant was built in 1935, with a further 40 being built after the Second World War.

12. The Bergius process, also developed in Germany during the Second World War, used concentrated (42%) hydrochloric acid in special acid-resistant equipment. It resulted in cleaner syrup, but was expensive because the acid had to be recovered. Nevertheless, the sugars from the Scholler and Bergius processes were used for the production of alcohol and for the growth of yeast for human consumption. By the end of the Second World War, approximately 9,000 tons of food yeast was being produced per year in Germany using these processes.

13. The Forest Products Laboratory of Madison, Wisconsin, in co-operation with the Cliff's Dow Chemical pilot plant at Marquette, Michigan, investigated acid hydrolysis of wood to sugar during and after the Second World War. The work resulted in a modification known as the Madison Wood Sugar Process which accelerated the hydrolysis process and produced higher yields. In this process, chopped wood waste was continuously percolated with 0.4-0.6% sulfuric acid at temperatures gradually rising to 150-185°C. The process yielded 4-5% sugar syrup in 45-55% yields. After neutralization, the sugar syrup was concentrated to 50% molasses, which was fed to dairy cattle as a silage additive or used to grow brewer's, baker's and food yeasts.

14. Immediately after the Second World War a plant incorporating the Forest Products Laboratory developments was built at Springfield, Oregon to produce ethyl alcohol for the manufacture of synthetic rubber via butadiene. After its completion, the Springfield plant demonstrated its functional usefulness but in cost-effective terms it could not compete with petrochemical derived ethanol.

15. Only one of the above processes have succeeded as a commercial operation in the almost fifty years since the Second World War. The percolation industry in the former Soviet Union using multifunctional, raw cellulosic materials yields a production not only of alcohol but also of yeasts and furfurol (when lignin was used as fuel). Nowadays this industry consists of 40 full-scale plants with a maximum capacity of 1,000 tons of wood material per plant per day. The annual production from this technology is 400,000 tons of fodder yeasts and 195 million liters of ethyl alcohol.

16. In recent years, the Brazilians have taken advantage of the Soviet experience for their wood hydrolysis plants. The Brazilian plants are not only making alcohol but also are manufacturing charcoal for use in the Brazilian steel industry. COALBRA, the Brazilian company responsible for building plants to make alcohol from wood, took its name from its two products, coke and alcohol. According to recent data, COALBRA has a pilot plant under construction which is expected to produce 30,000 liters per day of alcohol from wood. This work is under the jurisdiction of the National Alcohol Programme (PROALCOOL), established in 1975 as a means to partially substitute ethyl alcohol for petroleum derivatives. Impetus for the PROALCOOL program arose from Brazil spending half of its export revenue - about US $10 billion - to buy petroleum from abroad. Currently, Brazil has the largest alcohol-fuel industry in the world, with sugarcane as the raw material (Rothman et al., 1983), and produces 12 billion liters of alcohol annually (International Energy Agency Newsletter, 1992). Lignocellulose is not as yet a raw material for commercial ethanol production on a significant scale (Lynd, 1990).

17. Currently conversion of corn and lesser amount of other grains to ethanol is the basis for a substantial industry in the US (Lynd, 1990). Ethanol produced from these sources is used as a gasoline additive that raises octane. Approximately 7% of the total motor gasoline used in the US is blended with ethanol (National Advisory Panel on Cost-Effectiveness of Fuel Ethanol Production, 1987), and each year more than 3 billion liters of ethanol is being produces in the USA (International Energy Agency Newsletter, 1992). The economics of corn-derived ethanol are currently dependent on federal subsidies and the value of processing residues as feeds (Venkatasurbramanian & Keim, 1985).

18. In recent years investigators, mainly in the United States, have been working on the development of continuous hydrolyzers of cellulosic materials. These include Grethlein and Converse at Dartmouth, Church at American Can, and Rugg at New York University, who have all been developing continuous dilute acid hydrolysis technology. The two-stage continuous hydrolysis has some important advantages over percolation, such as effective fractionation, improved by-product recovery, and higher sugar concentration. However, the prevalent thought today is that although such continuous saccharification may be ready for first level pilot scale investigations, it is not yet ready for commercialization.

19. It should be noted here that alcohol production through the acid hydrolysis of wood also presents a series of disadvantages:

- to be economical, the scale of the process must be very large; in order to produce some 500,000 liters per day, an area of nearly 600,000 hectares is required to maintain a constant supply of raw material and fuel;

- it is extremely capital intensive, requiring an investment per liter of industrial capacity which is double or triple those of other raw materials, while production costs are 50 to 100% greater;

- the process requires a large amount of sulfuric acid (at the rate of one liter per three liters of alcohol produced);

- the process calls for rather severe conditions, and as a consequence, requires expensive corrosion-resistant equipment;

- the severe conditions of hydrolysis lead to a partial degradation of the glucose formed and, as a result, to a contamination of sugars by toxic by-products (e.g., furfural) which in turn are poisonous both for microorganisms, e.g., fermenting yeasts, and human beings.

20. Enzymatic hydrolysis is the most promising alternative to the use of dilute acid, but it is certainly not a replacement process. A future possibility is the development of a hybrid process which uses dilute acid to provide a pretreatment-fractionation which is coupled with enzymatic hydrolysis that converts the residual (resistant) cellulose to glucose. A choice between acid or enzymatic hydrolysis of cellulosics depends on certain circumstances, especially on the location of the respective plants, availability of raw materials and links with nearby chemical or biochemical factories able to supply mineral acids, culture broths, etc.

21. Another possibility, which is outlined below, is the use of a physico-chemical disruption of wood into a physical mixture of its major components with a subsequent fractionation of the mixture into individual components (wood refinery) using mild alkali and water. Clean, wet, and open to enzymatic attack cellulose will be susceptible to conversion into glucose.

22. Once the hemicellulose sugars and the lignin have been removed from raw materials such as wood, the remaining cellulose can be hydrolyzed. Both enzyme and acid catalysts are effective for this process. However, as a rule hydrolyzates after enzymatic conversion of cellulosics contain a substantially higher proportion of glucose than other products of cellulose degradation as compared to acid hydrolyzates. This may be particularly important when the aim is the production of pure glucose as food sugar, rather than its fermentation to alcohol, since glucose can be isomerized enzymatically into fructose (see Volume IV).


23. Before a successful industrial process for glucose production from cellulose can be realized the problems that must be resolved are the following:

(1) Selection of a readily available cellulosic raw material, the processing of which should be economically and technically feasible;

(2) Development of an effective pretreatment process which could significantly increase the subsequent rate of enzymatic hydrolysis and final product yield;

(3) Development of cellulolytic enzymes for glucose production for optimum conversion;

(4) Optimization of the glucose production process from cellulosic materials;

(5) Development of an optimal enzyme reactor for the most efficient conversion of cellulose into glucose in terms of a continuity of operation, insoluble cellulose residues and enzyme recycling;

(6) Designing and operation of a pilot plant for the enzymatic production of glucose.

24. In terms of their mechanism of action, cellulose-degrading enzymes can be subdivided into four groups regardless of the living organism from which they derive. These are one group of endoenzymes, two groups of exoenzymes and the fourth group consisting of cellobiases. "Endo" and "exo" are usually prefixed to the names of enzymes attacking polymer substrates in order to indicate whether they are acting on the internal or external portion of the polymer. For instance, if an enzyme attacks the chemical bonds far removed from the ends of a long polymer molecule it has the prefix "endo". It is "exo" when a short end group is cleaved off.

25. Hence, the longer the molecules making up the substrate (i.e., the higher the degree of its polymerization), the less pronounced is the role of the exoenzyme in the initial stage of the reaction since there only two ends to a linear chain but many internal bonds. On the other hand, an endoenzyme is most active when the conversion of the polymer begins and less so as the substrate molecules become shorter. That is why the degradation of polymer substrates in nature usually involves multi-enzyme complexes consisting of both "endo" and "exo", enzymes.

26. It can be seen that the enzymes of endo-wise mode of action begin the conversion process since the cellulose molecule consists of several thousand monomer units and the number of end glucose units in the native polymer are too few (compared to the number of intermediate glucosyl bonds in the cellulose) for the action of the exoenzymes to be noticeable initially. However, each successful attack of the endoglucanases breaks the polymer chain and forms two new ends in the shorter cellulose molecule, which can now be attacked by exoenzymes. Thus, the role of exoenzymes and the speed of their action increases as the endoenzymes degrade cellulose.

27. The exoenzymes acting on the partially split cellulose are of two types. One cleaves off glucose, the end-product of the hydrolysis, while the other, because of the specific structure of its active site, cleaves off cellobiose which is a dimer (a coupled glucose molecule). The first exoenzyme is an exoglucohydrolase, the second, exocellobiohydrolase. Recently experimental evidence has been obtained that some exoglucohydrolases in fact are endoglucanases, having a high transglucosidase activity. They attack long cellulose chains far from the ends, but are recycled from the reaction (as enzymes normally do) with the assistance not of water, as is usual for hydrolytic reactions, but apparently with the help of cellobiose, a final product of cellulose hydrolysis by endoglucanases. The resulting product of the "transglucosylation" reaction catalyzed by the endoglucanase is glucose.

28. Finally, the cellobioses are cleaved into two glucose molecules by the last of the enzymes in the cellulase complex - the cellobiases, i.e. - or are converted into glucose by some endoglucanases by means of transglycosylation, as described in the previous paragraph. More detailed descriptions of the dynamics of the enzymatic process are given in Volume II.

29. The reaction rate depends on the composition and quantity of the enzymes added to the cellulose, the state of the initial substrate (degree of its polymerization, degree of crystallinity, etc.), the quantity of native cellulose, the amount and the nature of constituents usually present in the native cellulose-containing material, the condition of the reaction, etc.

30. The formation of glucose and cellobiose during the course of the multienzymatic hydrolysis takes place at a regular rate when pure amorphous or crystalline cellulose is the starting substrate. Curves reflecting the yield or conversion at any given moment of the starting material to glucose and cellobiose have, therefore, been worked out along with the rate of accumulation of products. However, methods have not yet been developed to predict the dynamics of hydrolysis of heterogenous cellulosic material in an enzyme reactor.

31. There is another aspect to be considered. As noted above, in nature cellulose is mostly crystalline, and is rather resistant to all hydrolytic agents, including enzymes. To make crystalline cellulose amorphous and, therefore, more reactive toward cellulases, the very rigid, regular structure of its polymer chains must be broken. This can be done by subjecting the native materials to intensive milling, to treatment with phosphoric acid, or by dissolving it in special solutions. All these methods are technologically feasible.

32. But before deciding on the method, it is desirable to determine how enzymatic hydrolysis is affected by the native material's crystalline structure. The problem can be defined more comprehensively: what is the impact of the main structural characteristics of cellulose (i.e., specific surface, average particle size, degree of polymerization, and crystallinity) upon the rate of enzymatic hydrolysis.

33. This problem was resolved recently by a series of R&D projects in the U.S., Germany, Italy, and the former Soviet Union. Several dozen samples of cellulose, including native cellulose, were chosen for the experiments. All the pertinent structural parameters and the rate of enzymatic hydrolysis were registered and compared. Since the samples were of various origins, their structural factors also differ greatly. Thus, cotton was found to have the highest degree of polymerization - several thousand units - while that of cotton ground in a vibratory mill was lower - several hundred units (depending on the duration of treatment and types of mills). Cellulose samples treated with cobalt-60 isotopes or by electron accelerators had the lowest degree of polymerization - up to 20. However, as it turned out, samples of identical degrees of polymerization displayed different rates of hydrolysis. In other words, other structural factors were more important than polymerization in determining the rate of the enzymatic reaction.

34. It was also found that the average size of the particles (all other factors being the same) of the initial cellulose was not crucial in enzymatic hydrolysis. Cellulose, amorphous cellulose in particular, is a porous material with a developed, sponge-like internal surface. Thus, it is not the average size of the particles that normally determines its accessibility to enzymes, neither is it crucial to the efficiency of hydrolysis.

35. The accessible surface area is the main factor in determining the efficiency of the enzymatic attack on insoluble cellulose. This has been demonstrated by the linear relationship between the rate of hydrolysis and specific surface (square meters of surface per gram of material). It is not, however, the only factor. The rate of enzymatic hydrolysis is inversely proportional to the degree of crystallinity (which can be determined readily by X-ray diffractometric analysis).

36. There is no surprise in the observed relationship between the degree of the crystallinity of cellulose and its specific surface area, since both affect the hydrolysis rate to the same extent. The reason is that the greater the crystallinity the more packed the polymer chains of cellulose and the less accessible the surface of the substrate. Conversely, when the crystalline structure is destroyed, the inner chains of the cellulose give way and its accessible surface increases.

37. Consequently, these data may be used to predict the rate of enzymatic hydrolysis for virtually any cellulose sample, which must not, however, contain many heterogeneous admixtures (lignin etc.). It is simply a matter of measuring the specific surface area of the cellulose or the degree of its crystallinity. That, in turn, permits a standardization of cellulose-containing materials in industry; an important accomplishment. Thus, another bridge has been built between the structure of a substrate and its capacity to react - and the practical problem of technology, i.e., the enzymatic production of glucose from cellulose.

38. This treatise emphasizes the problem of enzymatic conversion of cellulosics into sugars and of producing ethanol from the latter by microorganisms. The production of ethyl alcohol will be mainly considered in Part C, which deals primarily with scaling-up technologies for the enzymatic conversion of cellulose into fuels.


39. Biomass in the form of agricultural and forest wastes accumulates every year in large quantities both in industrial and developing countries. This results in a deterioration of the environment and a loss of potentially valuable resources. Some of the potential biodegradable agricultural and agro-industrial cellulosic residues and their availability in several countries are listed below:
Agricultural crops
Cotton stalks, rice straw, corn cobs, bamboo dust, wheat straw, maize stalks, banana stem, tapioca stem, castor stem, planted forests
Sugar industry Bagasse pulp and pith
Rice-milling industry Rice husk, rice bran
Sawmill industry Sawdust, wood chips
Coconut industry Coconut husk, shell and pith

  Millions of Tons Per Annum

Rice husk 18.0
Rice bran 3.2
Rice straw 59.2
Bagasse 52.1
Jute sticks 2.5
Cotton stalks 12.0
Cotton linters 1.2
Wood wastes 5.5
Coconut shell/coir dust 0.6
Coconut shell/coir dust 0.6
TOTAL 154.3

Source: Bisaria & Ghose, 1981.

Assumed Cellulose
Content, %

Agricultural and food wastes 400 60 240
Manure 200 50 100
Urban refuse 150 45 68
Logging and other wood wastes 60 55 33
Industrial wastes 45 33 15
Municipal sewage solids 15 33 5
Miscellaneous organic waste 70 25 18

TOTAL 940   479

Source: Weigel, 1982.

  Tons Per
Content, %
Tons of

Wood-processing industry 61 40 24.4
Saw dust 15
Wood chips 28
Others 18
Wheat straw 10 30 3
Paper-making industry
and rayon manufacture
0.1-0.2 90-100 0.1-0.2
Rice straw 1.5 30 0.45
Rice husk 0.4 33 0.13
Corn cobs 2.0 28 0.6
Cotton-processing industry
Cotton stalks 6-10 35 2.1-3.5
Cottonseed hulls 1.5 50 0.75
Chiganak 0.8 40 0.3

TOTAL 84-88.1   32.4-33.9

Source: USSR Ministry of Light Industry, 1986

Growing Stock
Per Capita
(m3 biomass)

40. If just a fraction of these materials were to be converted into sugars and alcohol as well as into gas and feed protein, a significant contribution could be made to the overall problem of resource recycling and conservation. However, before using virtually any of these cellulosics it is necessary to convert them so that they are more reactive toward cellulolytic enzymes. As indicated above (paragraphs 31-37), it is necessary to decrease their crystallinity and to increase specific surface area and, of course, to remove as much as possible the non-cellulosic matrix of the cellulose, which may otherwise sterically block the accessibility of cellulose to cellulase enzymes.


41. Natural cellulose is a crystalline polymer generally associated with hemicellulose and lignin in a matrix and is highly resistant to enzymatic attack. Therefore, pretreatment is necessary. Most approaches separate the different types of pretreatment into mechanical, chemical, physical (other than mechanical), biological, or a combination of these methods:

For all these methods it is assumed that the cellulosic material is already in a form which can be processed readily.

42. Most pre-treatment processes have energy requirements that depend on the severity of the process. Severe mechanical and/or thermochemical processes have substantial energy requirements and when combined with those for product separation, can make some cellulosics bioconversion processes very inefficient. A special analysis performed recently by Datta, Exxon Research and Engineering Company, Linden, New Jersey, has shown that many of the proposed processes which use fine particles, severe conditions, etc., can consume a substantial amount of energy and lead to a generally inefficient process in terms of energy. Conversion processes that can use coarse particles, mild pretreatments, non-sterile conditions, etc., will have significant advantages in this respect.

43. Mechanical pretreatments (see paragraph 41) utilize shearing and impacting forces to yield a fine substrate possessing a low crystallinity index and high specific surface area, thus enhancing its susceptibility to enzyme action. Typically, mechanical pulp (stone groundwood pulp) is made by grinding logs or blocks of wood against a revolving abrasive stone in the presence of water. Chip groundwood, or refiner mechanical pulp, is provided by feeding chips or sawdust between a set of rotating, ridged plates (or disks) of a disk refiner.

44. An extension of the chip groundwood process is the thermomechanical process in which steam softens the chips prior to reduction in pressurized disk refiners. Mill and laboratory experiences regarding thermomechanical pulping at Smurfit Newsprint Corporation, Oregon, are described recently (Quick et al., 1991). Besides, among specially processed mechanical pulps steam defibrated and steam exploded pulps should be mentioned. Mechanical as well as thermomechanical pulps are practically identical in composition with wood.

        Degree of
Crystallinity, %

45. The use of fine substrates yields higher slurry concentrations, or higher bulk density, thus reducing the reactor volume. Using milling as a pretreatment method has the advantage of being relatively substrate insensitive but suffers from the major disadvantage of being energetically unfavorable (see below) and rendering the remaining lignin accessible as a significant inhibitor of the enzyme.

46. The estimated cost of mechanical pretreatment of wheat straw may vary from $0.01/kg for Fitz milling (model D comminutor, Fitzpatrick Company, Elmhurst, Illinois) to $2.24/kg for roller milling (based on the weight of the substrate).

47. Of the mechanical pre-treatments shown above, ball milling gives the most promising results in terms of hydrolysis rate and sugar yield. This pre-treatment is clean and easy to operate, but the long pre-treatment time makes its large-scale operation impractical.

48. The main difficulty with size reduction of cellulose is the fibrous nature of the material. This is evident from the comparatively low energy requirements for the size reduction of a brittle material such as coal, which takes only 5-7 KwHr/ton to reduce the particle size to 100-200 mesh by means of simple pulverization. For this reason, the energy content of feed is as low as 0.2-0.3% compared with 25-100% for fibrous cellulose.

49. Chemical pre-treatments have been used extensively as a means of lignin removal and to modify the structure of lignocellulosics. Conventional pulping processes, such as kraft, sulfite, and soda processes, are suitable for delignification, but these processes have been designed for removing lignin in order to preserve the quality of cellulose and are too expensive to be used as bioconversion pre-treatments.

50. Another, more promising pulping process involves the use of SO2, which causes a disruption in the lignin-carbohydrate association without the selective removal of either constituent. Typical process conditions are: temperature 120-150°C, 20-100 kg SO2/ton of dry biomass and the final solids content of 25-30%. 600-900 kg of steam per ton of dry biomass is required when about 50% of the energy is recovered to heat the boiler feed water. This translates to 9-18% of the energy content of the biomass being processed. It was shown that pre-treatment by pressure cooking for 2-3 hours at 120°C in an SO2 atmosphere results in nearly quantitative conversion of hardwoods to sugars while softwoods are hydrolyzed only slightly less readily. Apparently the presence of lignin in the pretreated wood substrate does not interfere directly with enzymatic hydrolysis, which can occur once cellulose is loosened from the lignin matrix, even though lignin is still present.

51. Caustic swelling is the most common chemical pretreatment. Pretreatment with caustic soda leads to an increased surface area due to swelling and disruption of lignin. Generally swelling occurs in two forms. Swelling agents such as water act at the "intercrystalline" level of cellulose, with a volume change approximately equivalent to the volume of water, resulting in a minor crystalline modification of the substrate. "Intercrystalline" swelling agents penetrate the crystalline as well as the amorphous region of the cellulose component and lead to a new crystalline modification, which is more reactive toward cellulase enzymes.

52. Sodium hydroxide, certain amines and anhydrous ammonia are some of the limited swelling agents. Unlimited swelling, which completely solubilizes cellulose, is induced by concentrated sulfuric and hydrochloric acids, cupram, cuen and cadoxen. Although the unlimited swelling agents efficiently increase the accessibility of cellulose substrates there are still problems with product separation, chemical recovery and interference by lignin. It is doubtful that any of the unlimited swelling agents provide an alternative to a more economically feasible pretreatment such as dilute alkali swelling.

53. Acid pretreatment uses dilute acids such as hydrochloric (HCl), sulfuric (H2SO4), and phosphoric (H3PO4) to remove hemicellulose by hydrolysis without causing significant glucose formation. This was reported recently by Grethlein at Dartmouth, who showed this to be an effective pretreatment for enzymatic hydrolysis of substrates such as newsprint, corn stover, poplar and oak. Following pretreatment and subsequent enzymatic hydrolysis, glucose yields as high as 100% have been obtained. The pretreatment is carried out in a continuous flow reactor at temperatures around 200°C, acid concentrations less than 1-1.5% by weight, and reaction times of the order of 12 seconds. Increasing the temperature to 220°C achieves approximately the same glucose yields; however, a smaller proportion of the glucose is produced by the enzymatic hydrolysis because the higher temperature pretreatment converts 15-28% of the cellulose to glucose. It should be noted that one major shortcoming of acid hydrolysis processes is the low total yield of sugars (50-55%) owing to side product formation.

54. Another technique for chemical pretreatment is the oxidation of lignin by an oxidizing agent (for example, peracetic acid or Fe2+/H202) which liberates cellulose for enzymatic hydrolysis. Considerable attention has also been paid to delignification using solvents such as ethanol, butanol, and acetone plus a suitable catalyst; the possibility of solvent recovery makes this alternative attractive. Although most chemical pretreatments are effective, waste chemicals are often difficult to dispose of or recycle.

55. The estimated costs of chemical pretreatment of wheat straw vary from $0.4/kg for caustic pretreatment to $11.25/kg for ethylene glycol treatment. In comparison, the costs of mechanical pretreatments vary from $0.01/kg to $2.24/kg (see paragraph 46).

56. Of the chemical pretreatments shown above, caustic pretreatment is a potential candidate for large-scale process development based on pretreatment cost, hydrolysis rate, and sugar yield. Chemical pretreatments, however, have disadvantages which cannot be ignored. These include the required use of specialized corrosion-resistant equipment, the need for extensive washing, and the difficulty of disposing of chemical wastes.

57. Physical pretreatments of cellulosics involve primarily g-irradiation and high-pressure steaming, either with or without fast decompression. Actually, steaming with a sharp reduction in pressure couples physical and chemical pretreatments; it will be considered in the last section of this section (paragraphs 66-70).

58. Steaming of biomass in the 150-200°C temperature range leads to increased enzymatic digestibility as a result of increasing pore size and the partial hydrolysis of hemicelluloses. The residence time at higher temperatures should be kept low to minimize reactions which produce by-products of a noncarbohydrate nature. According to some data and calculations, to reduce biomass containing 50% solids to a product containing 25-40% solids by steam treatment at 150-200°C, approximately 450-1300 kg of steam would be required per ton of dry biomass. This method has not yet proven effective in greatly increasing enzymatic hydrolysis.

59. γ-Irradiation was not effective as a pre-treatment of relatively pure cellulose (see below), but it slightly accelerated (up to 1.5-3 fold) the enzymatic hydrolysis of lignocellulose substances such as wheat and barley straw and bagasse. Other mechanical or chemical pretreatments, on the other hand, accelerated the conversion of bagasse 7- to 10-fold.

Glucose Formation

60. Lignin-utilizing microorganisms have been used for biological pre-treatment of lignocellulosic materials, and in "biological pulping" in paper manufacture in particular (Reid, 1991). This idea was inherent in the original concept of "white rot"; first thought to involve only lignin degradation. However, as subsequently shown, soft and brown-rot fungi degrade cellulose and hemicellulose in preference to lignin while white-rot fungi destroy all three wood components at the same time, and remove the lignin faster than other polymers. Unfortunately, evidence indicates that white-rot fungi do not use lignin as a growth substrate, so a complete selective removal of lignin by these organisms is not possible.

61. Nevertheless, partial delignification without cellulose loss is possible. Eriksson in Sweden has isolated cellulase-less mutants of white-rot fungi, which utilize a large fraction of xylan and mannan while degrading lignin from wood. For wood treatment with cellulase-less mutants of Phanerochaete sp. (= Sporotrichum pulverulentum) 35% of the xylan and all soluble substrates are utilized to degrade 30% of the lignin. However, a 10-14 day cycle time is a major problem.

62. Because of the time factor, removal of a substantial amount of lignin by fungi has not been seriously considered. For example, lignolytic fungus Pleurotus ostreatus when used in a 10-20-day trial to delignify wheat straw partially and to increase its enzymatic saccharification yields, did not increase the saccharification yields of the residue over the control. The yield, however, did increase 4-5 fold after 50 days of fermentation. Clearly, further studies are needed to explore the potential of biological delignification. Meaningful economic analyses require pilot-scale studies, which have not yet been conducted.

63. Of the combinations of pretreatment methods briefly considered here, biomechanical pulping and steam explosion are particularly attractive.

64. The properties of wood after partial bio-delignification (see paragraphs 60-62) have been examined, paying particular attention to the energy requirements for subsequent mechanical pulping. Preliminary studies with mill refining show that removing even small amounts of lignin (2.1% of the original) from pine chips resulted in substantial energy savings (20%). Moreover, enzymatic digestibility of the material is improved because the strength properties of the resulting mechanical pulp are lower than those of untreated pulp at a given degree of refining.

65. Modifying biomechanical pulping involves partial delignification of coarse thermomechanical pulp (TMP) prior to "post refining" for final pulp production. When coarse TMP (see par. 44) is treated with a white-rot fungus until it loses 2% in weight, a considerable decrease in energy consumption is observed in the post refining. Moreover, this decrease in energy consumption is accompanied by a loss in strength properties, especially when a wild-type rather than a cellulase-less mutant is employed. The fungal degradation rate of lignin appears to be faster in coarse TMP than in wood chips. Lignin degradation rates of an average 3% per day, over a 2-week period, have been observed; this, however, is preceded by a chip treatment. Technical problems with fungal treatment of TMP include maintaining correct environmental conditions on a large scale and the slow-paced degradation of lignin.

66. Steam explosion as a pre-treatment essentially involves steaming lignocellulosics at various temperatures and pressures for different retention times with a sudden sharp reduction in pressure to expel the material from the vessel. This treatment opens up the fiber, renders the hemicellulose soluble in hot water and appears to some extent to depolymerize the lignin. When conditions are optimized, the lignin becomes readily soluble in dilute sodium hydroxide solution from which it can be recovered by acidification as an active chemical.

67. Steam explosion includes both physical and chemical pretreatments. The high-pressure steaming of moist lignocellulosic substrates results in a partial decomposition of some of the hemicellulose components into acids, mainly acetic acid, which, in turn, catalyze the depolymerization of hemicellulose and lignin. Su from General Electric found the highest conversion when aspen wood chips are steamed at 195°C for approximately 20 minutes in the presence of SO2. Sulphur dioxide acts mainly as a catalyst.

68. Two Canadian companies also use steam explosion to pretreat lignocellulosics. Stake Technology operates a tubular, high-pressure, continuous reactor at temperature ranges of 200-240°C using various retention times. This equipment was initially developed for steaming hardwoods such as aspen and birch to make them digestible by ruminants. Other equipment, developed by the Iotech Corporation, uses a modified Masonite gun from which wood chips, after being steamed at approximately 200-250°C for 20-100 seconds, are exploded. The Iotech lignin from aspen wood becomes relatively soluble in ethanol after steam explosion and thus is a potentially valuable by-product for use as a chemical feedstock or in wood adhesives.

69. The major advantages and disadvantages of using steam explosion as a method of pretreatment are as follows:

70. Steam explosion has proven to be one of the most energy efficient methods of pretreating wood substrates as well as one of the most efficient methods for enhancing subsequent enzymatic hydrolysis of hardwood species.

71. In the context of contemporary understanding, a cellulase complex contains four groups of enzymes (see paragraphs 24-28). The overall flow chart of the enzymatic hydrolysis of cellulose is as follows:

Here endoglucanase attacks native cellulose (S), which is either amorphous or crystalline, and its action produces partially degrades cellulose (Gn). Then endoglucanase and/or cellobiohydrolase cleave the cellobiose units (G2) from the ends of the insoluble celluoligosaccharides (Gn). This cellobiose is later converted into glucose (G) by cellobiase. The final enzyme(s) in the cellulase complexes cleave glucose directly from the ends of long oligosaccharides. This enzyme(s) exhibits so-called exoglucohydrolase activity; in some cases it is an individual enzyme, but in some complexes its function is performed by endoglucanases (see Vol. II). In any case, this method of forming glucose, which does not include intermediate cellobiose hydrolysis, often plays a crucial role in the hydrolysis of cellulosics.

72. The composition of cellulase complexes in terms of the relative content of the individual cellulolytic components varies greatly from one biological source to another. Thus, cellulase complexes from species of Trichoderma fungi (an organism widely used throughout the world for basic and applied studies of enzymatic degradation of cellulose) are usually deficient in cellobiase. In order to increase the yield of glucose, at the expense of cellobiose accumulating in the reaction mixture, it is recommended that the Trichoderma complex be enriched by adding cellobiase isolated from another microbial source. On the other hand, cellulase complexes from Aspergillus fungi often have a relatively high content of cellobiase but lack cellobiohydrolase and exoglucohydrolase, both of which play an important role in accelerating cellulose degradation. The composition of some cellulase preparations is shown below:

  Activities of Individual Components

  Activities of Individual Components

  Activities of Individual Components

73. The main technical use of cellulases lies in the total conversion of cellulosic fractions from various cellulose-containing material into glucose. Therefore, it is in the interest of the industrial biochemist to measure the total cellulolytic activity, i.e., the activity of the cellulase complex which produces glucose from cellulose. Determinations of the total activity, however, are complicated by several factors, related to the nature of both the enzymes and the substrates:

  • The enzymes of cellulase complexes often act synergistically, so the activity measured is greatly influenced by the proportion in which various enzymes are present;
  • The substrates used, i.e., various forms of soluble or insoluble cellulosics, are macromolecules, which are difficult to standardize.

74. The most widely used substrates for the determination of total cellulolytic activity include filter paper, microcrystalline cellulose, cotton fibers and soluble carboxymethyl cellulose. Each method usually involves measuring the reducing sugars formed in the course of the enzymatic hydrolysis of the substrate. Filter paper hydrolysis, the so-called Mandels-Weber method, has been generally accepted for this purpose. This method determines cellulase activity in units of micromoles of reducing sugar (measured as glucose) liberated per one minute under standard assay conditions (filter paper units, FPU, or international units, IU).

75. For the last 30 years Trichoderma viride (= Trichoderma reesei) is known as the best source of "complete cellulase", i.e., cellulase which contains all the components necessary for the hydrolysis of crystalline cellulose. Using this microorganism in conjunction with the optimization of biosynthesis and mutation techniques, investigators at the U.S. Army Natick Research and Development Command, Massachusetts, and at Rutgers University, New Jersey have greatly increased enzyme yields and titers (in terms of total cellulase activity). The highest titer obtained to date, 14.8 I.U./ml, utilized fermentation of compression-milled cotton with the NG14 (Rutgers) mutant. Maximum productivity of 167 I.U./l/hr was recorded at Natick through the use of newer mutants. Recently it was reported that a commercial cellulase "Laminex" (T. reesei strain) manufactured by Genencor (San-Francisco based company) has a total activity of 84 IFPU/ml (International Filter Paper Units) (Philippidis et al., 1993).

76. With regard to cellobiase activity in a culture broth, 16.7 I.U./ml of cellobiase from Aspergillus phoenicis (QM 329) was produced at Natick with a maximum productivity of 135 I.U./l/hr over 119 hours, in a prepilot plant fermentation using a chemical pulp hydrolysis syrup. Commercial cellulase "Laminex" (see above) has beta-glucosidase activity of 91 I.U./ml (Philippidis et al., 1993).

77. To render these figures more meaningful, it should be noted that to produce 11% sugar syrup in 24 hours from compression-milled newspaper, the charge to the hydrolysis vessel would be 25% of cellulose containing the Trichoderma at an enzyme/substrate ratio of 10 I.U./gm. In this case the level of reducing sugars is high enough to permit direct practical fermentation to ethanol without an additional sugar concentration step. The production of glucose in the enzymatic hydrolysis of ball milled newspaper increases by 25-35% when cellobiase from Asp. phoenicis is added to the cellulase (T. reesei) broth at up to 3 I.U./ml.

NCU - Novo Cellulase Unit; the amount of enzyme which degrades carbomethyl cellulose to reducing carbohydrates with a reduction power corresponding to 1 mmol glucose per minute (international units).
EGU - viscosity method with Novo Nordisk standard.
L - liquid
T - granulate

78. The properties of cellulolytic enzymes from different organisms may vary in terms of heat stability, the dependence of the activity and the stability on pH, sensitivity to inhibition and activation by the reaction products, capacity of being adsorbed on the surface of an insoluble substrate, capacity for transglycosylation reactions (usually side reactions leading to the formation of unwanted by-products; in the case of cellulose hydrolysis, however, a major product of transglycosylation can be glucose, see Vol. II), and substrate specificity, etc. As yet, however, the relationships between cellulolytic enzyme sources, the nature and scale of variations and reaction conditions on the final enzymatic outcome are far from understood. Pertinent studies are still in their infancy. Adsorption of cellulases on the substrate surface will be briefly considered here as one of the most important properties of cellulases, since it determines the prerequisite step for the enzymatic hydrolysis of insoluble cellulose. As realized recently, the adsorption capacity of cellulases is extremely important for biotechnological application of the enzymatic hydrolysis of cellulose.

79. Scientists at Moscow State University and the Institute of Biochemistry, Moscow recently demonstrated that similar cellulase enzymes from different microorganisms vary dramatically in their capacity to be adsorbed on cellulose - the difference ranging from one hundred- to one thousand-fold. To achieve the optimal surface concentrations obtained by the most efficient cellulases (in terms of adsorption) it is necessary to use hundreds or thousands of times larger quantities of the "poorer" enzymes, which is hardly feasible. Consequently, the greater the adsorption capacity of the enzymes, the higher the yield of the end-product which results from the greater number of enzymes directly participating in the hydrolysis of cellulose.

80. More extensive investigations into this problem demonstrated that most endoglucanases consist of a number of isoenzymes (at least two) whose capacity to adsorb onto cellulose differs substantially. Two isoenzymes from the same source may differ a hundred-fold in binding to cellulose. On the other hand, tightly binding endoglucanases from various origins may differ in binding by an order of magnitude. Thus, tightly binding endoglucanase from T. reesei and poorly binding endoglucanase from Asp. niger differ in their adsorption constants by a factor of 1000. Clearly, these are important considerations for the biotechnologist. For example, poorly bound cellulases in a batch reactor may be used, but not for a column procedure, since weakly bound cellulases will be eluted immediately from the column by water or buffer.

81. The ability of a cellulase to solubilize cellulose is correlated directly to its capacity to be adsorbed onto it. The adsorption process itself is identical on both amorphous and crystalline cellulose but is related to the surface characteristics of the insoluble substrate. The more firmly enzymes bind to crystalline cellulose, the higher the rate of the reaction and the greater the yield of glucose, irrespective of the reaction time. Moreover, when cellulases are weakly adsorbed the degree of hydrolysis of cellulose does not exceed 8-9% and corresponds approximately to the percentage of amorphous cellulose in the substrate. Conversely, when the adsorption of the cellulases is tight, essentially total hydrolysis of crystalline cellulose takes place, as illustrated below:

82. Most striking in this respect was the difference in hydrolysis between amorphous and crystalline cellulose. In the case of amorphous cellulose, the degree of its conversion can be increased up to 100% simply by increasing the enzyme activity or concentration in the reaction mixture. For example, even in the case of cellulase from Asp. foetidus complete hydrolysis of amorphous cellulose is observed when the enzyme concentration in the solution is sufficiently high. In contrast, this is not the case with crystalline cellulose, which is highly resistant to weakly adsorbed cellulase.

83. Thus, if a cellulase binds weakly to a cellulose the crystalline substrate is virtually resistant to hydrolysis, irrespective of the amount of enzyme used for the reaction. On the other hand, if a cellulase binds tightly the reactivity of the crystalline cellulose increases such that it can reach 30% of the reactivity of the amorphous cellulose. This property, i.e., tight adsorption, when coupled with the high catalytic activity of enzymes toward soluble cellulosics, such as CM-cellulose, is crucial for the effective degradation of cellulose. These data indicate that along with measuring the enzymatic activity of cellulases, determination of their adsorption capacity on cellulose is crucial.

4. Enzyme Sources


84. Although many fungi can degrade cellulose, metabolic products consist usually of carbon dioxide and methane. In addition, although numerous fungi degrade soluble cellulose derivatives such as carboxymethyl cellulose, only comparatively few of them can produce high levels of extracellular cellulases capable of extensively degrading insoluble cellulose to soluble sugars in vitro. These fungi include Trichoderma reesei (= Trichoderma viride), T. koningii, T. lignorum, T. longibrachiatum, Phanerochaete chrysosporium (= Sporotrichum pulverulentum, = Chrysosporum lignorum), Geotrichum candidum, Penicillium funiculosum, P. iriensis, Eupenicillium javanicum, Schizophylium commune, Polyporus adustus, Fusarium solani, F. lini, Sclerotium rolfsii, Aspergillus wentii, Asp. terreus, Asp. niger, Asp. foetidus. Thermophilic microorganisms are viewed as a source of thermostable cellulases; however, cellulases from thermophiles may not necessarily be more heat-stable than cellulases from mesophiles.

85. Until recently, most of the applied work in the area of the enzymatic conversion of cellulose to glucose utilized strain Trichoderma viride QM 9414 as a source of cellulase. However, the development of hyper producing and catabolite repression resistant strains T. reesei Rut C-30 and Rut-P37 as well as strains T. reesei VTT-D-80132 and -80133 (VTT Biotechnical Laboratory, Finland), has led to a reevaluation of these processes. There obviously is a major potential in the area of biotechnological mutagenesis of cellulases for increasing the availability and choice of novel bioengineered cellulases, designed for specific technological purposes.

Trichoderma reesei: Relevant Facts


- Mutants are effective producers of a variety of enzymes, including those necessary for the hydrolysis of cellulose and xylan.

- The amount of extracellular enzyme protein excreted is exceptionally high.

- Cellulose is not necessary for the production of cellulase.

- Cellulase is already being produced on an industrial scale in many countries (including the United States, the Soviet Union, Japan and Denmark) and is commercially available.


- The specific activity of cellulase is low.

- End products' inhibition of cellulase activity is significant.

- Cellulase is not very thermostable compared with the enzyme produced by some thermophilic microorganisms.

- Catabolite repression is a limitation in the production of cellulases.

- Lower cellobiase activities are found compared to most other cellulolytic strains.
Source: Adapted from Proceedings, International Symposium on Ethanol from Biomass, 1983.


86. The mechanism of bacterial cellulose degradation is possibly similar to that of fungi. The endoglucanases of bacteria are found to be either cell bound, extracellular, or both. Bacterial cellobiases, however, are always cell bound.

87. The genus Cellulomonas is among the best characterized cellulolytic bacteria. Others are Bacteroides, Clostridium, Pseudomonas, Ruminococcus, Sporocytophaga, Streptomyces, and Thermomonospora. A cellulolytic enzyme preparation from Cellulomonas sp. is quite resistant to end-product inhibition by glucose, cellobiose, xylose and ethanol.

88. An attractive alternative to methods commonly used for the production of ethanol is a process in which cellulase production, cellulose hydrolysis and ethanol fermentation are carried out simultaneously in a single stage. When Clostridium thermocellum (a thermophilic and an obligate anaerobic bacterium) is used for this purpose either alone or in coculture with C. thermohydrosulfuricum, pure cellulose (Solka floc SW-100) is converted directly into ethanol along with acetic acid, hydrogen, and carbon dioxide formed as by-products. The yield of ethanol is about 0.3 grams per gram of consumed cellulose. While the continuous system of fermentation as well as the batch method is feasible, neither may be economical at the moment because C. thermocellum grows very slowly (Adney et al., 1991).

Marine Organisms

89. Many marine and fresh-water organisms contain cellulolytic enzymes that degrade cellulose in cell walls of aqueous plants. Cellulolytic enzymes are found in various types of Coelenterate, Vermes, Arthropoda, Mollusca and Echinodermata. The highest content of cellulases is found in crustacea, gastropods, bivalvia, and in the giant octopus. The very high activities of cellulases in some marine organisms make them attractive in the commercial manufacture of the enzyme, for at least bench-scale work. This might be economically significant, especially when wastes after processing edible marine organisms like crabs, lobsters, octopuses, squids, snails and oysters are utilized. As shown below, the amount of cellulases per organism per ml of cultural fluid by methods described above is sometimes comparable with that of a hyper-producing cellulolytic fungus (see paragraph 72).


90. It is a difficult problem to determine the number and types of cellulolytic organisms present in a complex ecosystem such as the rumen. However, there are many bacteria which can ferment cellulose to produce a variety of products, such as ethanol, lactate, succinate, or propionate, apart from the methanogenic precursors acetate, formate, hydrogen, and carbon dioxide. Co-cultures of such organisms with hydrogen - but not acetate-utilizing methanogens - produce methane via hydrogen, accompanied by an increase in acetate production.

91. Until recently the major cellulolytic microbes identified in the rumen are anaerobic bacteria, and they have long been accepted as the main agents of cellulose digestion. However, it was discovered recently that the fermentation of cellulose by the rumen anaerobic fungi also results in the formation of ethanol, lactate, acetate, formate, CO2 and H2. In co-culture the major products are acetate, carbon dioxide and methane.

92. Ruminococcus albus is one of the most important cellulolytic bacteria found in the rumen. It coexists with the cellulolytic bacteria Ruminococcus flavefaciens and Bacteroides succinogens in proportions that vary according to diet. With good quality diets ruminococci predominate; it is assumed that B. succinogens proliferate with fodders which are difficult to digest. R. flavefaciens and B. succinogenes can ferment the most highly ordered substrates such as cotton fiber; most strains of R. albus, in contrast, can only utilize substrates in which the cellulose is present in a more disordered form.

93. Of the cellulolytic bacteria found in the rumen, only R. albus can be cultured without rumen fluid in synthetic media to yield a reasonably large amount of extracellular cellulase. Reportedly, cell-free cellulases from R. albus can degrade the soluble derivatives of cellulose, or cellulose that has been partially disordered by physical or chemical treatment. However, ordered cellulose is not hydrolyzed to any significant extent. These enzymes have not been examined in relation to their behavior as potential industrial catalysts.


94. There are only a few research projects which focus on cellulases of plant origin. It seems that they only contain endoglucanases; therefore, the capacity of plant cellulases to produce glucose from cellulose is negligible.

95. Cellulase complexes from various sources differ substantially in their composition in relation to the individual cellulolytic enzyme components present (see paragraphs 71 and 72). Consequently, the dynamics and yields of glucose formation in the course of the enzymatic hydrolysis of cellulose vary for different cellulase preparations. If a complex is deficient in cellobiase, for example, as is the case for most Trichoderma reesei preparations, cellobiose accumulates in the reaction mixture at the expense of the end-product of the hydrolysis, i.e., glucose. It was reported for some T. reesei preparations that, after a reasonable time of enzymatic hydrolysis, roughly 80% of the soluble reaction product consisted of cellobiose. It was thus recommended that the cellulase broth be supplemented with cellobiase from other sources in order to increase glucose yield and decrease cellobiose inhibition of cellulolytic enzymes. A substantial increase in yield of glucose from the hydrolysis of cellulose can therefore be realized.

96. By using T. reesei cellulase with filter paper activity of 2-5 I.U./mi with a pretreated substrate present in concentrations of 5-25% by weight, a total sugar concentration of between 4-10% is obtained in 24 hours. In this case, the glucose production level is high enough to permit direct practical fermentation of ethanol without an additional sugar concentration step.

97. It is difficult, and at present impractical, to raise the total sugar concentration in the Trichoderma hydrolysates above 6-8% since cellulases from this source are inhibited by the reactor products, i.e. glucose and cellobiose. Thus, the rate of hydrolysis by T. longibrachiatum cellulase is halved in the presence of 1.1% glucose or 0.8% cellobiose, when the amount of cellulose in the reaction mixture is small. With an increase of the substrate concentration the critical level of products also increases although it hardly exceeds 6-10%, since beyond this the inhibition of the process is substantial. This leads to a lower efficiency of the enzymatic hydrolysis (in terms of g/l/hr) as a result of a higher sugar concentration in the reaction mixture. For example, during alkali pretreated cotton stalks hydrolysis in the continuous type reactor, hydrolytic efficiency is equal to 5 g/l/hr for a steady state level of glucose of 1.8%, but drops to 2.5 g/l/hr after the flow through the reactor is reduced in order to increase the glucose concentration at the outlet to 5.5%. A further increase of glucose content in the resultant syrups leads to an additional fall in the reactor productivity. Clearly, more research is required to find a cellulase-producing strain which is less susceptible to product inhibition. Otherwise a cost evaluation must be made between glucose production and the additional sugar concentration step.

98. During the hydrolysis process, an enzyme can be lost in any of three ways. The enzyme can be adsorbed strongly onto unhydrolyzed cellulose; some of it may remain in the solution containing glucose; and some may be inactivated during the course of the hydrolysis (as a result of the heat or of the shearing effects produced when an agitating reactor is used).

99. Methods of enzyme desorption from cellulose, such as those using phosphate ion gradients to adjust the pH to neutrality, may permit greater enzyme recovery. Fresh solids can be brought into contact with the hydrolysate to adsorb some of the enzyme remaining in the solution. However, scientists at the Natick Laboratories found that this method of enzyme recovery may not be economical. Nevertheless, other investigators believe that methods must be found for each particular substrate in order to achieve a satisfactory balance of activities in the blend of fresh and recycled cellulase.

100. Another approach to enzyme recycling is the use of adsorbed cellulase in the continuous conversion of cellulose into glucose in a column reactor. This technology has been scaled up in Russia. As cellulose is digested, the released enzyme is readsorbed on excess or newly added cellulose and retains activity. Adsorption results in a marked economy of enzyme reutilization in the continuous process. The substrate can be retained in the column reactor until a high-percent conversion (96% in the case of pretreated cotton stalks hydrolysis, or nearly 100% for paper-making wastes) has been achieved. The sugars are recovered in a clear aqueous solution free of enzyme, cellulose, and any insoluble impurities. The adsorbed enzyme is sufficiently active to digest cellulose for a relatively long time; only requiring replenishment once every 1-2 months in the form of a commercially available culture fluid.

101. Economic evaluations of a number of specific scale-up technologies for producing sugars and ethanol from cellulose are provided in Vol. II. The economic assessment performed at the University of California, Berkeley, is provided as an example. In light of the discussion below as well as general considerations pertaining to economic issues, some reference costs should be identified (see following table).

102. The cost analysis is based on a plant with a manufacturing capacity of 1 x 105 gallons/year of 95% fuel-grade ethanol. As substrate, 1376 tons of cellulose waste (corn stover) containing 58% glucose equivalents is supplied each day to the plant. Using 7.0 I.U./ml of cellulase, 40% of the substrate is hydrolyzed to fermentable sugars (12-15%) which, in turn, are converted by yeasts to ethanol in 46% yield. Continuous countercurrent recovery of the remaining enzyme is accomplished by adsorption on fresh pre-treated substrate. Following filtration, spent solids from the hydrolyzer are fed into the furnace of a steam power plant to provide steam and electricity for the process.

103. As summarized below, the economic analysis based on this process indicates that the manufacturing cost is 10 cents per pound for sugar provided the cost of corn stover is zero.

104. Assuming the cost of glucose to be 10.0 cents per pound, the processing cost and fixed capital distribution for 95% ethanol production are as follows:

105. If the corn stover is obtained free of charge, the cost of alcohol per gallon is $1.80. The predominant portion (76%) of the final ethanol cost is due to the cost of glucose. If the price of corn stover is taken into consideration, the minimum glucose production cost is obtained when lower substrate concentrations are used in the reaction system, since higher yields of sugars result due to product inhibition.

106. A comparison between the lowest manufacturing cost for sugar obtained using cellulase from the strain T. reesei QM 9414 versus that from a new mutant strain, T. reesei Rut C-30, is provided below. Processing with Rut C-30 results in a cost saving of 30-40% over that with QM 9414.

107. To produce one gallon of ethanol by fermentation, 12.88 pounds of sugar are required. To synthesize one gallon of ethanol from ethylene, four pounds of ethylene are needed. Based on material costs, the price of fermentable sugars must be reduced to approximately one third the price of ethylene (which is 10 to 20 cents per pound). In other words, the cost of glucose according to the above estimations has to be reduced 3- to 5-fold if the process is to be economically viable.

108. There is another approach to the problem. Without the concurrent addition of pentoses during ethanol production, it is unlikely that ethanol production from biomass would be economically feasible, especially from substrates such as hardwoods and agricultural wastes rich in pentosanes, i.e., corn stover. Therefore, organisms that can ferment pentoses to ethanol must be found in nature or be developed via the new techniques offered by genetic engineering (see Vol. II).


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