1993 CRB Lignocellulose Biotechnology Current and Future Prospects Dania

Critical Reviews in Biotechnology, 13(2):15 1- 172 (1 993)
Lignocellulose Biotechnology: Current and Future Prospects

Ramesh Chander Kuhad
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by UNAM on 11/22/11 For personal use only.
Ajay Singh
Biochemical Engineering Research Centre, I.I.T. Hauz Khas, New Delhi-110016, India
ABSTRACT: Lignocellulose is the most abundant biomass available on Earth. It has attracted considerable attention as an alternate feed stock and energy resource because of the large quantities available and its renewable nature. The potential uses of lignocelluloses are in pulp and paper industries, production of fuel alcohol and chemicals, protein for food, and feed using biotechnological means. The current industrial activity of lignocellulosic biomass fermentation is limited mainly because of the difficulty in economic bioconversion of these materials to value-added products. Considerable improvement in many processes related to lignocellulose biotechnology appeared during the last decade. Current uses of lignocellulosic biomass, process constraints, and areas of future research are discussed here. KEY WORDS: lignocellulose, biotechnology, microbes, bioconversion, chemical.
1. INTRODUCTION
Lignoceliuloses are the most abundant organic compounds in the biosphere, accounting for approximately 50% of the biomass in the world, which has been estimated with an annual production 10 to 50 X 10 tons.,?They occur in agricultural, forestry, and fruit and vegetable processing wastes. Biomass in the form of wastes accumulates every year in large quantities, causing a deterioration of the environment and loss of potentially valuable resources. A significant contribution could be made to the overall problem of biomass recycling and conservation. Biomass in the form of cellulose, hemicellulose, and lignin provides a means of collecting and storing solar energy and hence represents an important energy and material resource. It has been suggested that the waste lignin derived from the pulp and paper industry is of the order of 30 to 50 X lo6 tons per year.
0738-855 1/93/$.50 0 1993 by CRC Press, Inc.
The rising cost of fuel energy, need to control environmental pollution, and the changing social, potential, and cultural value of the world have challenged all countries to efficiently use renewable resources and also to put up increasing efforts to recycle traditional organic wastes. These include microbial delignification of wood pulp for preparation of cellulosic polymers, improved methanogenesis for biogas production, solid-state fermentation process, renewable energy from biomass, production of edible mushrooms, and chemical waste treatment processes for industrial and agricultural effluent including toxic chemical pollutants. An immense potential ha! been shown to create a technological breakthrough in bioproccesses for increasing the production of several useful products. Some of them include chemicals such as glucose, polyphenols, organic acids; fuels such as methane and ethanol; and solvents like acetone and butanol; and single cell protein (SCP). Figure 1 shows an integrated approach
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AGRICULTURAL WASTES
F#RESTRY
WASTES
LIGNIN
REWCING EQUIVALENTS
SOLVENT
ORGANI C ACIDS
CELLULAR ENZYMES
7
FEED
FIGURE 1. Integrated approach for bioconversion of lignocellulosic wastes into valueadded products.
for solvent fermentation and biomass production in feed and biocatalyst applications. The abundance of agricultural lignocellulosic residues from major foods such as grain crops and the forestry sector represent major prospects and have the potential of microbiological conversion to liquid fuel - e t h a n ~ l .This ~ . ~ can be achieved through the efficient utilization of both the cellulosic and hemicellulosic fraction of biomass. Much research has been performed on the saccharification of cellulosics, using Trichodermu cellulase enzyme complex."x Biotech152
nological processes have been utilized to solve the scrupulous task of lignocellulose utilization to start a new era of interventive industrial enzymatic conversion to fermentable sugar.9-' ' Enzymatic hydrolysis of cellulose, although not yet attractive commercially, is expected to be feasible in the long run such as in starch hydrolysis process. Improved processes are expected because of the recent advances made toward the understanding of cellulolytic enzymes and on the availability of powerful tools of molecular biochemistry for construction of novel organisms
capable of direct bioconversion. However, the scaling-up process of enzyme production and recovery, cellulose hydrolysis, and the production of value-added chemicals require further improvement to overcome economic constraint^.^ In this article, current uses of lignocellulosic biomass, process constraints, and areas of future research are discussed.
II. SOURCES OF LIGNOCELLULOSIC WASTES

Net productivity of dry biomass due to photosynthesis by plants on Earth is estimated to be 155 billion tons per year," of which about two thirds of the biomass is produced on land and about one third is found in the oceans. Most terrestrial plant materials occur in forests (65%), while more than 15% is generated in cultivated grasslands. About 1.25% of the total land biomass is used for human food, with about 9% lost during processing operations, and the rest accounting for the magnitude of availability of lignocellulosic wastes. l 2 Nonwood plant fibers represent a major resource of fibers for raw materials in many countries, particularly in the developing ones. Agricultural residues such as sugarcane bagasse, cereal, and pulse straw naturally growing plants like bamboos, papyrus, other grasses, and
plantation crops like hemp jute, ramie flax, cotton linters, etc. are of major importance. Type and availability of lignocellulosic wastes in any particular geographic region depend on the uses of climatic and environmental factors and culture type and nature of the local technology (Table 1). Thus, while wheat straw and maize byproducts are more prevalent in Europe and North America, rice straw is abundant in Asia. Large amounts of cellulosic agricultural wastes are produced each year in the U.S. and U.K. However, a considerable amount is disposed of by burning. Similarly, in many Asian countries, a large fraction of the available straw is used for burning, mulching, thatching for roofs, and cattle feed, while around half of the agricultural residue remains unutilized. According to Food and Agricultural Organi~ation'~ estimates, about 2946 million tons of cereal straws are produced worldwide. A number of pulse crops are cultivated in various parts of the world, and for every ton of pulse harvested, two to three times of the inedible plant residue is available. Similarly, after the harvesting of oil seeds, significant quantities of plant residues remain unutilized. Annually about 1.4 million tons of oil seed crop residues are available. The fibrous residue remaining after extracting juice from the sugarcane stalk is referred to as bagasse, which contains about 35% cellulose, 30% hemicellulose, and 30% lignin.
TABLE 1 Estimated Global Production of Lignocellulosic Wastes

Wastes (million tons) Pulse Oilseed Plantation crop crop crop
9 51 1 10 49 37 16 44 2 166 11 61 2 8 21 10 14 19 34 174 12 1 84 147 88 15 NAb 548
Continent/country Africa Asia Australia Europe Central America South America India
Cereal crop
165 1135 35 550 500 153 240 440 60 2946
us.
Canada World
a
<I
142
FA0 Production Year Book, 1989. NA = Not available.
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Citronela bagasse and lemon grass are some examples of industrial residues obtained after recovery of essential oil. Shive is a woody byproduct of flax, having little value. Distillery grape stalks and citrus peels, particularly orange juice, and industry wastes are produced in large quantities and cause serious disposal problems. Apple pomace i s also inexpensive and plentiful and can be exploited as substrate for the production of value-added chemicals, mushroom cultivation, and fuel solvents. Domestic solid wastes and effluent from paper and pulp industries constitute an important cellulosic resource. The most important feature of such wastes is that they have zero to negative cost at the site of origin; however, they are expensive to collect and too bulky to store. Thus, it is evident that on global basis the magnitude of available lignocellulosic waste is fairly high. With the improved bioconversion technology today, it would be possible to utilize these wastes economically.
111. MOLECULAR ARCHITECTURE OF LIGNOCELLULOSE
-cellulose,
It comprises three major group of polymers hemicellulose, and lignin (Table 2). However, the exact formulation is not known because it varies from source to source in terms of both its chemical constituents and their relative ratios. A typical wood cell is a multilayered structure consisting of external microfibril and a sec-
ondary wall containing three sublayers - S 1, S2, and S3. The concentration of cellulose is highest in sublayer S2 and diminishes toward the middle lamella. The concentration of hemicelhose and lignin is at its maximum in the middle lamella and decreases toward the lumen. Wood cells may contain up to 90% fiber, whereas only about 35 to 40% of the cells in straw are fibers. Within a given microfibril, lignin and hemicellulose may penetrate the space between cellulose molecules in the amorphous region. Cellulose, the most abundant cell wall component forming a supporting matrix for the cell membrane, is a regular linear hornopolymcr of repeating u-glucose units in (p 1-4) linkage. Although cellulose molecule has a high affinity for water, it is completely insoluble in it. In cellulose, the P-glucan chain always has extended ribbon-like conformations with two pyranose residues repeating in approximately 1.03 nm. The native cellulose crystallizes immediately after its biosynthesis. Industrial processing of cellulose frequently involves regeneration from solution or modification of native fibers by swelling agents. Cellulose contains both crystalline and amorphous regions. The proportion of crystalline fraction ranges between 50 to 90%. Cellulose fibrils surrounding the cell in regular parallel arrays in cross-layer are often cemented together by a matrix of another polymeric material hemicellulose. Unlike cellulose, hemicellulose shows variability in both structure and composition. Hemicelluloses are particularly heterogeneous poly-
TABLE 2 Composition (%) of Agricultural and Wood Residues

Substrate
Bagasse Barley straw Birch Corn cobs Corn stalks Groundnut shells Oat straw Pine Rice straw Rice hulls Saw dust Sorghum straw Wheat straw
Hexosans
33 40 40 42 35 38 41 41 32 36 55 33 30
Pentosans
30 20 33 39 15 36 16 10 24 15 14 18 24
Lignin
29 15 21 14 19 16 11 27 13 19 21 15 18
Ash
4 11 4 2 5 5 12 8 12 20 5 10 10
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mers in that they are composed mainly of the three hexoses - glucose, mannose, and galactose - and the two pentoses - xylose and arabinose - together with uronic acids. Three welldefined groups can be identified as:
1.
2.
3.
Xylans that have a basic backbone of polyp- 1,4-xylan with additional side links to arabinose, glucuronic acid, and arabino glucuronic acid. Mannans that are composed of glucomannans and galactomannans. Galactans appearing as arabinogalactans.
The type and amount of hemicellulose vary widely depending on plant materials, type of tissue, growth environment, and method of extraction. The degree of polymerization of shortchained heteropolymers of hemicelluloses is usually less than 200. Xylans of grasses and cereals are generally characterized by the presence of Larabinose linked as a side-chain to a D-xylose back bone. Wood xylans are characterized by the presence of 4-O-methyl-~-glucuronicacid linked to a D-xylose backbone. Hydrolysis of hemicelluloses in annual plants and agricultural wastes produces pentoses, primarily D-xylase, as the major products. Recalcitrant heteropolymer, lignin, the generic name for a diverse group of three-dimensional polymers of aromatic alcohol that associated with cellulose as lignocellulose provide the This structural rigidity to vascular plants. .Is complex high-molecular-weight biopolymer comprises about 25% of the photosynthetic biomass produced annually on Earth, and it contains 50% more carbon than cellulose. It is found in cell walls of all vascular plants. It fills the spaces between cellulose fibrils together with hemicellulose and pectin, and thus acts as binding material between cell wall components. The concentration of lignin in primary and secondary cell wall accounts for 70 to 90% of the total lignin present. The middle lamella contains about 10 to 30% of the total lignin found in wood fiber. Wood contains 20 to 30% lignin, while gramineous plants contain 10 to 15%. The monomeric units present in lignin vary with plant species. Lignin in Gymnosperms contains predominantly guaiacyl units, while in Angiosperms it has both guaiacyl and syringyl units in almost equal propor-
tions. Among Angiosperms, monocotyledons contain a large proportion of p-coumaryl units. Structurally, it is composed of three types of phenyl propane units linked through seven major types of C-C or C-0-C linkages. These aromatic rings are substituted with a 0, 1, or 2 methoxyl group and interunits lack stereo regularity. During the process of cell wall thickening, the phenyl propane precursors of lignin are oxidized by peroxidase to yield free radicals. The polymer is then formed from the coupling of various resonance forms of the free radicals. The resultant polymers contain a variety of interunit linkages, the guaiacylglycerol-f3-aryl ether being the most abundant. It has also been observed that some hemicelluloses are linked with lignin by covalent bonds. I Lignins of grasses and certain woods contain aromatic or cinnamic acid esterified through a side-chain hydroxyl group of lignin. 19.20 Through conventional means of isolation and cleavage, it is very difficult to study the structure of lignin. Several possible reasons for this complication include:
1.
2.
3.
4.
Lignins have irregular structure with no precise chemical grouping. Unlike nucleic acids, proteins, and polysaccharides, lignins do not have identical readily hydrolyzable linkages repeating as regular intervals. Because of the presence of C-C and diary1 ether-type bonds, which are difficult to cleave, lignins cannot be efficiently degraded. It is difficult to extract lignins in an unmodified state.
Thus, the best way to understand the structure of lignin is to retrace the steps involved in its biosynthesis. Conversion of I4C-labeled lignins to I4CO, has been found a useful assay for biodegradation of lignin.22Biosynthetically, lignin arises by free radical copolymerization from three precursor alcohols: p-hydroxycinnamyl (coumaryl) alcohol, 4-hydroxyl-3-methoxycinnamyl (coniferyl) alcohol, and 3,5-dimethoxy-4hydroxycinnamyl (sinapyl) alcohol . 2 3 In addition to cellulose, hemicellulose, and lignins, the plant cell wall contains extraneous materials, including extractives and nonextrac-
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tives. Wood contains 1 to 8% extractives on a dry weight basis; agricultural residues contain even more. They consist of waxes, fats, gums, starches, alkaloids, resins, tanning, essential oils, and various other cytoplasmic constituents. The nonextractives make up 0.2 to 0.8% of the dry weight and include inorganic compounds such as silica, carbonates, oxalates, etc. In agricultural residues, nonextractives may be up to 10% of the dry weight.
IV. BIOTECHNOLOGICAL APPROACH

The potential of lignocellulose as a renewable raw material is immense. It is produced and wasted annually in huge amounts and has several likely uses if suitable technology can be developed and operated economically. The main possibilities of its bioconversion are production of SCP, enzymes, liquid or gaseous fuels and solvents, and sugar syrup, which can be used as such or as feedstock for other microbiological processes. Table 3 exhibits the potential applications of products obtained by lignocellulose hydrolysis.
A. Single-Cell Protein
Single cell protein (SCP) production from extensive research and developmental efforts has been envisioned as the partial solution to the increasing demand for proteinaceous food in the world. Lignocellulosic waste from pulp and paper industries, as well as wastes from dairy and agricultural industries, are the potential substrate for use in the production of SCP.24-26 Cellulosic raw materials are abundant and cheap at the point of origin. However, the disadvantage lies in the economic hydrolysis process of cellulose to assimilable sugars. Conventional methods, involving high temperature and pressure treatment in the presence of mineral acid, are expensive. The successful utilization of cellulosic raw material for SCP production requires the development of simple and inexpensive hydrolysis technique. The current research in this field is focused on the economic production and utilization of cellulase enzyme for hydrolysis of cellulose. The increasing world demand for food and feed protein has spurred the search for noncon156
ventional protein sources to meet protein requirements. The impetus behind SCP production lies partly in the need for more protein and partly in the commercial increase in the economic advantages gained by substitution of microbial protein for the conventional protein supplements. SCP in various forms has attracted particular attention because of its amenability to controlled intensive cultivation and less dependence upon the vagaries of climate, weather, and soil characteristics. Several studies have involved various cellulolytic microbial species in producing SCP from waste cellulose and many of these are acceptable sources of edible protein.* The two possibilities of converting cellulose to SCP are (1) growing the microbe directly on cellulosic substrate for microbial protein production and (2) production of extracellular cellulase enzyme that can be used for saccharification of cellulosic substrates. Spent sulfite liquor (available as a byproduct of paper pulping) is widely used as a carbohydrate source and contains about 3% fermentable sugars. The microfungus Paecilomyces varioti is grown on waste sulfite liquor (the Pekilo process) for use as animal feed in Finland. Other organisms used for SCP include bacteria Cellulomonas and Lactobacillus; the molds Aspergillus, Penicillium, Chaetomium, and Polyporus; the yeasts Saccharomyces, Candida, and Rhodotorula; and the algae Spirulina and Chlorella. However, a major problem with Saccharomyces, a widely accepted food yeast, is that it is unable to grow on pentoses. Recently, some strains of Saccharomyces have been reported that can utilize pentose sugars under certain c ~ n d i t i o n s . ~ ~ Currently, SCP is viewed as an animal feed supplement, although projects have been undertaken to market SCP as a major proteinaceous food substitute. Use of SCP would release widely used food supplements such as soy meal, fish protein, and cottonseed flour, a protein isolated from peanut meal for human consumption in areas where conventional human foodstuffs are in short supply.3oTable 4 represents microorganisms commonly employed for SCP production using agro-industrial wastes. 31
6. Mushroom Cultivation
Based on the promises that ligno-cellulosic residue hold, it seems appropriate and not chau-
TABLE 3 Potential Uses of Lignocellulose Hydrolysis Products

Product Mixed sugar liquor Applications Fermentation processes, single cell protein, ethanol, butanol, organic acid, antibiotics, enzymes, etc. Animal feed carbohydrates, fermentation process, fructose syrups Ethylene, butadiene, hydroxymethyl furfural, laevulinic acid. Fermentation processes with selected organism, furfural adiponitrite, xylitol sweetener Fermentation with selected organism, animal feed carbohydrate Fuel, carbon black, sulfonates as dispersants and emulsifiers in drilling mud, dyes, etc. Chelating agents, humectant, resin extenders, phenol benzene, phenolic resin, vanillin, dimethyl-sulfoxide, methyl mercapton
Glucose
Xylose
Other sugar
Lignin
TABLE 4 Potential Microorganisms Used in Studies on SCP Production

Algae: Chlorella, Scenedesmus, Spirulina Bacteria: Bacillus, Hydrogenomonas, Methanomonas, Methylomonas, Pseudomonas Yeast: Candida, Rhodotorula, Saccharomyces Fungi: Aspergillus, Rhizopus, Fusarium, Penicillium
vinistic to promote the development of the resourceful field of mushroom cultivation. It offers well-proven, low-cost profitable technology of a good-quality human food.3 Currently, the most commercially viable process for bioconversion of agricultural lignocellulosic wastes is composting to provide substrate for growth of edible fungal fruiting bodies. Agaricus, Lentinus, Volvariella, and Pleurotus are the dominant taxa cultured for human consumption. In Europe, the organism of interest is Agaricus bisporus. In the Far East, a wide variety of mushrooms, especially the Oyster mushroom, Pleurotus ustreatus, and shitake are cultivated. Fungal fruiting bodies are rich in protein. Thus, production of enhanced protein-content lignocellulose for animal feeding
is feasible, P. ostreatus being the most favored organism for this purpose. 30 A . bisporus, the most widely cultivated fungus, accounts for over 75% of world mushroom production with growth substrates as composted straw. The U.S. leads the world in producing about 285,000 tons of the total Agaricus marketed. Certain fungi such as Pleurotus sp. can utilize lignocellulosic wastes such as wood with little or no p~etreatrnent.~~~ It has been cultivated on tree stumps and bed logs, but the discovery of its rapid growth on straw-based substrate paved the way for large-scale commercial production. Pleurotus is wide spread in Southern Europe and areas of Central Asia and North Africa. Pleurotus sajor-caju has been isolated in the foot hills of the
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Himalayas (India) growing on the stumps of EMphorbia. Coprinus fimetarius in recent years has been shown to grow quite easily on straw bales impregnated with a solution of calcium nitrate or a mixture of limestone and ammonium nitrate in In Japan Lentinus edodes is grown on whole oak pieces, whereas other species such as Volvariella Volvaceae, P. ostreatus, etc. are grown on straw or saw Table 5 lists potential species of mushrooms commercially cultivated for edible purposes.
2.
3.
4.
which will yield a higher-quality product with substantially less energy than used by conventional thermomechanical processes. Incorporation of these systems in bleaching schemes reduces the requirement of chlorine-based oxidizing agents. Color in bleach effluent resulting from the presence of complex hazardous aromatic compounds may be removed by lignolytic microorganisms. Partially purified enzyme preparation could also be utilized to accelerate retting of jute and similar processes.
C. Biological Pulping and Bleaching

Constant efforts of technologists and scientists to attain maximum efficiency through adopting the latest innovations have opened a number of avenues for the role of biotechnology in the different areas of pulp and paper manufacturing industries, forestry, bleaching, byproduct utilization, and waste management.jx '* Apart from the application of lignin bioconversion in improving animal feed resources, there is an immense potential of biolignolytic organisms in enhancing the production of several useful processes and products. Treatment of pulp with lignolytic white-rot fungi during pulping and bleaching process could be useful in many way s:'3-4x 1. Modification of partially coarse thermomechanical pulp, further processing of Biological pulping of wood chips using a cellulase negative mutant of Phnrierochaete chr-y soporium has demonstrated the reduction of the energy cost of pulping by 20%.'" In industrial applications, wood chips can be sterilized or pasteurized, inoculated with fungus, and placed in a series of silos from which the chips can be continuously removed. More recent studies show that pretreatment of aspen wood chips with P. hrrvispora decreased the energy requirements by 47%: compared with untreated chips.s" The application of this fungus also increased the terisil strength of pulp. Considering the high cost and uncertainty of energy supplies, especially in developing countries, an efficient biopulping process would be greatly advantageous from the perspective of both economy and ensuring continuous availability of good-quality pulp. Biopulping
TABLE 5 Some Commercial Mushroom Species Used for Cultivation on Lignocellulosic

Species Agaricus bisporus Auricularia sp. Production (million tons)
1.23 0.12
Substrate Composted straw Hardwood logs, sawdust, bran Straws Sawdust, bran Hardwood logs, sawdust, bran Hardwood logs Hardwood logs, sawdust plant wastes Wood logs Rice straws, composted cotton wastes
Coprinus firnetarius Flarnmulina velutipes Lentinus edodes

Pholeota nameko Pleurotus spp. Tremella fuciforrnis Volvariella volvacea
0.05 0.10
0.32
0.03 0.17
0.04 0.18
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would essentially eliminate the pollution hazards associated with chemical pulping processes. An examination of the pulp qualities and the energy savings coupled with the economic evaluation suggest that microbial treatment of wood chips for pulp production has a viable industrial future. In parallel to the alternative chemical methods, the use of enzymes in bleaching technology has also been con~idered.~' Enzymatic removal of hemicellulose has been shown to decrease the energy requirements for pulping." Indeed the treatment of crude pulp by xylanases causes cleavage reactions in the lignin-xylan matrix, which facilitates further delignification during Industrial research has shown that a limited treatment with xylanase modifies pulp properties, especially with respect to draining." Furthermore, xylan breakdown was also found desirable for several reasons, including the fact that xylan is closely associated with lignin and that the monomer xylose sugar is metabolized to provide necessary energy for lignin biodegradation. Decolorization of pulp and paper mill effluents has also been studied using P. c h r y o sporium. Treatment o f bleach plant effluents with P. chryso.spori~im resulted in 80% color reduction in a day.5uThe process has also been scaled-up and patented.s' Conventional processes such as aerated lagoons, activated sludge systems, and anaerobic bcd fermenters used to improve the biological oxygen demand (BOD) and chemical oxygen demand (COD) properties of wastewater seem to be ineffective in removing the polymeric degraded products that are primarily responsible for color. Extensive decolorization of E-1 effluent from a Kraft bleach plant has been achieved by using a granular-activated, carbon-packed anaerobic bioreactor. '(' An anaerobic-aerobic waste water treatment process, ANAMET system, has been found to be economical for the waste water treatment in the pulp and paper industries."' Chemical waste treatment processes frequently require large amounts of equipment; biological waste treatment including fungal fermentations and enzyme preparations could be used to decolorize industrial effluent stream and breakdown toxic chemicals economically. Eriksson6' has recently developed a combined aerobic and anaerobic treatment for bleaching plant effluents.
The reactors are complemented with an ultrafiltration system. The overall system is useful in removing odor problems that normally occur in anaerobic treatment. The results were improved threefold when compared with those with aerated lagoons or activated sludge techniques. The aimed process is a closed system of wastewater from bleach plants and circulation of the purified water back to the plant. Another interesting approach is to modify lignin content and quality in forest trees by genetic manipulation in order to facilitate the pulping and bleaching process.h2The plant material generally decreases from hardwoods to softwoods to grasses depending on the relative degree of lignin condensation. More precise techniques and understanding are, however, necessary to assess lignin quantity and quality during morphogenesis and maturation. Eriksson and D i n u P studied forest trees with low lignin content and with lignin of modified methoxyl content to manipulate lignin structure. Information on distribution of enzymes involved in the lignin biosynthesis and lignification is collected by taking samples from cell culture, embryos, and seedlings. Use of monoclonal antibodies in conjunction with cell and tissue cultures will be helpful in studying the structure, quality, and quantity of lignin more precisely and also for studying genes controlling lignin biosynthesis.62 It is expected that the creation of trees easier to pulp and bleach would avoid damaging effluents.h2 Interestingly, the lignolytic system of P. chrysosporium appears capable of degrading several recalcitrant organohalides, including D.D.T. ( 1 , I-bis[4-chlorophenol]-2,2,2,-trichloroethane), polychlorinated biphenols, polychlorinated dibenzo (p) dioxins, and lindane (1,2,3,4,5,6-hexachloro c y c l ~ h e x a n e )The . ~ ~ involvement of ligninases in the degradation/ dehalogenation of benzopyrene and DDT has been demonstrated.*' The limitation of the enzymatic process is, however, the high redox potential of substrate, thus resulting in incomplete degradation. The use of whole fungi may be useful in such cases as CO, is likely to be the end product.&l Recently, Cyathus butleri and C . stercoreus have been studied for their ability to degrade various dyes, for example, crystal violet, methylene blue, malachite green.65
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D. Upgradation of Low-Quality Fodder

Possibility of converting lignocellulosic substrate to more digestible and nutritious feed stuff for ruminants has concentrated research into upgrading animal feeds by solid-state fermentation p r o c e s ~ e ~A .~ large ' ~ ~number ~ of white-rot fungi have been studied for solid-state bioconversion of lignocellulosic materials because of their ability to degrade lignin. P. clirysosporium. Cyathus sp., Coriolus hirsutus, Dichomitus squalens, and Pleurotus spp. are the extensively studied organisms. I n vitro digestibility of wheat straw, up to 75% was achieved by Agosin and OdieP7 by using C . stercoreus or D . squaleris. The mode of attack was mineralization of lignin leaving carbohydrates in situ. In a study using P. sajurcuju for fermentation of wheat straw, 20% increase in in vitro digestibility was achieved using sterilized straw.'* The average improvement by 30 to 40% of feed stuffs in terms of feed intake as a result of biological treatment has been reported by Hahn and Garret."' Enzymatic methods have also been used to delignify lignocellulosic substrates to increase their feed ~ a l u e . ~ " . ~ ' Important progress in the knowledge of the biodelignification has been made during the last d e ~ a d e . ~ ' However, -~' structural changes in straw lignin during straw delignification and the mechanism of lignin depolymerization have only been partially established. The low molecular size lignin fractions disappear after fungal treatment. Cinnamic acids are involved in lignin-polysaccharide linkages, and their degradation could increase lignin extractability. Breakdown of p-04 and p-1 dimers has been investigated extensively and simultaneous degradation of condensed and uncondensed lignin and biphenyl structures has been demonstrated. Recently, Valmaseda et al.82studied the kinetics of straw solidstate fermentation with P. ustreatus and T. vprsicofor to characterize the biodelignification process. Decrease in syringyl/guaicyl and syringyl/ p-hydroxyphenyl ratios and cinnamic acid content was noticed during fungal treatment. This was accompanied with a strong increase in respiratory activity and decrease in free sugars, extractives, and in vitro digestibility. Most of the low-quality feed stuff contains high concentrations of cellulose, small amounts of protein and fat, and relatively high ash con-
tents when compared with high-quality feed stuffs. However, they contain large amounts of total digestible nutrients and energy values. The nutritional value of these low-quality feed stuffs can be improved significantly if they are effectively processed before feeding. Considerable potential exists in upgrading fodder using solidstate fermentation employing lignolytic fungi. However, as in many biotechnological processes, scaling-up creates some problems. In transferring the process from laboratory scale to the pilot and finally industrial scale, it becomes progressively more difficult to sterilize the substrate. Pasteurization has been found effective in pretreating straw to allow development of an effective fermentation process.'X Optimization of the process will, however, require a thorough understanding of the physiology of lignolytic fungi so that lignin and cellulose degradation processes can be manipulated to favor delignification. Apart from improving digestibility and feed value of straw, the mycostraw obtained after solidstate fermentation has potential for biogas production." After microbial removal of lignin, the straw components are more accessible for anaerobic digestion. Mycostraw may constitute a technical substrate for many biotechnological processes. Thus, the beneficial products in this process are fungi in the first step and methane in the second step.
E. Production of Solvents and Chemicals from Lignocellulosics

Lignocellulosic biomass can be converted to value-added products either by thermochemical or biological Higher temperature and pressure are required in thermochemical processes, which further resulted in a complex mixture of products. On the other hand, biological processes (fermentation) using microorganisms operate mostly at lower temperatures and produce very few major products. The technology for solventkhemical production from cellulosic biomass includes pretreatment of substrate, hydrolysis of pretreated substrate, fermentation, and product recovery. The most important features for the choice of microbial system used in the fermentation process are the range of substrates to be utilized and product yields in the entire
160
process. Microorganisms that have received much attention include several groups of yeasts such as Saccharomyces, Pachysolen, Candida, and Pichia; thermophilic bacteria, Clostridium; mesophilic bacteria, Zymomonas; and mold, Fusarium.
1. Pretreatment of Substrate
The lignocellulosic substrate needs pretreatment so as to make it more susceptible to the action of enzymes or microorganisms. The main objective of the pretreatment is to loosen the structure of lignocellulosic material, thus a large surface area can be available for the action of cellulolytic enzymes. This can be achieved using several means such as mechanical, physical, chemical, and biological techniques. Among all the pretreatment methods, steam pretreatment has gained much interest and wide acceptance as a highly efficient and economically feasible method.xh A high degree of enzymatic digestibility can be achieved using the steam pretreatment te~hnique.*~-~' Chemical pretreatment using alkali is also an equally effective method; however, the effluent treatment problem is a major drawback. Further, more expensive materials in the process equipment may also be needed.8h
2. Enzymatic Hydrolysis
After pretreatment substrate is subjected to enzymatic hydrolysis. The generally acceptable mechanism of cellulose degradation by microorganism is that by the synergistic action of at least (EC three enzymes: endo- I ,4-P-~-glucanase 3.2.1.4), exo- 1,4-P-~-glucanase (EC 3.2.1.91), and P-u-glucosidase (EC 3.2.1.21).'" Endoglucanase cleave p-1,4 linkage of cellulose randomly, whereas exoglucanase releases cellobiose or glucose units from the nonreducing end of cellulose polymer. The structure of hemicelluloses are more complicated. They involve linear 1,4+-linked chains of xylose or mannose, substituted with other sugars or acetyl groups, and thus make up branched heteropolysaccharides. A more complex set of enzymes are required for their hydrolysis. The endoxylanases are the most widely studied and characterized hemicellulolytic enzymes. However, for complete saccharifica-
tion of hemicellulosic polymers, several other hydrolytic enzymes - p- 1,4-xylosidase, a-glucosidase, a-arabinosidase, and acetyl xylan esterase - are also required. Cellulases have a high affinity for cellulose; therefore, they have received a great deal of attention to study the adsorption profile of enzymes on substrate. Two steps are believed to be involved in enzymatic hydrolysis of cellulose: first, the adsorption of cellulase onto the surface of cellulose, and second, the breakdown of cellulose to fermentable s ~ g a r s . ~ 'The - ~ * process of adsorption of the enzyme onto the substrate seems to influence and control the rate and degree of saccharification. 93-y5 These enzymes can be recovered and recycled. It has been projected that recycling of 60% of the cellulolytic enzymes may have a major impact on the cost of because one of the major bottlenecks associated with the enzymatic hydrolysis process is the cost of enzymes, which account for about 60% of the ~' methods have total cost of the p r o ~ e s s . Several been attempted for this purpose using an immobilization te~hnique,~* ultrafiltration,9y washing the residue by buffer of neutral pH,'O" or by contacting the hydrolysate with fresh subStrateYs.l~" ,102 However, an economical technique for recycling of enzymes during hydrolysis of cellulosic substrates is yet to be developed.xh Forty-eight to ninety-six h are required to achieve complete saccharification of cellulosic substrate. The rate of hydrolysis is usually high during the first few hours of the process and decreases thereafter. This may be due to several factors, including competitive inhibition of enzymes and degradation of substrate, which decreases the susceptibility to enzymatic attack. Inhibition of enzymes is caused mainly by glucose and cellobiose. Simultaneous saccharification and fermentation process has been proposed to minimize the competitive inhibition. Io3 Alternatively, glucose can be removed continuously using ultrafiltration, which relieves the inhibition. Immobilized enzymes are more stable than the soluble enzymes and are resistant to inof hibition by glucose and c e l l o b i o ~ eRemoval .~~ hydrolysate at an early or intermediate stage of saccharification and addition of fresh buffer can result in higher sac~harification.~~ The choice of substrate and enzyme concentration is an important consideration with respect to process economics. A low dosage of enzyme
161
is preferred because the cost of enzyme is high. The cost of raw materials and energy, the extent of enzyme recovery, and the value of byproducts are other important factors.86,'04
3 . Fermentation
Fermentation of cellulose with conventional methods is usually achieved in two steps: (1) enzymatic hydrolysis of polysaccharides to monosaccharides, and (2) bioconversion of monosaccharides to solvents and chemicals. To improve process efficiency, a combination of these two \teps in the same bioreactor has been attempted using different cellulases and microbial spe(.ies. 1(15.106 Solvent production from cellulose in a simultaneous saccharification and fermentation process eliminates the problem of end-product inhibition, because glucose does not accumulate in this system and is converted to ethanol or other products immediately following saccharification. ' 0 7 Bioconversion of glucose to ethanol is a wellestablished technology; the main problem is encountered with the fermentation of pentose fractions present in acid or enzymatic hydrolysate of lignocellulosic materials. The hydrolysis products of wood contain large amounts of pentose sugar$, mainly xylose. Pentoses are not easily utilized by the common yeast Saccharornyces cerevisiae. After a search for pentose-fermenting organisms, few yeast species belonging to the genera Pachysolen, Candida, and Pichia appear to have potential. 'OH.i"V Certain filamentous fungi may also be potential such as Fusarium oxysporum. Mucor, and Paecilomyces. '"-' I' Xylose can be fermented in two ways: direct conversion to ethanol or other chemicals or isomerizing to xylulose by xylose isomerase and then fermenting to ethanol. Growth rates and yields are variable, attributing to strain variation and different environmental conditions employed. Pichia stiptis has been reported to give an ethanol yield of 0.39 g/g x y l o ~ e . "Concentrations ~ of 2 to 3% were obtained with the most efficient strains. Efficient strains of F. oxysporum have been reported to yield 0.4 to 0.5 g ethanol per gram of However, rate of xylose fermenxylose.' ''-I tation is slow with molds and overall productiv-
ities have been found low when compared with yeast."' Ethanol yields reported are low from xylose fermentation with yeast and bacteria, and another difficulty is to achieve a complete conversion of xylose. On the other hand, molds give high ethanol yields from xylose, but the conversion rates are too slow to be considered economical and thus need further improvement. Several studies report on the product concentration in the range of 20 to 80 gil, yields higher than 0.4 gig substrate, and productivities higher than 0.5 gilih from lignocellulosic hydrolysate (Table 6). A high value of ethanol concentration, 84 gil, with productivity of I . 17 glli h has been obtained using Cundid~i shrhntue from steam-pretreated hydrolysate of whole barley. "" The differences in product yield and tolerance in bioconvcrsion of lignocellulosic hydrolysatc may be influenced by several factors, including choice of substrate and its pretreatment, sugar composition of hydrolysate, presence of inhibitors, and the choice of organism. Unlike enzymatic hydrolysate, acid hydrolysate of lignocellulosic substrate sometimes contains other substances that are more or lcss inhibitory to the microorganisms. There are at least four potential sources for the formation of inhibitory compounds in wood hydrolysate: sugar decomposition products (furfurals), soluble lignin products (phenols), wood extractives (tannic acid, terpenes), and heavy metal ions (nickel, iron, chromium). This problem may be dealt with by avoiding their formation using safe pretreatment techniques or by using adapted strains. I Several species of saccharolytic bacteria produce neutral solvents, including butanol, acetone, butanediol, isopropanol, and ethanol in addition to volatile fatty acids and gaseous products from charbohydrate fermentation. Lignocellulosic materials are one of the most attractive substrates for solvent production as they are inexhaustible and less expensive raw materials. Few attempts have been made to use cellulosic substrates for the production of solvents other than ethanol. A simultaneous saccharification and fermentation process has been studied to produce butanol and acetone from alkali-pretreated wheat straw using Trichoderma reesei cellulase and Clostridium acetobutylicurn . A final butanol concentration of 10 g/l could be achieved from
'('~
162
TABLE 6 Fermentation of Lignocellulosic Hydrolysate by Various Microbial Species

Sugar type present (9/1)
126 G, 54 X 45X 64 G, 28 X
Microorganism
Candida shehatae Pachysolen tannophilus Pichia stiptis Saccharornyces cerevisiae Fusariurn oxysporurn Mucor sp.
a
Substrate
Whole barley Wheat straw Aspen Soft wood Wheat straw Bagasse
Pretreatment
Enzymatic Trifluoroacetic acid Steam, enzymes Na, SO, Acid Acid
Maximum ethanol (dl)

84.0 13.5
Productivity (g/l/h)
0.47 0.30 0.45 0.34 0.29 1.17
Ref.
73 93 94 95 96 65
0.08
0.85 0.26
41.o 12.7 3.2 19.0
6G,27.5 X
2G,9X
0.03
0.16
13G,43X
0.33
Glucose = G, Xylose = X.
140 g of straw. Simultaneous utilization of both cellulosic and hemicellulosic components was observed. Mes-Hartree and Saddler, and Maddox and M ~ r r a y also ~ studied butanol production from wood hydrolysate and hemicellulose hydrolysate, respectively, for butanol production. Brrcillus tnaceruns has been found to use a wide variety of substrates such as starch, xylan, hexoses, pentoses for the production of acetone. 12 Butanediol is produced by facultative anaerobic bacteria, such as Aerobucrrr, Aerotnotim, Klc+siellu, Bacillus, and Serrutia. A lot of work was done during World War 11; the interest in butanediol fermentation was renewed during the last 10 years, mainly because of the ability to ferment pentose sugars and sugars present in wood hydrolysate.x -O However, the market value of butanediol is uncertain. Recent interest has been focused on the direct fermentation of cellulose and hemicellulose in which a single organism carries out both hydrolysis and fermentation into ethanol or acetic acid in a single step. Thus, this direct approach eliminates the need for separate enzyme production and hydrolysis reactor. Some potential thermophilic anaerobic bacteria and filamentous fungi have been identified recently that are found to produce simultaneously hydrolytic enzymes and ethanol in a single-step process. Thermophilic production of ethanol has several advantages that are summarized in Table 7.
Clostridium thermocellum is the most widely studied thermophilic anaerobic bacterium for bioconversion of cellulose. The cellulolytic enzyme system differs from that of fungi and aerobic bacteria. Ethanol yields of 0.3 to 8.0 mol/mol of glucose equivalents in the cellulose utilized have been found using different strains. 1 1 1 . 1 3 2Besides ethanol they produce acetic acid and lactic acid. One of the most interesting developments in this field has been the use of cellulolytic co-culture (mixed culture) for increased efficiency of substrate utilization together with enhanced product yiclds. Several workers have investigated the use of C. thermocellum with various xylose-fermenting thermophiles. Mixed culture of C. thermocellum and C. thermohydrosu@ukwn has been found effective in fermenting pure as well as natural cellulosic and hemicellulosic substrates. Using a system of C. thermocellum and C . thermo.sacc.hurofyticum, ethanol yields up to 240 m M were achieved with a conversion of 85% of solka floc or 52% of total carbohydrates in corn cobs. Other thermophilic anaerobic bacterial strains employed for ethanol production in coculture with C. rhermocellum and Thermoanaerohium brockii and Thermoanaerohacter ethanolicus. Mixed culture of thermophilic bacteria has been shown to be quite useful for the production of ethanol from cellulose- and hemicellulosecontaining materials. However, for industrial ap-
163
TABLE 7 Advantages of Thermophilic Anaerobic Fermentation Process

Advantages 1. Fermentation of a wide range of substrates including cellulose and starch
2. Less biomass production with high product yields
3. Rapid fermentation due to high metabolic activity

4. Little risk of contamination by undesired microbes 5. Production of thermostable polysaccharidases
6. Possibility of the continuous recovery of products directly from bioreactor during fermentation
Disadvantages 1. Lower end product tolerance when compared with yeasts
plication studies on pilot fermentations, utilizing the available lignocellulosic biomass as carbon sources arc needed. Recently some fungal strains of Fusarium oxysporum, Neurospora crassa, and Monilia sp. were found to have potential in fermenting cellulosic substrates to ethanol in a one-step process. Ninety percent conversion of alkali-treated bagasse to ethanol was achieved using N . crussa by Rao et al. I Ethanol concentration of 16 gil from solka-floc has been obtained in 8 d using Mnniliu sp. isolated from bagasse compost. Fusarium species are regarded as potential decomposers of plant biomass, including cellulose, hemicellulose, lignins, and pectins. Several groups studied ethanol and acetic acid production from various cellulosic substrates using cellulolytic strains of F. o.rysporum. I ? . 1f.13hDetailed investigations on pentose fermentation and metabolism has been made recently by Singh et al. 1 3 7 - ~ 7 y No available fungal strain is, however, completely satisfactory for the fermentation of cellulosic biomass to ethanol, because the most limiting factor is the slow conversion rate which necds further improvement using strain selection and recombinant DNA technology techniques.
I.
2.
3.
A better understanding of the regulation and catalytic functioning of cellulases and ligninases. Insight into the nature of synergistic intcractions between different enzymes. The development of economically feasible system for the conversion of waste biomass into value-added products.
4. Genetic Engineering Aspects In order to get a better understanding of lignocellulose biodegradation at the molecular level and to develop the bioprocessing potential of lignocellulolytic microorganisms, recombinant DNA technology is expected to facilitate: I4O.I4l
l64
Our understanding of molecular biochemistry of cellulase biosynthesis and secretion have advanced manyfold in recent years; however, still much remains to be done to make cellulose hydrolysis a practically feasible process. It is expected that for industrial application cellulases must have high adsorption capacities and catalytic efficiencies, high thermal stabilities, and low-end product inhibition. 4 2 . 1 4 3 It is, therefore. essential that efforts should be made into cloning cellulase genes with desirable molecular properties. Cloning of genes coding for individual components of cellulase complex would allow the biosynthesis of pure components, and their potential for cellulose hydrolysis would be as.sessed. A large number of fungal and bacterial genes have been cloned in recent years (Table 8). Escherichiu coli has been the most commonly used host with plasmid or phage vector. However, cellulase genes have also been expressed efficiently in other bacteria such as Bacillus suhtilis,145 Zymomonus m ~ b i l i s 14 ~ ~ Pseudomoilas . fluorescens, 148 B . megaterium, 149,150and Azotobacter vinelandii,,52and in the yeast Saccharomyces cerevisiae. 153-155 The expression of cel-
TABLE 8 Cloning of Microbial Cellulase Gene

Donor cell
Bacillus subtilis B. subtilis Cellulomonas fimi C. fimi Clostridium therrnocellum Pseudomonas fluorescens P . fluorescens Ruminococcus fla vefaciens Trichoderma reesei T: reesei
Cellulase gene
p-1,4-Glucanase Endoglucanase Cellulase Exoglucanase Endoglucanase Endoglucanase Endoglucanase Endoglucanase Endoglucanase I Cellobiohydrolase I
Host cell
Escherichia cob B. megaterium Brevibacteriurn lactoferrnentum Saccharomyces cerevisiae S. cerevisiae
E. coli
Ref.
135 114 128 129 124 118 132 117 124 136
Z.rnobilis
E. coli
S. cerevisiae E. coli
lulase genes in heterologous hosts must ensure transcription of the cloned gene through cloned promoters, efficient translation of the gene, and subsequent export of the cellulase protein to the extracellular medium. '44 The fungus Trichoderma rersei is generally considered as the most efficient and best characterized producer of cellulases. In view of reducing enzyme production costs, genetic improvement programs have been undertaken in numerous laboratories throughout the world. With the availability of suitable vectors and an efficient transformation system, it is possible to clone genes of cellulase having desired properties for cellulose hydrolysis. ' 5 6 , ' 5 7 This offers the possibility of constructing Trichoderma strains producing cellulases with higher specific activities as well as for the production of bulk quantities of enzyme. However, thermoresistance of these enzymes needs improvement. Protein engineering offers the technology, if not yet the rationale, for doing this. Crllulomonus fimi cellulase genes were introduced into Brevibacterium lactofermenturn, a producer of glutamic acid and lysine at industrial scale. "* This approach might provide a cellulolytic B. lactofermmtum for producing glutamic acid and lysine from cellulosic materials. S . cercvisiae and Z . rnobilis are known for efficient conversion of glucose to ethanol. They have been used as hosts for the expression of cellulase genes,
the presence of which enable these organisms to directly ferment cellulose to ethanol. Several studies have demonstrated that both S. cerevisiac1.59- Ihl and 2. r n ~ b i l i s ' ~ can ~,~ express ~ * cellulase activity. However, a considerable amount of additional work is required before efficient conversion of cellulose to glucose and finally to ethanol within a fermentative organism is achieved, 163-16s because bioconversion of lignocellulose to ethanol requires a substantial input of energy for either the physical or chemical pretreatment of the substrate before enzymatic hydrolysis of cellulose. Potentials for cloning and expression of ligninase genes in various host organisms have been studied in recent years. The major hosts used for the expression of recombinant protein are E. coli and S . cerevisiae. Ligninase genes encoding the main isoenzyme H8 of P. chysosporium has been cloned in E . coli.I6' The specific activities of recombinant H8 ligninase were equivalent to those of P. chrysosporiurn H8. The recombinant ligninase demonstrated substrate-dependent peroxide uptake in the presence of Kraft lignin and milled wood lignin. Although higher expression of ligninase has been demonstrated in E. coli, the disadvantage is its inability to glycosylate the ligninase protein.2' The drawback of yeast host is relatively low expression level. Various other hosts are under trial for the expression of gene product, for example, Pseudomonas sp., Asper-
gillus niger, and A. nidulans. A. niger appears to be a potential host organism for expression of ligninase because of its ability to recognize heterologous promoters and secretion signal as well as capacity for accumulating relatively higher amounts of protein in extracellular medium.2' The advent of recombinant DNA technology has accelerated research in the field of lignocellulose biotechnology. In the past 5 years, large numbers of cellulase and ligninase genes have been characterized and expressed in a variety of hosts to develop new organisms that can be exploited for utilization of enormous amounts of lignocellulosic wastes.
VII. FUTURE PROSPECTS

Increase in the cost and decreasing feedstock of fossil fuel since 1970 has attracted world-wide attention of scientists to examine the alternative resources. Among these alternatives is the utilization of a wide range of sugar, starch, and cellulosic plant biomass using microorganisms for the production of a variety of value-added chemicals and protein-rich food and feed materials. Utilization of sugar and starch-rich agricultural commodities is, however, considered to compete with food production and thus have a less favorable energy balance in comparison to cellulosic materials.I Ix Lignocellulosic residues are the sources of cheap raw materials. However, the difficulty in economically converting these components is responsible for the incomplete success in developing practical processes for lignocellulose utilization. lh7 The current industrial activity of lignocellulosic biomass fermentation is limited mainly because of two reasons. First, the process cost of raw material is a critical factor. Lower substrate treatment cost and maximum carbon yields will improve the economic viability of the fermentation process. Second, present fermentation productivities when compared with chemical processes for fuel and chemical production are slow. It can be improved by searching for new isolates with desirable properties, understanding physiology of the strain, making accessible to genetic manipulation using advanced recombinant DNA techniques to manipulate carbon flow to the de-
sired products, and application on an industrial scale. Thus, the overall emphasis of future genetic engineering research would be to produce better fermentation biocatalysts with wider substrate ranges, improved kinetics of product synthesis, and the ability to function under environmental conditions more suited to bioprocess engineering. Plans for future research in this area and evaluation of results are directly concerned not only with the efficiency of process variables, but also with the energy and environmental aspects. The economic importance of utilization of natural cellulosic substrates mostly depends on the bioconversion of both hexose and pentose sugars present in the hydrolysate. Fermentation of pentoses is slower than hexose fermentation. Thus, improvement in pentose-converting organisms in terms of yield, productivity, and end product tolerance are likely to be concerned in the overall process. Direct fermentation of cellulosic biomass by certain microbial systems that produce solvent plus polysaccharidases is an attractive and exciting approach. The advantages of direct fermentation include the use of a single bioreactor that simplifies the process and reduces capital cost; increase in overall rate of conversion because the intermediate products are removed as soon as they are formed, relieving feedback inhibition; and catabolite repression of polymerases. This can be achieved by further improvements in thermoanaerobic fermentations or by genetic engineering manipulation of yeast, Zyrnornonas, Fusariurn, or other anaerobic and mesophilic bacteria. In addition to the increased efficiency in the use of raw materials, value of byproducts are increased by producing commercial polysaccharidases and microbial biomass that have higher value added as either a feed because of its protein, lipid, or vitamin content, or as biocatalyst for further biochemical synthesis and transformation. The processing of lignocellulosic biomass into value-added chemicals via fermentations is at a primitive stage of development when compared with the chemical processing of petroleum and natural gas. We strongly believe that given time for process improvements in this field, bioconversions of lignocellulosics will be in high demand.
166
The help of Mr. Vikas Pruthi and Mr. Deepak Nehlani, Department of Microbiology, and Mr. Rajiv Chawla, JTPA, U.D.S.C., New Delhi, in the preparation of the manuscript is duly acknowledged.
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Critical Reviews in Biotechnology, 13(2):15 1- 172 (1 993)

Lignocellulose Biotechnology: Current and Future Prospects

II. SOURCES OF LIGNOCELLULOSIC WASTES

TABLE 1 Estimated Global Production of Lignocellulosic Wastes

FA0 Production Year Book, 1989. NA = Not available.

111. MOLECULAR ARCHITECTURE OF LIGNOCELLULOSE

TABLE 2 Composition (%) of Agricultural and Wood Residues

IV. BIOTECHNOLOGICAL APPROACH

TABLE 3 Potential Uses of Lignocellulose Hydrolysis Products

TABLE 4 Potential Microorganisms Used in Studies on SCP Production

C. Biological Pulping and Bleaching

TABLE 5 Some Commercial Mushroom Species Used for Cultivation on Lignocellulosic

Coprinus firnetarius Flarnmulina velutipes Lentinus edodes

D. Upgradation of Low-Quality Fodder

E. Production of Solvents and Chemicals from Lignocellulosics

TABLE 6 Fermentation of Lignocellulosic Hydrolysate by Various Microbial Species

Maximum ethanol (dl)

41.o 12.7 3.2 19.0

TABLE 7 Advantages of Thermophilic Anaerobic Fermentation Process

3. Rapid fermentation due to high metabolic activity

TABLE 8 Cloning of Microbial Cellulase Gene

VII. FUTURE PROSPECTS

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