Enzyme and Microbial Technology 40 (2007) 801805

Production of xylanase by Trichoderma longibrachiatum on a mixture of wheat bran and wheat straw: Optimization of culture condition by Taguchi method
Mehrdad Azin , Roya Moravej, Davood Zareh
Biotechnology Research Center, Iranian Research Organization for Science & Technology, P.O. Box 15815-3538, Tehran, Iran Received 14 June 2006; accepted 14 June 2006

Abstract The usefulness of Taguchi method for optimization of culture condition, designed for production of xylanase by Trichoderma longibrachiatum was investigated. L16 orthogonal array, by which the effect of ve factors in four levels, could be tested, was chosen. Analysis of variance (ANOVA) was performed on the obtained results and optimum condition suggested by statistical calculations was tested in a verication test. An increase of 41.9% in xylanase production was observed, after performing optimization techniques, including one-factor-at-a-time (OFAT) and Taguchi method, which indicates suitability of this method in microbiological processes optimizations. 2006 Elsevier Inc. All rights reserved.
Keywords: Optimization; Orthogonal array; Taguchi method; Trichoderma longibrachiatum; Xylanase

1. Introduction Microbial enzymes like xylanases are important additives in numerous areas from food processing to paper and pulp industries. These enzymes improve nutrient digestibility in certain diets both in ruminants and poultries, where no digesting enzymes which can digest complex cell wall carbohydrates, exist. The positive performance of these enzymes is demonstrated clearly in nutrition of poultries [13], biobleaching of paper pulp [4] and improving the baked products [5,6]. Xylanase hydrolyzes xylosidic linkages in xylan polymers [712]. Solid-state fermentation (SSF), whereby an insoluble substrate is fermented with sufcient but no free moisture [13], typically uses agricultural residues such as wheat bran, wheat straw, rice bran, etc. for production of larger amounts of microbial metabolites at a lower cost [1417], although, normally the production of industrial enzymes, like xylanase is performed by submerged culture [18,19]. Optimal environmental condition is a prerequisite for promotion of maximum growth and production of enzymes where SSF is used [20,21].

Corresponding author. Tel.: +98 21 88838350; fax: +98 21 88838350. E-mail address: azin@irost.org (M. Azin).

Trichoderma spp., major agents of agricultural waste decomposition and decay, possess a broad range of enzymes, and hence are known as good producers of lignocellulolytic enzymes [17,19,22]. However, use of other microorganisms from kingdom of bacteria such as Bacillus sp. [21], Cellulomonas [23], Streptomyces sp. [24], Thermomonospora [25], Thermotoga [11], and other thermophiles [26] and many species from fungi like white- or brown-rot fungi [27] and even anaerobic fungi [28] is documented. Optimization of production of xylanase is a prerequisite for its economical manufacturing. In this regard, normally the response surface method (RSM) is preferred by biologists [29]. Even for enhancement of xylanase production, this method has been used [30,31]. Taguchi method, as another way for designing fractional factorial experiments [32], is somehow not very well known for optimization of biotechnological processes. In this article, the effect of different proportions of substrates, growth temperature, initial pH, moisture level, inoculums size, and nitrogen source, each in four levels, were optimized by Taguchi method. The results of experiments performed for obtaining optimum levels of xylanase by Trichoderma longibrachiatum, using easily available substrates, like wheat straw and wheat bran in SSF, are discussed.

0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.06.013

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M. Azin et al. / Enzyme and Microbial Technology 40 (2007) 801805 Table 1 Factors and their levels which were studied by Taguchi method Factor Moisture % (w/w) Temperature ( C) pH Inoculuma Nitrogen source
a b

2. Materials and methods 2.1. Microorganism

The organism used in this study, T. longibrachiatum PTCC 5140, was obtained from Persian Type Culture Collection, Iranian Research Organization for Science & Technology, Tehran, Iran. The stock cultures were maintained on potato dextrose agar (PDA) slants at 4 C and subcultured at monthly intervals [33].

Level 1 50 25 4.5 105 Peptone

Level 2 55 28 5.5 106 CSPb

Level 3 60 31 6.5 107 (NH4 )2 SO4

Level 4 65 33 7 108 (NH4 )2 HPO4

2.2. Chemicals
All chemicals were of pure grade made by known manufacturers. Birch wood xylan, which was used for enzyme assay, was from Sigma Co.

Inoculum: spore number/g dry substrate. CSP: spray dried (powdered) corn steep liquor.

activity (U) is that amount of activity which released 1 mol d-xylose min1 [41].

2.3. Culture media

As substrate for SSF, air-dried and milled wheat straw [WS] (40-mesh size), obtained from local producers, and wheat bran [WB] (18-mesh size), Dine Pharmaceutical Co., Tehran, Iran, were utilized [3437]. The composition of mineral salts solution (g/l) was: 0.5 MgSO4 7H2 O; 0.5 CaCl2 2H2 O and 3.0 KH2 PO4 . Ten grams of wheat straw and wheat bran in different proportions (9:1 to 1:9) along with controls (10:0 and 0:10) were used as carbon source. As nitrogen source, 0.1 g of four different substrates: peptone (Merck Co., Germany), corn steep powder (spray dried corn steep liquor), from Glucosan Co., Ghasvin, Iran, (NH4 )2 SO4 and (NH4 )2 HPO4 were used and their effects were evaluated [38]. Corn steep liquor was delivered from manufacturer as a 6% (g dry weight/100 ml) liquid, and spray dried by a Compact Spray Dryer, APV Anhydro AS, Denmark, at 150 C inlet and 75 C outlet air temperature settings.

3. Experimental design and statistical analysis

One factor at a time [29] and Taguchi methods [32] were used for optimization of culture condition. The amount of nitrogen, ratio of mineral salts to solid substrate and ratio of wheat bran to wheat straw, were determined by one factor at a time method. Optimization of ve other factors: moisture percent (w/w), temperature of incubation, pH, inoculum density and type of nitrogen source, as shown by Table 1, were studied by Taguchi method. To perform the Taguchi method, 16 different experiments, by using L16 orthogonal array, were run, as shown in Table 2. Based on the primary results, a verication test was also performed to check the optimum condition. An analysis of variance (ANOVA) for the obtained results was investigated. For designing the experiments, analysis of variance and optimization of process, Qulitek-4 software, Nutek Inc., was used.

2.4. Growth condition

Fermentations were carried out in 250 ml capacity asks containing 10 g carbon source. Flasks were autoclaved at 121 C for 15 min, cooled and inoculated by fungal spores, which were grown on PDA for 4 days. To obtain the desired moisture content, nitrogen sources were added to carbon sources and mixed with buffer (at initial preset pH values) before sterilization. Inocula were prepared by washing the conidiospores from surface of a PDA slant, by addition of 25 ml sterile distilled water, containing 0.1% Tween 80 . Spore densities were determined using a counting chamber slide (Neubar style) before inoculation. Inocula were added such a way that desired moisture content was not affected; that is, total added liquid was calculated to reach the desired moisture content. Inoculated asks were incubated at different temperatures for 96 h. Each treatment was performed in four replicates to reduce the experimental errors.

4. Results 4.1. Effect of carbon source As the starting point, wheat straw (40-mesh size) was chosen as carbon source in SSF. An inoculum of 105 spores/g substrate was added to a medium of 50% (w/w) moisture, pH 5.5, and incubated in 26 C for 3 days. The xylanase activity was shown to be 417.2 U/g substrate, at the end of the fermentation. A mixture of different proportions of wheat bran (WB)/wheat straw (WS) was studied as next step. Between various ratios of WB/WS, highest level of xylanase, 479.7 U/g substrate, was produced at the ratio of 7:3 (Fig. 1). So, an increase of 14.9% in enzyme production was caused.

2.5. Enzyme extraction

Crude enzyme was extracted by adding 170 ml citratephosphate buffer (pH 5) containing 0.1% Tween 80 to each ask. The asks were then placed on a gyratory shaker at 200 rpm for 1 h at 25 C. The extract was ltered through nylon cloth to remove the biomass and culture medium residuals. After centrifugation of ltered extract at 7000 rpm for 30 min, the supernatant was used for determination of enzyme activity [39].

2.6. Enzyme assay

Xylanase activity was determined by measuring total reducing sugars released from 1% (w/v) birch wood xylan in 0.5 ml citrate buffer, 50 mM, pH 4.8, when 0.5 ml of a suitably diluted enzyme was added. A standard curve of d-xylose was used as reference. The mixture was incubated at 54 C for 30 min. The reaction was stopped by adding dinitrosalicylic acid (DNS) and the enzyme activity was determined as mentioned by Miller [40]. Each unit of xylanase

Fig. 1. Effect of different proportions of wheat bran/wheat straw on production of xylanase. Error bars correspond to standard deviation (three replicates); WB, wheat bran; WS, wheat straw.

M. Azin et al. / Enzyme and Microbial Technology 40 (2007) 801805 Table 2 Levels of ve different factors, applied in each of 16 trials, and obtained resultsa Trial number Levelsb of factors used in each trial Moisture % (w/w) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
a b

803

Temperature ( C) 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

pH 1 2 3 4 2 1 4 3 3 4 1 2 4 3 2 1

Inoculum (spore/g substrate) 1 2 3 4 3 4 1 2 4 3 2 1 2 1 4 3

Nitrogen source 1 2 3 4 4 3 2 1 2 1 4 3 3 4 1 2

Resultsa (unit enzyme/g substrate) 572.32 531.25 514.84 420.93 559.84 507.69 527.39 517.21 502.41 426.23 462.23 499.46 494.3 482.44 444.79 489.02 10.92 6.25 6.84 9.8 10.23 9.06 6.76 7.34 10.55 11.71 8.71 8.45 13.43 8.33 10.58 12.76

1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4

Shown gures are the average of results of four replicates standard deviation. For description of levels, refer to Table 1.

4.2. Effect of minerals The effect of amount of minerals, was studied in 1, 3, 5, and 7 concentrations, which were added in equal liquid volumes, so no changes were occurred in moisture content of the cultures. Not only no increase in xylanase production was observed by addition of mineral salt solution, but also slight decreases were recorded in higher concentrations of minerals (results not shown). 4.3. Experimental design by Taguchi method Effects of moisture, temperature, pH, inoculum size and type of nitrogen source were studied by Taguchi method, which is a fractional factorial experimental design. The results of experiments performed in this section showed that the maximum average yield of xylanase was 572.3 U/g substrate, which occurred when experiments conditions were as follows: moisture 50% (w/w); pH 4.5; temperature 25 C; inoculums size 105 spores/g substrate; where peptone was used as nitrogen source. Carbon source was a ratio of 7:3 of WB:WS. Table 2 shows the results thus obtained after performing the experiments. Fig. 2 depicts the main effect of each of these factors. By the term main effects, the average of obtained results
Table 3 Analysis of variance of main effects of factors Factor Moisture % (w/w) Temperature ( C) pH Inoculum (spore number/g substrate) Nitrogen source Other (error) Total d.f. (f) 3 3 3 3 3 0 15 Sum of squares (S) 35074.69 25761.80 20133.45 21913.05 10319.50 26484.25 139686.74 Variance (V) 11691.56 8587.27 6711.15 7304.35 3439.83 551.755 Fraction (F) 21.19 15.56 12.16 13.24 6.23 Pure sum (S ) 33419.42 24106.53 18478.19 20257.78 8664.24 Percent of participation P (%) 23.94 17.26 13.23 14.5 6.2 24.89 100.00 Fig. 2. Main effects of factors or average of obtained results (as U/g substrate), in which each factor is at a given level. For a description of levels, refer to Table 1.

(as unit of enzyme, produced per g of substrate), in which each factor is at a given level, is meant. Analysis of variance (ANOVA) of obtained results (Table 3) showed that, changing the moisture content of medium has been the most important factor in causing differences in obtained results. By the same way, the least important factor was the type of nitrogen source.

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M. Azin et al. / Enzyme and Microbial Technology 40 (2007) 801805

Table 4 Optimum conditions suggested by statistical calculations after performing the tests Factor Moisture % (w/w) Temperature ( C) pH Inoculum (spore/g substrate) Nitrogen source Total contribution of all factors Current grand average of performance Expected result at optimum condition Level description 55 25 5.5 105 CSP Level 2 1 2 1 2 Contributiona 30.08 34.28 10.88 22.57 14.64 112.45 497.88 610.33 Fig. 4. Changes in media dry weight (substrate plus biomass) during fermentation. Error bars correspond to standard deviation (four replicates).

a The amount of increasable enzyme units after performing the optimization, in relation to the average amount of produced enzyme in the experimental design.

When these results were analyzed, an optimum condition was suggested by calculations. Table 4 shows the suggested condition. Statistical calculations predicted that if the conditions were chosen as shown in Table 4, the enzyme production should reaches 610.33 U/g substrate. However, after performing the fermentation at said condition, the produced xylanase was about 592.7 U/g substrate, but since the difference between predicted and actual result was only about 2.97%, it should be regarded as acceptable. Therefore, comparing with 573.3 U/g substrate, produced before, a further increase of about 3.4% was achieved. 4.4. Time course of the fermentation In a separate experiment, it was shown that the highest amount of xylanase was produced after 96 h (Fig. 3). In this experiment, a comparison was also performed between the condition of trial number 1, which showed the best result among 16 trial conditions, and the optimum condition, which was found after statistical calculations. As it is shown, the new fermentation con-

dition was obviously better, for the rate of increasing the amount of enzyme in rst days was higher. As it was shown, in the rst and second days, about 2.7 times more enzyme was produced and the nal production of the enzyme was about 12% higher, too. 4.5. Dry weight loss Results showed that a sharp decrease in dry weight of cultures, in rst and second days of fermentation have occurred, which was followed by a gentle increase in dry weight in remaining days (Fig. 4). After 2 days of inoculation, there remained only 3.6 g from 10 g dry substrate, which indicated that about 64% of the substrate was consumed by the mould. It should be noted that, since separation of moulds mycelium and the solid particles of the substrate was practically impossible, measuring the weight of remaining dry substance, as a whole, after drying for 24 h at 95 C, was used as a means for following up the growth. After 6 days, the dry weight was gently increased to 4.78 g. 5. Discussion Taguchi method is not a usual way for optimizing biotechnological processes. Other methods such as response surface and PlakettBurman may be preferred because researchers are more familiar with them [2931]. Nevertheless, there are few references showing usefulness of Taguchi method in optimization of biotechnological processes [42]. According to the ndings here mentioned, this method may be used quite easily, since it has the ability to include categorical factors (e.g. nitrogen type) along with continuous ones (e.g. temperature). While at the beginning of the experiments the production of xylanase was about 417.2 U/g substrate, after primary optimization of the culture conditions, it was raised to 479.7 U/g substrate (14.9% increase). By using Taguchi optimization process, the enzyme produced was increased to 592.7 U/g substrate, indicating a further increase of about 23.5% in production of xylanase. As a result, enzyme production was nally increased about 41.9%, in relation to the initial step.

Fig. 3. Time course of xylanase production. (A) Culture condition as trial number 1 (moisture content, 50% (w/w); temperature, 25 C; pH 4.5; inoculum, 105 spores/g substrate; nitrogen source, peptone). (B) Culture condition as used in verication test (moisture, 55% (w/w); temperature, 25 C; pH 5.5; inoculum, 105 spores/g substrate; nitrogen source, CSP). Error bars correspond to standard deviation (three replicates).

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Acknowledgements The authors wish to thank Iranian Research Organization for Science & Technology (IROST) for its nancial support to the project. Effective assistance of Dr. Ranjit K. Roy, from Neutek Inc. for supplying the Qualitek-4 software is acknowledged in particular. References
[1] Almirall M, Esteven-Garcia E. In vitro stability of a -glucanase preparation from Trichoderma longibrachiatum and its effect in barley-based diet feed to broiler chicks. Anim Feed Sci Technol 1995;54:14958. [2] Annison G. Commercial enzyme supplementation of wheat-based diets raises ileal glucanase activitites and improves apparent metabolisable energy starch and pentosan digestibilities in broiler chickens. Anim Feed Sci Technol 1992;38:10521. [3] Classen HL. Cereal grain starch and exogenous enzymes in poultry diets. Anim Feed Sci Technol 1996;33:7914. [4] Viikari L, Kantelinen A, Sundquist J, Linko M. Xylanases in bleaching. From an idea to industry. FEMS Microbiol Lett 1994;13:33550. [5] Maat J, Roza M, Verbakel J, Stam H, Santos H, da Silva MJ, et al. Xylanases and their application in bakery. In: Visser J, et al., editors. Progress in biotechnology, vol. 7. Amsterdam: Elsevier; 1992., ISBN 0-444-89477-2 p. 34960. [6] Martnez-Anaya MA, Jim nez T. Physical properties of enzyme e supplemented doughs and relationship with bread quality parameters. Z Lebensm Unters Forsch A 1998;206:13442. [7] Bennet JW. Mycotechnology: the fungi in biotechnology. J Biotechnol 1998;66:1017. [8] Fogarty WM, Kelly. Microbial enzymes and biotechnology. Elsevier Applied Science; 1990, ISBN 1-851-66486-6. pp. 173 and 3489. [9] Ridder ER, Nokes SE. Optimization of solid-state fermentation parameters for the production of xylanase by Trichoderma longibrachiatum on wheat bran. Trans ASAE 1998;41:14539. [10] Ridder ER, Nokes SE. Optimization of solid-state fermentation parameters for the production of xylanase by Trichoderma longibrachiatum on wheat bran in a forced aeration system. Trans ASAE 1999;42:178590. [11] Sunna A, Antranikian G. Growth and production of xylanolytic enzymes by the extreme thermophilic anaerobic bacterium Thermotoga thermarum. Appl Microbiol Biotechnol 1997;45:6716. [12] Sunna A, Antranikian G. Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol 1997;17:3967. [13] Chahal DS. Solid-state fermentation with Trichoderma reesei for cellulase production. Appl Environ Microbiol 1985;49:20510. [14] Jecu L. Solid-state fermentation of agricultural waste for endoglucanase production. Ind Crop Prod 2000;11:15. [15] Waites MJ, Morgan NL. Industrial microbiology an introduction. Black Well Science; 2001, ISBN 0-632-05307-0. pp. 14950. [16] Lequart C, Nuzillard JM, Kurek B, Debeire P. Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl oligo saccharides. Carbohydr Res 1999;319:14. [17] Smits JP, Rinzema A, Tramper J, Van Sonsbeek HM, Knol W. Solid-state fermentation of wheat bran by Trichoderma reesei QM9414: substrate composition changes, C balance, enzyme production, growth and kinetics. Appl Microbiol Biotechnol 1996;46:48996. [18] Adamsen AK, Lindhagen J, Ahring BK. Optimization of extracellular xylanase production by Dictyoglomus sp. B1 in continuous culture. Appl Microbiol Biotechnol 1995;44:32732. [19] Beg QK, Kapoor M, Mahajan L, Hoondal GS. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 2001;56:32638.

[20] Deschamps F, Hute MC. Xylanase production in SSF: study of its properties. Appl Microbiol Biotechnol 1985;22:17780. [21] Gessesse P, Mamo G. High level xylanase production by alkaliphilic Bacillus sp. by using SSF. Enzyme Microb Technol 1999;25:68 72. [22] Xiong H, Turunen O, Pastinen O, Leisola M, von Weymarn N. Improved xylanase production by Trichoderma reesei grown on l-arabinose and lactose or d-glucose mixtures. Appl Microbiol Biotechnol 2004;64: 3538. [23] Vega-Estrada J, Flores-Cotera LB, Santiago A, Maga a-Plaza I, Montesn Horcasitas C. Draw-ll batch culture mode for production of xylanases by Cellulomonas avigena on sugar cane bagasse. Appl Microbiol Biotechnol 2002;58:4358. [24] Bega QK, Bhushanb B, Kapoora M, Hoondala GS. Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp. Enzyme Microb Technol 2000;27:45966. [25] Sudeep PG, Ahamd A, Rao MB. A novel thermostable xylanase from Thermomonospora sp.: inuence of additives on thermostability. Bioresource Technol 2001;78:2214. [26] Niehaus F, Bertoldo C, K hler M, Antranikian G. Extremophiles as a source a of novel enzymes for industrial application. Appl Microbiol Biotechnol 1999;51:71129. [27] Milagres AMF, Sales RM. Evaluating the basidiomycetes Poriamedula panis and Wolporia cocos for xylanase production. Enzyme Microb Technol 2001;28:5226. [28] Dijkerman R, Op den Camp HJM, Van der Drift C. Cultivation of anaerobic fungi in a 10-l fermenter system for the production of (hemi-)cellulolytic enzymes. Appl Microbiol Biotechnol 1996;46:8591. [29] Haaland DP. Experimental design in biotechnology. New York: Marcel Dekker Inc.; 1989, ISBN 0-8247-7881-2. [30] Souza MCde O, Roberto IC, Milagres AMF. Solid-state fermentation for xylanase production by Thermoascus aurantiacus using response surface methodology. Appl Microbiol Biotechnol 1999;52:76872. [31] Park YS, Kang SW, Lee JS, Hong SI, Kim SW. Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs. Appl Microbiol Biotechnol 2002;58:7616. [32] Roy RK. Design of experiments using the Taguchi approach. WileyInterscience; 2001, ISBN 0-471-36101-1. [33] Royer JC, Nakas JP. Xylanase production by Trichoderma longibrachiatum. Enzyme Microb Technol 1989;11:17882. [34] Abdullah AL, Tengerdy AF, Murphy VG. Optimization of solid-state fermentation of wheat straw. Biotechnol Bioeng 1985;27:207. [35] Acebal C, Castilon MP, Estrada P, Mata I. Enhanced cellulase production from Trichoderma reesei QM9414 on physically treated wheat straw. Appl Microbiol Biotechnol 1986;24:21823. [36] Gawande PV, Kamat MY. Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application. J Appl Microbiol 1999;87:5119. [37] Prasertsan P, Kittikul AH. Optimization for xylanase and cellulase production from Aniger niger ATTC 6275 in palm oil mill wastes and its application. World J Microbiol Biotechnol 1997;13:5559. [38] Shmala TR, Sreekantiah KR. Production of cellulase and d-xylanase by some selected fungal isolates. Enzyme Microb Technol 1986;8:17882. [39] Bailey MJ, Biely P, Putanen K. Interlaboratry testing of methods for assay of xylanase activity. J Biotechnol 1992;23:25770. [40] Miller G. Use of dinitro-salicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:4268. [41] Haapala R, Parkkinen E, Suominen M, Linko S. Production of endo1,4-beta-glucanase and xylanase with nylon-web immobilized and free Trichoderma ressei. Enzyme Microb Technol 1996;81:495501. [42] Lee M-T, Chen W-C, Chou C-C. Medium optimization by orthogonal array designs for cholesterol oxidase production by Rhodococcus equi number 23. Process Biochem 1997;32:697703.