PERSPECTIVES FOR THE PRODUCTION LIGNOCELLULOSIC MATERIALS

OF

BIOETHANOL

FROM

Petia Petrova, Viara Ivanova University of Food Technologies, Department of Organic Chemistry and Microbiology, Plovdiv, Bulgaria E-mail: vn.ivanova@abv.bg

ABSTRACT
The most common renewable fuel today and suitable alternative to replace fossil fuels is ethanol that can be blended with petrol or used as neat alcohol in engines. Ethanol is currently produced from sugar (Brazil) or grain (starch, USA). However, this raw material base will not be sufficient because the increasing demand for fuel ethanol and the lower than expected reduction of greenhouse gases. An alternative is the production of bioethanol from agroindustrial wastes containing abundant cellulosic fibers and carbohydrates such as grape pomace, sugar beet pomace, barley and rice straw, corncobs, sunflower stalks and heads, cotton waste, brewer's spent grain, forest residues etc. Lignocellulosic raw materials and agroindustrial wastes minimize the potential conflict between land use for food (and feed) production and energy feedstock production. This review summarizes recent developments in the bioconversion processes, the new technologies required and the advances achieved in recent years to bring agricultural feedstock and lignocellulosic ethanol towards industrial production. Keywords: bioethanol, lignocellulosic feedstock, processes, microroganisms Nonfood plants rich in cellulose or even residual waste diverted from landfills may define the biofuel future. The raw material is less expensive than conventional agricultural feedstock and can be produced with lower input of fertilizers, pesticides, and energy. Biofuels from lignocellulose generate low net greenhouse gas emissions, reducing environmental impacts, particularly climate change.

Introduction
Biofuels are an attractive alternative to current petroleumbased fuels as they can be utilized as transportation fuels with little change to current technologies and have significant potential to reduce greenhouse gas emissions. The liquid biofuels most widely used today are ethanol and biodiesel. Liquid or gaseous (methane or hydrogen) biofuels are derived from organic materials such as starch, oilseeds and animal fats, or cellulose. The other types of biomass-derived fuels under development are green diesel, cellulosic ethanol, butanol, pyrolysis liquids, diesel from algae, hydrocarbons from biomass. Biofuel sources are geographically more evenly distributed than the fossil fuels; thus, the sources of energy will, to a larger extent, be domestic and provide security of supply.

Global liquid biofuels production and main feedstocks

Bioethanol is the most widely used liquid biofuel. The largest producers in the world are the United States, Brazil, and China. Production of bioethanol from sugarcane in Brazil in 2004 accounted nearly 18% of the countrys automotive fuel needs. In Brazil, ethanol-powered and flexible-fuel vehicles are manufactured for operation with hydrated ethanol (around 93% v/v ethanol and water 7%). As a result of this, coupled to the development of domestic deep-water oil sources, Brazil has achieved complete self-sufficiency in oil (6).

The global production and use of biofuels has increased dramatically in the past few years, primarily due to increasing World ethanol production (all grades) has reached a oil prices, national security concerns, environmental considrecord 62x109 liters in 2007, with the United States and erations, and the efforts to revitalize rural communities. The Brazil as dominant producers (approximately 70%) (33). question today is not whether biofuels will be a part of the Recently the United States surpassed Brazil as the worlds energy mix, but rather what economic, social, and environlargest producer of bioethanol. In 2009, the US produced mental implications they will have. 529 BIOTECHNOL. & BIOTECHNOL. EQ. 24/2010/SE SECOND BALKAN CONFERENCE ON BIOLOGY
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39.5x109 liters of ethanol using corn as a feedstock (first generation of ethanol production) while the second largest producer, Brazil, created about 30x109 liters of ethanol using sugarcane. Europe is the most important biodiesel producer in the market, with European rapeseed accounting for 58 percent of the global biodiesel produced in the world. Germany, France, the US, and Italy are the leading producers of biodiesel. Over 90% of the worlds bioethanol derives from crops (60% from cane sugar and beet sugar and the remainder from grains, mainly corn starch using the (first generation of ethanol plants. The US ethanol industry uses corn as its main feedstock (32). The share of the US corn crop that is consumed by the ethanol industry has grown from around 5% to more than 25% in 10 years. Brazilian ethanol is produced from sugarcane on land that could be used for food production. Practically all biofuels in the world are produced from feedstocks that could be used to produce food or that are produced on land that could produce food (2). Expansion of biofuel production in the United States, Europe, and South America has coincided with recent sharp increases in prices for food grains, feed grains, oilseeds, and vegetable oils. Producing biofuels in the second generation biofuel plants out of feedstocks that cannot be used directly for food production or do not reduce the amount of land that can be used to produce food can be accomplished in two ways (3): i/ The most straightforward way is to capture biomass that is currently treated as either waste or that is a co-product of existing production processes with very low or negative current economic value. Examples of waste streams that could potentially be converted into biofuels include perennial grasses, agricultural wastes (e.g., wheat straw), a portion of municipal trash and garbage (e.g., waste paper, waste food scrapes, used cooking oils), crop residues (in particular corn (maize) stover, wheat and rice straw), wood pulp residues, macroalgae, and forest residues (e.g., wood pieces leftover after timber extraction). Currently these streams often generate negative value in that consumers and firms must pay for disposal. A recent study estimated that a city of one million people could provide enough organic waste (1300 tons per day) to produce 430 000 litres of bioethanol a day. Horticultural waste biomass (e.g., tree trunks, twigs, and leaves) could also be a potential source of cellulosic

feedstock (23). The authors estimated that the 50,000 156,000 tons of horticultural biomass collected each year from about 1 million planted trees in Singapore can be used to produce 1458 million liters of bioethanol that can displace 1.66.5% of the countrys transport gasoline demand. New technology that allows for economic conversion of these potential sources of feedstock for biofuels offers the double benefit of a reduction in global waste and the generation of valuable transportation fuels. In addition, tapping waste streams places no burden on the worlds ability to produce food. ii/ The second way that biomass can be created without competing for food land is to use land that is not suitable for producing food or to grow the biomass without using land. There are large areas in US and Europe that once produced food crops but are now in pasture or trees. Conversion of these lands to the production of woody biomass to be used for cellulosic biofuels would not affect food prices. The candidate grass species for cellulosic ethanol production include switch grass, miscanthus (Miscanthus spp.), reed canary (Phalaris arundinacea), and giant reed (Arundo donax) (30, 31). Most of these crops can be cultivated on marginal or agriculturally degraded lands, and thus may not compete with food production. High-diversity mixtures of grassland species can even provide greater bioenergy yields and greenhouse gas (GHG) reductions than certain conventional bioethanol or biodiesel production systems. Forest plantations and agroforestry systems can also serve as potential sources of cellulosic feedstocks for bioethanol production. Over the past four decades, new forest plantations in the United Kingdom have been increasing at an average rate of 25,000 ha per year mostly in Scotland, northern England, and Wales (38). The planted species in these forests include Sitka spruce (Picea sitchensis), Scots pine (Pinus sylvestris), lodgepole pine (Pinus contorta), hybrid larch (Larix spp.), Douglas fir (Pseudotsuga spp.), and noble fir (Abies procera). Although these forests have been planted for timber, they could also be harvested to supply biofuel production. An example of the third-generation biofuel plants is to produce biomass without extensive use of land using macroalgae as another potential source of biofuel feedstock. Aquatic unicellular green algae, such as Chlorella spp., are typically considered for biodiesel production owing to their

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high growth rate, population density, and oil content (7). Algae have much higher productivity (90,000 l of biodiesel per hectare) than soybean (450 l/ha), rapeseed (1200 l/ha), or oil palm (6000 l/ha) (12). In addition to their high yields, macroalgae cultures are not land-intensive and may provide further benefits of wastewater remediation or nutrient reduction (7). Bioethanol application Ethanol, (C2H5OH, ethyl alcohol) is produced by the fermentation of carbohydrate materials. Ethanol is used for production of alcoholic beverages, for industrial purposes (as a solvent, disinfectant, or chemical feedstock) and, in recent years, as a blending agent with gasoline to increase octane and reduce carbon monoxide and other smog-causing emissions. Fuel ethanol can be used in a variety of ways. Ethanol is commonly used as an oxygenated fuel additive to reduce emissions of carbon monoxide, nitrous oxides and hydrocarbons (70). Numerous common ethanolic motor-fuel formulations are being used with increasing frequency. Ethanol has a higher octane rating than petroleum fuels enabling combustion engines to run at higher compression ratios and thus give superior net performance (70). In addition, ethanol exhibits higher vapour pressure and heat of vaporization than gasoline and therefore increased power outputs are observed while using ethanol (73). Low-level ethanol blends such as E10 (10% ethanol/90% gasoline) can be used in conventional vehicles; while highlevel blends, such as E85 (85% ethanol/15% gasoline) can only be used in specially designed vehicles, such as flexible fuel vehicles (FFVs). Ethanol demand and production perspectives The demand for bioethanol is expected to increase dramatically until 2020. In 1999 the US signed an executive order specifying a tripling in the production of biobased products and bioenergy by the year 2010. As a consequence, US oil imports will be reduced by nearly 4 billion barrels over that time. Efforts to decrease greenhouse gas (GHG) emissions are expected to spur the production of renewable energy sources by 6% within the European Union by 2010 (73). In France, the approval of a clean air act could increase ethanol production to 500 million liters. Similar projects in Spain, Sweden and the Netherlands are expected to increase the utilization of ethanol to account for 15 % of BIOTECHNOL. & BIOTECHNOL. EQ. 24/2010/SE
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transportation fuels by 2010 (36). The EU market for fuel ethanol will grow considerably in the coming years, as a result of the EU policy to substitute 8% of fossil transport fuels by renewable biofuels by the year 2020. The cost of raw material dominates the cost of total ethanol production. To attain commercial interest, the costs of bioethanol production must be reduced, and a sufficient amount of cheap and readily available raw material is a necessity. Currently, the lowest cost routes are to produce bioethanol from US corn or Brazilian sugar cane. Process options which involve the importation of intermediate products (sugar concentrate) prior to processing are less costeffective. None of the biofuel options are currently cost competitive with petrol or diesel on a pre-tax basis. The lowest cost biofuel, bioethanol from Brazilian sugar cane, is about 40% more expensive than gasoline on an energy basis. According to some studies (64) by 2020, minimum costs of bioethanol are expected to fall by about 10% compared to 2002 values. The perspective for the fuel pathways for bioethanol production up to 2020 are (Table 1): In the EU countries for bioethanol production from wood, straw, wheat or corn. for the North America for bioethanol from wood, straw, wheat or corn. In South America for bioethanol from sugar cane. In Eastern Europe for bioethanol from wood, straw, wheat or corn.

Feedstocks for bioethanol production In the first-generation technology bioethanol is produced by converting sugars directly (first-generation technology) from crops like sugarcane or sugar beets, indirectly through starch from corn, wheat, potatoes, or cassava into ethanol via fermentation followed by distillation (67). In the second generation technology ethanol is produced through cellulose from biomass (second-generation technology). The largely used substrate, however, are the food crops sugarcane and sugar beet. They contain large amounts of sucrose, which can be converted into its monomeric components (Table 2) and contribute to 60% of the worlds bioethanol production (73). Sugarcane and sugar beet byproducts including wastes like bagasses and molasses, and fruit juice are also used for alcohol production. Other crops, including corn (maize) and cereal crops like

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wheat contain starch that is converted into glucose during fermentation. Tapioca, barley, oats, sweet sorghum, rice and potato are also used. 11.7 kg of corn starch can produce about seven liters of ethanol. The expansion of biofuels production, particularly in the United States, together with increased world-wide demand for grains and increased energy costs, has led to drastically higher grain prices (14, 16). Agricultural wastes including corn (maize) stover, barley, wheat and rice straw, corncobs, sunflower stalks and heads, cotton waste, brewer's spent grain, grape pomace, tomato and orange peels etc., and wood constitute the source of cellulosic and lignocellulosic materials (51). Wheat straw is an abundant agricultural residue in Europe and Asia, and can be used as a raw material for bioethanol production (50, 60). TABLE 1

Lignocellulosic biomass typically contains 50%-80% (dry basis) carbohydrates that are polymers of 5C and 6C sugar units (Table 2). Yield of ethanol from lignocellulose however is low, because of lack of suitable technology. Lignocellulosic raw materials minimize the potential conflict between land use for food (and feed) production and energy feedstock production. The raw material is less expensive than conventional agricultural feedstock and can be produced with lower input of fertilizers, pesticides, and energy. Biofuels from lignocellulose generate low net greenhouse gas emissions, reducing environmental impacts, particularly climate change. Lignocellulosis biofuels might also provide employment in rural areas.

Fuel pathways considered for production of bioethanol up to 2020; adapted from (64) Option 1 2 3 4 5 6 Fuel type Bioethanol Bioethanol Bioethanol Bioethanol Bioethanol Bioethanol Raw material Wood Straw Wheat Corn Sugarcane Sugar beet Processing Acid hydrolysis+fermentation Acid hydrolysis+fermentation Malting+fermentation Fermentation Fermentation Fermentation

TABLE 2 Sources of sugars for ethanol production Sources 1. Crops 2. Lignocellulose C. Cellulose D. Hemicellulose E. Lignin Glucose Glucose + Galactose + Mannose + Xylose + Arabinose + Other (L-Rhamnose, L-Fucose, Uronic acids) Lignols (coniferyl, sinapyl, coumaryl) Carbohydrates A. Sucrose B. Starch Hydrolysis products Fructose + Glucose Glucose

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TABLE 3 Process conversion efficiencies; data adapted from (16, 64). Option Fuel type Process Conversion ciency effi2002 efficiency (GJ bioethanol/ GJ feedstock) 1 2 3 4 5 Bioethanol Bioethanol Bioethanol Bioethanol Bioethanol Wood-acid hydrolysis Straw- acid hydrolysis Wheat Sugarcane Sugar beet 47% 40% 349 l/t 80 l/t 85 kg/t 0.47 0.40 0.53 0.38 0.13

TABLE 4 Future process conversion efficiencies; data adapted from (16, 64) Option Fuel type Process Estimated efficiency improvement to 2020 (%) 1 2 3 4 5 6 7 Bioethanol Bioethanol Bioethanol Bioethanol Bioethanol Bioethanol Bioethanol Wood-acid hydrolysis Straw- acid hydrolysis Wheat Corn-wet milling Corn-dry milling Sugarcane Sugar beet +5% +5% +10% +20% +20% 0 +5% 0.49 0.42 0.59 0.67 0.66 0.38 0.13 2020 efficiency (GJ bioethanol/ GJ feedstock)

Fig. 1. Production of bioethanol from sugar, starch, wood or straw

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TABLE 5 Co-product yields; data adapted from (16, 64) Option 1 Fuel type Bioethanol Process Acid hydrolysis straw Co-product Electricity Ash Acetic acid 2 Bioethanol Wheat Straw Bran Animal feed 3 Bioethanol Corn-wet milling Corn oil Corn feed Corn meal 4 5 Bioethanol Bioethanol Sugarcane Sugar beet Electricity Pulp Animal feed Co-product yield 1.83 GJ/t Ethanol 0.38 t/t Ethanol 0.12 t/t Ethanol 18.5 t/t Ethanol 0.12 t/t Ethanol 1.51 t/t Ethanol 1.89 kg/GJ corn 12.85 kg/GJ corn 3.07 kg/GJ corn 100 kWh/t sugarcane 2.75 t/t Ethanol 0.75 t/t Ethanol

Processes used for bioethanol production

Summaries of process conversion efficiencies and co-product yields are presented in Tables 3-5. Table 3 summarises the data available for 2002 on process conversion efficiencies (product yields) for the developed biodiesel and bioethanol pathways. Efficiencies are expressed in original units from the literature and as GJ biofuel per GJ feedstock. Table 4 shows projected improvements in process conversion efficiencies for 2020 in comparison to 2002. The information about the co-product yields is summarised in Table 5 and should be considered as illustrative only. Bioethanol from fermentation sugarcane or sugar beet using

up to 110C to reduce microbial contamination, decanted, sometimes concentrated by evaporation and then fermented. In combined sugarethanol plants, sucrose crystals that are formed after cane-juice concentration are removed by centrifugation, leaving syrup (molasses) that contains up to 65% w/w sugars. Both sugarcane juice and molasses (after adjusting the sugar concentration) normally contain sufficient minerals and organic nutrients to be immediately suitable for ethanol production by fermentation with Saccharomyces cerevisiae. Sugarcane has an energy production per hectare that is substantially higher than the other feedstocks, but the process conversion efficiency is only about 0.35-0.40 GJ bioethanol per GJ feedstock and yield of 80 l ethanol/ton (Table 3). The sugarcane residue or bagasse can be burned to generate electricity, producing about 0.08 GJ electricity per GJ feedstock (Table 5).

This is the simplest of all the processes for producing bioethanol by fermentation (Fig. 1). The harvested sugarcane or sugar beet is crushed and then soluble sugars are extracted by washing through with water. Yeast is added and fermentation takes place under similar conditions to that in the above processes (17).

During the fermentation of sugarcane, up to 10% of available sugar can be diverted away from ethanol and converted to glycerol and succinic acid. Attempts to improve Sugarcane (Saccharum officinarum) contains 1217% ethanol yields by reducing the synthesis of these unwanted total sugars on a wet-weight basis (90% sucrose and 10% metabolites has resulted in reduced yeast competitiveness glucose or fructose). The average extraction efficiency to within fermentors (70). Although yeasts with improved produce cane juice by crushing is approximately 95% and the properties such as ethanol and temperature tolerances have remaining solid residue is cane fibre (bagasse) (16). In been genetically engineered, such strains are not yet used factories that only produce ethanol, the cane juice is heated widely by the fuel ethanol industry (73). It is now considered 534 BIOTECHNOL. & BIOTECHNOL. EQ. 24/2010/SE SECOND BALKAN CONFERENCE ON BIOLOGY
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that the process could be not more improved ( Table 4). Bioethanol from wheat from malting and fermentation The process is similar to that for other methods producing bioethanol by fermentation, but an initial milling and malting (hydrolysis) process is necessary. The wheat is first crushed or milled. The valuable bran and germ are usually removed first by dry processing in a flour mill before steeping in water. In its passive form, malting is a process by which under controlled conditions of temperature and humidity, enzymes present in the wheat break down starches into C6 sugars. However, this process is very slow, and the commercial process introduces artificial enzymes to break down the starch into sugar (17). These sugars are washed out of the wheat with water, whilst the leftover residue can be sold for animal feed (Table 5). The C6 sugars are then fermented using yeast at between 32 and 35oC and pH 5.2. Ethanol is produced at 10-15% concentration and the solution is distilled to produce ethanol at higher concentrations. The current conversion efficiency of the process is about 0.53 GJ of ethanol per GJ wheat (349 liters of ethanol/t; Table 3) (50) with a possibility to increase up to 0.59 GJ of ethanol per GJ wheat in 2020 (Table 4). Bioethanol from corn using fermentation This is similar to the process for wheat, but with small differences in the initial processing of the corn ( Fig. 1). Firstly, the maize (Zea mays) must be milled, either by wet milling or dry milling. The United States is the main producer of alcohol from corn, and the split between the use of wet and dry milling is fairly even. The milling produces co-products of residues which can be sold as animal feed. For wet milling, several types of residues are produced; dry milling produces only one type of animal feed product ( Table 5). In the wet milling the grain is soaked (steeped) in water with sulfur dioxide for up to 40 h, followed by grinding and separation of starch and co-products. The starch fraction is gelatinized by cooking at low and high temperatures, followed by the addition of -amylase, which yields dextrin oligosaccharides. In the final saccharification process, glucoamylase converts the starch to glucose, which can eventually be fermented to ethanol. The main difference with dry milling is that the entire grain is milled to a median diameter of approximately 1 mm and the different components of the cereal grain are not fractionated before the BIOTECHNOL. & BIOTECHNOL. EQ. 24/2010/SE
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water and enzyme are added; the slurry is processed as in wet milling. In both systems, the sugar-containing juice that leaves the processor is essentially sterile, and this is a crucial point in the subsequent successful downstream processing (16). The current conversion efficiency of both wet and dry milling process routes is about 0.53 GJ of ethanol per GJ wheat (64) (Table 3). The processes are well established but there is some limited scope for efficiency improvements (Table 4). Bioethanol from wood or straw using acid hydrolysis and fermentation The process of producing ethanol from wood or straw feedstocks requires the production of ethanol from both C5 and C6 sugars - unlike the only the C6 sugars in conventional ethanol production from sugarcane (Fig. 1).This process is technically feasible but is complex and expensive and there are few industrial examples. Ongoing research and development in the US aims to address cost issues and develop a more efficient process (17, 64). This is thought by many to be a step on the way to the eventual goal of an enzyme hydrolysis process. Hydrolysis can also be done using dilute sulfuric acid through layers of sawdust and wood chip at high temperature and pressure. Sugars produced by hydrolysis must be quickly removed since they are destroyed by acids. The hexoses and pentoses obtained by hydrolysis are subjected to anaerobic fermentation by yeast, bacteria and filamentous fungi. The current conversion efficiency is about 0.47 GJ of ethanol per GJ wood (Table 3). It is expected that the process could be improved up to 2020 and then conversion efficiency could reach 0.49 GJ of ethanol per GJ wood (Table 4). Bioethanol from wood or straw by enzyme hydrolysis and fermentation This process is not yet well developed enough to be put into large volume practice, but is expected to be commercially viable by 2020 (16). It is essentially similar to the process by which ethanol is produced from wood and straw through acid hydrolysis and fermentation, except that enzymes instead of acids are used for the hydrolysis process. The process is classified as the carbohydrate platform where the biomass (woodchips) is pretreated (broken down) into cellulose, hemicellulose and lignin using heat, pressure and catalyst to expose the sugar polymers. The pretreatment step is followed SECOND BALKAN CONFERENCE ON BIOLOGY
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by separate enzymatic hydrolysis of cellulosic and lignocellulosic materials for converting them to C5 and C6 sugars by the respective enzymes produced on a large scale by microorganisms and then the simple sugars can be fermented into ethanol by fermentation (Fig. 1). Separate enzymatic hydrolysis and fermentation (SHF) could be used. These two steps could be carried out also simultaneously in a process known as simultaneous saccharification and fermentation (SSF). The latter has been claimed to be superior to SHF, owing to a higher resulting ethanol yield and lower capital cost (17). The lignin from the wood is separated from the ethanol and dewatered sufficiently to burn in a boiler as fuel (Table 5). There is enough energy content in the lignin to permit generation of all process steam and electricity required by the process and still have surplus for export to the electrical grid. Since the source of the fuel for the electricity is from the tree (as opposed to a fossil fuel), the electricity is considered green.

than starch (e.g. corn) and sucrose (e.g. sugarcane) producing crops and available in large quantities. Agricultural lignocellulosic residues are abundant renewable resources for bioconversion to sugars, which can then be fermented to fuel ethanol. The most important benefit of fuel ethanol production from biomass is reduced CO2 emissions, thus reducing the greenhouse effect (28, 29). Lignocellulosic biomass is a complex substrate (Table 6), typically contains 50%-80% (dry basis) carbohydrates that are polymers of 5C and 6C sugar units. The two types of polysaccharides, cellulose (~45% of dry weight) and hemicellulose (~30% of dry weight) (Table 6), are bound together by a third component lignin (~25% of dry weight) (60). The lignin, which is a complex three-dimensional polyaromatic matrix, is partly covalently associated with hemicellulose, thus preventing hydrolytic enzymes and acids from accessing some regions of the holocellulose. Specifically, cellulose is a linear, insoluble biopolymer composed of repeating D-glucopyranose residues linked by -1,4 glycosidic bonds. In contrast to other glucan polymers, such as starch, the repeating unit of cellulose is not glucose, but cellobiose, a disaccharide. Cellulose exhibits a high degree of polymerization: the individual glucan chains, or cellodextrins, can reach lengths of greater than 25 000 glucose residues (34). Cellulose produced by plants is composed of both highly amorphous regions containing large voids and other irregularities as well as tightly packed crystalline regions. The crystalline structure of the cellulose however makes it difficult to hydrolyze. Hemicellulose is a highly branched heteropolymer containing sugar residues such as hexoses (D-galactose, Lgalactose, D-mannose, L-rhamnose, L-fucose), pentoses (Dxylose, L arabinose), and uronic acids (D-glucuronic acid) (Table 2). Hemicellulose is more easily hydrolyzed than cellulose. The composition of hemicellulose depends on the source of the raw material. Lignin, the most abundant aromatic polymer in nature, is a macromolecule of phenolic character, being the dehydration product of three monomeric alcohols (lignols) ( Table 2), trans-p-coumaryl alcohol, trans-p-coniferyl alcohol, and trans-p-sinapyl alcohol, derived from p-cinnamic acid (73). For lignocellulose to be amenable to fermentation, it needs to undergo treatments that release its monomeric sugars, which then can be converted by a microorganism. The SECOND BALKAN CONFERENCE ON BIOLOGY
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Bioethanol from lignocellulose

Lignocellulose-to-ethanol production technology has been investigated intensively in the US, Canada, Sweden, Finland, and Netherlands in the last two decades (17). Lignocellulosic biomass feedstock The cellulosic resources represent the most abundant global source. Cellulose is the most abundant biopolymer on earth; an estimated 7.51010 tons are annually synthesized through photosynthetic processes. It has been estimated that approximately half of the carbon fixed annually within terrestrial ecosystems is stored as cellulose. Found primarily in plant cell walls (plant cell walls are about 75 percent complex sugars), cellulose is embedded in a hetero-matrix composed of xylan, other hemicelluloses and lignin ( Table 6). Cellulose synthesis is primarily associated with plants; however some animals, bacteria and algal species can also produce the polymer (34). Cellulosic biomass including forestry residue, agricultural residues, pulp mill refuse, switch grass and lawn, garden wastes and municipal solid wastes (MSW), is a potential feedstocks for the synthesis of biofuels. Lignocellulosic biomass is a renewable resource and has great potentials for the production of fuel ethanol because it is less expensive

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two main steps are: (i) a pretreatment (by physical or chemical procedures) that releases hexoses and pentoses from hemicellulose, and (ii) an enzymatic treatment (or, alternatively, hydrolysis by chemical procedures) that generates glucose from cellulose. The cellulose hydrolysis is the main bottleneck in cellulosic fuel production. There is no microorganism currently available that can utilize lignin monomers for ethanol production. The hydrolysis processes used in the past are essentially chemical processes, but the acid recovery costs and the formation of toxic products make them uncompetitive. They are now substituted by enzymatic processes, which are more specific and allow higher hydrolysis yields under less severe conditions. The following problems could be solved in the lignocellulose-to-ethanol process: 1. Efficient de-polymerization hemicellulose to soluble sugars; of cellulose and

requirements, which make up a large part of the production cost. Pretreatment techniques have generally been divided into three distinct categories, including physical, chemical, and biological pretreatment. Physical pretreatment does not use chemical agents, and typically includes uncatalyzed steam explosion, liquid hot water pretreatment (LHW), mechanical comminution, and high energy radiation (13, 14). Pretreatment with proton beam irradiation, steam explosion and microwave reactors are also under investigation. Uncatalyzed steam-explosion is also named autohydrolysis, in which only steam water is used. It is one of the most common pretreatment methods for lignocellulosic biomass (16). In this method, biomass particles are rapidly heated by high-pressure saturated steam for a period time to promote the hemicellulose hydrolysis. This process is terminated by swift release of pressure, which renders the biomass undergo an explosive decompression. During the pretreatment, the hemicellulose is often hydrolyzed by organic acids such as acetic acids and other acids formed from acetyl or other functional groups, released from biomass. A problem for most pretreatment methods is the generation of compounds that are inhibitory towards the fermenting microorganisms, primarily phenols. Degradation products that could have inhibitory action in later fermentation steps are avoided during pretreatment by wet oxidation. Wet oxidation, a reaction involving oxygen and water at elevated temperature and pressure, was presented in the early 1980s to pretreat lignocellulose (wood) as an alternative to the well-studied steam explosion (52). Compared to other pretreatment processes, wet oxidation has been proven to be more efficient for treating some lignocellulosic materials, because the crystalline structure of cellulose is opened during the process. Organic molecules, including lignin, decompose to CO2, H2O, and simpler and more oxidized organic compounds, mainly to low-molecular-weight carboxylic acids (16). Wet oxidation appears to have the advantage of producing fewer byproducts, such as furfural and hydroxymethylfurfural (34). Under the conditions of wet oxidation, aliphatic aldehydes and saturated carbon bonds are very reactive; hence, the sugar degradation products, which are known inhibitors of microbial growth (13), are not

2. Efficient fermentation of a mixed-sugar hydrolysate containing six-carbon (hexoses) and five-carbon (pentoses) sugars; 3. Advanced process integration to minimize process energy demand; 4. Cost-efficient use of lignin. Pretreatment Conventional production of ethanol from cellulose via fermentation involves a complex process of pretreatment in attempt to recover a maximum amount of sugars from the hydrolysis of cellulose and hemicellulose, and to ferment them into ethanol. Pretreatment is required to alter the biomass macroscopic and microscopic size and structure as well as its submicroscopic structural and chemical composition and to facilitate rapid and efficient hydrolysis of carbohydrates to fermentable sugars. The pretreatment aims to increase pore size and reduce cellulose crystallinity. In acid-catalyzed pretreatment, the hemicellulose layer is hydrolyzed, whereas in alkali-catalyzed pretreatment, mainly, a part of the lignin is removed and hemicellulose has to be hydrolysed by the use of hemicellulases. Hence, pretreatment is necessary to expose the cellulose fibres to the enzymes or to at least make the cellulose more accessible to the enzymes. An efficient pretreatment can substantially reduce the enzyme

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expected to be produced at high concentration. In liquid hot water pretreatment (LHW), pressure is utilized to maintain water in the liquid state at elevated temperatures. It has been shown to remove up to 80% of the hemicellulose and to enhance the enzymatic digestibility of pretreated biomass materials such as corn fiber (17) and sugarcane bagasse. Lignin is typically removed after pretreatment and then burned in the refinerys boiler, replacing some fossil fuel use. A size reduction step is required before most chemical and thermochemical pretreatment processes. Biomass materials can be comminuted by various chipping, grinding and milling. The shredded, chopped or pelletized biomass is typically mixed with dilute acids or ammonia. Chemical pretreatments with acidic or basic catalysis, especially at high temperature (including steam explosion), are effective methods. During these processes, hemicellulose and lignin may be hydrolyzed to their monomeric constituents and lignin-cellulose-hemicellulose interactions are partially disrupted, thus increasing the enzymatic digestibility of cellulose (14). The acid pretreatment uses concentrated or diluted acids. When diluted acid hydrolysis is applied the xylose yield is significantly higher. Acid-catalyzed pretreatment primarily solubilizes the hemicellulose fraction into the liquid phase. For softwood, the liquid mainly contains solubilized mannose in addition to small amounts of xylose, arabinose, galactose and glucose. The solid phase comprises lignin and cellulose, the latter of which is subjected to enzymatic hydrolysis. The maximum cellulase activity of most fungal-derived cellulases and glucosidases is observed at ~50 C and at a pH of 4.05.0; however, the optimal conditions vary with the hydrolysis time and are dependent on the source of the enzymes (62). However, the production cost of enzymes is still too high and requires further reduction. One way to achieve this is to use a fraction of the feedstock and/or the hydrolysate for in situ enzyme production by fungi or other microorganisms. In catalyzed steam-explosion some acidic chemicals (gases and liquids), primarily including SO 2, H2SO4, CO2, oxalic acid, etc. are used as catalysts to impregnate the biomass. Steam pretreatment of SO2 impregnated spruce chips yields a material that is relatively easy to hydrolyze and ferment, whereas dilute acid impregnation results in a material harder to ferment because of the generation of

inhibitory compounds (58). With present bench-scale technology, it is possible to obtain around 300 liters of ethanol per metric ton spruce, which is ~70% of the theoretical overall yield based on hexose sugars (58, 71). Steam pretreatment with the addition of a catalyst for hydrolysis and improved enzymatic digestibility is the closest to commercialization It has been widely tested in pilot-scale equipment, for example, in the Iogen pilot plant (Canada) (55), the Souston pilot plant (France) (52) and in the pilot plant in rnskldsvik (Sweden), and is used in a demonstration-scale ethanol plant at Iogen (Canada). It is also to be used in Salamanca (Spain), in a plant constructed by the company Abengoa. Alkaline pretreatment is basically a delignification process, in which a significant amount of hemicellulose is solubilized as well. In comparison with other pretreatment technologies, alkali pretreatment usually uses lower temperatures and pressures, even ambient conditions. Pretreatment time, however, is recorded in terms of hours or days which are much longer than other pretreatment processes. In ammonia fiber/freeze explosion (AFEX), the biomass is exposed to hot liquid ammonia under high pressure for a period time, and then the pressure is suddenly released. This swift reduction of pressure opens up the structure of lignocellulosic biomass leading to increased digestibility of biomass. AFEX pretreatment simultaneously delignify and solubilize some hemicellulose while decrystallizing cellulose (11). Biologic pretreatments apply lignin-solubilizing microorganisms including white-, brown-, soft-rot fungi, and bacteria to modify the chemical composition and/or structure of the lignocellulosic biomass so that the modified biomass is more amenable to enzyme digestion. These methods are environmentally friendly and energy saving but are relatively slow, and most lignin-solubilizing microorganisms also solubilize or consume hemicellulose and cellulose to grow (14). Different types of pretreatment have been studied, but three combined and chemical methods appear more efficient: dilute acid hydrolysis, steam explosion with catalyst addition and thermohydrolysis. These pretreatments could result in high hydrolysis yields of the cellulose fraction (close to 100%), and in a maximum recovery of the sugars from the

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hemicellulosic fraction. Most probably, there will not be one general method because different types of raw material require different pretreatments. So far, methods such as ammonia fiber explosion (AFEX), wet oxidation and liquid hot water (LHW) treatment seem to be more successful for agricultural residues (11, 65, 66), whereas steam pretreatment has resulted in high sugar yields for both forestry and agricultural residues. Glucose yields >90% and xylose yields >80% were obtained after enzymatic hydrolysis, both with and without the addition of an acid catalyst (43, 46, 54, 58). Enzymatic hydrolysis Cellulose can be hydrolytically broken down into glucose in a Separate hydrolysis and fermentation (SHF) process either enzymatically by cellulytic enzymes or chemically by sulfuric or other acids. Hemicellulose, a branched polymer composed of pentose (5-carbon) and hexose (6-carbon) sugars, can be hydrolyzed by hemicellulases or acids to release its component sugars, including xylose, arabinose, galactose, glucose and/or mannose. Hexoses such as glucose, galactose, and mannose are readily fermented to ethanol by many naturally occurring organisms, but the pentoses including xylose and arabinose are fermented to ethanol by few native strains, and usually at relatively low yields (16). A consortium of enzymes - endoglucanase, exoglucanase and -glucosidase (cellobiase), collectively known as cellulase, are needed to break down cellulose into its constituent glucose monomers. Although they are commercially available, usually derived from the fungus Trichoderma reesei, their high cost is a major concern (14). The cost of the enzymes is about 1020% of the ethanol production cost. Various approaches to reducing costs include improving cellulase production by fungi, recycling the cellulase, binding the cellulase to inert supports, cloning the relevant genes into bacteria such as Escherichia coli and the use of bacterial species such as Klebsiella oxytoca, which naturally possess some of these genes. Employment of enzymes for the hydrolysis of lignocellulose is considered the prospectively most viable strategy to offer advantages over other chemical conversion routes of higher yields, minimal byproduct formation, low energy requirements, mild operating conditions, and environmentally friendly processing. Although the enzymatic BIOTECHNOL. & BIOTECHNOL. EQ. 24/2010/SE
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route has the highest cost at present, it has long-term potential for cost reductions compared to other more established routes such as concentrated acid and two-stage dilute acid hydrolysis. Despite recent advances in engineering cellulases to be more efficient and less costly, the complete saccharification of pretreated lignocellulose still requires a very long time for digestion and high loadings of enzyme (3050 mg enzyme per g of crystalline cellulose). Today, only fungi naturally produce the needed titers of cellulase to meet this need (23). Substrate utilization and fermentation The last technological barrier of the process concerns the ethanolic fermentation of the pentoses. Contrary to sucroseand starch-based ethanol production, lignocellulose-based production is mixed-sugar fermentation in the presence of inhibiting compounds low molecular weight organic acids, furan derivatives, phenolics and inorganic compounds released and formed during pretreatment and/or hydrolysis of the raw material (27). Cellulose monomers are principally glucose and cellobiose, whereas hemicellulose monomers are a mixture of hexoses (principally glucose, with some mannose and galactose) and substantial amounts of pentoses (principally xylose, with some arabinose). Lignocellulosic raw materials, in particular hardwood and agricultural raw materials, can contain 520% (or more up to 40%) of the pentose sugars xylose and arabinose. Xylose is by far the most abundant pentose sugar, whereas arabinose can constitute as much as 1415% in corncob hulls and wheat bran, respectively. The five sugars of interest for fermentation are glucose, mannose, galactose, xylose, and arabinose ( Table 2). The hexoses can be fermented to ethanol by the most commonly used industrial fermentation microorganism S. cerevisiae and the ethanologen bacterium Zymomonas mobilis. Although Z. mobilis can produce ethanol from glucose at final concentrations of 130 g/L, and S. cerevisiae fermentations can reach final concentrations of 160 g/L, these microorganisms do not ferment xylose or arabinose. Nobody has yet identified a microorganism which is able to ferment the pentoses into ethanol with performances similar to those of Saccharomyces cerevisiae on glucose (24).

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Fermentation processes configuration

Separate hydrolysis and fermentation (SHF) In this system the substrate flow from the pretreatment and hemicellulose hydrolysis is subjected to enzyme hydrolysis by the cellulose enzyme complex. Then the flow enters the glucose fermentation reactor. The mixture is after that distilled to remove the ethanol. In a second reactor the xylose is fermented to ethanol, and the ethanol is again distilled. The cellulase production uses substrate from the hemicellulose hydrolysis (16). Simultaneous saccharification and fermentation (SSF) Current strategies to produce fuel ethanol from cellulose, referred to as second-generation biofuels, utilize simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and co-fermentation (SSCF) (34, 35). Both SSF and SSCF require extensive pretreatment of the cellulosic feedstock by steam-explosion and/or acid treatment, followed by addition of exogenously produced cocktails of cellulolytic enzymes to hydrolyse cellulose chains and release the glucose monomers required for fermentation. Simultaneous saccharification and fermentation (SSF) is a process option for production of ethanol from lignocellulose and consolidates hydrolysis of cellulose with the direct fermentation of the produced glucose. Both hydrolysis of cellulose by the cellulase complex and fermentation of hexoses by the ethanologenic microorganism are coupled in one vessel (21). The pentoses are fermented before the hydrolysis of cellulose in a separate fermenter. Cellulase is produced in a separate fermentor using hemicellulose hydrolysate. The principal benefits of performing the enzymatic hydrolysis together with the fermentation, instead of in a separate step after the hydrolysis, are the reduced end-product inhibition of the enzymatic hydrolysis, and the reduced investment costs. SSF is today important in the dry-milling process in the cornbased ethanol industry in the US. The yield from the SSF are in the range of 8085% on the basis of total carbohydrates. The simplest and original SSF is a batch process in which substrate, enzymes and yeast are all present in the reactor initially, and at the intended concentrations (34, 48).

activity, in order to achieve higher efficiencies. The SSF process improves the enzyme efficiency by reducing the feed-back inhibition from the hydrolysis products. Combining cellulose hydrolysis and glucose fermentation in one vessel could improve rates, yields, concentrations. The big benefit of SSF is that it reduces sugar inhibition to enzymes, realizing high-solids fermentation, improved cellulose conversion rates, increased ethanol concentration, low enzyme loadings. The screening of efficient fermentative microorganisms under high temperature conditions has to be further implemented because the optimal saccharification temperature is 45C, and the optimal fermentation temperature is 30C (23). The disadvantages of SSF in comparison to the separate hydrolysis and fermentation (SHF) process are (16, 35, 48): i/ The optimum temperature for enzymatic hydrolysis is typically higher than that of fermentation at least when using yeast as the fermenting organism. In an SHF process, the temperature for the enzymatic hydrolysis can be optimized independently from the fermentation temperature, whereas a compromise must be found in an SSF process. ii/ Furthermore, the yeast cannot be reused in an SSF process due to the problems of separating the yeast from the lignin after fermentation. Therefore, the yeast will necessarily represent a yield loss in an SSF process, if the yeast is produced from carbohydrates within the process or a running cost if it is externally supplied. iii/ The enzymes are equally difficult to reuse, also in an SHF process. The enzymes are either produced within the process thereby representing a loss of substrate or are externally supplied and thereby add to the chemical costs. iiii/ Re-circulation of enzymes is equally difficult since the enzymes bind to the substrate, although a partial desorption can be obtained after addition of surfactants (48). Simultaneous (SSCF) saccharification and co-fermentation

Enzymatic hydrolysis has to be improved in order to reduce the cost of consumption of the enzymes (48). Research works will have to focus upon the enzyme specific 540 BIOTECHNOL. & BIOTECHNOL. EQ. 24/2010/SE
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This process represents hydrolysis of the cellulose and cofermentation of pentose and hexose sugars by xylose- and glucose-fermenting microorganisms in one vessel. Cellulase is produced separately using a hemicellulose hydrolysate. The microorganisms are genetically engineered. Progress is rapid in the field of xylose fermentation, but few industrial yeast strains have yet the demonstrated capability of fermenting xylose in lignocellulosic hydrolyzates efficiently.

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Hahn-Hgerdal et al. (15) recently presented information on the performance of industrial xylose fermenting strains in lignocellulosic hydrolyzates. All strains covered in their summary were xylose reductase (XR) and xylitol dehydrogenase (XDH) expressing strains. Ethanol concentrations reaching 40 g/l and yields up to 80% of the theoretical based on xylose and glucose have been achieved. By-product formation decreased the ethanol yield from xylose with xylose fermenting strains of S. cerevisiae. However, less xylitol was formed by XR/XDH-carrying strains in fermentation of lignocellulosic hydrolyzates (37, 39-41) compared to defined medium, probably due to additional electron acceptors present in the media. Recently, simultaneous saccharification and co-fermentation (SSCF) using cellulase (Spezyme CP) and recombinant Escherichia coli (ATCC-55124) were recently applied to paper mill sludge (21). Ethanol yields of 7581% of the theoretical maximum were obtained from the SSCF on the basis of total carbohydrates. Consolidated bioprocessing (CBP) Breaking down cellulose with enzymes is usually a separate step from fermentation and a very costly one. An alternative route to production of bioethanol is the utilization of microorganisms that can both convert biomass to fermentable sugars and ferment the resultant sugars to ethanol in a process known as consolidated bioprocessing. Consolidated bioprocessing (CBP) is a system in which cellulase production, substrate hydrolysis, and fermentation are accomplished in a single process step by cellulolytic microorganisms in microbial community (72). CBP offers the potential for lower biofuel production costs due to simpler feedstock processing, lower energy inputs, and higher conversion efficiencies than separate hydrolysis and fermentation processes, and is an economically attractive near-term goal for third generation biofuel production. To date, the heterologous production of cellulases has been pursued primarily with bacterial hosts producing ethanol at high yield (engineered strains of Escherichia coli, Klebsiella oxytoca and Zymomonas mobilis) and the yeast Saccharomyces cerevisiae. Cellulase expression in strains of K. oxytoca resulted in increased hydrolysis yields (but not growth without added cellulase) for microcrystalline cellulose (Avicel), and anaerobic growth on amorphous cellulose (75, 76). Kondo and coworkers expressed cellulases

(10), xylanases (22), and amylases (57) on the cell surface of different S. cerevisiae strains. High cell density suspensions of the recombinant strains fermented amorphous cellulose, raw starch, and birchwood xylan to ethanol with yields of 0.45, 0.44 and 0.3 g ethanol/g substrate, respectively. Several cellobiohydrolases have been functionally expressed in S. cerevisiae (1, 19, 48, 61, 77). The specific activity of recombinant cellobiohydrolase was shown to be comparable to that of the native enzyme (61). Several strains of S. cerevisiae with expanded substrate utilization capability have been developed: a xylose-isomerase-expressing strain that grows well on xylose (26); a strain expressing the genes of the bacterial L-arabinose utilization pathway and overexpressing the yeast galactose permease allowing the fermentation of arabinose (4); and a -glucosidase-expressing strain able to grow anaerobically on cellobiose at rates comparable to glucose (61). Microorganisms The industrial fermentation of lignocellulose hydrolysate to ethanol requires microorganisms, which have a broad substrate range, and which produce ethanol with high yield and productivity. Such microorganisms must also tolerate ethanol and inhibitors formed in the pretreatment process. Most research efforts have been devoted to the development of efficient xylose-fermenting microorganisms (20, 24). Two groups of microorganisms - enteric bacteria and some yeasts - are able to ferment pentoses, but with low ethanol yields. Furthermore, in the case of xylose fermenting yeasts (Pachysolen tannophilus, Candida shehatae , and Pichia stipitis), large-scale utilization is hampered by their sensitivity to high concentrations of ethanol (40 g/l), the requirement for carefully monitored microaerophilic conditions, high sensitivity to inhibitors, and the inability to ferment xylose at low pH. The biochemical pathways involved in xylose metabolism are different in enteric bacteria and yeast. Thus, in bacteria, a xylose isomerase (XI) converts xylose to xylulose, which after phosphorylation, is metabolized through the pentose phosphate pathway (PPP) (13). In yeasts, xylose is converted into xylitol and subsequently to xylulose in reactions catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, with NAD(P)H and NAD+, respectively, acting as cofactors. Some filamentous fungi have been shown to ferment most SECOND BALKAN CONFERENCE ON BIOLOGY
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of the sugars found in pretreated biomass hydrolysates, such as glucose, mannose, galactose, xylose, and arabinose. Some fungi, such as Monilia, Fusarium, Rhizopus, Aspergillus, Neocallimastix, and Trichoderma (59), have been reported to possess the ability to convert cellulose to ethanol. Recent genetic improvements focused on the transformation of Saccharomyces cerevisiae and Zymomonas mobilis could result in good fermentative performances on pentoses (Table 7). Three approaches have been attempted to enable the xylose to be utilized: (i) to clone genes from pentose utilizing species into S. cerevisiae; (ii) to co-culture two different strains of genetically modified Zymomonas mobilis; and (iii) to clone pentose-utilizing genes into ethanol-resistant strains of E. coli. Efforts have been made to obtain recombinant strains of

bacteria and yeast able to meet the requirements of industrial lignocellulose fermentation (Table 7). The first xylosefermenting S. cerevisiae strain was generated through the introduction of genes for xylose-metabolizing enzymes from P. stipitis (24). Later xylose-fermenting strains of S. cerevisiae were constructed by introducing the genes encoding xylose isomerase from the bacterium Thermus thermophilus (69) and the anaerobic fungus Piromyces sp. (25), respectively. A xylose fermenting Z. mobilis was generated by introducing a xylose-metabolizing pathway from E. coli (74). More recently, the obligatory anaerobic bacterium Thermoanaerobacterium saccharolyticum has been genetically engineered for improved ethanolic fermentation (13).

TABLE 6 The contents of cellulose, hemicellulose, and lignin in common agricultural residues and wastes (adapted from the literature (50, 60) Lignocellulosic materials Hardwoods stems Softwood stems Nut shells Corn cobs Grasses Paper Wheat sraw Sorted refuse Leaves Cotton seed hairs Newspaper Waste papers from chemical pulps Primary waste water solids Swine waste Solid cattle manure Coastal Bermuda grass Switch grass Cellullose (%) 40-55 45-50 25-30 45 25-40 85-99 30 60 15-20 80-95 40-55 60-78-15 6 1.6-4.7 25 45 Hemicellulose (%) 24-40 25-35 25-30 35 35-50 0 50 20 80-85 5-20 25-40 10-20 not available 28 1.4-3.3 35.7 31.4 Lignin (%) 18-25 25-35 30-40 15 10-30 0-15 15 20 0 0 18-30 5-10 24-29 not available 2.7-5.7 6.4 12.0

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TABLE 7 Fermentation results on lignocellulose hydrolysate from various recombinant and native xylose-fermenting organisms; data adapted from (14, 16) Organism Escherichia coli KO11 E. coli FBF5 Zymomonas mobilis 8b Pichia stipitis P. stipitis CBS 5773 Saccharomyces cerevisiae 424A S. cerevisiae TMB 3006 S. cerevisiae TMB 3400
1

Hydrolysate Bagasse hemicellulose Corn fiber Rice hull Corn stover

7 2 1

Yield g/g inv. sugar 0.49 0.39 0.46 0.43 0.42 0.41 0.35 0.36 0.41 0.37 0.43 0.33
2 3

Fermentation time (h) n/a 93-102 n/a 64 n/a n/a 48 24 n/a n/a 96

Productivity g/g cells.h 0.37 n/a 0.214 n/a n/a 0.44 0.05 0.072 0.044 0.66 0.25 0.036
7

Ref. (14, 16) (45) (13) (53) (14, 42) (13, 44 (14, 16) (56) (56) (14) (14) (47)

Corn stover

Wheat straw Spent sulfite liquor Corn fiber Corn stover Spruce Spruce Corn stover

Supplemented with 2.5% (w/v) corn steep liquor; supplemented at 100 g/l glucose; n/a, not available

supplemented with 5% (w/v) corn steep liquor;

80% hydrolysate

Bakers yeast Saccharomyces cerevisiae is the most commonly used microorganism for industrial ethanol production (5). It has also been shown to efficiently ferment lignocellulosic hydrolysate to ethanol (46). However, S. cerevisiae cannot utilize xylose for growth and ethanol production. Saccharomyces cerevisiae strains with improved capacity to utilize xylose have been acquired through choice of parental strain, random mutagenesis, adaptation and/or breeding (59, 68). When the P. stipitis genes XYL1 and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, were introduced in S. cerevisiae in combination with the endogenous gene XKS1 encoding xylulokinase (XK) (9,18, 63) the resulting strains were able to utilize xylose for growth and ethanol production. Industrial S. cerevisiae strains transformed with genes for XR, XDH and XK are capable to ferment nondetoxified lignocellulose hydrolysates (13). Recently, a haploid laboratory strain of S. cerevisiae has been successfully transformed with a xylose isomerase gene (25).

Conclusions
Ethanol produced from various lignocellulosic materials such as wood, agricultural and forest residues has the potential to be a valuable substitute for, or complement to, gasoline. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. The amount of bioethanol that can be produced from an acre of land varies from 1500 liters per acre for US corn and 2500 liters per acre for Brazilian sugarcane. Cellulosic ethanol could raise yields to more than 3800 liters per acre, significantly reducing land requirements. Cellulose is the worlds most widely available biological material, present in such low-value materials as wood chips and wood waste, fast growing grasses, crop residues like corn

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stover, and municipal waste. US cellulosic fuel production costs are now estimated at more than $0.7 per liter, compared with $0.4 per liter for corn ethanol. Venture capital and government subsidies are supporting companies interested in making cellulosic ethanol commercially viable, primarily in the United States, but also in several other countries, including Canada, Brazil, China, Japan, and Spain. Technological advances and efficiency gainshigher biomass yields per acre and more liters of ethanol per ton of biomasscould steadily reduce the economic cost and environmental impacts of bioethanol production. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes, the enzyme loading and especially the production of cellulases can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis. Although bioethanol production has been greatly improved by new technologies, several research challenges remain in order to further improve the overall yield of ethanol, increase the productivity in the conversion steps and to reduce the production cost. These challenges include also maintaining a stable performance of the genetically engineered yeasts in commercial scale fermentation operations, developing more efficient pretreatment technologies for lignocellulosic biomass, improving fermentation of all sugars available in wood and to make the fermenting organism more tolerant to inhibitors, increasing process integration to reduce the number of process steps, the energy demand and to re-use process streams in order to minimize the use of fresh water and reduce the amount of waste streams.

3.

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