Ethanol from Lignocellulosic Materials ... - ACS Publications

Chapter 3

Ethanol from Lignocellulosic Materials: Pretreatment, Acid and Enzymatic Hydrolyses, and Fermentation 1,2

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Downloaded by UNIV OF GUELPH LIBRARY on July 2, 2012 | http://pubs.acs.org Publication Date: July 29, 2004 | doi: 10.1021/bk-2004-0889.ch003

Mohammad J. Taherzadeh and Claes Niklasson 1

Department of Chemical Engineering and Environmental Science, Chalmers University of Technology, SE-412 96, Göteborg, Sweden Department of Chemical Engineering, Isfahan University of Technology, Isfahan, Iran

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Ethanol production from lignocellulosic materials is a major global task in producing liquid fuel by sustainable processes. The structure of the lignocellulose is usually opened by dilute-acid hydrolysis or steam explosion in a pretreatment step, while the resulting cellulose and hemicelluloses can be cleaved to the monomers (sugars) by acid or enzymatic hydrolyses. The hydrolyzates are then fermented to ethanol by using baker's yeast or other microorganisms. The acid hydrolysis suffers from a number of inhibitory by-products including furans, phenolic compounds and carboxylic acids, whereas the enzymatic one is still expensive and slow. Very good progress has been made within the last two decades on development of pretreatments, hydrolyses, fermentation techniques and recombinant microorganisms. These advances are briefly reviewed here.

© 2004 American Chemical Society

In Lignocellulose Biodegradation; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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Introduction Lignocellulosic materials represent an abundant, and largely unused, source of raw materials for the production of fuel ethanol. These materials can be obtained at a low cost from a variety of resources, e.g. forest residues, municipal solid waste, waste paper and crop residue resources (4). The amounts of the three major groups of polymers, i.e. cellulose, hemicellulose and lignin, depend on the type of material. Garotte et ai (5) present a compilation of compositions of lignocelluloses of different hardwoods, softwoods and agricultural residues reported in publications. The hardwoods (white birch, aspen, red maple, Eucalyptus, Populus and oak) contain 39%-54% cellulose, 14%-37% hemicellulose and 17%-30% lignin. The corresponding values for softwoods (pines and firs) are 41%-50% cellulose, 11-27% hemicellulose and 20-30% lignin. The composition of different agricultural residues varies widely. For instance, wheat straw may consist of up to 50% cellulose, whereas the cellulose content reported for sunflower seed hulls is only 24%. The basic steps necessary for obtaining fermentable sugars include a pretreatment, followed by one or several hydrolysis steps, in which the actual hydrolysis of the polymers into monomeric sugars takes place. The sugars are then fermented to ethanol by using several types of microorganisms. These three steps are discussed separately below.

Pretreatment The purpose of the pretreatment is primarily to open up the structure of the material to facilitate access to the cellulose structure. However, a small amount of sugarsfromthe hemicellulose may be formed already during the pretreatment process. The pretreatment process can be carried out with a variety of methods (6, 7): 1. Physically by e.g. ball-milling, two-roll milling, colloid milling, hammer milling, vibro energy milling, high pressure steaming, extrusion, expansion, pyrolysis, and high energy radiation. 2. Chemically by e.g. alkalis (NaOH, N H , (NH ) S0 ), acids (H S0 , HC1, H3PO4), gases (C10 , NO , S0 ), oxidizing agents (H 0 , 0 ), cellulose solvent (Cadoxen - ethylene diamine and water, or CMCS - sodium tartarate, ferric chloride, sodium sulfite and sodium hydroxide solution) and solvent extraction by ethanol-water, benzene-ethanol, ethylene glycol, or butanol-water. 3. Biologically by e.g. enzymes or fungi. 3

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However, not all of these methods are desired for pretreatment of the lignocellulosic materials because of technical or economic feasibility. In some cases, a method is used to increase the efficiency of another method. For instance, milling could be applied to create a better steam explosion by reducing the chip size (8). Furthermore, it should be noticed that the selection of pretreatment method should be compatible with the selection of hydrolysis. For example, if acid hydrolysis is to be applied, a pretreatment with alkali may not be beneficial. The most commonly applied pretreatment methods will be discussed here.

Steam Explosion Steam explosion is among the mostfrequentmethods for the pretreatment of lignocellulosic materials. The method involves treatment of the chipped biomass with high-pressure saturated steam followed by a rapid reduction of the steam pressure to obtain an explosive decompression. Either high temperature and low residence time (eg. 270°C and 1 min) or lower temperature and longer residence time (e.g. 175°C and 30 min) could be applied for an optimal pretreatment (9). Early processes were running at high pressure (220-270°C) and short residence time (40-90 s), whereas recent investigators use lower temperature (190-200°C) and a longer residence time of about 10 min (10). However, the type of lignocellulose, residence time, temperature, chip size and humidity are important factors in optimizing the treatment conditions of a steam explosion process (11). The variation in these factors results in various degrees of degradation of lignin, hemicellulose and cellulose (12, 13). Gregg and Saddler (14) give examples of recommended time and temperature/pressure to be used for various feedstocks.

Dilute-acid Pretreatment

It is known that the addition of any kind of acid or gases that results in acidic conditions for steam explosion, such as H S0 , S 0 (or H S0 ), or C 0 (or H C0 ), will successfully improve the enzymatic hydrolysis, decrease the formation of inhibitory compounds, and lead to a better removal of hemicellulose (15). The effects are almost similar for hardwoods, softwoods and herbaceous materials. S 0 presumably diffuses into the wood chips as sulfiirous acid, H S0 , which turns into sulfuric acid during steaming (16). Sulfuric acid is the most extensively studied, apparently because of its low price and high efficiency (17). In addition to the factors affecting steam explosion, the acid concentration plays an important role in dilute-acid pretreatment. There are a number of examples of the treatment conditions for various feedstocks in the literature. As examples we 2

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may mention 220°C, 30 s residence time, and 1% H S 0 for the treatment of sugar cane bagasse (18) and 210°C, 2 min residence time and 0.175% H S 0 for the treatment of Eucalyptus grandis (19). More severe conditions (e.g. higher acid concentration, higher temperature and/or longer residence time) will result in degradation of cellulose (2), which will be discussed later in more detail. 2

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Ammonia Fiber (or Freeze) Explosion (AFEX) The concept of AFEX is similar to steam explosion, except in the type of gas: lignocelluloses are exposed to ammonia instead of water vapor. In a typical treatment, one kg dry biomass is exposed to 1-2 kg liquid ammonia at high temperature (e.g. 50-90°C) and high pressure for a period of time (e.g. 30 min), and then the pressure is swiftly reduced (7, 20). The efficiency of the AFEX method in cellulose decrystallization, with increased accessible surface area, dramatically increases lignocellulose susceptibility to enzymatic attack in the hydrolysis step (21). In spite of acid pretreatment, AFEX does not dissolve hemicellulose, which is an advantage for the enzymatic hydrolysis (22). Furthermore, no inhibitory compounds for the fermentation step are formed (23), and the method is not affected by the chip size so that milling is unnecessary. There are many adjustable parameters in the AFEX process: ammonia loading, water loading, temperature, time, blowdown pressure, and number of treatments (21). A lower lignin content of the lignocellulose results in a higher efficiency of the AFEX-pretreated enzymatic hydrolysis. While 90% hydrolysis of cellulose and hemicellulose of Bermuda grass (5% lignin) was obtained, the efficiencies for the hydrolysis of newspaper (18-30% lignin) and aspen chips (25% lignin) were less than 40% and 50%, respectively (24).

Alkaline Pretreatment Some bases such as NaOH, Ca(OH) , NH OH can be used for pretreatment of the lignocelluloses (25). Dilute sodium hydroxide with concentration of 0.8 to 50 g/1 (26) or 5-10 g NaOH/100 g feedstock (6) has primarily been used for this purpose. The mechanism behind this method is believed to be saponification of intermolecular ester bonds crosslinking xylan hemicelluloses and other components (7). Consequently, the base causes swelling, leading to an increased internal surface area, decrease in the degree of polymerization, decrease in the crystallinity, separation of structural linkages between lignin and carbohydrates, and disruption of the lignin structure (6). NaOH showed more effective results in 2

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53 increasing the susceptibility of the residual corn husk towards enzymes, compared to sulfuric and phosphoric acids, yielding 83-96% reducing sugars (26).


Hydrolysis Hydrolysis is the second step in production of ethanolfromlignocellulosic materials. It involves cleaving the polymers of cellulose and hemicellulose. The cellulose usually contains only glucan, whereas hemicellulose contains polymers of several sugars such as mannan, xylan, glucan, galactan and arabinan. Consequently, the main hydrolysis product of cellulose is glucose, whereas the hemicellulose gives rise to several pentoses and hexoses. However, softwood hemicellulose is mainly composed of mannose, whereas the dominant sugar in hardwood hemicellulose is xylose (2). The hydrolysis can be obtained chemically or enzymatically.

Chemical Hydrolysis Chemical hydrolysis involves exposure of lignocellulosic materials to a chemical for a period of time at a specific temperature, and results in sugar monomers from cellulose and hemicellulose polymers. Acids are dominantly applied in chemical hydrolyses. Sulfuric acid is the most investigated acid (27), although other acids such as HC1 (28) have also been used. Acid hydrolyses can be divided into two groups: (a) concentrated-acid hydrolysis and (b) dilute-acid hydrolysis.

Concentrated-acid hydrolysis Hydrolysis of lignocellulose by concentrated sulfuric or hydrochloric acids is a relatively old process. Braconnot in 1819 first discovered that cellulose could be converted to fermentable sugar by concentrated acid (29). Concentrated-acid processes are generally reported to give higher sugar and ethanol yield, compared to dilute-acid processes. However, dilution and heating of the concentrated acid during the hydrolysis process make it extremely corrosive. Therefore, the process requires either expensive alloys or specialized nonmetallic constructions, such as ceramic or carbon-brick lining. The high investment and maintenance costs have greatly reduced the commercial potential for this process. Furthermore, the environmental impact strongly limits the application of hydrochloric acid (30).


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Dilute-acid hydrolysis Among the chemical hydrolysis methods, dilute-acid hydrolysis is probably the most commonly applied. It is a method that can be used either as a pretreatment preceding enzymatic hydrolysis, or as the actual method of hydrolyzing lignocellulose to the sugars (31). The first established dilute-acid hydrolysis process was probably the Scholler process. This was a batch process, in which the wood material was kept in 0.5% sulfuric acid at 11-12 bar for approximately 45 minutes (32). Nowadays, almost all dilute-acid hydrolysis processes are performed in a batch mode with a retention time of a few minutes. However, there have been some studies concerning continuous hydrolysis in plug flow reactors (33). A recent study (2) presents data for one-stage dilute-acid hydrolysis, where 0.5% sulfuric acid was used at temperatures of 188-234°C and a retention time of 7 minutes. A major part of the hemicellulose (more than 80%) could be hydrolyzed by dilute-acid hydrolysis at temperatures less than 200°C, but the maximum overall glucose yield occurred at a hydrolysis temperature higher than 220°C. This is due to the larger recalcitrance of cellulose to hydrolysis. In no case was a better yield than 40% of the theoretical glucose yield from glucan obtained (Figure 1). 1 0.8 0.6 Φ

> 0.4 0.2 ?80

200 220 240 Temp (°C) Figure 1. Glucose yield from glucan (Ώ) and mannose yield from mannan (M) in a one-step dilute-acid hydrolysis of (25% dry weight) spruce as a function of hydrolysis temperature (1). To avoid degradation of monosaccharides at high temperatures, the diluteacid hydrolysis can be carried out in two (or more) stages. In the first stage, which should be carried out at relatively mild conditions, hemicellulose is converted to sugar monomers. It is considered as equivalent to a dilute-acid



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pretreatment step. In the second stage, the residual solid is hydrolyzed at more severe conditions allowing cellulose to be hydrolyzed (34). When dilute-acid hydrolysis is used for pretreatment of lignocellulosic materials, considerably milder conditions than those mentioned above are applied. In a one-stage pretreatment, a temperature between 140 and 170 °C can be used, but two treatments at about 120°C for a longer time may also be used (35). The main drawback of the acid hydrolysis processes is the formation of undesirable by-products. This not only lowers the yield of sugars, but several of the by-products severely inhibit the formation of ethanol in the fermentation process. Potential inhibitors are furfural, 5-hydroxymethylfurfural (HMF), levulinic acid, acetic acid, formic acid, uronic acid, 4-hydroxybenzoic acid, vanillic acid, vanillin, phenol, cinnamaldehyde, formaldehyde, etc. (I, 36). Some inhibitors, such as terpene compounds, are initially present in the wood, but apparently most of the inhibitors are formed in the hydrolysis process.

Enzymatic Hydrolysis Enzymatic hydrolysis of cellulose and hemicellulose can be carried out by highly specific cellulase and hemicellulase enzymes (glycosylhydrolases). This group includes at least 15 protein families and some subfamilies (37). A complete cellulase system consists of three classes of enzymes: 1,4-P-D-glucan cellobiohydrolases, endo-1,4-p-D-glucanases and 1,4-p-D-glucosidases. While the first and second enzymes cleave the cellulose to cellobiose, it is further split to glucose by the third enzyme, β-glucosidase is not a cellulase, but its action is very important to complete depolymerization of cellulose to glucose. Since hemicellulose contains different sugar units, the hemicellulytic system is more complex and involves at least endo-l,4-p-D-xylanases, exo-l,4-p-D-xylosidases, endo-l,4«p-D-mannanases, p-mannosidases, α-glucuronidases, acetyl xylan esterases, a-L-arabinofiiranosidases, and a-galactosidases (38). Several species of bacteria including Clostridium, Cellumonas, Bacillus, Thermomonospora, Ruminococcus, Bacteriodes, Erwinia, Acetovibrio, Microbispora, and Streptomyces and fungi including Tricoderma, Pénicillium, Fusarium, Humicola, Phanerochaete and Schizophillum spp. are able to produce cellulases and hemicellulases (7, 37). Among these microorganisms, Trichoderma was mentioned as the most efficient cellulose-hydrolysing organism (17). The maximum cellulase and p-glucosidase activities occur at 40-60°C and a pH of 4.0 to 5.0. However, the optimal condition may change with the hydrolysis residence time (39, 40). The order of magnitude for the incubation time is 1-3 day. There are several advantages and disadvantages of dilute-acid and enzymatic hydrolyses compared to each other (Table I). Enzymatic hydrolysis is carried out under mild conditions, whereas high temperature and low pH result in corrosive



56 conditions for the acid hydrolysis. While it is possible to obtain a cellulose hydrolysis of close to 100% by enzymatic hydrolysis after a pretreatment (41), it is difficult to achieve such high yield with the acid hydrolysis. Furthermore, the previously mentioned inhibitory compounds are formed during acid hydrolysis, whereas this problem is not so severe for enzymatic hydrolysis. Enzymatic hydrolysis has its own problems in comparison to the dilute-acid hydrolysis. A hydrolysis of several days is necessary for enzymatic hydrolysis (39), whereas a few minutes are enough for the acid hydrolysis (2). The prices of the enzymes are still very high and could be decreased dramatically by increasing the enzyme-specific activity (42). Another problem of the enzymatic hydrolysis is that the sugars released inhibit the enzymes during hydrolysis. In order to overcome this latter problem, simultaneous saccharification and fermentation (SSF) was invented, as distinct from a separate hydrolysis and fermentation (SHF). In SSF, the glucose produced by the hydrolysis is consumed immediately by the fermenting microorganism, which avoids end-product inhibition of βglucosidase. However, since the optimal temperatures of the hydrolysis and fermentation are 45-50°C and 30°C, it is difficult to obtain the entire process at optimum conditions (17).

Comparing variable

Mild hydrolysis conditions High yield of hydrolysis Avoiding formation of inhibitory by-products Avoiding product inhibition during hydrolysis Low cost of hydrolysis Short time of hydrolysis

Dilute-acid Enzymatic hydrolysis hydrolysis

No No No Yes Yes Yes

Yes Yes Yes No No No

Fermentation of the hydrolyzates Fermentation of the lignocellulosic hydrolyzates is more difficult than the well-established processes of ethanol production from e.g. molasses and starch. Hydrolyzates contain a broader range of inhibitory compounds, where the composition and the concentration of these compounds depend on the type of lignocellulosic materials and the chemistry and nature of the pretreatment and hydrolysis processes. Secondly, the hydrolyzates of hemicelluloses contain not only hexoses but also pentoses, where xylose is the dominant sugar in the hydrolyzates from hardwood hemicelluloses. Therefore, the fermenting microorganism should be able to produce ethanol from the hydrolyzates with a


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high yield and productivity, withstand potential inhibitors, and produce ethanol from pentoses, as well as being safe for humans. Baker's yeast (Saccharomyces cerevisiae) is the most commercially used microorganism for ethanol production, but it cannot take up xylose. This section is devoted to the effect of the inhibitory compounds, different fermentation techniques, and the xylose-fermenting microorganisms.


The effects of inhibitory compounds on the fermentation

The by-products mentioned earlier inhibit the fermentation by different mechanisms. As a function of conditions and method of hydrolysis, different byproducts will dominate. Most likely, a combination of the action of several substances is the reason for observed inhibition (43). Furthermore, it is not only the quantitatively dominant inhibitors which determine the fermentability of a hydrolyzate. The toxicity of a hydrolyzate is found to differ from that of a synthetic medium with the same amount of the major hydrolyzate inhibitors added, indicating the importance of other inhibitors present in small amounts (44).

Carboxylic acids Acetic acid, formic acid and levulinic acid are the most common carboxylic acids found in the hydrolyzates. Acetic acid is not only a by-product of hydrolysis (45) but is also a well-known by-product in fermentation (46). Acetic acid is mainly formed from acetylated sugars in the hemicellulose, which are cleaved off already at mild hydrolysis conditions. Therefore, the acetic acid yield in the hydrolysis does not significantly depend on the severity of the hydrolysis process (2). Hydrolysis of hardwoods (alder, aspen and birch) at 198-234°C, 0.5 g/1 H S0 , and 33% wood dry materials for 7 min resulted in approximately 10 g/1 acetic acid, whereas the hydrolysis of softwoods (pine and spruce) produced 3 g/1 acetic acid at similar conditions (2). Acetic acid is sometimes mentioned as an important inhibitor (47, 48). Since acetic acid has a dissociation constant of 4.75 in water, it will be partly dissociated at a pH of 5-5.5 (typical values for the fermentation). It is generally accepted that the effect of the undissociated part of the acid is larger than the effect of the dissociated part (49). The undissociated carboxylic acids can diffuse through the cell membrane (50, 51). Since the intracellular pH is higher than that of the extracellular medium (52), the undissociated acid which has diffused into the cell is partly dissociated into acetate and hydrogen ions, thereby potentially lowering the intracellular pH (53, 54). The optimum extracellular pH for growth 2

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of S. cerevisiae is about 5 (55), although growth is possible down to a pH as low as 2.5. Without added acetic acid, there is no clear effect on the specific growth rate of the yeast cells down to a pH of 3.5 (3). However, a clear effect on both YATP/X and the minimum pH of growth can be observed in the presence of acetic acid in the medium (Figure 2). Formic and levulinic acids are other weak carboxylic acids (dissociation constants 3.75 and 4.4 respectively), which are found in hydrolyzates. Formic acid is most likely formed from degradation of HMF (56, 57), although other parallel formation routes are possible. Formic acid is a stronger inhibitor than acetic acid (57), and acts inhibitory to the fermentation process above a concentration of about 1 g/1 (58). Levulinic acid is a degradation product of HMF (56), and was shown to have a negative effect on fermentability of the hydrolyzates (57). However, due to the low concentration of formic and levulinic acid normally found in hydrolyzates, they are probably of secondary importance with respect to inhibitory effects.

Undissociated acetic acid [g/1]

Total acetic acid [g/1]

Figure 2. (a) The ATP demand for the growth of S. cerevisiae in media containing different concentration of undissociated acetic acid, and (b) region of anaerobic growth of S. cerevisiae as a function of medium pH and total acetic acid concentration. Under the lower line no growth occurs and above the upper line μ £0.2 h" (Reproduced from reference (3). Copyright 1997 Elsevier) 1

Furans Furfural and HMF are the only furans usually found in hydrolyzates in significant amounts (2, 59). The inhibitory effects of these furans have previously not been quite clear, and partly contradictory results have been reported in the literature (see e.g. (60-68)). However, studies in the past few years have provided a better understanding of the physiological effects of these inhibitors (69-74).



59 Furfural has been found to inhibit the in vitro activity of several important enzymes in the primary carbon catabolism such as hexokinase, aldolase, phosphofructokinase, triosephosphate dehydrogenase and alcohol dehydrogenase. Of these, triosephosphate dehydrogenase and alcohol dehydrogenase appeared to be the most sensitive enzymes (64). However, the inhibition of certain non-glycolytic enzymes, such as pyruvate dehydrogenase and aldehyde dehydrogenase, is even more severe (72). Consequently, cell growth is more sensitive to the presence of furfural than the ethanol production from glucose. Interestingly, furfural can be converted by the yeast, forming mainly furfwyl alcohol and furoic acid. This conversion has been reported not only for Saccharomyces cerevisiae, but also for a number of other yeasts of the genera Torulopsis, Pichia and Rhodotorula (70, 75-77). The yeast S. cerevisiae has a capacity to convert furfural at a maximum specific conversion rate of 0.6 g/g-h. However, at that conversion rate, cell growth stops. The maximum conversion rate with a maintained cell growth is approximately 0.15 g/g-h. The clearly dominating product is furfuryl alcohol, and less than 1% of the furfural is converted to furoic acid (70). In addition to furfuryl alcohol and furoic acid, an acyloin product of furfural has been reported (I). This metabolite, 3-(2-furfuryl)2-hydroxy-2-methyl-3-oxo-propanoic acid, is produced when the cells are growing on glucose and a high concentration of furfural (above 2 g/1) is present in the medium. HMF is not as severely toxic to S. cerevisiae as furfural (69). This is in line with the observation that the in vitro inhibition of the enzymes pyruvate dehydrogenase and aldehyde dehydrogenase is smaller by HMF than by furfural. On the other hand, the conversion rate of furfural is about 4 times faster than the conversion rate of HMF. This means that HMF remains much longer than furfural in the medium, and consequently, the effects of HMF last longer than those of furfural Furthermore, when furfural and HMF are present simultaneously in the medium, the conversion rates of both compounds are lowered. This results in the presence of inhibitors for a longer time in the medium, and in a higher toxicity for the cells (69). The main products of HMF conversion by S. cerevisiae were shown to be hydroxymethyl-furfuryl alcohol, 5hydroxymethyl-furan-carboxylic acid and a condensation product of HMF and pyruvic acid. Each of these compounds could be the dominant product of HMF depending on the cultivation condition.

Phenolic compounds A large number of phenolic compounds have been found in hydrolyzates (78, 79). However, reported concentrations are normally a few milligrams per liter (57). This could be due to the low water solubility of many of the phenolic


60 compounds, or a limited degradation of lignin during the hydrolysis process. Among the phenolic compounds, less heavily substituted phenolics are probably the most inhibitory materials in the hydrolyzates (43, 80). The inhibition effects of some of the phenolic compounds are summarized in Table II. Like furans, many of the phenolic substances can be converted by microorganisms. For instance, vanillin, hydroxybenzaldehyde, and syringaldehyde are assimilated by S. cerevisiae in the fermentation process (81), and conversion of vanillin to vanillyl alcohol by Klebsiella pneumoniae has also been reported (80).


Table IL The inhibition effects of some phenolic compounds

Name Phenol Vanillin Vanillin Vanillic acid Hydroxybenzaldehyde Syringaldehyde Syringaldehyde 4-hydroxybenzoic acid

Concentration 1 5 1

Ethanol from Lignocellulosic Materials ... - ACS Publications

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