Production of xylanase by Trichoderma longibrachiatum on a mixture of wheat bran and wheat straw: Optimization of culture condition by Taguchi method

Enzyme and Microbial Technology 40 (2007) 801–805

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 five 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 verification 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 [1–3], biobleaching of paper pulp [4] and improving the baked products [5,6]. Xylanase hydrolyzes xylosidic linkages in xylan polymers [7–12]. Solid-state fermentation (SSF), whereby an insoluble substrate is fermented with sufficient 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 [14–17], 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: [email protected] (M. Azin).

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

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 it’s 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, inoculum’s 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.

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2. Materials and methods

Table 1 Factors and their levels which were studied by Taguchi method

2.1. Microorganism

Factor

Level 1

Level 2

Level 3

Level 4

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].

Moisture % (w/w) Temperature (◦ C) pH Inoculuma Nitrogen source

50 25 4.5 105 Peptone

55 28 5.5 106 CSPb

60 31 6.5 107 (NH4 )2 SO4

65 33 7 108 (NH4 )2 HPO4

a

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.

2.3. Culture media

b

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 min−1 [41].

3. Experimental design and statistical analysis

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 [34–37]. 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.

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 five 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 verification 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

4. Results

Fermentations were carried out in 250 ml capacity flasks 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 flasks were incubated at different temperatures for 96 h. Each treatment was performed in four replicates to reduce the experimental errors.

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 citrate–phosphate buffer (pH 5) containing 0.1% Tween 80® to each flask. The flasks were then placed on a gyratory shaker at 200 rpm for 1 h at 25 ◦ C. The extract was filtered through nylon cloth to remove the biomass and culture medium residuals. After centrifugation of filtered 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.

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Table 2 Levels of five different factors, applied in each of 16 trials, and obtained resultsa Trial number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 a b

Levelsb of factors used in each trial Moisture % (w/w)

Temperature (◦ C)

pH

Inoculum (spore/g substrate)

Nitrogen source

Resultsa (unit enzyme/g substrate)

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

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

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

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

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

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

Shown figures 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 experiment’s conditions were as follows: moisture 50% (w/w); pH 4.5; temperature 25 ◦ C; inoculum’s 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

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.

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)

Sum of squares (S)

3 3 3 3 3 0

35074.69 25761.80 20133.45 21913.05 10319.50 26484.25

15

139686.74

Variance (V)

Fraction (F)

Pure sum (S )

11691.56 8587.27 6711.15 7304.35 3439.83 551.755

21.19 15.56 12.16 13.24 6.23

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

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Table 4 Optimum conditions suggested by statistical calculations after performing the tests Contributiona

Factor

Level description

Level

Moisture % (w/w) Temperature (◦ C) pH Inoculum (spore/g substrate) Nitrogen source

55 25 5.5 105

2 1 2 1

30.08 34.28 10.88 22.57

CSP

2

14.64

Total contribution of all factors Current grand average of performance Expected result at optimum condition

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 first days was higher. As it was shown, in the first and second days, about 2.7 times more enzyme was produced and the final 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 first 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 mould’s 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

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 verification 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).

Taguchi method is not a usual way for optimizing biotechnological processes. Other methods such as response surface and Plakett–Burman may be preferred because researchers are more familiar with them [29–31]. Nevertheless, there are few references showing usefulness of Taguchi method in optimization of biotechnological processes [42]. According to the findings 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 finally increased about 41.9%, in relation to the initial step.

M. Azin et al. / Enzyme and Microbial Technology 40 (2007) 801–805

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