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Biomassand BioenergyVol. 10, Nos S/6, pp. 367-375,1996 Copyright 0 1996ElsevierScience Ltd Printed in Great Britain. All rights reserved 0961-9534(95)00114-X 0961-9534/96 $15.00+ 0.00




Department of Industrial Microbiology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland
(Received 15 September 1994; accepted 30 September 1995)

Abstract-Current trends in production of fuel ethanol from lignocellulosic materials are reviewed. Particular emphasis has been laid on the microbial synthesis of cellulases, enzymatic hydrolysis, pretreatment of lignocellulosics, and their simultaneous saccharification and fermentation to ethyl alcohol. Some pilot-scale plants producing alcohol from biomass are also presented. Copyright 0 1996 Elsevier Science Ltd. KeywordsBiomass conversion; lignocellulose pretreatment; cellulolysis; fermentation; fuel ethanol.



The progressive expansion of civilization and the related ever-developing science and technology present us with many new problems. It is primarily a question of energy, food and the pollution of our natural environment. The shortage of liquid fuels is more than predictable. Also, for political reasons, many countries have taken up the search for alternative resources of energy, like bituminous shales and sands, solar energy, sea-tide power, geothermal and nuclear energy, wind power, coal conversion, and biomass. Plant biomass seems to offer greater possibilities in this aspect. This is due to its abundance and renewability as well as the possibility of producing various chemical by-products, fuels, fodder and food products from this source. Biomass includes any kind of plant matterstarting with wood machining wastes and ending with selected crops rich in organic compounds, such as sugars or oils. The global production of plant biomass-of which over 90% is lignocellulose-amounts to about 200 x lo9 t per year, where about 8 to 20 x lo9 t of the primary biomass remains potentially accessible. However, the effective utilization of the lignocellulosic feedstock is not always practical because of its seasonal availability, scattered stations, and the high costs of transportation and storage of such large amounts of organic material. Lignocellulosic biomass can be converted into useful products both via physicochemical or

biological processing (Fig. 1). So far, the most commonly applied technologies are evidently chemical, which are unfortunately quite expensive and produce additional waste. Therefore, it to consider biological seems worthwhile methods for utilizing lignocellulosic wastes, which rely on microbial or enzymatic conversion. Intelligent ways of utilizing biological processes, which occur very slowly in nature, must be found. Accelerating these processes is critical to render them economically viable. Despite many studies carried out in this field, satisfactory results which would make these methods more effective on a larger scale have not been achieved so far.

Biological conversion of lignocellulosic biomass into ethyl alcohol is one of the possibilities to utilize its energy. Biobased alcohol production is the subject of intensive research because ethanol can be either blended with gasoline as a fuel extender and octane enhancing agent, or used as a neat fuel in internal combustion engines. A 1:9 or 2:8 mixture of ethanol and gasoline, commonly known as gasohol, is successfully applied today in Brazil and the U.S.A. The results of research, as well as practical applications, have shown that the addition of ethanol to gasoline enables a 4-fold reduction of tetraethyl lead supplement, or even its elimination, without risk of affecting



engine longevity. This factor is of primary significance for environment protection. 3.4 Biomass-based ethanol production includes the following stages: feedstock acquisition and pre-processing, biosynthesis of cellulases, enzymatic hydrolysis and fermentation, and alcohol recovery. The production of cellulolytic enzymes appears to be the most expensive process, ranging up to 43.7% of the total costs of production.s Feedstock acquisition also seems to appear as a limiting factor. To reduce the cellulase production costs, intensive research is being carried out on the enhancement cellulases synthesis by micro-organisms using various genetic techniques, such as mutagenization6 protoplast fusion, or genetic engineering.8 Optimizing growth conditions, as well as using cellulase inducers . lo is a major factor that should not be neglected in this process. An intensive search is being made for cheap sources

of carbon for the biosynthesis of these enzymes by replacing highly purified cellulose substrates with pretreated lignocellulosic waste. I.I* Among the various means of reducing the costs of cellulases production, one should include attempts to recycle and recover them from cellulose hydrolyzate, immobilization, 14. I5 or the use of more effective methods of cultivation of micro-organisms for the synthesis of these enzymes, such as fed-batch cultureP and solid state fermentation. It was observed that good results in hydrolysis of lignocellulosic wastes can be achieved when using cellulases produced by mixed cultures of micro-organisms. * Many micro-organisms capable of biosynthesis of cellulolytic enzymes are known, but only a few of them are of any use in large scale production. This is due to the fact that it is necessary to synthesize a full complex of enzymes needed for biodegradation of native





Fig. 1. Biomass utilization pathways

Technology for conversion of lignocellulosic biomass to ethanol


cellulose. The former consists of various extracellular exo-B 1,4-D-glucanases (EC, endo-B-1,4-D-glucanases (EC and g-1,4-D-glucosidases (EC, the combined effect of which leads to cellulose hydrolysis to glucose. 9 At present the best cellulase producers are the mutants of wild strain of Trichoderma reesei QM6a isolated by Reese in 1950, and since then, they have constantly been improved by multistage mutagenization in many countries. And thus T. reesei QM 9414, MCG 77, Rutger C-30 strains were isolated in the USA; T. reesei VTT-D-79124 in Finland; T. reesei CL-847 in France; T. reesei SVG 17 in Yugoslavia and in Czechoslovakia a series of MHC was obtained. These mutants differ in their activity of the particular components in the cellulase complex, and in some cases, they also differ in the type of carbon source required as a substrate for their growth. Cellulolytic preparations are obtained from post-culture broths following concentration by ultrafiltration or vacuum evaporations, precipitation with ammonium sulfate or ethanol, or by freeze-drying. Crude preparations are used for enzymatic hydrolysis of cellulose wastes. The enzymatic hydrolysis yield is governed by many factors, which include: type of substrate pretreatment, inhibition of enzymatic activity by the end-products of the biodegradation, thermostability of enzymes, their concentration and adsorption on the substrate, duration of the hydrolysis, medium pH, substrate concentration in the medium and rate of its agitation. Therefore it is necessary to optimize the hydrolysis conditions to achieve satisfactory run of the whole saccharification processes. Cellulose hydrolysis is due to synergistic action of endo- and exoglucanases, and D-glucosidases. As a result of the action of the first two groups of enzymes on cellulose, cellobiose and glucose are obtained, and as their concentration in the reaction medium gradually increases, the activities of the respective cellulases are inhibited because of endproduct inhibition, resulting in a final decrease in rate and yield of the saccharification process. Cellobiose has also been found to be a stronger cellulase inhibitor than glucose.23 The high activity of D-glucosidase in the cellulase complex (the enzyme degrading cellobiose to glucose) removes cellobiose from the reaction mixture which increases the efficiency of the enzymatic hydrolysis of cellulose. The inhibition of hydrolysis can also be prevented by performing

the cellulose saccharification with a simultaneous fermentation (SSF) of the produced glucose to ethanol using yeast or bacteria.14 The present market offers many cellulase preparations (including those obtained from T. reesei) containing low levels of f3-glucosidase,2s which leads to an increased accumulation of cellobiose in the enzymatic hydrolyzates of the cellulose. The inability of industrial glucosefermenting yeasts to ferment cellobiose26 results in incomplete conversion of cellulose hydrolyzate to ethanol, significantly diminishing its final yield. These drawbacks may be overcome by selection of T. reesei mutants with higher B-glucosidase activity,27 supplementation of the cellulase complex with a D-glucosidase from other sources (often an Aspergiflus SP.)*~ or selection of yeast mutants that are capable of fermenting glucose and cellobiose simultaneously.29 The main difficulty in solving the problem of enzymatic depolymerization of the lignocellulosic materials is used to their complexity. Cellulose fibrils are embedded in an amorphous matrix of lignin and hemicelluloses which render the plant tissue resistant to micro-organisms and their enzymes. Therefore, all large-scale processes designed for bioconversion of cellulosic and lignocellulosic feedstock require a pretreatment stage of the native feedstock. The objective of such a procedure is to increase the specific surface area of the substrate, reduce crystallinity of the cellulose, and breakdown the lignocellulosic complex.3 Although many methods of pretreatment of the lignocellulosic biomass have been developed (Table l), no universal, technically and financially viable, method is known.32-39 Alkaline maceration, autohydrolysis, and acid-catalyzed steam explosion methods appear economically feasible and have attracted many investigators. When treated with dilute sodium hydroxide lignocellulosic feedstocks undergo partial delignification combined with swelling of the cellulose, a decrease in the degree of polymerization and crystallinity, and an increase in enzyme accessibility. A more effective modification of this method is ethanol-alkali pulping of the substrate at 170C. At high temperature, the organic solvents reduce the surface tension of the cooking liquors, thus promoting the diffusion of the lignin breakdown products from the substrate into the liquor, enabling the base to get to the carbohydrates and lignin found in the substrate. Sodium


J. SZCZODRAK andJ. FIEDUREK Table 1. Pretreatment methods of lignoceilulosics for enzymatic hydrolysis

Method Physical

Operations (factors) causing changes in the substrate structure Milling and grinding (ball, vibro energy, two-roll, pressure, hammer); Irradiation (electron beam, y-rays, microwaves); High temperature (pyrolysis, steam explosion)

Kind of changes Increase in specific surface area and size of pores, decrease of the degrees of polymerization of cellulose and its crystallinity, hydrolysis of hemicelluloses, partial depolymerization of lignin Delignification, decrease of the degree of polymerization and crystallinity of cellulose associated with its swelling, porosity growth Delignification and reduction in degree of polymerization of cellulose and hemicellulose Degradation of hemicelluloses, delignification, increase of the specific surface area and size of pores

References 32-34


Alkalis, acids, gases, oxidizers, reducers, organic solvents White-rot fungi (Pleurorus, Pycnoporus,
Ischnoderma, Phlebia, etc.)

11,35, 36



Alkali-pulping associated with steam explosion, grinding followed by alkaline or acid treatment


hydroxide, beside converting lignin to its more soluble sodium derivative, removes low molecular weight carbohydrate components from the cell wall structure, thereby causing an enlargement of the pores in the lignocellulosic substrate and an improvement in the accessibility to reagents.40 Autohydrolysis consists in heating the cellulosic biomass with water or steam at temperatures of 185-210C for 5-20 min, or at temperatures of 240-260C for 20 s followed by the explosive expansion of the mixture.4 This method enables a separation of the substrate into a water-soluble fraction containing hemicellulose hydrolyzate, a fraction containing lignin, and a cellulose fraction containing 70-90% cellulose. As a result of such hydrothermal pretreatment, the hemicellulose undergoes a deep hydrolysis and lignin is partially depolymerized. Acetic acid produced during the heating of the substrate proved to be a positive catalyst of these reactions.4 This method has also been shown not to alter the crystalline structure of the cellulose. Another approach for pretreatment of biomass is dilute acid process, which at present appears to be close to near-term commercial application, In this process, about 0.5% sulfuric acid is added to the feedstock, and the mixture is heated to about 140-160C for 5-20 min. Most of the hemicellulose is hydrolyzed leaving a porous structure of primarily cellulose and lignin that is more accessible to enzymatic action.4 Minimization of by-product formation that could impede the subsequent fermentation steps is desirable. Based on the information given in the

relevant literature, one can hopefully expect that the problem of enzymatic hydrolysis of plant feedstock will be partially solved in the future by applying lignin biosynthesis inhibitors in the farming stage (e.g. grass crops). One type of such an inhibitor is a-aminooxy-g-phenylpropionic acid which inhibits the key enzyme in the phenylalanine-cinnamic acid pathway for the biosynthesis of lignin.44 Research on lignin-free grass culture has been under way on greenhouse scale for some time at Jeallot s Hill in Berkshire, U.K. As such plants are stripped of their lignin barrier and contain mainly cellulose in their biomass, they should display better digestibility.45 Progress in the utilization of cellulosic feedstock for ethanol production can be readily evaluated by the evolution of the production cost with time. Fifteen years ago, this cost amounted to US$1.06 1~ (USS4.0 per gallon, but in recent years this cost has been lowered to US$O.47 1-I (US$1.8 per gallon). By putting into effect the results of recent scientific research, the cost of such a production can go below USSO. 1-I (US$l.O per gallon). When the cellulolytic enzymes are recirculated in the hydrolysis process, combined with a more complete utilization of the by-products, the costs of ethanol production can run even below US$O.21 I- (US30.8 per gallon), a level that is competitive when any oil prices exceed US$25 per barrel. For comparison, the calculated selling price of one liter of alcohol from chemical synthesis (where ethylene is the feedstock) was USZGO.44 (USS1.65 per gallon) and that of corn fermentation was US$O.50 l- (US$1.88 per gallon). 46.47

Technology for conversion of lignocellulosic biomass to ethanol 3. DEVELOPED POTENTIAL BIOSYSTEMS FOR ETHANOL PRODUCTION


Cellulose-to-ethanol biotransformation can be conducted either by direct microbial conversion (DMC) of this polysaccharide, or by its successive or simultaneous saccharification and fermentation (SSF). The direct conversion of cellulose to ethanol appears to be an economically viable method, as all stages necessary for ethanol production (cellulase biosynthesis, enzymatic hydrolysis, and fermentation) are interconnected. DMC is based on the utilization of mono- or co-cultures of micro-organisms which ferment cellulose to ethanol. This type of activity is shown by various anaerobic thermophilic bacteria, such as Clostridium thermocellum ,48 as well as by some filamentous fungi, including Monilia SP.,~~ Neurospora crassa, and Paecilomyces SP.~ Studies on the fermentation process utilizing these micro-organisms have shown this process to be very slow (3-12 days) with a poor yield (0.8-60 g 1-l of ethanol), which most probably is due to the low resistance of micro-organisms to higher concentrations of ethyl alcohol. Another disadvantage of this process (particularly in the case of bacterial fermentation) is the production of various by-products, primarily acetic and lactic acids. 5 .53 The direct conversion of modified wheat straw to ethanol can be also conducted by utilizing co-cultures of C. thermocekm strains and anaerobic bacteria fermenting pentoses, and C. namely C. thermosaccharolyticum
thermohydrosulphuricum .54

To obtain a higher yield from the lignocellulosic substrate in the ethanol production process by DMC intensive research for new thermophilic strains are under way. Micro-organisms are sought which do not produce organic acids and are more resistant to higher ethanol concentrations5 The approach which offers the highest flexibility in terms of operating conditions followed by control is saccharification cellulose fermentation, in which all stages (enzyme biosynthesis, hydrolysis, and fermentation) are separated. Unfortunately, the endenzymatic hydrolysis products of the (glucose and cellobiose) inhibit the enzymes in the cellulase complex, which limits the rate of saccharification and final concentration of the sugars and leads to a low yield of ethanol.3, SSF (simultaneous saccharification and fer-

mentation), combines enzymatic hydrolysis of cellulose with simultaneous fermentation of the sugars obtained to ethanol. In comparison to the process where these two stages are sequential, the SSF method enables attainment of higher (up to 40%) yields of ethanol by removing end-product inhibition, as well as by eliminating the need for separate reactors for saccharification and fermentation. Other advantages of this approach are a shorter fermentation time and a reduced risk of contamination with external microflora, due to the high temperature of the process, presence of ethanol in the reaction medium, and anaerobic conditions.3,47 In spite of the obvious advantages presented by the SSF, it has, however, some drawbacks. These lie mainly in different temperature optima for hydrolysis (45-50C) and fermentation (28-35C). Besides, ethanol itself and some toxic substances arising from pretreatment of the lignocellulose inhibit the action of fermenting micro-organisms, as well as the cellulase activity. 56. 57 Achieving micro-organismenzyme compatibility becomes a major issue in the SSF, since some compounds (e.g. proteolytic enzymes) that are released on cell lysis or are secreted by a particular strain can degrade the cellulases; alternately, components in the enzyme preparation can reduce microbial viability leading to cell lysis. To overcome the problems related to SSF, many species of yeasts, as well as the bacterium Zymomonas mobilis, have been tested with 59 cellulases produced by T. reesei mutants.24, 58. In order to make the SSF process more effective, it has been found necessary to search for thermostable strains capable of producing substantial amounts of ethyl alcohol at temperatures optimal for saccharification and suitably resistant to ethanol.60 Roychoudhury et aI.6 have developed a notable way of eliminating the negative effects which excessive concentrations of ethanol have on yeast activity and cellulases within the SSF system. They used a vacuum cycling reactor where the concentration of ethanol was kept at a relatively low level by its removal in the flash chamber.

In spite of extensive research on fuel ethanol production from biomass, not a single plant capable of converting cellulosic feedstock to ethanol, via biological processing on the






industrial scale, has been put into operation anywhere in the world, although some pilot scale plants have been commissioned. The first experimental plant to produce ethanol from cellulosic feedstock based on the SSF method was put into service in Pittsburg, PA in 1976 where one ton of material (solid municipal waste) was treated daily. In the early 1980s Gulf Oil carried out a precise techno-economic evaluation, and designed a plant with a daily yield of 567,750 1 (150,000 US gallons) of ethanol produced from 2000 t of cellulose waste. This process yielded an end product sale price of US$O.25 1-l (US$O.95 per gallon) estimated in 1983. The costs involved with this project were estimated to be US$l12m (data from 1981) however the project has not been started so far. 46. 47. 6 A cooperative agreement between NREL (National Renewable Energy Laboratory) in Golden, CO, and New Energy Company, IN, promises to aid both the corn ethanol and the biomass ethanol industry. New Energy operates a production facility to produce fuel-grade ethanol from corn where the NREL-developed technology is used to convert the fiber (which is mainly cellulose) in corn to ethanol. New Energy will design, construct, and operate a pilot plant at its production facility to test the process. If successful, ethanol production is expected to increase by at least 15% for the same quantity of corn and at the same time demonstrate cellulose conversion technology on a pilot scale. In another pathway for production of alcohol fuels, an alliance between NREL and AMOCO Corporation has been made to convert municipal solid waste (MSW) to ethanol. The user facility at NREL is a l-ton per day process development unit (PDU). If the process is promising, AMOCO will build a 40-100 ton per day engineering development unit to demonstrate the process on a large scale. 63.64 Currently, the Tennessee Valley Authority (TVA) is developing technology for dilute acid conversion of MSW into ethanol.65 In Canada a 1 tonne per day pilot plant has been constructed at the cost of 4.2 million dollars in 1984. The wood feedstock was pretreated by a steam explosion process. The Rut C30 Trichoderma reesei strain has been used to produce cellulases.66 In 1983, a pilot plant station was started in Japan, within the RAPAD program (Research Association for Petroleum Alternatives Devel-

opment), to treat 720 kg of material daily. Enzymes were produced in fermenters of about 4000 1, using T. reesei strains. Partial recovery of enzymes was conducted by ultrafiltration. Ethanol production capacity was 150200 1 day- . New strain Kluyveromyces cellobiovorus KY 5199 capable of fermenting xylose as well as cellobiose and glucose to ethanol was obtained.67-69 One larger plant capable of processing up to 4 t h - of material, applying the continuous method, was commissioned in 1988 in Soustons, France. Cellulosic feedstock is processed by autohydrolysis-steam explosion. Enzyme production is conducted in a 30 m3 fermenter. The T. reesei (CL-847 strain) is cultured on medium containing lactose. The strain productivity . According to amounts up to 25 1 FPU 1- h the authors who developed this new method of cellulase complex production, the running cost of the enzymes required to produce 1 kg of sugar is approximately 3e. The enzymatic hydrolysis is conducted in a 25 m3 reactor. 3800 kg of sugar can be obtained from a single cycle. The ethanol fermentation is in a 50 m3 tank. Overall yields of 160-190 kg of ethanol were obtained from 1000 kg of poplar wood (100% dry matter). It is expected to produce ethanol, and to carry out acetone-butanol fermentation. . I It is foreseen that within the next 5-10 years renewable, alternative transportation fuels from biomass and wastes will be competitive with fuels derived from petroleum at about USSO. 1~ . 5.

None of the above-mentioned systems of lignocellulose-to-ethanol bioconversion meets the requirements of industry due to their low rates of cellulose hydrolysis, which is the stage limiting the rate of alcohol production. Another problem arises from the fact that most micro-organisms used for converting cellulosic feedstock cannot utilize xylose, a hemicellulose hydrolysis product. Some significant progress can be noted in this field, however. Mutants with higher cellulolytic activity and resistance to ethanol, such as Clostridia, have been isolated. Moreover, these mutants do not produce by-products.53 Several options have been investigated for the process of conversion of xylose into ethanol. One of the methods uses yeasts, such as Pichia stipitis,


for conversion

of lignocellulosic


to ethanol


Candida shehatae and Pachysolen tannophilus, to convert xylose directly into ethanol in micro-aerophilic conditions. However, the control of the oxygen at a proper level to achieve high yields and productivity is quite difficult. Moreover, these strains often cannot tolerate as high ethanol concentrations as conventional glucose fermenting strains. Another strategy for obtaining ethanol from xylose is to produce an enzyme known as xylose isomerase that will convert xylose into a keto-isomer called xylulose, which undergoes ethanolic fermentation by yeasts. Although this process has the advantage of employing anaerobic yeast on a large scale, the necessity of adding xylose isomerase enzyme to the broth, combined with different pH optima for the enzyme and yeast, complicates the technology.73.74 Another option is to incorporate the xylose isomerase gene into the yeast genome to enable the modified organism to ferment hexoses and pentoses.75 A more recent development has been the genetic engineering of several bacteria including Escherichia coli, Klebsiella oxytoca and Erwinia sp. to allow them to directly ferment xylose into ethanol. Key genes from the glucose-fermenting bacterium Zymomonas mobilis have been incorporated into these organisms to allow ethanol production. The result is a single organism that can ferment xylose into ethanol. Similarly, the ethanol-producing bacterium Z. mobilis was metabolically engineered to broaden its range of fermentable substrates to include the pentose sugar xylose. Two operons encoding xylose assimilation and pentose phosphate pathway enzymes were constructed and transformed into Z. mobilis. The recombinant efficiently fermented both glucose and xylose, which is essential for economical conversion of lignocellulosic biomass to ethano1.778 Currently, bacteria modified by this approach must operate at neutral pH where control of invasion by other organisms is more difficult then at the more acidic pH levels typical of most yeasts. To reduce the costs of enzymatic hydrolysis, T. reesei mutants, constitutive and sugar derepressed, have been selected for synthesizing cellulases on sugar (e.g. lactose) solutions.9.79 Moreover, genetic engineering made it possible to transfer cellulase genes from Trichoderma to Saccharomyces cerevisiae. 80.8 However, the cellulases were produced at a concentration too low to be useful. There is a group of micro-organisms (Clostridium, Cellulomonas,

Trichoderma , Penicillium , Neurospora , Fusar ium, Aspergillus, etc.) showing a high cellu-

lolytic and hemicellulolytic activity, which are also highly capable of fermenting monosaccharides to ethanol. It may be possible, within this group of micro-organisms, to produce superstrains via genetic engineering capable of hydrolyzing cellulose and xylan along with fermentation of glucose and xylose to ethanol. A review of current world-wide effort on biomass-based fuel ethanol production has shown that in spite of numerous difficulties encountered, it remains a vital biotechnological problem. A positive solution to this challenging issue could bring economic advantage not only for the fuel and power industry, but also benefit to environmental protection. As world deposits of crude oil shrink, causing its price to rise, the interest in technologies enabling the processing of biomass into liquid fuel will grow stronger, particularly in those countries which do not have their own crude oil deposits. This is the stimulus that keeps many laboratories working on improving the technology of lignocelluloseto-ethanol conversion, whether it be on the level of basic research or pilot plant tests.
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