Enzyme and Microbial Technology 39 (2006) 897902

Enzymatic saccharication of wheat straw for bioethanol production by a combined cellulase xylanase and feruloyl esterase treatment
M.G. Tabka a, , I. Herpo el-Gimbert a , F. Monod b , M. Asther a , J.C. Sigoillot a
a

UMR 1163 INRA/Universit es de Provence et de la M editerran ee de Biotechnologie des Champignons Filamenteux, IFR86-BAIM, ESIL, 163 Avenue de Luminy, Case Postale 925, 13288 Marseille Cedex 09, France b Institut Fran cais du P etrole 1&4, Avenue de Bois-Pr eau 92852, Rueil-Malmaison Cedex, France Received 7 October 2005; received in revised form 18 January 2006; accepted 18 January 2006

Abstract The focus of this study was to improve conditions of use of fungal lignocellulolytic enzymes for conversion of lignocellulosic biomass to fermentable sugars for the production of bioethanol. Wheat straw was pre-treated by acid treatment with diluted sulfuric acid followed by steam explosion. Several enzymatic treatments implementing hydrolases (cellulases and xylanases from Trichoderma reesei, recombinant feruloyl esterase (FAE) from Aspergillus niger and oxidoreductases (laccases from Pycnoporus cinnabarinus) were investigated to the saccharication of exploded wheat straw. A synergistic effect between cellulases, FAE and xylanase was proven under a critical enzymatic concentration (10 U/g of cellulases, 3 U/g of xylanase and 10 U/g of FAE). The yield of enzymatic hydrolysis was enhanced by increasing the temperature from 37 C to 50 C and addition of a non-ionic surfactant, Tween 20. Optimisation of enzymatic hydrolyses allowed the use of lower quantities of enzymes and improved the cost effectiveness of the process. 2006 Elsevier Inc. All rights reserved.
Keywords: Fungal enzyme; Saccharication; Wheat straw

1. Introduction Production of fuel ethanol from renewable lignocellulosic materials has been extensively studied in the last decades [1]. Lignocellulosic materials to be considered for ethanol production include wood, hardwood rather than softwood which is more difcult for enzymatic hydrolysis [2], crops from annual plants, agricultural residues and waste paper. Wheat straw is one of the most abundant crop residues in European countries with a production of 170 million tonnes and seems to be the cheapest and the most useful raw material for ethanol production [3]. It is comprised mainly of cellulose (40%), hemicelluloses (26%) and about 20% of lignin [4]. Hydrolysis of cellulose and hemicelluloses is hampered by the overlapping of polymer embedded microbrils. Several methods have been designed to increase hydrolysis yield, including acidic and steam pre-treatments, and steam explosion [5]. These treatments result in partial degradation of the polysaccharidic cell wall, opening bres and

Corresponding author. Tel.: +33 4 91 82 86 52; fax: +33 4 91 82 86 01. E-mail address: gtabka@esil.univ-mrs.fr (M.G. Tabka).

allowing the penetration of chemicals or enzymes inside the structure. Enzymatic degradation of plant cell wall has been extensively studied. Cellulases (EC 3.2.1.4) from Trichoderma reesei are mostly used, as several mutant strains of this fungus produce high levels of extracellular cellulolytic enzymes, up to 40 g/l [6]. The fungus produces a complete set of cellulases: cellobiohydrolases (EC 3.2.1.91), endoglucanases (EC 3.2.1.4) and -glucosidases (EC 3.2.1.21) that are necessary to efciently hydrolyse cellulose [7]. In addition, T. reesei is able to produce hemicellulases, mainly xylanases (EC 3.2.1.8), depending on the growth conditions and substrate [8]. It is well known that conjugated action of cellulases and hemicellulase results in a higher nal sugar production as compared to cellulases alone. The major hemicellulose polymer in cereal and hardwood is xylan. Xylan consists of a -1,4-linked d-xylose backbone and can be substitued by different side groups such as l-arabinose, d-galactose, acetyl, feruloyl, p-coumaroyl and glucuronic acid residues [9]. Ferulic acid is the most abundant hydroxy cinnamic acid in the cell wall of cereal [10]. It is covalentely cross-linked to arabinoxylans by ester bonds and to components of lignin mainly by ether bonds [11]. Accessory enzymes such as feruloyl

0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.01.021

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M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897902 Esterase activity was assayed as previously described by Ralet et al. [23] as a function of time, using methyl ferulate (MFA) or methyl sinapinate (MSA) as the substrate. Exoglucanase (FPase; Exo-1, 4--d-glucanase, EC 3.2.1.91) activity was determined according to Mandels et al. [24]. The assay mixture (2 ml) consisted of a 1.9 ml citrate buffer (50 mM, pH 4.5), Whatman no. 1 lter paper (50 mg, 1 cm 3 cm) and 0.1 ml of a suitably diluted enzyme. The reaction mixture was incubated at 50 C for 60 min. Endoglucanase (CMCase, Endo-1, 4--d-glucanase; EC 3.2.1.4) activity was determined according to Mandels et al. [24] with slight modication. The total reaction of 1 ml contained a 0.5 ml sample of a suitably diluted enzyme and 0.5 ml of 1% (w/v) carboxymethyl cellulase (CMC) solution in a citrate buffer (50 mM, pH 4.5) and incubated at 50 C for 30 min. Xylanase (1,4--d-xylanase, EC 3.2.1.8) activity was determined under similar conditions as described above, except that 1% xylan solution (oat spelts, SigmaAldrich, Saint Quentin Fallavier, France) was used as the substrate in place of CMC. Reducing sugars were determined by the dinitrosalicylic acid (DNS) using Millers method [25]. All activities were expressed in international units (U) dened as 1 mol of glucose or xylose produced per minute. Experiments were performed in triplicate and standard error was lower than 10% of the mean.

esterase (FAE; EC 3.1.1.73) should also act in synergy with xylanases by cleaving diferulic bridges between xylan chains, opening the structures and releasing lignin [12,13]. In Trichoderma strains no FAE activity was detected. This enzyme occurs naturally in Aspergillus niger exoenzymes but homologous cloning of the FAE gene enhances the production of about 25-fold, allowing application experiments [14]. Removal of lignin could be conducted by alkaline/oxydative extraction [15] but extensive delignication requires very extreme conditions as in chemical paper pulp production. Enzymatic delignication is achieved naturally by white-rot fungi. These organisms, belonging to the basidiomycete family, produce oxydative enzymes such as laccases that generate radicals able to cleave covalent bonds in lignin. These enzymes could also act in synergy with cellulases and hemicellulases by removing the lignin layer on the bre surface. In this study, we will examine the role of some accessory enzymes in the extensive hydrolysis of wheat straw by the cellulases and xylanases of T. reesei. Feruloyl esterase from A. niger, and laccases from Pycnoporus cinnabarinus were chosen and will be used alone or in association with cellulases and xylanases in order to enhance wheat straw saccharication.
2. Materials and methods 2.1. Fungal strain and enzyme production
The strain P. cinnabarinus BRFM 137 (Banque de Ressources Fongiques de Marseille, Marseille, France) and described as a hypersecretory laccase strain [16] was used in this study. Large scale fungal culture was grown in a 15-l bioreactor as described by Herpo el et al. [17,18] using ethanol as an inducer [19]. Ethanol was added gradually to the medium to avoid inhibition of the fungal growth. Ethanol at 8 g/L was added to the culture broth on the day of inoculation. This concentration was maintained for 3 days by adding ethanol every day. The ethanol concentration was then increased to 10 g/l and maintained at this level until the end of the culture. After eight days, the culture supernatant was harvested and concentrated by ultraltration using a 10 kDa membrane (Millipore S.A., Molsheim, France). The strain A. niger BRFM 451 obtained in our laboratory by homologous overexpression of the feruloyl esterase A gene (faeA) [14] was used for the production of feruloyl esterase in 15 l bioreactor. Culture conditions were transposed from Erlenmeyers culture previously described by Record et al. [14]. The bioreactor was lled with 12 l (working volume) of basal medium containing 40 g/l of glucose and were inoculated with 105 conidiospores of A. niger per ml. The pH of the medium was maintained at 5.2 using 4N citric acid solution. The fermentation was carried out at a stirrer speed of 350 rpm and an aeration rate of 0.5 VVM (volume air per volume reactor per minute) at 30 C. The culture supernatant was harvested after 6 days and concentrated by ultraltration using a 10 kDa membrane (Millipore S.A.). Two enzymatic preparations containing hydrolases from T. reesei CL 847 were kindly provided by SAF-ISIS (Souston, France) from the LESAFFRE group. The enzyme mixtures containing mainly cellulases or xylanases were obtained by fed-batch fermentation with different carbon sources [8,20,21]. The rst sample exhibited only cellulase specic activity as high as 0.664 U/mg of exo-glucanase and 25.6 U/mg of endo-glucanase. No Xylanase activity was detected, while the second sample of hydrolases preparation presented mainly xylanase activity (27.3 U/mg).

2.3. Origin of the steam exploded wheat straw

Acidic steam-pretreated wheat straw was used as substrate in the hydrolysis experiments. The pretreated substrate was kindly supplied by IFP (Institut Franc ais du P etrole-Rueil-Mallmaison, France). The raw material for the pretreatement was chopped wheat straw furnished by VALAGRO (Poitiers-France). The acidic impregnation of the shopped wheat straw was achieved by soaking it overnight in a solution of 0.08N H2 SO4 . After chemical reaction, the substrate was drained and pressed. The spined straw was maintained for 1 min at a pressure of 100 bars. The wet material was then exploded under steam pressure at 18 bars in a batch cooker. At the end, the pre-treated material was washed twice for 30 min and stored as such in deep freeze (20 C).

2.4. Enzymatic hydrolysis experiments

Experiments were carried out on 10 g of pretreated wheat straw (dry weight) in a stainless steel vessel (500 ml working volume) incubated in a water bath to 50 C, the substrate was suspended to 10% (w/v, dry) in 100 mM sodium acetate buffer at pH 4.8 and enzymes were used alone or in association in a one-stage treatment. The concentrations of enzyme used were: 5 U/g (d.w.) of xylanase, 10 U/g (d.w.) of cellulase, 20 U/g (d.w.) of FAE and 20 U/g (d.w.) of laccase. Treatment with laccase was performed under 150 kPa oxygen. The reaction medium was stirred at 150 rpm by mechanical agitation (marine propeller) for 24 h. Assays were carried out in triplicate. Supernatants were analysed for glucose and reducing group contents. Wheat straw dry weight after enzymatic treatment was estimated by ltration on glass-bre (GF/D Wathman, Maidstone, UK) and drying at 110 C until the mass was constant. Optimisation of the saccharication conditions was carried out on 0.2 g of pretreated wheat straw (dry weight) in 15 ml screw-cap tubes. Experiments were carried out at 2% consistency in 100 mM sodium acetate buffer at pH 4.8. Substrate was treated with one-stage or sequential enzymatic treatment. Enzyme concentrations were: 3 U/g (d.w.) of xylanase, 10 U/g (d.w.) of FAE, 10 U/g (d.w.) of cellulase and 20 U/g (d.w.) of laccase. Treatment with laccase was performed in presence of 1 or 10% of N-hydroxy-N-phenylacetamide (NAH) or 3% of hydroxylbenzotriazole (HBT) as chemical mediator for 4 h. In sequential treatment, after the rst enzymatic stage performed under 150 kPa oxygen, substrate was ltered, washed twice with distilled water and centrifuged at 12,000 rpm for 15 min at 4 C. Hydrolysis experiments were performed in an incubator at 37 or 50 C with gentle rotative agitation for 48 h.

2.2. Enzyme activity assays 2.5. Chemical hydrolysis of pre-treated wheat straw
Laccase activity was determined as previously described by Sigoillot et al. [22] using 2,2 -azino-bis-[3-ethylthiazoline-6-sulphonate] (ABTS) as the substrate. Before any treatment, pre-treated wheat straw was washed thoroughly with distilled water overnight; afterwards it was milled in a Waring Blendor to obtain

M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897902 a coarse powder. This was net ltered and conserved at 20 C for enzymatic hydrolysis essays. In order to estimate the total glucose concentration in wheat straw, a total acidic hydrolysis analysis was performed. A second step of crushing was carried out using 0.3 g (dry weight) of straw coarse powder. The ne powder obtained was suspended in 2 ml of 72% H2 SO4 and kept at 25 C for 30 min. The acidic mixture was diluted by adding distilled water and kept at 100 C in a heating dry block for 2 h, after centrifugation at 5000 g for 15 min, the supernatant was neutralized with 25% ammoniac and analysed to estimate the total glucose concentration. Glucose present in hydrolysis samples was estimated using the combined enzymatic test, i.e. glucose oxidase type II-S from A. niger (GOD) and Horseradish peroxidase type II (POD) (SigmaAldrich) with the chromogen ABTS. 5 ml of ABTS/GOD/POD reagent (1g/l ABTS, 14,000 U/l GOD and 20 mg/l POD in phosphate 0.1 M pH7 buffer) were added to 200 l of sample or dilution. After an incubation time of 40 min at room temperature, the detection of the dye formed was carried out at 610 nm.

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2.6. Sugar analysis

The concentration of glucose, arabinose, xylose, rhamnose, galactose, mannose and cellobiose was determined by high-performance liquid chromatography (HPLC) using a HPLC model 1050 (Hewlett-Packard, Rockville, MD, USA), equipped with a refractive index detector. After a two step ltration on glass-bre (GF/D Wathman) and on 0.45 m Millex ltration units (Millipore Corporation, MA, USA) the samples were mixed with acetonitril 50% (v/v) and centrifuged at 140,000 rpm for 10 min, 25 l of the supernatant were injected on HPLC for sugar analysis. The internal standard was inositol.

Efcacy of the enzymatic treatment was evaluated by measuring sugar yield and residual dry weight. Glucose was mainly released during the hydrolysis. However, other sugars were found in hydrolysate in a concentration that did not exceed 20% of the glucose level, mainly xylose, and in small quantities rhamnose, arabinose, mannose and galactose (data not shown). Results of enzymatic hydrolysis are given Table 1. In all experiments, as it could be expected, the higher the glucose yield the lower the residual dry weight. Cocktails of cellulase, xylanase and FAE were particularly studied as they gave the best results while the use of laccase in association with hydrolases gave lower results. This can be due to the release of phenolic inhibitory compounds in the medium that decreases activity of cellulase and other hydrolases as showed by Ganble et al. [26]. This problem should be solved and yield enhanced by a sequential treatment with a rst step of a lignolytic enzyme (laccase with redox mediator) followed by a washing of the substrate and a second step of hydrolytic enzymes. A statistical analysis on the whole set of data following the model: z = a0 + a1 x + a2 y + a3 x2 + a4 y2 + a5 xy where x and y are xylanase and FAE added activities, respectively, and z is the response (glucose yield or dry residual weight) gave the equation for glucose production: z = 1.402 + 0.2179x + 0.0888y + 0.0149x2 + 0.00096y2 0.0220xy The result is signicant as the coefcients are signicantly different from 0 for x, y and xy and critical probability in test within a 5% range. Considering only experiments involving hydrolases (Table 2), the following model was obtained: z = 0.5632 + 0.5467x + 0.1489y 0.02810xy which was consistent with the previous model.

3. Results and discussion Several one-stage enzymatic saccharication treatments using enzymes (FAE, laccase and xylanase alone or in combination) in association with cellulases were evaluated for the conversion of lignocellulose of steam exploded wheat straw to monomeric sugars. An experimental factorial design was made in order to study the effect of laccase and hydrolase on the saccharication of wheat straw. All enzymes were added in a mixture containing 10 g (d.w.) of steam exploded wheat straw suspended in a phosphate buffer pH5 (10%, w/w) with 10 IU/g of cellulase. Incubation time was 24 h in an agitated vessel at a temperature of 50 C.

Table 1 Glucose yield and residual dry weight from hydrolysis of steam exploded wheat straw by mixture of fungal enzymes (xylanase, ferulic acid esterase (FAE A) and laccase Assay number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Xylanase (UI/g) 0 5 0 5 5 0 5 5 3 10 3 10 15 0 FAE (UI/g) 0 0 20 20 0 20 20 40 10 10 30 30 20 0 Laccase (UI/g) 20 0 0 20 20 0 0 0 0 0 0 0 0 0 Glucose yield (% dry weight) 21.6 39.5 34.7 18.2 21.0 32.2 36.5 32.4 33.2 35.4 49.0 18.6 38.4 1.4 Residual dry weight (g) 7.38 5.46 5.85 7.34 7.3 6.19 5.73 5.85 6.07 5.96 4.19 7.85 5.54 8.67

All experiments were conduced using 10 U/g of cellulase from Trichoderma reesei.

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M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897902

Table 2 Table of experimental values involving only hydrolases X 5 0 0 5 5 3 10 3 10 15 0 Y 0 20 20 20 40 10 10 30 30 20 0 X2 25 0 0 25 25 9 100 9 100 225 0 Y2 0 400 400 400 1600 100 100 900 900 400 0 XY 0 0 0 100 200 30 100 90 300 300 0 Z 3.95 3.47 3.22 3.65 3.24 3.32 3.54 4.9 1.86 3.84 0.14

X, xylanase (UI/g); Y, ferulic acid esterase (FAE A) (UI/g). All experiments were conduced using 10 U/g of cellulase from Trichoderma reesei.

In both cases, the model exhibited a critical point (Fig. 1A and B) corresponding to: d2 z d2 z = 2 = 0, for x = 5 and y = 20. 2 d x d x The critical point is roughly the same in the two models. Its practical signicance is that under these concentrations, xylanase and FAE act in synergy. As a consequence, there is an economical interest to use fewer enzyme in association rather than a high enzyme concentration. In such a case (above the critical point), there is a decrease in yield when enzymes are used in association, probably due to an overlapping of the actions of the two enzymes. As a consequence, for high enzyme concentration, better results should be obtained using only one of these enzymes.

Fig. 2. Glucose yield (%dry weight) from enzymatic hydrolysis of steam exploded straw (CEL: 10 UI/g of cellulase; XYL: 3 UI/g of xylanase, FAE A: 10 UI/g of Ferulic acid esterase A; Tween 20: 0.1% (w/v)).

Fig. 1. (A) Quantities of released glucose surface contours as function of xylanase and feruloyl esterase A activities. (B) Wheat straw dry residual weight surface contours as function of xylanase and feruloyl esterase (FAE A) activities.

These results provided a basis for further experiments aiming to improve the yield of the enzymatic hydrolysis of wheat straw using low enzyme concentration. Xylanase and FAE concentrations used were 3 and 10 IU/g, respectively, values chosen under the critical point allowing a synergistic effect of the two enzymes. The effect of temperature and addition of surfactant on the quantity of glucose released after 48 h of enzymatic hydrolysis were studied (Fig. 2). Tween 20 was chosen as the surfactant as it had previously been recognised to be a good enhancer of enzymatic cellulose hydrolysis, non-toxic and suitable for biotechnical use [27]. The effects of cellulase alone or in combination with xylanase, on glucose released was similar, reaching 18% and 35% at 37 C and 50 C, respectively. Treatment of wheat straw with cellulase and FAE A at 50 C had no benecial effect on the release of glucose compared to treatment with cellulase alone. This result is in good agreement with the results of Yu et al. [28] who reported that feruloyl esterase (III) alone from A. niger was unable to inuence the release of sugars from destarched wheat bran. However, the same experiment carried out at 37 C gave better results than cellulase alone or cellulase with xylanase. The highest release of glucose was obtained when cellulase, xylanase and FAE were used in mixture. The percentage of released glucose was increased from 37% to 51.4% when raising the temperature from 37 C to 50 C. The results obtained conrm the synergistic effect demonstrated above between xylanase and FAE A when enzymatic levels were located under the critical points. Whatever the enzymatic treatment, the increase in temperature from 37 C to 50 C improved the hydrolysis of cellulose. This could be explained by an increase of enzymatic activities which have an optimal temperature close to 50 C [29]. However, the increase is more signicant for the treatment with xylanase than with FAE. This increase, combined with the better result observed at 37 C for FAE, suggests that the xylanase activity is more dependent on temperature for this substrate. This correlates with the Arrhenius plots of enzyme activities on the test substrates (i.e. methyl ferulate for FAE and oat-spelt xylan

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Craig DANIELS for his helpful suggestions during the writing of this paper. References
[1] Eriksson T, Karlsson J, Tjerneld F. A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei. Appl Biochem Biotechnol 2002;101:4160. [2] Palonen H, Tjerneld F, Zacchi G, Tenkanen M. Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin. J Biotechnol 2004;107:6572. [3] Fang JM, Fowler P, Tomkinson J, Hill CAS. Preparation and characterisation of methylated hemicelluloses from wheat straw. Carbohyd Polym 2002;47:28593. [4] Lequart C, Nuzillard JM, Kurek B, Debeire P. Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides. Carbohyd Res 1999;319:10211. [5] Nunes AP, Pourquie J. Steam explosion pretreatment and enzymatic hydrolysis of eucalyptus wood. Bioresour Technol 1996;57:10710. [6] Durand H, Clanet M, Tiraby G. Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme Microb Technol 1988;10:3416. [7] Ilmen M, Saloheimo A, Onnela ML, Penttila ME. Regulation of cellulase gene expression in the lamentous fungus Trichoderma reesei. Appl Environ Microbiol 1997;63:1298306. [8] Warzywoda M, Larbre E, Pourquie J. Production and characterization of cellulolytic enzymes from Trichoderma reesei grown on various carbon sources. Bioresour Technol 1992;39:12530. [9] Mazeau K, Moine C, Krausz P, Gloaguen V. Conformational analysis of xylan chains. Carbohyd Res; in press. [10] Mueller-Harvey I, Hartley RD. Linkage of p-coumaroyl and feruloyl groups to cell-wall polysaccharides of barley straw. Carbohyd Res 1986;148:7185. [11] Akin DE, Morrison WH, Rigsby LL, Gamble GR, Sethuraman A, Eriksson K-EL. Biological delignication pf plant components by the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus. Anim Feed Sci Technol 1996;63:30521. [12] Yu P, Maenz DD, McKinnon JJ, Racz VJ, Christensen DA. Release of ferulic acid from oat hulls by Aspergillus ferulic acid esterase and Trichoderma xylanase. J Agric Food Chem 2002;50:162530. [13] Faulds CB, Williamson G. Release of ferulic acid from wheat bran by a ferulic acid esterase (FAE-III) from Aspergillus niger. Appl Microbiol Biotechnol 1995;43:10827. [14] Record E, Asther M, Sigoillot C, et al. Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application. Appl Microbiol Biotechnol 2003;62:34955. [15] Curreli N, Agelli M, Pisu B, Rescigno A, Sanjust E, Rinaldi A. Complete and efcient enzymic hydrolysis of pretreated wheat straw. Process Biochem 2002;37:93741. [16] Herpoel I, Moukha S, Lesage-Meessen L, Sigoillot JC, Asther M. Selection of Pycnoporus cinnabarinus strains for laccase production. FEMS Microbiology Letters 2000;183:3016. [17] Herpo el I, Jeller H, Fang G, et al. Efcient enzymatic delignication of wheat straw pulp by a sequential xylanaselaccase mediator treatment. J Pulp Paper Sci 2002;28:6771. [18] Herpo el I, Asther M, Sigoillot JC. Design and scale up of a process for manganese peroxidase production using the hypersecretory strain Phanerochaete chrysosporium I-1512. Biotechnol Bioeng 1999;65:46873. [19] Lomascolo A, Record E, Herpo el-Gimbert I, et al. Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer. J Appl Microbiol 2003;94:61824. [20] Pourqui e J, Warzywoda M, Chevron F, Th ery M, Lonchamp D, Vandecasteele JP. Scale up of cellulase production and utilization. In: Aubert JP, Beguin P, Millet J, editors. Biochemistry and Genetics of Cellulose Degradation FEMS Symposium, vol. 43. Oval Road, London: Academic Press Ltd.; 1987. p. 7186.

Fig. 3. Arrhenius plots of xylanase and FAE A. Slope for xylanase () and FAE A ( ) expressed as the logarithm of relative activities vs. inverse of enzyme incubation temperatures ranging from 30 C to 50 C.

for xylanase, Fig. 3). Slope for xylanase expressed as the logarithm of relative activities versus inverse of temperature is more negative than that of FAE. Addition of Tween 20, a non-ionic surfactant signicantly improved the hydrolysis of wheat straw by the enzymatic mixture. Glucose recovery reached 34% at 37 C and 46% at 50 C in the presence of cellulase and FAE A after addition of Tween 20. The mechanism of surfactant activation is probably due to its adsorption on hydrophobic surfaces mainly composed of lignin fragments. According to Eriksson et al. [1], this could prevent unproductive binding of cellulase or accessory enzymes (xylanase and FAE A) to lignin layers. This hypothesis is in strong accord with the work of Kaya et al. [30], which found that the enzyme activities increased with both non-ionic and cationic surfactants, that would be due to rather physical than chemical properties of surfactants. The level of glucose recovery (51% of dry weight) represents about 81% of the maximum amount of glucose recovery. Steam expolsion with acidic pre-treatment gave good results on Eucalyptus chips using commercial mixture of cellulase [31]. Curreli et al. [15] obtained better results but with more drastic pre-treatment and high level of puried cellulase. 4. Conclusion Enzymatic hydrolysis of wheat straw for ethanol production is bottle-necked by the cost of enzymes and the limitation of their efcacy due to the covalent linkages and physical binding between lignin and hemicellulosic grasses cell walls components [32]. Steam explosion seems the best suitable physical pretreatment of straw as it partially hydrolyses hemicellulose and increase the enzymatic digestibility of cellulose remaining in biomass residues. On the other hand, the optimisation of enzymatic treatment, including the use of accessory enzyme such as xylanases and laccase can reduce the concentrations of the enzymes needed and enhance the cost-effectiveness of ethanol production by enzymatic hydrolysis of lignocellulosic materials. Acknowledgments This research was supported by IFP (Institut franc ais du p etrole), by ADEME (Agrice program), and by grants from Tunisian Ministry of Higher Education. The authors thank Mr

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M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897902 [27] Ooshima H, Sakata M, Harano Y. Enhancement of enzymatichydrolysis of cellulose by surfactant. Biotechnol Bioeng 1986;28:1727 34. [28] Yu P, McKinnon JJ, Maenz DD, Olkowski AA, Racz VJ, Christensen DA. Enzymic release of reducing sugars from oat hulls by cellulase, as inuenced by Aspergillus ferulic acid esterase and Trichoderma xylanase. J Agric Food Chem 2003;51:21823. [29] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 2002;83:111. [30] Kaya F, Heitmann JA, Joyce TW. Inuence of surfactants on the enzymatic-hydrolysis of xylan and cellulose. TAPPI J 1995;78: 1507. [31] Ramos JP, Breuil C, Saddler JN. The use of enzyme recycling and the inuence of sugar accumulation on cellulose hydrolysis by Trichoderma cellulases. Enzyme Microb Tech 1993;15:1925. [32] Bidlack J, Malone M, Russel B. Molecular structure and component integration of secondary cell walls in plants. Proc Okla Acad Sci 1992;72:516.

[21] Pourqui e J, Warzywoda M. Cellulase production by Trichoderma reesei. In: Bioconversion of Forest and Agricultural Plant Residues. In: Saddler JN, editor. Biotechnology in Agriculture, vol. 9. Wallingford: CAB, International; 1993. p. 10025. [22] Sigoillot JC, Herpo el I, Frasse P, Moukha S, Lessage-Meessen L, Asther M. Laccase production by a monokaryotic strain of Pycnoporus cinnabarinus derived from a dikaryotic strain. World J Microbiol Biotechnol 1999;15:4814. [23] Ralet MC, Faulds CB, Williamson G, Thibault JF. Degradation of feruloylated oligosaccharides from sugar-beet pulp and wheat bran by ferulic acid esterases from Aspergillus niger. Carbohyd Res 1994;263:25769. [24] Mandels M, Hontz L, Nystrom J. Enzymatic hydrolysis of waste cellulose. Biotechnol Bioeng 1974;16:147193. [25] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:4268. [26] Gamble GR, Snook ME, Henrikson G, Akin DE. Phenolic constituents in ax bast tissue and inhibition of cellulase and pectinase. Biotechnol Lett 2000;22:7416.