Bioresource Technology 93 (2004) 110

Review

Alternatives for detoxication of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review
Solange In^ es Mussatto, In^ es Conceic ao Roberto ~
*
Department of Biotechnology, Faculty of Chemical Engineering of Lorena, Rodovia Itajub a-Lorena, Km 74,5, 12600-970 Lorena, S~ ao Paulo, Brazil Received 8 January 2003; received in revised form 14 October 2003; accepted 14 October 2003

Abstract Acid hydrolysis of lignocellulosic materials produces several inhibitory compounds, such as sugar and lignin degradation products, compounds derived from the lignocellulosic structure, and heavy metal ions. Their toxicity is a major factor limiting bioconversion processes that utilize hydrolyzates. The identication of these compounds and the choice of the best hydrolyzate detoxication method are important for improving the eciency of the fermentative processes. A variety of biological, physical, and chemical techniques have been proposed to reduce the concentration of these compounds in lignocellulose hydrolyzates. However, the eciency of any detoxication method depends both on the hydrolyzate composition, which diers according to the raw material used, and on the hydrolysis conditions employed. This review describes the eects of the inhibitory compounds on fermentation yield and productivity, as well as various detoxication methods for treating the hydrolyzates. 2003 Elsevier Ltd. All rights reserved.
Keywords: Lignocellulosic hydrolyzates; Inhibitors; Detoxication; Fermentative processes

Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lignocellulosic hydrolyzates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitors of lignocellulosic hydrolyzates . . . . . . . . . . . . . . . . . . . . . . 3.1. Sugar degradation products . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Lignin degradation products . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Compounds derived from lignocellulosic structure . . . . . . . . . . . 3.4. Heavy metals ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Combined action of several toxic compounds: synergistic eect Hydrolyzate detoxication methods . 4.1. Biological methods . . . . . . . . 4.2. Physical methods . . . . . . . . . . 4.3. Chemical methods . . . . . . . . . 4.4. Combined treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 2 3 3 3 4 4 4 5 5 5 6 7 7 7 8 8

4.

5. Activated charcoal treatment . . . . . . . . . . . . . . 5.1. Eect of pH . . . . . . . . . . . . . . . . . . . . . 5.2. Eect of contact time . . . . . . . . . . . . . . . 5.3. Eect of temperature . . . . . . . . . . . . . . . 5.4. Eect of activated charcoal concentration

Corresponding author. Tel./fax: +55-1231-533165. E-mail address: ines@debiq.faenquil.br (I.C. Roberto).

0960-8524/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2003.10.005

S.I. Mussatto, I.C. Roberto / Bioresource Technology 93 (2004) 110

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 8 9

1. Introduction Forestry and agricultural residues are abundant, renewable, and inexpensive energy sources. When hydrolyzed, these lignocellulosic materials release sugars (D -glucose, D -galactose, D -manose, D -xylose, and L arabinose) and several compounds derived from sugar and lignin degradation (furfural, hydroxymethylfurfural, acetic acid, syringic acid, p-hydroxybenzoic acid, vanillin, and other compounds). The lignocellulose hydrolyzates can be used as fermentation media to obtain xylitol, ethanol, and other useful products. However, the by-products of sugar and lignin degradation negatively aect fermentation eciency, because some of them are toxic to fermentative microorganisms and inhibit their metabolism. Therefore, knowing about these inhibitors and how to minimize their eects is very important. This review summarizes the inhibition mechanisms of several toxic compounds found in lignocellulosic hydrolyzates, as well as the most recent detoxication methods used to partially or completely remove these inhibitors, consequently improving the eciency of fermentation processes.

2. Lignocellulosic hydrolyzates Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin; the contents vary according to plant species. The complex structure of lignocellulose in plants forms a protective barrier to cell destruction by bacteria and fungi. To make this structure suitable for conversion in fermentative processes, cellulose and hemicellulose must be hydrolyzed into their corresponding monomers (sugars) for utilization by microorganisms (Iranmahboob et al., 2002). Although several methods for hydrolyzing lignocellulose have recently been proposed, this review is focused on dilute-acid hydrolysis, one of two major processes for hydrolyzing agricultural and wood wastes (the other is enzymatic hydrolysis). In general, acid hydrolysis of lignocellulose is conducted with mineral acids such as dilute H2 SO4 or HCl (in the range of 2 5%), at temperatures of about 160 C and pressures of about 10 atm (Sun and Cheng, 2002). In this process, acid concentration and temperature are crucial factors for forming toxic compounds. Moderate temperatures

(<160 C) have proven adequate for hemicellulose hydrolysis, promoting little sugar decomposition. On the other hand, temperatures above 160 C favor cellulose hydrolysis, generating a high quantity of sugars and lignin decomposition products (McMillan, 1994). From their studies on hydrolysis of lignocellulosic materials, Sun and Cheng (2002) concluded that concentrated acids including H2 SO4 and HCl are toxic, corrosive, and hazardous, requiring corrosion-resistant reactors. Besides, such acids must be recovered after hydrolysis to make the process economically feasible. Another important factor in the hydrolysis processes is reaction time. If the reaction time is longer than 1 h, xylose concentration decreases due to degradation (Cruz et al., 2000). Palmqvist and Hahn-H agerdal (2000b) related the main products and by-products generated during the hydrolysis process. According to these authors, cellulose can be degraded into glucose, and hemicellulose can be degraded into xylose, mannose, acetic acid, galactose, and glucose. At high temperatures and pressures, glucose and xylose can be degraded into furfural and hydroxymethylfurfural, respectively. When furfural and hydroxymethylfurfural are degraded, formic acid is formed. Levulinic acid is formed by hydroxymethylfurfural degradation, and phenolic compounds are generated from the partial breakdown of lignin. Besides these compounds, other substances are formed during hydrolysis, namely syringic, vanillic, caproic, caprylic, pelargonic, and palmitic acids, which are toxic to fermenting microorganisms. The following sections describe their inhibitory action and some hydrolyzate detoxication methods.

3. Inhibitors of lignocellulosic hydrolyzates The kind of toxic compounds and their concentration in lignocellulose hydrolyzates depend on both the raw material and the operational conditions employed for hydrolysis. Moreover, fermentation variablessuch as cell physiological conditions, dissolved oxygen concentration, and pH of the mediumare also associated with the toxicity of these compounds, in several cases accentuating their toxic eect (Taherzadeh et al., 2000). Toxic compounds can stress fermentative organisms to a point beyond which the ecient utilization of sugars is reduced and product formation decreases. According to

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 et al. Olsson and Hahn-H agerdal (1996) and Parajo (1998), toxic compounds can be divided in four groups: sugar degradation products, lignin degradation products, compounds derived from lignocellulose structure, and heavy metal ions. 3.1. Sugar degradation products During hydrolysis, pentose sugars can degrade to furfural, a toxic compound that, depending on its concentration in the fermentation medium, can inhibit cells and aect the specic growth rate and cell-mass yield per ATP (Palmqvist and Hahn-H agerdal, 2000b). When studying ethanol production by the yeast Pichia stipitis, Roberto et al. (1991b) observed that furfural concentrations lower than 0.5 g/l had a positive eect on cell growth, whereas concentrations above 2 g/l inhibited cell growth almost completely. Delgenes et al. (1996) showed that furfural concentrations of 0.5, 1.0 and 2.0 g/l reduced P. stipitis growth by 25%, 47% and 99% respectively. Nigam (2001) found that a furfural concentration of 0.25 g/l in fermentation medium was not enough to reduce the ethanol yield and productivity, but a concentration as high as 1.5 g/l interfered in respiration and growth of the microorganism. Ethanol yield and productivity were decreased by 90.4% and 85.1% respectively. Regarding hydroxymethylfurfural, a toxic compound originating from hexose degradation, Palmqvist and Hahn-H agerdal (2000b) reported that its inhibitory effect is similar to that of furfural, causing a longer lagphase during growth. However, hydroxymethylfurfural is considered less toxic than furfural, and its concentration in hemicellulose hydrolyzates is normally low due to three factors: (1) the low quantity of hexose in hemicellulose, (2) the conditions employed in the hydrolytic processes, which normally do not degrade hexoses in large quantities, and (3) the high reactivity of this compound. According to Delgenes et al. (1996), P. stipitis growth was reduced by 43%, 70% and 100% when the hydroxymethylfurfural concentration in the medium was 0.5, 0.75 and 1.5 g/l respectively. Azhar, cited by Alves et al. (1998), found that hydroxymethylfurfural, at a concentration of 1 g/l, was enough to inhibit cell growth and fermentation by Saccharomyces cerevisiae. Martinez et al. (2000) observed that ethanol production by E. coli from sugarcane bagasse hemicellulose hydrolyzates was aected by furans (hydroxymethylfurfural and furfural) only when their concentrations were higher than 900 mg/l. Individually, these compounds were only partially responsible for the hemicellulose hydrolyzate toxicity. Additionally, there was a synergistic eect when these two compounds were combined with several other compounds, including a variety of phenolic and aromatic compounds derived from lignin degradation, and several types of acids (acetic, formic and levulinic).

Vogel-Lowmeier et al. (1998) reported that furfural, hydroxymethylfurfural, and acetateall derived from acid hydrolysis of lignocelluloseinhibited cell growth and xylose fermentation by Pachysolen tannophilus and P. stipitis, the latter yeast being the most sensitive. They also reported that the eect of toxic compounds on xylose metabolism of yeasts is extremely complex, and found some evidence that fermentation inhibition is due to a synergistic eect of these compounds. 3.2. Lignin degradation products A large variety of compounds (aromatic, polyaromatic, phenolic, and aldehydic) are released from lignin during hydrolysis of lignocellulosic materials. Phenolic compounds have a considerable inhibitory eect on the fermentation of lignocellulosic hydrolyzates, and those with low molecular weight are the most toxic. Phenolic compounds cause a partition and loss of integrity of biological membranes, thereby aecting their ability to serve as selective barriers and enzyme matrices. In consequence, both cell growth and sugar assimilation are reduced (Palmqvist and Hahn-H agerdal, 2000b).  et al. (1998) reported that lignin degradation Parajo products are more toxic to microorganisms than furfural and hydroxymethylfurfural, even when their concentrations are low. They also reported that xylose metabolism of S. cerevisiae from wood hydrolyzate was totally or half inhibited by vanillin at concentrations of 5 or 3.7 g/l, respectively. In investigating the eects of dierent concentrations of phenols (from 0.1 to 4 g/l) on xyloseto-xylitol bioconversion by Candida guilliermondii, Villa et al. (1998) observed that phenol at concentrations as low as 0.1 g/l, aected neither xylose consumption nor cell growth nor xylitol production, but at higher concentrations it was strongly inhibitory. 3.3. Compounds derived from lignocellulosic structure Several inhibitory products, such as raw material extractives (acidic resins, taninic, and terpene acids) and acetic acid derived from acetyl groups present in the hemicellulose, are discharged into the hydrolyzate during the hydrolytic process. According to McMillan (1994), these extractives produce less inhibition of microbial growth than lignin derivatives or acetic acid. Lawford and Rousseau (1998) reported that when the pH of the medium is low, acetic acid (pKa 4:75) appears in the undissociated form, is liposoluble, and diffuses across the plasma membrane. Once in the cell interior, where pH is 7.4, this acid dissociates and accumulates in the cytoplasm, discharging protons. As a consequence, the internal pH drops inhibiting cell activity, and even causing death. The toxicity of acetic acid varies according to the cultivation conditions employed in the fermentative

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process. Van Zyl et al. (1991) used P. stipitis to produce ethanol from sugarcane bagasse hemicellulosic hydrolyzate and found that the degree of inhibition caused by acetic acid depended not only on its concentration, but also on the oxygen concentration and on the pH of the medium. At pH 6.5 and with acetic acid concentrations above 4 g/l, the ethanol production started to decrease, dropping to 50% when the acid concentration reached 15 g/l. At pH 5.1, however, the same drop in ethanol concentration (50%) was observed when the acetic acid concentration was only 1 g/l, and no ethanol was produced with 10 g of acetic acid per liter of hydrolyzate. According to Felipe et al. (1995), acetic acid concentrations up to 1.0 g/l improved the xylose-to-xylitol bioconversion, but concentrations higher than 3 g/l were harmful to the fermentation process. On the other hand, ethanol production was stimulated by acetic acid concentrations up to 10 g/l in medium free of other toxic compounds (Palmqvist et al., 1999). 3.4. Heavy metals ions Heavy metal ions (iron, chromium, nickel and copper) can originate from corrosion of hydrolysis equipment, and their toxicity inhibits enzymes in the microorganisms metabolic pathways. Watson et al. (1984) evaluated the eects of metal cations on the cellular growth of P. tannophilus and on the activity of the enzymes involved in xylose metabolism. They employed synthetic media containing concentrations of metal cations similar to those normally found in hydrolyzates. Microbial activity was slightly reduced when copper, nickel, chromium, and iron ions were present in the media in quantities below than 4, 5, 100 and 150 mg/l, respectively. On the other hand, a 60% reduction was produced by nickel ions at a concentration of 100 mg/l. 3.5. Combined action of several toxic compounds: synergistic eect The maximum concentration of each inhibitor that a microorganism can withstand cannot be established because inhibition strongly depends on factors such as the kind of microorganism, its adaptation to the medium, the type of fermentative process employed, the number of inhibitors present in the medium, and their synergistic eect. According to Palmqvist et al. (1999), ethanol production was stimulated by acetic acid (up to 10 g/l) in medium without furfural, and by furfural (up to 2 g/l) in medium without acetic acid. However, the combination of these two compounds negatively aected the growth rate, cell mass yield, and ethanol yield. Zaldivar et al. (2000) demonstrated that the toxicity of hemicellulosic hydrolyzates results from the aggregation of several toxic compounds (alcohols, aldehydes, and acids) but not from individual compounds.

Nigam (2001) tried to obtain ethanol from wheat straw hemicellulosic hydrolyzate and from synthetic medium, both formulated with the same concentrations of acetic acid, furfural, and lignin derivatives. In the synthetic medium, the ethanol yield and productivity were respectively 74.4% and 83% lower than in the hydrolyzate. The same author also tested three other synthetic media, each containing only one kind of toxic compound, and found that the percentages of reduction in yield and productivity were, respectively, 30.2% and 59.6% for the medium with acetic acid, 7.0% and 12.8% for the medium with furfural and 14.0% and 44.7% for the medium with lignin derivatives. Thus the inhibitory eect of these compounds is higher when they are combined, due to their synergistic eect.

4. Hydrolyzate detoxication methods When compared with the fermentation of commercial sugars or detoxied hydrolyzates, the fermentation of non-detoxied hemicellulosic hydrolyzates is characterized by slow kinetics, with limited yield and productivity. This is due to the presence of a variety of compounds that act as potent inhibitors of microbial metabolism. Therefore, the lignocellulosic substrates need to be pretreated and neutralized to attain the fermentation pH, thereby becoming more suitable for microorganism metabolism (Roberto et al., 1991a; Kuhad and Singh, 1993; Winkelhausen and Kuzmanova, 1998; Mussatto, 2002). Several studies employing hemicellulose hydrolyzates have been conducted to identify toxic compounds and to minimize their negative eects on fermentation. According to Taherzadeh et al. (2000), there are four dierent approaches for minimizing the presence of inhibitors in hemicellulosic hydrolyzates: (1) to avoid formation of inhibitors during hydrolysis; (2) to detoxify the hydrolyzate before fermentation; (3) to develop species of microorganisms able to resist inhibitors; (4) to convert toxic compounds into products that do not interfere with metabolism. These authors also reported that new metabolically engineered microbial species better tolerate inhibitors and can decrease the need to detoxify hydrolyzates. However, the compounds to which these species gain resistance are still not dened. A number of detoxication methodsincluding biological, physical, and chemical oneshave been proposed to transform inhibitors into inactive compounds or to reduce their concentration. The eectiveness of a detoxication method depends both on the type of hemicellulose hydrolyzate and on the species of microorganism employed, because each type of hydrolyzate has a dierent degree of toxicity, and each species of microorganism has a dierent degree of tolerance to inhibitors (Larsson et al., 1999). Before choosing a

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detoxication method, we must consider the composition of the hydrolyzate, which varies according to the raw material and the hydrolysis conditions employed. 4.1. Biological methods Biological methods of treatment involve the use of specic enzymes or microorganisms that act on the toxic compounds present in the hydrolyzates and change their composition. Wood hydrolyzates detoxied with laccase and peroxidase enzymes of the white-rot fungus Trametes versicolor promoted an increase in glucose consumption and ethanol productivity, due to the action of these enzymes on acid and phenolic compounds nsson et al., 1998). The detoxication mechanism of (Jo these enzymes probably involves oxidative polymerization of low-molecular-weight phenolic compounds. The use of microorganisms has also been proposed to selectively remove inhibitors from lignocellulose hydrolyzates. Schneider (1996) reported that the concentration of acetic acid in the medium dropped from 6.8 g/l to less than 0.4 g/l in wood hydrolyzate due to selective removal by a mutant species of S. cerevisiae. Adaptation of a microorganism to the hydrolyzate is another interesting biological method for improving the fermentation of hemicellulosic hydrolyzate media (Felipe et al., 1996; Olsson and Hahn-H agerdal, 1996;  et al., 1998; Silva and Roberto, 2001; Sene et al., Parajo 2001). This method, which is based on successive fermentations, utilizes the microorganism of each experiment as the inoculum of the next one. According to Silva and Roberto (2001), the adaptation of Candida guilliermondii to rice straw hemicellulose hydrolyzate for xylitol production is an eective and inexpensive method to alleviate the inhibitory eect of toxic compounds on the xylose-to-xylitol bioconversion. 4.2. Physical methods Hydrolyzate concentration by vacuum evaporation is a physical detoxication method for reducing the contents of volatile compounds such as acetic acid, furfural and vanillin, present in the hydrolyzate. However, this method also moderately increases the concentration of non-volatile toxic compounds (extractives and lignin derivatives) and consequently the degree of fermentation  et al. (1997) used this method with inhibition. Parajo wood hydrolyzate and observed an increase in the concentrations of lignin derivatives and extractives. The volume of hydrolyzate was reduced to about 1/3 and the fermentation time necessary for the yeast to consume about 90% xylose increased from 24 to 94 h. Larsson et al. (1999) attained a total removal of furfural from wood hemicellulosic hydrolyzate by reducing its volume by 90% through vacuum evaporation. On the other hand, the hydroxymethylfurfural concentration de-

creased only by 4%. Silva and Roberto (1999) utilized vacuum-evaporated with rice straw hydrolyzate as a substrate for microbial conversion of xylose (90 g/l) into xylitol, and noticed that the production process was drastically hindered by the increase in concentration of non-volatile compounds, which are toxic to the microorganism and strongly interfere with fermentation. Converti et al. (2000) also reported that evaporation is suitable to remove acetic acid, furfural and other volatile compounds from hemicellulose hydrolyzates, improving the fermentative process for xylitol production. Rodrigues et al. (2001) employed the vacuum-evaporation method either before or after treating sugarcane bagasse hemicellulose hydrolyzate with activated charcoal. The result was that 98% of furfural was removed whereas acetic acid was only partially eliminated, because this compound is volatile in its undissociated form. 4.3. Chemical methods Chemical methods include precipitation of toxic compounds and ionization of some inhibitors under certain pH values, the latter being able to change the degree of toxicity of the compounds (Van Zyl et al., 1998; Roberto et al., 1991a; Martinez et al., 2001; Mussatto, 2002). Toxic compounds may also be adsorbed on activated charcoal (Dominguez et al., 1996; Silva et al., 1998; Lee et al., 1999; Mussatto and Roberto, 2001), on diatomaceous earth (Ribeiro et al., 2001), and on ionexchange resins (Van Zyl et al., 1991; Lee et al., 1999; Larsson et al., 1999; Nilvebrant et al., 2001). According to Roberto et al. (1991a), pH adjusting with a combination of bases and acids is a low-cost treatment that gives good results. By adjusting the pH of sugarcane bagasse hemicellulosic hydrolyzate rst to 10 with Ca(OH)2 , and then to 6.5 with H2 SO4 , these authors obtained a partial removal of phenolic and other compounds, and a xylitol yield of 0.48 g/g. Van Zyl et al. (1998) adjusted the pH of sugarcane bagasse hemicellulose hydrolyzate rst with Ca(OH)2 and then with NaOH, and found that the former treatment enhanced the hydrolyzate fermentability, whereas the latter increased the precipitation of toxic compounds. Nilvebrant (cited by Palmqvist and Hahn-H agerdal, 2000a) used NaOH and Ca(OH)2 to adjust the pH to 10 of dilute-acid hydrolyzate of spruce, and obtained a decrease of about 22% in the concentration of Hibberts ketones and a reduction of 20% in the concentrations of furfural and hydroxymethylfurfural. On the other hand, the concentration of acetic acid was not aected by this treatment. Martinez et al. (2001) reported that using Ca(OH)2 to adjust the pH of sugarcane bagasse hemicellulose hydrolyzate (overliming treatment) to 9.0, proved to be a very ecient detoxication method. For their experiment, they heated the hydrolyzate to 25 and 60 C. The latter temperature was more appropriate for

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this kind of treatment, because it removed about 51% of total furans and 41% of phenolic compounds, with a loss of only 8.7% of sugars. Among the chemical methods of treating hemicellulose hydrolyzates, activated charcoal stands out because it is a low-cost material with a high capacity to adsorb compounds (Gong et al., 1993; Dominguez et al., 1996; Silva et al., 1998; Mussatto and Roberto, 2001). In studying ethanol production by yeasts of Candida sp., Gong et al. (1993) submitted sugarcane bagasse hemicellulose hydrolyzate to three distinct methods of treatment: (1) pH adjustment, (2) addition of activated charcoal, and (3) use of ion-exchange resins. Adjusting the pH (to 8.5 with CaO and to 7.0 with H3 PO4 ) produced the highest cell inhibition during the fermentative process. Using ion-exchange resins resulted in the best ethanol productivity (0.463 g/l h) after 96 h of fermentation, but because resins are expensive, the same authors considered this detoxication method to be economically unviable. Treating the hydrolyzate with activated charcoal proved the best method, because it provided a good process productivity (0.403 g/l h) at a lower cost. Dominguez et al. (1996) also studied dierent types of treatments for sugarcane bagasse hemicellulose hydrolyzate (neutralization, activated charcoal with neutralization, and ion-exchange resins with neutralization). Most of the toxic compounds were removed and the xylitol volumetric productivity was the highest (0.205 g/l h) when the hydrolyzate was treated with activated charcoal. Besides activated charcoal (powdered or granulated), diatomaceous earth has also been used to adsorb compounds. Ribeiro et al. (2001) used both treatments to remove some impurities (pigments, free fatty acids, and oxidation products) from a solution of crude oliveresidue oil in n-hexane, and obtained better results with powdered activated charcoal. Adsorption on ion-exchange resins is also an eective technique, but its cost is high compared to the cost of other treatments (Lee et al., 1999). According to Van Zyl et al. (1991), fermentation of untreated sugarcane bagasse hemicellulose hydrolyzate containing 9 g/l of acetic acid resulted in an ethanol productivity of 0.15 g/ l h, with a yield of 0.27 g/g. When the hydrolyzate was treated with anionic-exchange resins, 84% of acetic acid was removed, the ethanol productivity and yield reaching 0.56 g/l h and 0.37 g/g, respectively. Similar results (0.58 g/l h and 0.35 g/g, respectively) were attained with synthetic medium formulated with the same concentrations of sugars and without acetic acid. Larsson et al. (1999) compared four dierent detoxication methods for wood hemicellulose hydrolyzate: (1) pH change with NaOH or Ca(OH)2 , (2) evaporation, (3) adsorption on anion-exchange resins, and (4) the use of microorganisms. Anion-exchange resins removed high percentages of toxic compounds from the hydro-

lyzate: acetic acid (96), phenolic compounds (91), furfural (73), and hydroxymethylfurfural (70). Besides, the hydrolyzate treated with anion-exchange resins had the highest ethanol yield (0.49 g/g), volumetric productivity (1.42 g/l h) and biomass yield (0.080 g/g). Nilvebrant et al. (2001) treated wood hydrolyzate with anion- and cation-exchange resins for ethanol production. Anionexchange resins were more eective, removing a higher quantity of phenols, furans, aldehydes, and aliphatic acids from the hydrolyzate, and providing an ethanol productivity and yield of 1.71 g/l h and 0.46 g/g, respectively. Cation-exchange resins gave a similar yield (0.45 g/g) but a low productivity (0.65 g/l h). For untreated hydrolyzates the productivity and yield were 0.21 g/l h and 0.42 g/g respectively. 4.4. Combined treatments Dierent combinations of treatment methods have been amply used to detoxify lignocellulosic hydrolyzates. In their experiments on xylose-to-xylitol bioconversion, Alves et al. (1998) employed dierent combinations of bases (CaO or Ca(OH)2 ) and acids (H2 SO4 or H3 PO4 ) to change the initial pH of sugarcane bagasse hemicellulose hydrolyzate, with or without activated charcoal. The best results were attained when the pH was increased from 0.5 to 7.0 with CaO and then decreased to 5.5 with H3 PO4 , before 2.4% activated charcoal was added to the hydrolyzate. In these conditions, the values of xylitol yield and productivity were 0.79 g/g and 0.47 g/l h, respectively, after 63 h of fermentation. On the other hand, the pH increase from 0.5 to 7.0 with CaO and then decrease to 5.5 with H2 SO4 , without the addition of activated charcoal, was the worst condition for hydrolyzate treatment, resulting in a xylitol yield and productivity of 0.58 g/g and 0.35 g/l h respectively. Converti et al. (1999) utilized several species of yeast to produce xylitol from hardwood hemicellulose hydrolyzates and observed that the presence of both lignin degradation compounds and acetic acid hindered the fermentation process. To purify the hydrolyzates and enhance the fermentation kinetics, they employed a combination of dierent treatments. Initially, the pH was adjusted to 10.0 with Ca(OH)2 and then decreased to 5.5 with H2 SO4 . Subsequently, activated charcoal was added to the hydrolyzates, which were then evaporated. The combination of pH adjustment and activated charcoal adsorption was especially good for removing lignin derivatives, but charcoal alone removed 95.4% of these compounds. The evaporation process reduced the acetic acid concentration to a level below the inhibitory threshold (3 g/l) and also removed furfural and other volatile compounds. The combination of all these treatments allowed ecient conversion of xylose into xylitol, with a volumetric productivity of 0.41 g/l h and a product yield of 0.63 g/g.

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Converti et al. (2000) pursued detoxication of wood hydrolyzate for xylitol production by P. tannophilus. They used the following treatments: pH adjustment to 10.0 with Ca(OH)2 , acidication to pH 5.5 with H2 SO4 , addition of sodium sulte (1 g/l), boiling to 100 C, and activated charcoal adsorption. Partial results showed that pH adjustment and activated charcoal removed large quantities of lignin degradation products (60% and 95% respectively), whereas boiling reduced the acetic acid and furfural concentrations to levels below those that inhibit the microbial metabolism (acetic acid was reduced from 31.2 to 1.0 g/l and furfural from 1.2 to 0.5 g/l). The hydrolyzate fermentation was also very eective, because a high concentration of xylitol (39.5 g/l) was produced from a xylose concentration of 89.0 g/l after 96 h of fermentation. On the other hand, when boiling (100 C) and activated charcoal adsorption were excluded from this sequence of treatments, xylitol production after 96 h of fermentation was only 3.1 g/l. Rodrigues et al. (2001) attempted to remove volatile and non-volatile compounds from sugarcane bagasse hemicellulose hydrolyzate employing activated charcoal treatment either before or after vacuum evaporation. According to these authors, treating the hydrolyzate before evaporation eectively removed phenolic compounds, whereas treating it after evaporation eectively removed acetic acid. To produce xylitol, Mussatto (2002) employed evaporation, pH adjustment, and activated charcoal adsorption to detoxify rice straw hemicellulose hydrolyzate. Evaporation removed furfural and some acetic acid, whereas pH adjustment and activated charcoal removed the non-volatile compounds (hydroxymethylfurfural and lignin degradation products). According to this author, pH adjustment and activated charcoal produced better detoxication when they were used in combination. In general, before choosing a detoxication method or a sequence of methods, it is important to identify the main inhibitors present in the hydrolyzate. This knowledge helps not only to choose a low-cost and ecient detoxication method, but also to establish the hydrolysis conditions that best minimize inhibitor formation. Because hydrolyzate detoxication increases the cost, it should be employed only with strongly inhibiting hydrolyzates or with sensitive organisms. Further, the detoxication method should be inexpensive, easy to integrate into the process, and able to remove inhibitors selectively.

variables used for the adsorption process: pH, temperature, contact time, and activated charcoal concentration. The eects of these variables are described in the next sections. 5.1. Eect of pH Inhibitor adsorption on activated charcoal is quite sensitive to changes in pH. If the solutes to be removed are either weakly acidic (such as phenols or carboxylic acids) or weakly basic (such as amines), then the pH of the medium aects their adsorption (Fox and Kennedy, 1985). Weak organic acidics are most readily adsorbed in the non-ionized state and consequently a low pH (acids) favors adsorption. For example, phenols are weak acids, and at low pH, the neutral or non-ionized phenolic molecules are highly adsorbed, whereas at high pH, the phenols are anions (phenolate ions) and are adsorbed poorly. According to Weber (1985), neutral species (acetic acid, phenol molecules) tend to adsorb more strongly from the aqueous phase than do their corresponding ionic forms (acetate, phenolate ions) because physical and chemical properties of compounds change upon ionization, which aects their adsorbability. On the other hand, weakly basic compounds are also most readily adsorbed in their non-ionized state and therefore, absorption is favored by high pH (alkaline). As a rule, organic acids are best adsorbed from acidic solutions, and organic bases adsorbed in basic solutions (Fox, 1985). Mussatto (2002) used two pH values (2.0 and 8.0) while treating rice straw hemicellulose hydrolyzate with activated charcoal. A major removal of lignin degradation products (particularly of phenolic compounds) was attained with pH 2.0. From a statistical analysis, this author concluded that pH is a variable that strongly inuences adsorption processes using activated charcoal. 5.2. Eect of contact time Contact time is an important variable in activated charcoal treatment. Adequate contact time between the charcoal and the hydrolyzate uid is essential to allow the charcoal to approach equilibrium with the adsorbate. During the adsorption process, the charcoal surface is progressively blocked by the adsorbate, becoming completely covered after some time. When this happens, the charcoal cannot adsorb any more compounds. As each charcoal particle puries a certain volume of liquid, increasing the charcoal dosages (or the ratio of charcoal:hydrolyzate) rapidly promotes an equilibrium between adsorbate and adsorbent, because the number of charcoal particles to treat the same volume of liquid is increased (Bernardin, 1985).

5. Activated charcoal treatment To purify or recover chemicals, activated charcoal is widely used to remove compounds from the liquid phase by adsorption. Nevertheless, the eectiveness of activated charcoal treatment depends on the following

S.I. Mussatto, I.C. Roberto / Bioresource Technology 93 (2004) 110

 et al. (1995) evaluated the inuence of contact Parajo time on removing toxic compounds from wood hydrolyzate treated with activated charcoal. They observed that removal of lignin degradation products was maximum after 20 min, ceasing afterwards, even when the contact time was increased to 90 min. Gurgel et al. (1995) tested dierent contact times (30, 45, and 60 min) between fermented sugarcane bagasse hemicellulosic hydrolyzate and activated charcoal, and found that varying the contact time exerted only a small inuence on  et al. (1996) treated the clarication process. Parajo wood hydrolyzate with dierent activated charcoal concentrations (varying from 20 to 400 g hydrolyzate per g charcoal) using contact times ranging from 1 to 5 days, and observed that one day of contact time was enough to reach the adsorption equilibrium in all the experiments. 5.3. Eect of temperature The adsorption of compounds increases with higher temperature, because high temperatures provide a faster rate of diusion of adsorbate molecules from the solution to the adsorbent (Bernardin, 1985). Gurgel et al. (1995) used fermented sugarcane bagasse hemicellulose hydrolyzate to evaluate the eects of three dierent temperatures (35, 50, and 80 C) on the hydrolyzate clarication resulting from the adsorption of compounds on activated charcoal. The residual color decreased up to 70% when the temperature was raised from 35 to 80 C. Moreira et al. (2000) reported that temperature inuences adsorption of colorant molecules on charcoal. As these molecules are relatively large, and their orientation on the charcoal surface can be transversal under a certain temperature and longitudinal under another, the activated charcoal capacity for adsorption can either decrease or increase. Chiang et al. (2001) found that the adsorption of benzene on activated charcoal was temperature-dependent, improving when the temperature was raised to 80 C, because the higher the temperature, the better the interaction between the benzene and charcoal molecules. Mussatto (2002) observed that, besides pH, temperature also greatly inuences the removal of toxic compounds (mainly lignin degradation products) from rice straw hydrolyzate treated with activated charcoal. When the temperature was raised from 25 to 45 C, the removal rates of these compounds increased about sixfold, probably due to a relatively more ecient packing of the molecules in the charcoal pores at higher temperatures. 5.4. Eect of activated charcoal concentration According to several authors, the proportion of activated charcoal used to treat the hydrolyzate can

 strongly inuence the removal of compounds (Parajo et al., 1995, 1996; Silva et al., 1998; Lee et al., 1999;  et al. (1996) obMussatto and Roberto, 2001). Parajo served that removing phenolic compounds from wood hydrolyzates increased from 15% to 75% when the ratio of hydrolyzate to activated charcoal was reduced from 400 to 10 g/g. Silva et al. (1998) treated sugarcane bagasse hemicellulose hydrolyzate with activated charcoal in proportions varying from 1% to 30% (w/w), and observed that 1% charcoal was sucient to eliminate 94% of phenolic compounds, causing a sugar loss of only 0.47%. On the other hand, 30% of charcoal reduced the sugars by 31.3%, which was an undesirable result. To minimize the inhibitory eect of compounds produced during the pretreatment of wood hydrolyzate, treatment with activated charcoal was also conducted by Lee et al. (1999). The highest removal rate was observed when the charcoal:glucose ratio (g:g) was increased from 0.05 to 0.20. Mussatto and Roberto (2001) utilized rice straw hemicellulose hydrolyzate to study the eect of activated charcoal treatment on xylose-to-xylitol bioconversion and found that the degree of phenolic compound removal increased when the hydrolyzate:charcoal ratio was decreased from 120 to 24 g/g. However, the best values of fermentative process were achieved with the ratio of 40 g/g, which removed 27% of phenolic compounds.

6. Conclusions Because several inhibitory compounds are formed during hydrolysis of the raw material, the hydrolytic process has to be optimized so that inhibitor formation can be minimized. When low concentrations of inhibitory compounds are present in the hydrolyzate, detoxication is easier and fermentation is cheaper. The choice of a detoxication method has to be based on the degree of microbial inhibition caused by the compounds. As each detoxication method is specic to certain types of compounds, better results can be obtained by combining two or more dierent methods. Another factor of great importance in the fermentative processes are the cultivation conditions, which, if inadequate, can stimulate the inhibitory action of the toxic compounds.

Acknowledgements The authors acknowledge nancial support from Fundac ao de Amparo  a Pesquisa do Estado de S~ ao ~ Paulo (FAPESP) and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), Brazil. The authors also wish to thank Maria Eunice M. Coelho for revising this manuscript.

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