VOLUME III
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SSF + xylose |
355. The above table also shows that the biological steps dominate the cost of production and that reduced overall costs correlate with their reduced costs. The economic importance of the poorly defined biological steps makes them a logical priority for future research (Lynd, 1990). Improvement of these procedures is likely to result from both engineering and enzymological (cellulases) and microbiological (fermentation) approaches. A process development unit is being put into place at NREL to establish a fully-integrated process operation at the pilot side.
B. CANADA
1. Iotech Process
356. The Iotech Corporation Limited, Ottawa, Ontario, a Canadian-owned company founded in 1975, has underway a multidisciplinary program to investigate the conversion of cellulosic feedstocks to ethanol, chemicals and feed, and to commercialize the process. According to available data, the work was financed with $1 million (U.S.) per year from private investors and government grants with the DOE providing $397,000.
357. The Iotech process uses steam explosion for the pretreatment of lignocellulosics (see paragraphs 66-70), producing as an essential component a high quality lignin suitable for chemical production. The lignin is a sterilized powder that can be converted to resins, plastics, detergents and petrochemicals. A one ton/day pilot plant, designed and built by Iotech, used aspen chips as feedstock and converted 90% of cellulose and 80% of hemicellulose into sugars. The glucose obtained was converted into ethanol at 95% of the theoretical yield. A yield of 68 gallons of ethanol per ton of wood was achieved. If a feasible technology for xylose fermentation is developed by Iotech, the yield is likely to approach 90 gallons per ton.
358. A detailed design of a 250 ton/day (7.5 million gallons per year) demonstration plant and a preliminary design for a 1,000 ton/day (30 million gallons per year) commercial plant has been prepared. The 1980 market price of $1.70 per gallon (1983, $2.08 per gallon) of ethanol was projected as achievable assuming lignin is burned as fuel. A 250 ton/day demonstration plant was scheduled for construction in the United States by the mid-1980s; a similar one was planned for Canada. These plans have apparently been postponed.
2. Stake Process
359. Stake Technology Limited, Ottawa, Ontario, has become well known following the disclosure of its efficient, low-cost conversion of waste lignocellulosics biomass through continuous "autohydrolysis". The Company's patented process and equipment accepts waste biomass, such as hardwood chips, sugarcane bagasse or straw, without using any chemicals or other additives. Raw materials are fed continuously via a plug-forming feeder into a steam-pressurized cylindrical vessel, which contains a helical screw conveyor, with steam pressures of 500 psi or more. Autohydrolized materials are discharged intermittently to atmospheric pressure through an orifice. The product is then subjected to an aqueous extraction, followed by an alkaline extraction, resulting in the recovery of a raw material containing three major components: cellulose, hemicellulose, and lignin. The cellulose fraction is further saccharified by acid or enzymatic hydrolysis to glucose which is fermented to ethanol. The pentose-rich hemicellulose fraction can be converted to furfural or xylitol, or may undergo a specific fermentation to ethanol. The lignin fraction has high fuel value or it may be used as a starting material for producing chemicals. Alcohol yields greater than 80% of theoretical have been achieved.
360. A joint venture was formed between Stake and Technip (France's largest engineering and construction firm) to integrate engineering, construction and marketing activities for alcohol plants outside North America. Vulcan Cincinnati had an exclusive license for the use of Stake's technology within North America.
361. According to the latest information (Yu et al., 1992), the Company's major research interests include biomass chemicals, high yield pulping and wastepaper recycling. In the area of biomass chemicals, StakeTech's Biomass Conversion Process emphasized process and product flexibility. StakeTech has been collaborating with the University of Sherbrooke to achieve effective fractionation of hardwood and softwood residues based on steam-explosion technology. It has also carried out technology development for the co-production of alcohol/furfural/lignin from various wood and agricultural residues. StakeTech is currently in the process of jointly evaluating steam-explosion technology for the conversion of waste paperboard to ethanol. In the production of alcohol, the Company has been involved in the evaluation of commercial and developmental cellulase preparations for the economically effective enzymatic hydrolysis of steam-exploded material to produce fermentable sugars (Yu et al., 1992). Two new integrated pilot plant facilities using StakeTech's continuous steam-explosion reactors were scheduled to be in operation in Trisaia, Italy and Ohio, USA, in the summer of 1992.
3. University of Toronto
362. The objective of the group at the Department of Chemical Engineering, University of Toronto, was to develop and optimize an autohydrolysis extraction process in order to use under-utilized woods and agricultural residues to produce alcohol. Steps in the basic process are: autohydrolysis; hemicellulose extraction; lignin extraction; hydrolysis, with clarification of the wood sugar solution; and fermentation and distillation. According to the research group, acid hydrolysis is preferred in most cases. Some aspects of the process have been tested at the pilot stage.
363. The Toronto University program was concerned specifically with the problem of sugar survival in acid hydrolysis. Partial acid hydrolysis was studied with as low as 20% conversion of glucose with the remainder being recycled. Using this process, glucose yields of 70 - 80% are possible. Enzymatic hydrolysis was another method under study. To overcome the problem of glucose inhibition, a partial hydrolysis-recycle process has been investigated, similar to that proposed for acid hydrolysis. It was concluded that this recycle reaction will become practical when immobilized enzymes become available.
4. Canertech Inc.
364. The interests of the Canadian Crown Corporation Canertech Inc. lie in energy conservation and in renewable energy development. Part of the latter interest was its Ethanol-From-Cellulose Program, which was a special project within the National Energy Program. Initially, the Program aimed to complete a 1 - 5 ton/day pilot plant by April 1984 and a 50 - 100 ton/day demonstration plant in 1986/1987. The estimated cost of the overall program was $21 million, of which $7 million was allocated for the pilot plant. The need for extensive process technology development apparently delayed the construction of the pilot plant.
365. A detailed comparative assessment of five alternative acid hydrolysis processes was undertaken with the aim of producing ethanol fuel that was competitive in price with gasoline. In regard to the economics, the Company found that by using aspen feedstock, assuming reasonable process development and allowing no commercial credit for lignin, three of the five processes could have led to a competitively-priced fuel in Canada by 1990-1995. Two of the processes, though in early stages of development, appeared technically promising. One of these may have an economic edge and is, therefore, likely to be selected for pilot scale development. However, proprietary complexities associated with one of these two processes has delayed action and probably eliminated one of the alternatives from further consideration.
C. RUSSIA
1. Celloglucose Process (A.N. Bach Institute of Biochemistry/Moscow State University/Research Institute of Bioengineering)
366. The National Program in Biotechnology, started at the end of 1981, called for the large-scale production of glucose in the form of glucose syrups and crystalline glucose by means of enzymatic hydrolysis of cellulosic wastes. The development of an appropriate industrial level process should have been attained by the end of the 1980s. Work along this line was coordinated by the Commission of Biotechnology of Cellulose at the USSR Academy of Sciences and by the National Council on Biotechnology.
367. The Program's initial goals were to complete two prepilot plants capable of producing 100 kilos of crystalline glucose per day by 1984 and two demonstration plants producing 1 - 5 tons/day of crystalline glucose by 1985/1986. A continuous prepilot plant was running at the A.N. Bach Institute of Biochemistry, USSR Academy of Sciences, in 1984-1988, producing glucose and ethanol from cellulosic wastes (USSR Program, that is Utilization of Sugar Syrups from Refuse). Some raw materials were used directly and included cotton stalks and sawdust; others required chemical pretreatment. Among the materials were cotton linters and other cotton ginning residues, and also under-utilized slurries from paper-making consisting of tiny cellulose particles. The source of enzymes used by the prepilot plant was solutions regularly donated by the nearest industrial biochemical unit which produced 600 m3 per year of cellulase culture in the form of a non-concentrated microbial culture fluid with a filter paper activity of 1 I.U./ml.
368. The original hydrolyzer unit was a countercurrent continuous reactor with an average glucose productivity of 2 - 5 g/liter/h. The productivity of the reactor can be increased, according to bench-scale experimental results, up to 15 g/liter/h, which corresponds to approximately 24,000 tons per year of glucose per industrial 200 m3 reactor. The cost structure shows that the three major components (raw materials with their pretreatment, enzyme production and use, and the purification and crystallization of glucose) contribute approximately equally to the cost of crystalline glucose production.
369. The high-efficiency, continuous process of enzymatic conversion of cellulose to glucose was made possible as a result of detailed investigation into cellulases' adsorption on cellulose. As a result, optimal conditions were determined for the adsorption of the enzymes on pretreated cellulosics. The adsorption was found to be so strong that researchers could use a column-type reactor for scaling-up the process. Other accomplishments include:
- The development of specific methods for determining activity (in absolute units) for all four principal components of the cellulase complexes (see paragraphs 24-28, 71-77) without their resolution;
- The development of a kinetic theory of action of a multi-enzyme cellulase system on cellulose useful for prediction of the kinetic behavior of glucose formation from cellulose while taking into account the activity of individual components of the cellulase complex;
- An increased understanding of adsorption of different cellulases (from different microbial and other sources) to cellulose and the quantitative evaluation of the availability of the enzymes for effective hydrolytic action toward cellulosics;
- Clarification of the role of adsorption in the effectiveness and conversion of amorphous crystalline cellulose to glucose;
- The development of a formula relating the quantitative relationships between the major physicochemical and structural factors of cellulose and the effectiveness of its conversion into glucose.
370. These approaches allowed researchers to properly select the microbial source of cellulases and cellulosic materials for practical conversion of cellulose into glucose. In 1988 the prepilot testing was transferred to a Privolzhsky Biochemical Plant near Moscow, where the T. viride liquid culture was produced and where a larger pilot plant was under construction. Turmoil in the USSR microbiological industry with regard to safety issues resulted in reorganization of the entire industry in general and the Biochemical Plant in particular, and the ensuing political and economical crises in the country led to a complete cessation of work at the plant in 1990-1991.
D. INDIA
1. The Indian Institute of Technology
371. At this Institute an integrated approach to the bioconversion of lignocellulosics to sugars, organic feedstock and liquid fuels was developed. Within the framework of this objective, two pretreatment processes for lignocellulosics were used. The first, a two-step process, involves dilute alkali treatment followed by steam treatment at 120°C in the presence of alkali. The second process employs treatment with butanol as a catalytic solvent (see paragraph 339) for effective delignification. It was shown that hemicellulose, the solvent, and lignin can be recovered to the extent of 95, 96, and 80%, respectively, by using steam distillation and solvent extraction.
372. Furthermore, a process of simultaneous saccharification and fermentation was also developed. Using cellulase and Pichia etchelsii at 40°C, ethanol yields of up to 32 grams per liter are obtained at 140 grams per liter of bagasse concentration. The direct conversion of cellulose into ethanol and other chemicals by C. thermocellum is also being studied. Ethanol yields of 0.20 and 0.25 g/g substrate degraded are observed using raw and mild alkali pretreated bagasse. A process to utilize the xylose component in bagasse (about 30%) via enzymatic isomerization to xylose and subsequent conversion of both glucose and xylose into ethanol is being developed. The energy requirements for producing 190 proof (95%) from a feed concentration of 4% have been estimated to be nearly half that of the distillation process.
E. FINLAND
1. Technical Research Center/Helsinki University of Technology/State Alcohol Monopoly
373. A project was undertaken to develop an industrial process for producing ethanol from wood and other cellulosic materials based on enzymatic hydrolysis. Areas of research include the development of mutants of T. reesei for the production of cellulases, cellobiases and hemicellulases; pretreatment of cellulosic materials; hydrolysis of pretreated cellulosic materials; enzyme recycling; fermentation of glucose to ethanol by yeasts or bacteria (mainly Zymomonas sp.) in a separate process or simultaneously with hydrolysis of cellulose, fermentation of pentose to ethanol by molds (Fusarium sp.); and process design. The production of cellulolytic enzymes was investigated at pilot scale and tested at an industrial fermentation plant. According to the investigators, the economic feasibility of the processes based on enzymatic hydrolysis of cellulosic materials is as yet uncertain.
F. SWEDEN
374. The work of the Swedish Forest Products Research Laboratory, Stockholm, focused on enzyme mechanisms involved in fungal cellulose and lignin degradation. For the fungi Phanerochaete sp. and T. reesei, the pattern of attack on cellulose was elucidated in some detail. In addition, a group at the Biomedical Center, University of Uppsala, focused for a long time on studying cellulases from Trichoderma strains with the general aim of obtaining information of the greatest possible value to applied research. In recent years, amino acid sequence determination of the individual cellulolytic components as well as their X-ray crystallographic studies were begun by the Uppsala group. Currently the Swedish Forest Products group in co-operation with a major Swedish company is trying to develop a process for ethanol production based on biomass. This project has only recently begun and is still at an investigative stage.
G. ITALY
1. Comitato Nazionale Energia Nucleare, Rome
375. The development of advanced biological technologies for the conversion of cellulosic wastes, including textile wastes, paper pulp, wheat straw, corn cobs, and olive shoots to ethanol was the primary objective. Pretreatment utilized sodium hydroxide at low temperatures. The saccharification of cellulosics was performed by a mixture of commercial T. viride and A. niger cellulases, the latter being immobilized in gelled alginate beads. An improvement in conventional batch fermentation was achieved by continuous fermentation with immobilized living cells packed in a column reactor; S. cerevisiae cells are immobilized with a 10% (w/v) suspension in 3% sodium alginate. Ethanol is produced continuously from a medium containing 15% (w/v) glucose with a yield of 90% of the theoretically determined maximum amount. The work has thus far been performed on a bench scale.
H. SOUTH AFRICA
376. The Council for Scientific and Industrial Research in Pretoria coordinates a national program for converting cellulose and hemicellulose from bagasse to liquid fuels and chemicals. Of the three million tons of bagasse (with cellulose contents of 43 - 45%), 1,300,000 tons per year of cellulosic fraction could be recovered. The conversion of this material to ethanol could contribute 10% of the country's liquid fuel needs. The following research organizations in South Africa are involved in the part of the bagasse program dealing with the bioconversion of cellulosics to alcohol:
- National Food Research Institute, Pretoria: Isolation and improvement of aerobic cellulolytic organisms; optimization of cellulase production on a pilot scale and the development of large-scale enzyme production process technology; a promising technology using T. reesei QM 9414 or MCG 77 is planned for transfer to a pilot scale 150-liter fermenter;
- University of Natal, Pietermaritzburg: Hydrolysis of untreated and pretreated bagasse by using T. reesei or mutant strains of locally isolated microorganisms; fermentation of the resulting glucose and pentoses to ethanol;
- Sugar Milling Research Institute, Durban: Pretreatment of bagasse to enhance enzymatic hydrolysis;
- University of Orange Free State, Bloemfontain: Enzymatic saccharification of acid extracted bagasse; fermentation of glucose and pentose to ethanol;
- University of Fort Hare, Alice: The study of the cellulase complex of T. reesei;
- University of Cape Town: Ethanol fermentation from bagasse hydrolysate.
I. JAPAN
377. Ethanol production from cellulosic materials has been established in Japan since 1980 as a national project of the Ministry of International Trade and Industry. Twelve companies belonging to the Research Association for Petroleum Alternatives Development that worked on this project have payed particular attention to the development of a practical delignifying process, physical or chemical pretreatment of cellulosics, isolation of active cellulolytic microorganisms, economical production of cellulase, and innovative ethanol separation methods as well as the selection of new cellulosic biomass and its cultivation in large quantities.
378. Japanese researchers attempted to devise a very simple procedure for enzymatic saccharification of cellulosic materials with the intent to produce sugar and ethanol at low cost with considerable savings in fuel. They claim that the production of 10 - 20% sugar solutions, comparable to extracts of sugar cane and sugar beet, is now feasible from cellulosic resources such as rice straw, grass, bagasse, sawdust, corrugated paper and newspaper, by saccharification with T. viride cellulase preparations. Cellulases are recycled during the course of hydrolysis by means of ultrafiltration or by the locally developed tannic acid-polyethylene glycol method. The simultaneous saccharification-fermentation at temperatures of 15 - 30°C in open tanks in the presence of citric or lactic acids to prevent microbial contamination, i.e. the Oriental brewing method, gives 5 - 7% ethanol solutions after a 7-day incubation. According to the researchers this is now a practical way of using sugar solutions from cellulosics. The experiments have apparently been performed on a laboratory scale.
379. On a relatively larger scale, the chemical delignification (by boiling with a 1% sodium hydroxide solution for 3 hours or by autoclaving at 120°C with 1% NaOH for one hour) of eight tons of air-dried rice straw usually yields four tons of holocellulose. After enzymatic saccharification of the holocellulose, one ton of Candida utilis yeasts or a corresponding amount of ethanol is obtained while providing 50% saccharification. On the other hand, the nonhydrolyzable residue provides two tons of a mixture consisting mainly of lignin. This mixture, when further mixed with two tons of the residual root and stump of rice plant, is useful as new compost.
380. For the saccharification of delignified Carpinus shavings or delignified sawdust the economic substrate concentration was estimated to be 9 - 10%. After four days of incubation with 1 - 3% T. viride cellulase solution at 45°C the sugar concentration was 8.6%, and the degree of saccharification 77%. In another series of experiments, 25% shredded tissue paper, delignified rice straw, or delignified bagasse incubated with 1 - 3% T. viride cellulases at 45°C for four to eight days yielded 13 - 22% sugar solutions with a 40-66% degree of saccharification. These experiments have been performed on a laboratory scale. Whether scale-up technologies have been developed and assessed economically is not clear from the available sources. lt seems, however, that the high enzyme concentrations and the long incubation time would not result in an economically feasible process.
J. POTENTIAL FOR BIOTECHNOLOGY OF CELLULOSE IN DEVELOPING COUNTRIES
381. The shortage of food and fuel has become increasingly serious in many developing countries as a result of increased population density. From this point of view the enzymatic saccharification of cellulosic materials, primarily renewable agricultural residues, presents a possible partial solution to this problem, especially given the extremely large quantity of cellulosic resources being produced each year in subtropical and tropical developing countries.
382. In those developing countries where the technique of microbial technology is not fully established, enzymatic hydrolysis of cellulosic materials and fermentation of the resulting sugars into ethanol should be performed on a relatively small scale with facilities that are already available.
383. In some cases, existing branches of industry typical for developing countries can produce cellulosic material as wastes that are ready for bioconversion and could, when utilized, be beneficial for the industry. For example, in the manufacture of tobacco substantial amounts of waste filter material and cigarette paper are generated. Such waste materials generally find no use in cigarette manufacture, but instead are typically disposed of by burning after separation from tobacco components. On the other hand, sugars, particularly glucose, are employed in a number of tobacco treatment processes. Sugars are used as a carbon source during tobacco fermentations and as tobacco casing materials or in the production of tobacco flavorants.
384. Of the numerous cellulosic residues, rice straw is one of the most promising renewable resources in many developing countries. Cotton and sugar cane residues are also promising materials since they have been studied extensively and the findings have been published widely. With the data accumulated in the literature it is easier to select an appropriate approach to processing the material. The biological pretreatment that uses lignin utilizing microorganisms (paragraphs 60-62) should then be considered a highly promising means for pretreatment of lignocellulosics, particularly in developing countries. The potential of this approach is far from exhausted even in industrial countries which can utilize highly instrumented, capital-intensive equipment for the pretreatment of lignocellulosics. Of course new and more efficient lignin-utilizing microorganisms lacking cellulose should be discovered, thereby increasing the efficiency of the biotechnological processing of lignocellulose.
385. Clearly, commercial cellulase preparations, such as T. reesei cellulases, are too expensive for developing countries to purchase on large scale for the saccharification of their cellulosic resources. The cellulases produced by solid culture using, for example, wheat bran or rice straw, as well as solid culture methods rather than submerged cultivations are recommended for fungal cellulase production in developing countries, at least during the initial period of transition to modern biotechnology. Such cultures have been successfully developed in Japan and the Soviet Union and are suitable in terms of both enzymatic activity and costs.
386. It appears likely that the enzymatic saccharification of cellulose to glucose followed by its refinement or conversion to edible sugars (e.g., fructose) or its fermentation into liquid fuels, or its conversion to fodder, will gradually become a new rural industry in developing countries. From a practical standpoint, uncomplicated equipment together with manual labor to provide employment to a large number of rural people should be used. Hopefully, renewable cellulosic resources will be used as industrial raw material for producing sugar, yeast, and liquid fuel in the near future. Importantly, education may well prove to be the critical determinant for the future developments foreseen in this treatise.
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