Enzyme and Microbial Technology 39 (2006) 897–902

Enzymatic saccharification of wheat straw for bioethanol productionby a combined cellulase xylanase and feruloyl esterase treatment

M.G. Tabka a,∗, I. Herpoel-Gimbert a, F. Monod b, M. Asther a, J.C. Sigoillot a

a UMR 1163 INRA/Universites de Provence et de la Mediterranee de Biotechnologie des Champignons Filamenteux, IFR86-BAIM, ESIL,163 Avenue de Luminy, Case Postale 925, 13288 Marseille Cedex 09, France

b Institut Francais du Petrole 1&4, Avenue de Bois-Preau 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 tofermentable 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 implementinghydrolases (cellulases and xylanases from Trichoderma reesei, recombinant feruloyl esterase (FAE) from Aspergillus niger and oxidoreductases(Fe

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laccases from Pycnoporus cinnabarinus) were investigated to the saccharification of exploded wheat straw. A synergistic effect between cellulases,AE 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 ofnzymatic 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.

eywords: Fungal enzyme; Saccharification; Wheat straw

. Introduction

Production of fuel ethanol from renewable lignocellulosicaterials has been extensively studied in the last decades [1].ignocellulosic materials to be considered for ethanol produc-

ion include wood, hardwood rather than softwood which is moreifficult for enzymatic hydrolysis [2], crops from annual plants,gricultural residues and waste paper. Wheat straw is one ofhe most abundant crop residues in European countries with aroduction of 170 million tonnes and seems to be the cheapestnd the most useful raw material for ethanol production [3]. It isomprised mainly of cellulose (40%), hemicelluloses (26%) andbout 20% of lignin [4]. Hydrolysis of cellulose and hemicel-uloses is hampered by the overlapping of polymer embedded

icrofibrils. Several methods have been designed to increaseydrolysis yield, including acidic and steam pre-treatments, andteam explosion [5]. These treatments result in partial degra-ation of the polysaccharidic cell wall, opening fibres and

∗ Corresponding author. Tel.: +33 4 91 82 86 52; fax: +33 4 91 82 86 01.

allowing the penetration of chemicals or enzymes inside thestructure.

Enzymatic degradation of plant cell wall has been exten-sively studied. Cellulases (EC 3.2.1.4) from Trichoderma reeseiare mostly used, as several mutant strains of this fungus pro-duce high levels of extracellular cellulolytic enzymes, up to40 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 efficientlyhydrolyse cellulose [7]. In addition, T. reesei is able to producehemicellulases, mainly xylanases (EC 3.2.1.8), depending onthe growth conditions and substrate [8]. It is well known thatconjugated action of cellulases and hemicellulase results in ahigher final sugar production as compared to cellulases alone.

The major hemicellulose polymer in cereal and hardwood isxylan. Xylan consists of a �-1,4-linked d-xylose backbone andcan be substitued by different side groups such as l-arabinose,d-galactose, acetyl, feruloyl, p-coumaroyl and glucuronic acidresidues [9]. Ferulic acid is the most abundant hydroxy cinnamicacid in the cell wall of cereal [10]. It is covalentely cross-linkedto arabinoxylans by ester bonds and to components of lignin

E-mail address: gtabka@esil.univ-mrs.fr (M.G. Tabka). mainly by ether bonds [11]. Accessory enzymes such as feruloyl

141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2006.01.021

898 M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897–902

esterase (FAE; EC 3.1.1.73) should also act in synergy withxylanases by cleaving diferulic bridges between xylan chains,opening the structures and releasing lignin [12,13].

In Trichoderma strains no FAE activity was detected. Thisenzyme occurs naturally in Aspergillus niger exoenzymes buthomologous cloning of the FAE gene enhances the production ofabout 25-fold, allowing application experiments [14]. Removalof lignin could be conducted by alkaline/oxydative extraction[15] but extensive delignification requires very extreme condi-tions as in chemical paper pulp production. Enzymatic deligni-fication is achieved naturally by white-rot fungi. These organ-isms, belonging to the basidiomycete family, produce oxydativeenzymes such as laccases that generate radicals able to cleavecovalent bonds in lignin. These enzymes could also act in syn-ergy with cellulases and hemicellulases by removing the ligninlayer on the fibre surface.

In this study, we will examine the role of some accessoryenzymes in the extensive hydrolysis of wheat straw by the cel-lulases and xylanases of T. reesei. Feruloyl esterase from A.niger, and laccases from Pycnoporus cinnabarinus were cho-sen and will be used alone or in association with cellulases andxylanases in order to enhance wheat straw saccharification.

2. Materials and methods

2.1. Fungal strain and enzyme production

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Esterase activity was assayed as previously described by Ralet et al. [23] asa function of time, using methyl ferulate (MFA) or methyl sinapinate (MSA) asthe substrate.

Exoglucanase (FPase; Exo-1, 4-�-d-glucanase, EC 3.2.1.91) activity wasdetermined according to Mandels et al. [24]. The assay mixture (2 ml) consistedof a 1.9 ml citrate buffer (50 mM, pH 4.5), Whatman no. 1 filter paper (50 mg,1 cm × 3 cm) and 0.1 ml of a suitably diluted enzyme. The reaction mixture wasincubated at 50 ◦C for 60 min.

Endoglucanase (CMCase, Endo-1, 4-�-d-glucanase; EC 3.2.1.4) activitywas determined according to Mandels et al. [24] with slight modification. Thetotal reaction of 1 ml contained a 0.5 ml sample of a suitably diluted enzyme and0.5 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 sim-ilar conditions as described above, except that 1% xylan solution (oat spelts,Sigma–Aldrich, Saint Quentin Fallavier, France) was used as the substrate inplace of CMC.

Reducing sugars were determined by the dinitrosalicylic acid (DNS) usingMiller’s method [25]. All activities were expressed in international units (U)defined as 1 �mol of glucose or xylose produced per minute. Experiments wereperformed in triplicate and standard error was lower than 10% of the mean.

2.3. Origin of the steam exploded wheat straw

Acidic steam-pretreated wheat straw was used as substrate in the hydroly-sis experiments. The pretreated substrate was kindly supplied by IFP (InstitutFrancais du Petrole-Rueil-Mallmaison, France). The raw material for the pre-treatement was chopped wheat straw furnished by VALAGRO (Poitiers-France).The acidic impregnation of the shopped wheat straw was achieved by soaking itovernight in a solution of 0.08N H2SO4. After chemical reaction, the substratews1f

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The strain P. cinnabarinus BRFM 137 (Banque de Ressources Fongiques dearseille, Marseille, France) and described as a hypersecretory laccase strain

16] was used in this study. Large scale fungal culture was grown in a 15-lioreactor as described by Herpoel et al. [17,18] using ethanol as an inducer19]. Ethanol was added gradually to the medium to avoid inhibition of theungal growth. Ethanol at 8 g/L was added to the culture broth on the day ofnoculation. This concentration was maintained for 3 days by adding ethanolvery day. The ethanol concentration was then increased to 10 g/l and maintainedt this level until the end of the culture. After eight days, the culture supernatantas harvested and concentrated by ultrafiltration using a 10 kDa membrane

Millipore S.A., Molsheim, France).The strain A. niger BRFM 451 obtained in our laboratory by homologous

verexpression of the feruloyl esterase A gene (faeA) [14] was used for theroduction of feruloyl esterase in 15 l bioreactor. Culture conditions were trans-osed from Erlenmeyers culture previously described by Record et al. [14]. Theioreactor was filled with 12 l (working volume) of basal medium containing0 g/l of glucose and were inoculated with 105 conidiospores of A. niger per ml.he pH of the medium was maintained at 5.2 using 4N citric acid solution. The

ermentation was carried out at a stirrer speed of 350 rpm and an aeration ratef 0.5 VVM (volume air per volume reactor per minute) at 30 ◦C. The cultureupernatant was harvested after 6 days and concentrated by ultrafiltration using10 kDa membrane (Millipore S.A.).

Two enzymatic preparations containing hydrolases from T. reesei CL 847ere kindly provided by SAF-ISIS (Souston, France) from the LESAFFREroup. The enzyme mixtures containing mainly cellulases or xylanases werebtained by fed-batch fermentation with different carbon sources [8,20,21]. Therst sample exhibited only cellulase specific activity as high as 0.664 U/mgf exo-glucanase and 25.6 U/mg of endo-glucanase. No Xylanase activity wasetected, while the second sample of hydrolases preparation presented mainlyylanase activity (27.3 U/mg).

.2. Enzyme activity assays

Laccase activity was determined as previously described by Sigoillot etl. [22] using 2,2′-azino-bis-[3-ethylthiazoline-6-sulphonate] (ABTS) as theubstrate.

as drained and pressed. The spined straw was maintained for 1 min at a pres-ure of 100 bars. The wet material was then exploded under steam pressure at8 bars in a batch cooker. At the end, the pre-treated material was washed twiceor 30 min and stored as such in deep freeze (−20 ◦C).

.4. Enzymatic hydrolysis experiments

Experiments were carried out on 10 g of pretreated wheat straw (dry weight)n a stainless steel vessel (500 ml working volume) incubated in a water batho 50 ◦C, the substrate was suspended to 10% (w/v, dry) in 100 mM sodiumcetate buffer at pH 4.8 and enzymes were used alone or in association in ane-stage treatment. The concentrations of enzyme used were: 5 U/g (d.w.) ofylanase, 10 U/g (d.w.) of cellulase, 20 U/g (d.w.) of FAE and 20 U/g (d.w.) ofaccase.

Treatment with laccase was performed under 150 kPa oxygen. The reac-ion medium was stirred at 150 rpm by mechanical agitation (marine propeller)or 24 h. Assays were carried out in triplicate. Supernatants were analysed forlucose and reducing group contents. Wheat straw dry weight after enzymaticreatment was estimated by filtration on glass-fibre (GF/D Wathman, Maidstone,K) and drying at 110 ◦C until the mass was constant.

Optimisation of the saccharification conditions was carried out on 0.2 g ofretreated wheat straw (dry weight) in 15 ml screw-cap tubes. Experiments werearried out at 2% consistency in 100 mM sodium acetate buffer at pH 4.8. Sub-trate was treated with one-stage or sequential enzymatic treatment. Enzymeoncentrations were: 3 U/g (d.w.) of xylanase, 10 U/g (d.w.) of FAE, 10 U/gd.w.) of cellulase and 20 U/g (d.w.) of laccase. Treatment with laccase was per-ormed in presence of 1 or 10% of N-hydroxy-N-phenylacetamide (NAH) or 3%f hydroxylbenzotriazole (HBT) as chemical mediator for 4 h. In sequential treat-ent, after the first enzymatic stage performed under 150 kPa oxygen, substrateas filtered, washed twice with distilled water and centrifuged at 12,000 rpm

or 15 min at 4 ◦C. Hydrolysis experiments were performed in an incubator at7◦ or 50 ◦C with gentle rotative agitation for 48 h.

.5. Chemical hydrolysis of pre-treated wheat straw

Before any treatment, pre-treated wheat straw was washed thoroughly withistilled water overnight; afterwards it was milled in a Waring Blendor to obtain

M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897–902 899

a coarse powder. This was net filtered and conserved at −20 ◦C for enzymatichydrolysis essays.

In order to estimate the total glucose concentration in wheat straw, a totalacidic hydrolysis analysis was performed. A second step of crushing was carriedout using 0.3 g (dry weight) of straw coarse powder. The fine powder obtainedwas suspended in 2 ml of 72% H2SO4 and kept at 25 ◦C for 30 min. The acidicmixture was diluted by adding distilled water and kept at 100 ◦C in a heatingdry block for 2 h, after centrifugation at 5000 × g for 15 min, the supernatantwas neutralized with 25% ammoniac and analysed to estimate the total glucoseconcentration. Glucose present in hydrolysis samples was estimated using thecombined enzymatic test, i.e. glucose oxidase type II-S from A. niger (GOD)and Horseradish peroxidase type II (POD) (Sigma–Aldrich) with the chromogenABTS. 5 ml of ABTS/GOD/POD reagent (1g/l ABTS, 14,000 U/l GOD and20 mg/l POD in phosphate 0.1 M pH7 buffer) were added to 200 �l of sample ordilution. After an incubation time of 40 min at room temperature, the detectionof the dye formed was carried out at 610 nm.

2.6. Sugar analysis

The concentration of glucose, arabinose, xylose, rhamnose, galactose, man-nose and cellobiose was determined by high-performance liquid chromatog-raphy (HPLC) using a HPLC model 1050 (Hewlett-Packard, Rockville, MD,USA), equipped with a refractive index detector. After a two step filtration onglass-fibre (GF/D Wathman) and on 0.45 �m Millex filtration units (MilliporeCorporation, MA, USA) the samples were mixed with acetonitril 50% (v/v) andcentrifuged at 140,000 rpm for 10 min, 25 �l of the supernatant were injectedon HPLC for sugar analysis. The internal standard was inositol.

3. Results and discussion

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Efficacy of the enzymatic treatment was evaluated by mea-suring sugar yield and residual dry weight. Glucose was mainlyreleased during the hydrolysis. However, other sugars werefound in hydrolysate in a concentration that did not exceed 20%of the glucose level, mainly xylose, and in small quantities rham-nose, arabinose, mannose and galactose (data not shown).

Results of enzymatic hydrolysis are given Table 1. In allexperiments, as it could be expected, the higher the glucoseyield the lower the residual dry weight. Cocktails of cellulase,xylanase and FAE were particularly studied as they gave the bestresults while the use of laccase in association with hydrolasesgave lower results. This can be due to the release of phenolicinhibitory compounds in the medium that decreases activity ofcellulase and other hydrolases as showed by Ganble et al. [26].This problem should be solved and yield enhanced by a sequen-tial treatment with a first step of a lignolytic enzyme (laccasewith redox mediator) followed by a washing of the substrateand a second step of hydrolytic enzymes.

A statistical analysis on the whole set of data following themodel:

z = a0 + a1x + a2y + a3x2 + a4y

2 + a5xy

where x and y are xylanase and FAE added activities, respec-tively, and z is the response (glucose yield or dry residual weight)gave the equation for glucose production:

z

dwh

z

w

TG at stral

A se (U

11111

A sei.

Several one-stage enzymatic saccharification treatmentssing enzymes (FAE, laccase and xylanase alone or in com-ination) in association with cellulases were evaluated for theonversion of lignocellulose of steam exploded wheat straw toonomeric sugars.An experimental factorial design was made in order to study

he effect of laccase and hydrolase on the saccharification ofheat straw. All enzymes were added in a mixture contain-

ng 10 g (d.w.) of steam exploded wheat straw suspended inphosphate buffer pH5 (10%, w/w) with 10 IU/g of cellulase.

ncubation time was 24 h in an agitated vessel at a temperaturef 50 ◦C.

able 1lucose yield and residual dry weight from hydrolysis of steam exploded whe

accase

ssay number Xylanase (UI/g) FAE (UI/g) Lacca

1 0 0 202 5 0 03 0 20 04 5 20 205 5 0 206 0 20 07 5 20 08 5 40 09 3 10 00 10 10 01 3 30 02 10 30 03 15 20 04 0 0 0

ll experiments were conduced using 10 U/g of cellulase from Trichoderma ree

= 1.402 + 0.2179x + 0.0888y + 0.0149x2

+ 0.00096y2 − 0.0220xy

The result is significant as the coefficients are significantlyifferent from 0 for x, y and xy and critical probability in testithin a 5% range. Considering only experiments involvingydrolases (Table 2), the following model was obtained:

= 0.5632 + 0.5467x + 0.1489y − 0.02810xy

hich was consistent with the previous model.

w by mixture of fungal enzymes (xylanase, ferulic acid esterase (FAE A) and

I/g) Glucose yield (% dry weight) Residual dry weight (g)

21.6 7.3839.5 5.4634.7 5.8518.2 7.3421.0 7.332.2 6.1936.5 5.7332.4 5.8533.2 6.0735.4 5.9649.0 4.1918.6 7.8538.4 5.54

1.4 8.67

900 M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897–902

Table 2Table of experimental values involving only hydrolases

X Y X2 Y2 XY Z

5 0 25 0 0 3.950 20 0 400 0 3.470 20 0 400 0 3.225 20 25 400 100 3.655 40 25 1600 200 3.243 10 9 100 30 3.32

10 10 100 100 100 3.543 30 9 900 90 4.9

10 30 100 900 300 1.8615 20 225 400 300 3.84

0 0 0 0 0 0.14

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

In both cases, the model exhibited a critical point (Fig. 1Aand B) corresponding to:

d2z

d2x= d2z

d2x= 0, for x = 5 and y = 20.

The critical point is roughly the same in the two models. Its prac-tical significance is that under these concentrations, xylanaseand FAE act in synergy. As a consequence, there is an econom-ical interest to use fewer enzyme in association rather than ahigh enzyme concentration. In such a case (above the criticalpoint), there is a decrease in yield when enzymes are used inassociation, probably due to an overlapping of the actions of thetwo enzymes. As a consequence, for high enzyme concentra-tion, better results should be obtained using only one of theseenzymes.

Fxs

Fig. 2. Glucose yield (%dry weight) from enzymatic hydrolysis of steamexploded 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)).

These results provided a basis for further experiments aim-ing to improve the yield of the enzymatic hydrolysis of wheatstraw using low enzyme concentration. Xylanase and FAE con-centrations used were 3 and 10 IU/g, respectively, values chosenunder the critical point allowing a synergistic effect of the twoenzymes. The effect of temperature and addition of surfactanton the quantity of glucose released after 48 h of enzymatichydrolysis were studied (Fig. 2). Tween 20 was chosen as thesurfactant as it had previously been recognised to be a goodenhancer of enzymatic cellulose hydrolysis, non-toxic and suit-able for biotechnical use [27]. The effects of cellulase alone orin combination with xylanase, on glucose released was similar,reaching 18% and 35% at 37 ◦C and 50 ◦C, respectively. Treat-ment of wheat straw with cellulase and FAE A at 50 ◦C had nobeneficial effect on the release of glucose compared to treat-ment with cellulase alone. This result is in good agreement withthe results of Yu et al. [28] who reported that feruloyl esterase(III) alone from A. niger was unable to influence the release ofsugars from destarched wheat bran. However, the same experi-ment carried out at 37 ◦C gave better results than cellulase aloneor cellulase with xylanase. The highest release of glucose wasobtained when cellulase, xylanase and FAE were used in mix-ture. The percentage of released glucose was increased from37% to 51.4% when raising the temperature from 37 ◦C to 50 ◦C.The results obtained confirm the synergistic effect demonstratedabove between xylanase and FAE A when enzymatic levels werel

tTwttoict

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

ocated under the critical points.Whatever the enzymatic treatment, the increase in tempera-

ure from 37 ◦C to 50 ◦C improved the hydrolysis of cellulose.his could be explained by an increase of enzymatic activitieshich have an optimal temperature close to 50 ◦C [29]. However,

he increase is more significant for the treatment with xylanasehan with FAE. This increase, combined with the better resultbserved at 37 ◦C for FAE, suggests that the xylanase activ-ty is more dependent on temperature for this substrate. Thisorrelates with the Arrhenius plots of enzyme activities on theest substrates (i.e. methyl ferulate for FAE and oat-spelt xylan

M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897–902 901

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

for xylanase, Fig. 3). Slope for xylanase expressed as the loga-rithm of relative activities versus inverse of temperature is morenegative than that of FAE. Addition of Tween 20, a non-ionicsurfactant significantly improved the hydrolysis of wheat strawby the enzymatic mixture. Glucose recovery reached 34% at37 ◦C and 46% at 50 ◦C in the presence of cellulase and FAE Aafter addition of Tween 20. The mechanism of surfactant activa-tion is probably due to its adsorption on hydrophobic surfacesmainly composed of lignin fragments. According to Erikssonet al. [1], this could prevent unproductive binding of cellulaseor 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 bothnon-ionic and cationic surfactants, that would be due to ratherphysical than chemical properties of surfactants.

The level of glucose recovery (51% of dry weight) repre-sents about 81% of the maximum amount of glucose recovery.Steam expolsion with acidic pre-treatment gave good results onEucalyptus chips using commercial mixture of cellulase [31].Curreli et al. [15] obtained better results but with more drasticpre-treatment and high level of purified cellulase.

4. Conclusion

Enzymatic hydrolysis of wheat straw for ethanol productionitb[

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Craig DANIELS for his helpful suggestions during the writingof this paper.

References

[1] Eriksson T, Karlsson J, Tjerneld F. A model explaining declining rate inhydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A)and endoglucanase I (cel7B) of Trichoderma reesei. Appl BiochemBiotechnol 2002;101:41–60.

[2] Palonen H, Tjerneld F, Zacchi G, Tenkanen M. Adsorption of Tricho-derma reesei CBH I and EG II and their catalytic domains on steampretreated softwood and isolated lignin. J Biotechnol 2004;107:65–72.

[3] Fang JM, Fowler P, Tomkinson J, Hill CAS. Preparation and characteri-sation of methylated hemicelluloses from wheat straw. Carbohyd Polym2002;47:285–93.

[4] Lequart C, Nuzillard JM, Kurek B, Debeire P. Hydrolysis of wheat branand straw by an endoxylanase: production and structural characterizationof cinnamoyl-oligosaccharides. Carbohyd Res 1999;319:102–11.

[5] Nunes AP, Pourquie J. Steam explosion pretreatment and enzymatichydrolysis of eucalyptus wood. Bioresour Technol 1996;57:107–10.

[6] Durand H, Clanet M, Tiraby G. Genetic improvement of Trichodermareesei for large scale cellulase production. Enzyme Microb Technol1988;10:341–6.

[7] Ilmen M, Saloheimo A, Onnela ML, Penttila ME. Regulation of cellulasegene expression in the filamentous fungus Trichoderma reesei. ApplEnviron Microbiol 1997;63:1298–306.

[8] Warzywoda M, Larbre E, Pourquie J. Production and characterization ofcellulolytic enzymes from Trichoderma reesei grown on various carbonsources. Bioresour Technol 1992;39:125–30.

[

[

[

[

[

[

[

[

[

[

[

s bottle-necked by the cost of enzymes and the limitation ofheir efficacy due to the covalent linkages and physical bindingetween lignin and hemicellulosic grasses cell walls components32].

Steam explosion seems the best suitable physical pre-reatment of straw as it partially hydrolyses hemicellulose andncrease the enzymatic digestibility of cellulose remaining iniomass residues. On the other hand, the optimisation of enzy-atic treatment, including the use of accessory enzyme such

s xylanases and laccase can reduce the concentrations of thenzymes needed and enhance the cost-effectiveness of ethanolroduction by enzymatic hydrolysis of lignocellulosic materials.

cknowledgments

This research was supported by IFP (Institut francais duetrole), by ADEME (Agrice program), and by grants fromunisian Ministry of Higher Education. The authors thank Mr

[9] Mazeau K, Moine C, Krausz P, Gloaguen V. Conformational analysisof xylan chains. Carbohyd Res; in press.

10] Mueller-Harvey I, Hartley RD. Linkage of p-coumaroyl and feruloylgroups to cell-wall polysaccharides of barley straw. Carbohyd Res1986;148:71–85.

11] Akin DE, Morrison WH, Rigsby LL, Gamble GR, Sethuraman A, Eriks-son K-EL. Biological delignification pf plant components by the whiterot fungi Ceriporiopsis subvermispora and Cyathus stercoreus. AnimFeed Sci Technol 1996;63:305–21.

12] Yu P, Maenz DD, McKinnon JJ, Racz VJ, Christensen DA. Releaseof ferulic acid from oat hulls by Aspergillus ferulic acid esterase andTrichoderma xylanase. J Agric Food Chem 2002;50:1625–30.

13] Faulds CB, Williamson G. Release of ferulic acid from wheat bran bya ferulic acid esterase (FAE-III) from Aspergillus niger. Appl MicrobiolBiotechnol 1995;43:1082–7.

14] Record E, Asther M, Sigoillot C, et al. Overproduction of the Aspergillusniger feruloyl esterase for pulp bleaching application. Appl MicrobiolBiotechnol 2003;62:349–55.

15] Curreli N, Agelli M, Pisu B, Rescigno A, Sanjust E, Rinaldi A. Com-plete and efficient enzymic hydrolysis of pretreated wheat straw. ProcessBiochem 2002;37:937–41.

16] Herpoel I, Moukha S, Lesage-Meessen L, Sigoillot JC, Asther M. Selec-tion of Pycnoporus cinnabarinus strains for laccase production. FEMSMicrobiology Letters 2000;183:301–6.

17] Herpoel I, Jeller H, Fang G, et al. Efficient enzymatic delignification ofwheat straw pulp by a sequential xylanase–laccase mediator treatment.J Pulp Paper Sci 2002;28:67–71.

18] Herpoel I, Asther M, Sigoillot JC. Design and scale up of a process formanganese peroxidase production using the hypersecretory strain Phane-rochaete chrysosporium I-1512. Biotechnol Bioeng 1999;65:468–73.

19] Lomascolo A, Record E, Herpoel-Gimbert I, et al. Overproduction oflaccase by a monokaryotic strain of Pycnoporus cinnabarinus usingethanol as inducer. J Appl Microbiol 2003;94:618–24.

20] Pourquie J, Warzywoda M, Chevron F, Thery M, Lonchamp D, Vande-casteele JP. Scale up of cellulase production and utilization. In: AubertJP, Beguin P, Millet J, editors. Biochemistry and Genetics of CelluloseDegradation FEMS Symposium, vol. 43. Oval Road, London: AcademicPress Ltd.; 1987. p. 71–86.

902 M.G. Tabka et al. / Enzyme and Microbial Technology 39 (2006) 897–902

[21] Pourquie J, Warzywoda M. Cellulase production by Trichoderma reesei.In: Bioconversion of Forest and Agricultural Plant Residues. In: Sad-dler JN, editor. Biotechnology in Agriculture, vol. 9. Wallingford: CAB,International; 1993. p. 100–25.

[22] Sigoillot JC, Herpoel I, Frasse P, Moukha S, Lessage-Meessen L,Asther M. Laccase production by a monokaryotic strain of Pycno-porus cinnabarinus derived from a dikaryotic strain. World J MicrobiolBiotechnol 1999;15:481–4.

[23] Ralet MC, Faulds CB, Williamson G, Thibault JF. Degradation of feru-loylated oligosaccharides from sugar-beet pulp and wheat bran by ferulicacid esterases from Aspergillus niger. Carbohyd Res 1994;263:257–69.

[24] Mandels M, Hontz L, Nystrom J. Enzymatic hydrolysis of waste cellu-lose. Biotechnol Bioeng 1974;16:1471–93.

[25] Miller GL. Use of dinitrosalicylic acid reagent for determination ofreducing sugar. Anal Chem 1959;31:426–8.

[26] Gamble GR, Snook ME, Henrikson G, Akin DE. Phenolic constituentsin flax bast tissue and inhibition of cellulase and pectinase. BiotechnolLett 2000;22:741–6.

[27] Ooshima H, Sakata M, Harano Y. Enhancement of enzymatic-hydrolysis of cellulose by surfactant. Biotechnol Bioeng 1986;28:1727–34.

[28] Yu P, McKinnon JJ, Maenz DD, Olkowski AA, Racz VJ, ChristensenDA. Enzymic release of reducing sugars from oat hulls by cellulase,as influenced by Aspergillus ferulic acid esterase and Trichodermaxylanase. J Agric Food Chem 2003;51:218–23.

[29] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol pro-duction: a review. Bioresour Technol 2002;83:1–11.

[30] Kaya F, Heitmann JA, Joyce TW. Influence of surfactants onthe enzymatic-hydrolysis of xylan and cellulose. TAPPI J 1995;78:150–7.

[31] Ramos JP, Breuil C, Saddler JN. The use of enzyme recycling and theinfluence of sugar accumulation on cellulose hydrolysis by Trichodermacellulases. Enzyme Microb Tech 1993;15:19–25.

[32] Bidlack J, Malone M, Russel B. Molecular structure and componentintegration of secondary cell walls in plants. Proc Okla Acad Sci1992;72:51–6.