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Optimization of nutrient medium containing agricultural wastes for xylanase production by Aspergillus niger B03 using optimal composite experimental design
Bioresource Technology 98 (2007) 2671–2678
Optimization of nutrient medium containing agricultural wastes for xylanase production by Aspergillus niger B03 using optimal composite experimental design Georgi Todorov Dobrev a,*, Ivan Genov Pishtiyski a, Veselin Stanchev Stanchev b, Rositza Mircheva c b
a University of Food Technologies, Department of Biochemistry and Molecular Biology, 26 Maritza Boulevard, Plovdiv 4002, Bulgaria University of Food Technologies, Department of Automatics, Information and Control Equipment, 26 Maritza Boulevard, Plovdiv 4002, Bulgaria c Biovet Ltd., Research and Development Analytical Department, 39 Petar Rakov, Peshtera 4550, Bulgaria
Received 23 May 2006; received in revised form 31 July 2006; accepted 17 September 2006 Available online 7 November 2006
Abstract The xylanase biosynthesis is induced by its substrate – xylan. The high xylan content in some of the wastes like corn cobs and wheat bran makes them an accessible and cheap source of inducers. Nutrient medium for xylanase biosynthesis in submerged cultivation of Aspergillus niger B03 has been optimized. The optimization process was analyzed using optimal composite experimental design and response surface methodology. The predicted by the regression model optimum components of nutrient medium are as follows (g/l): (NH4)2HPO4 2.6, urea 0.9, corn cobs 24.0, wheat bran 14.6 and malt sprout 6.0. Five parallel experiments have been carried out, at definite, optimum components concentrations of the nutrient medium, and a mean value of the activity Y = 996.30 U/ml has been obtained. The xylanase activity, obtained with the optimized nutrient medium is 33% higher than the activity, achieved with the basic medium. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Xylanase; Optimization; Agricultural waste; Aspergillus
1. Introduction The hemicelluloses unite a big group of high molecular polysaccharides, insoluble in water but soluble in alkaline solutions. These polysaccharides are associated with cellulose and lignin and play an important structurally-supportive role in building up of plant cell walls (Bidlack et al., 1992; Nakamura, 2003). The composition of hemicelluloses, includes branched heteropolymers of pentoses (xylose, arabinose), hexoses (mannose, galactose, glucose), and uronic acids. Xylan is the main hemicellulolytic polysaccharide found in plant cell walls. Xylan is the second
*
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one in its distribution polysaccharide in nature after cellulose. It comprises 20–30% dry weight of the plants (Saha, 2003). The molecule of xylan consists of a b-1,4-linked D-xylose backbone and can be substituted by different side groups such as L-arabinose, D-galactose, acetyl, feruloyl, p-coumarouyl and glucuronic acid residues (Izydorczyk and Biliaderis, 1995; Kulkarni et al., 1999; Subramanyan and Prema, 2002). For the total hydrolysis of xylan the action of several enzymes is needed: xylanase (endo-1,4-b-D-xylanase EC 3.2.1.8), b-D-xylosidase (EC 3.2.1.37), a-L-arabinofuranosidase (EC 3.2.1.55), a-L-glucuronosidase (EC 3.2.1.139), acetylxylan esterase (EC 3.1.1.72), ferulic acidic esterase (EC 3.1.1.73) and q-coumaric acidic esterase (EC 3.1.1.-). Xylanase plays a key role in xylan hydrolysis to xylooligosaccharides (Colins et al., 2005; Coughlan and Hazlewood,
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1993; Kulkarni et al., 1999; Subramanyan and Prema, 2002). Xylotriose is the lowest molecular olygosaccharide that can be obtained under the action of the most studied by now xylanases (Ryan et al., 2003). The interest towards xylanase enzyme preparations has been dictated by their wide applications in various fields of industry. The enzyme hydrolysis of xylan lies in the basis of its utilization as an energy source in animal feed or in different biotechnological processes (Coughlan and Hazlewood, 1993; Kulkarni et al., 1999; Subramanyan and Prema, 2002). The partial enzyme hydrolysis of xylan changes its physical and chemical properties, which concerns the quality of different products of the food and flavour industry. Xylanase finds applications in fruit juices and wines clarifying (Colins et al., 2005; Coughlan and Hazlewood, 1993). In brewing, xylanase is applied in filtering improvement (Yin et al., 2005). The utilization of xylanase in bread-making significantly improves the desirable texture, loaf volume and shelf life of bread (Courtin and Delcour, 2002; Dutron et al., 2004). Xylanase enzyme preparations are also widely used in bio-bleaching in paper industry. They facilitate the delignification of the plant pulp in the production of high-quality paper. In that way, the chlorine-containing bleaching agents, that are a serious ecological problem, can be reduced (Gupta et al., 2000). The industrial production of xylanase enzyme preparations is based on a microbial biosynthesis. Most often industrial xylanase producing strains moulds of the species Aspergillus sp. and Trichoderma sp., as well as bacterial strains of the species Bacillus sp. are used. There are two possibilities for cultivation of microbial enzyme producing strains: solid-state and submerged cultivation (Gawande and Kamat, 1999). At the present moment, the submerged cultivation is used more widely, allowing a higher degree of processes intensification and a better level of automation. Xylanases from moulds are extra-cellular, inducible enzymes. That determines the great significance of nutrient medium selection. The xylanase biosynthesis is induced by its substrate – xylan (Subramanyan and Prema, 2002; Kulkarni et al., 1999). The high xylan content in some of the wastes like corn cobs and wheat bran makes them an accessible and cheap source of inducers. Annually about seven million tons of wastes are deposited from the member-countries of the EU, in the form of corn and wheat bran (Bonnin et al., 2001). The inclusion of these components in the nutrient media composition is the main strategy in microbial xylanase biosynthesis (Davidov and Atev, 1996; Marinova et al., 1993). In order to improve the nutrient medium for xylanase production, conventional methods based on the ‘‘changeone-factor-at-a-time’’ in which, one independent variable is studied while fixing all others at a specific level, may lead to unreliable results and inaccurate conclusion. This experimental procedure is also expensive and time consuming for large number of variables. The mathematical design finds wide application in nutrient media optimization for microbial enzyme production. The aim is to obtain mathematical
models showing the dependence of the enzyme activity on independent variables (the concentration of the separate components of the nutrient medium). The mathematical dependences obtained are used for prediction of the optimum values of the independent variables, ensuring the maximum enzyme activity (Bocchini et al., 2002; Park et al., 2002; Techapun et al., 2002). The aim of this study is the optimization of the nutrient medium for xylanase production in submerged cultivation of the strain Aspergillus niger BO3, by using the methods of mathematical modeling and statistical processing of the results. 2. Methods 2.1. Analytical methods Moisture, ash, fat, protein content of wheat bran, corn cob and malt sprout were determined by ICC standard methods No. 109/1, 104/1, 136 and 105/2 (ICC, 1991). The b-glucan content was determinate by a mixed-linkage b-glucan assay procedure (Megazyme, 1988). Total sugars were determined by the method of Dubois (Dubois et al., 1956). The amounts of individual sugars in agricultural wastes were quantified by gas chromatography. The agricultural wastes sample (3 mg) was hydrolyzed with 2.0 M trifluoroacetic acid (1.5 ml) for 2 h at 120 °C. The excess trifluoroacetic acid was evaporated at about 40 °C with a stream of nitrogen. After drying, the sample was dissolved in 1 ml solution of internal standard phenyl-b-D-glucopyranoside (1 mg/ml) in pyridine. The received methyl glycosides were derivatized with hexamethyldisilazane (0.5 ml) with trifluoroacetic acid (0.15 ml) as a catalyst at 80 °C for 5 min. After cooling, the solution was analyzed on GS/FID system (Hewlett-Packard 6890) with column HP-5 5% phenyl methyl siloxane (30 m; 0.32 mm; 0.25 lm). The following temperature conditions were used: the initial temperature 190 °C was raised to 280 °C at 10 °C/min. Derivatized methylglycosides (the standards) were used for the identification of sugars. 2.2. Microorganism The experiments were carried out with the strain A. niger BO3 provided from company Biovet Ltd. The preservation and sporulation of the strain were done on a solid nutrient medium with the following composition (g/l): glucose 4.0, yeasts extract 4.0, malt extract 10.0 and agar 20.0. The pH was corrected up to 6.20–6.40 and the medium was sterilized at 121 °C for 30 min. The inoculated test-tubes with slant agar were incubated at 29 °C for seven days. They are stored at 4 °C for 3–4 months. 2.3. Inoculum preparation For inoculum preparation the nutrient medium described by Davidov and Atev (1996) was used. The cul-
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tivation was done in 500 ml flasks, containing 100 ml nutrient medium, inoculated with 2.5 ml spore suspension, containing 3.107–3.108 spores/ml. The cultivation was performed at 29 °C on a shaker, at 180 rpm for 22 h. 2.4. Xylanase biosynthesis Xylanase biosynthesis was performed into 500 ml flasks, containing 50 ml nutrient medium with the following basic composition (g/l): corn cobs 20.0, wheat bran 10.0, malt sprout 10.0, (NH4)2HPO4 2.5 and urea 1.0. pH was corrected up to 6.70–6.80 and the medium was sterilized at 121 °C for 30 min. The flasks were inoculated with 10% inoculum. The strain was cultivated at 28 °C for 64 h at 180 rpm shaking. The xylanase activity, obtained with the basic nutrient medium is 750.37 U/ml. For the optimization experiments, the nutrient medium was composed of different concentrations of corn cobs, wheat bran, (NH4)2HPO4, urea, malt sprout (Table 1). 2.5. Xylanase activity It was determined by the method of Bailey et al. (1992). One unit (U) of xylanase activity is defined as the amount of enzyme releasing 1 lmol of xylose equivalent per minute under pH 5.0 and temperature 50 °C. The substrate of the enzyme under is 1% solution of oat spelt xylan (Sigma) in acetate buffer with pH 5.0. 2.6. Optimal composite design Response surface methodology and 251 fractional factorial design was used to optimize of nutrient medium for the production of xylanase by A. niger B03. Optimal composite design (OCD) was used to find the optimum concentration of the nutrients components for xylanase production. The OCD contained 251 fractional factorial matrix with ‘‘star points’’ around the center points. The distance from the center of design space to a factorial point is a = ±1. The quadratic regression models are one of the most widely used in practice. They allow description of the object in a comparatively wide area of the input variables change (Vuchkov and Stoyanov, 1980). They are expressed as follows: Table 1 Value of independent variables at different levels of the 251 factorial design Independent variables (g/l) X1-(NH4)2HPO4 X2-Urea X3-Malt sprout X4-Corn cobs X5-Wheat bran
Levels 1
0
1
2.6 0.9 6.0 12.0 6.0
4.0 1.5 12.0 18.0 11.0
5.4 2.1 18.0 24.0 16.0
Yb ¼ bo þ
m X
bi x i þ
i¼1
m X
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bij xi xj þ
i¼1;j¼iþ1
m X
bii x2i
ð1Þ
i¼1
where Yb is the response variable, b the regression coefficients of the model and x the coded levels of the independent variables. MATLAB v.5.2 software from MathsWorks Inc. was used for regression and graphical analysis. The independent variables participating in the 251 OCD and their values are presented in Table 1. The coding of the independent variables is done by using: xi ¼
ðxi xio Þ Dxi
ð2Þ
xi is the current value of the i-factor, xi0 is the current value of the i-factor in the center point of the plan, and Dxi is the step of variation of the i-factor. 3. Results and discussion Corn cobs and wheat bran are the agricultural wastes which are most often included in nutrient media for microbial xylanase production. They are characterized with indefinite composition, depending on the climate and agro technical conditions (Techapun et al., 2003). Wheat bran is a by-product of wheat–flour processing and accounts for 15–20% of the weight of the grain. It contains 20–30% arabinoxylan. Corn cobs do not vary greatly in chemical composition, except for starch content (Hespell, 1998). The composition of the agricultural wastes used for xylanase production by submerged fermentation of A. niger B03 is presented in Table 2.
Table 2 Chemical constituents of agricultural wastes Composition
Agricultural wastes Wheat bran
Corn cobs
Malt sprout
Water (%) Ash (%)a Fat (%)a Protein (%)a Total sugars (%)a Total (%)a
12.10 3.98 2.59 14.87 79.12 100
11.70 8.60 3.17 3.13 81.31 96.21
11.90 5.79 0.94 32.55 56.03 95.31
Sugars (%)a: Starch b-Glucans Arabinoxylansb
24.69 13.22 17.30
n.d. 9.57 35.56
n.d. 20.47 14.29
Neutral sugars (%)a: Glucose Xylose Arabinose Mannose Galactose
49.15 12.65 7.01 1.48 8.19
17.49 22.93 17.48 3.28 20.70
26.44 8.15 8.09 2.11 8.59
n.d. – Not detected Data are mean of duplicates analyses. a Expressed as percent of dry material-% (dm). b Arabinoxylans = 0.88(%Xyl + %Ara).
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The composition of the wheat bran used is: 79.12% (dm) total sugars, 14.87 % (dm) protein, 2.59 % (dm) fat and 3.98 % (dm) ash. The composition of the total carbohydrates includes 17.30 % (dm) arabinoxylan, 24.69 % (dm) starch and 13.22 % (dm) b-glucans (Table 2). The ratio arabinose/xylose (Ara/Xyl) is an important characteristic, determining degree of substitution in arabinoxylans macromolecule. The arabinoxylans in the investigated wheat bran are characterized with Ara/Xyl = 0.55. The corn cobs used have the following composition (Table 2): total sugars 81.31 % (dm), protein 3.13 % (dm), fat 3.17 % (dm) and ash 8.60 % (dm). In the composition of carbohydrates the absence of starch is noticed. However, the content of arabinoxylans is considerably high – 35.56 % (dm) and that of b-glucans is 9.57 % (dm). The arabinoxylans in the investigated corn cobs are characterized with a higher ratio Ara/Xyl = 0.75 and a higher degree of branching in comparison to arabinoxylans in wheat bran. Malt sprout, wastes of the brewing industry are also included in the nutrient medium for xylanase biosynthesis. They are characterized with high protein content – 32.55 % (dm). The content of total sugars is 56.03 % (dm), ash 5.79 % (dm) and fat 0.94 % (dm) (Table 2). Arabinoxylans – 14.29 % (dm) and b-glucans – 20.47 % (dm), are included in the composition of total carbohydrates. No starch was detected. The achievement of high xylanase activity in the inclusion of these plant wastes in nutrient medium composition
has an exceptionally important economical significance in xylanase production. The investigated agricultural wastes contain vastly different quantities of arabinoxylan which determine their inducing effect on xylanase biosynthesis. For optimization of nutrition medium 251 OCD has been carried out. The results at each point based on the experimental design are shown in Table 3. After the regression analysis for this experiment, the model was simplified by elimination of the statistically insignificant coefficients. At the 5% probability level, the linear coefficient X4 and the coefficient of interaction X4 Æ X5 were found to be significant. However, the coefficient X5 (p = 0.252) was maintained in the model because of its magnitude. The quadratic coefficients X42 (p = 0.377) and X52 (p = 0.339) were maintained in the model because they describe nonlinearity in the area of the maximum. The mathematical model was reduced to: Yb ¼ 654:25 þ 87:52 X 4 þ 31:91 X 5 þ 79:68 X 4 X 5 64:75 X 24 69:89 X 25
ð3Þ
The predicted values of xylanase activity Yb are presented in Table 3. The statistical significance of the second-order model Equation (3) was evaluated by the F-test analysis of variance (ANOVA), which showed that this regression is statistically significant at 99% confidence level (significance F = 1.14 Æ 106). The coefficient of determination of the model (R2 = 0.82) was calculated to be indicating that
Table 3 Experimental design and results of the 251 factorial design Run number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 a
Coded levels
Xylanase activity (U/ml)
X1
X2
X3
X4
X5
Ya
ˆ Y
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 1 1 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 1 1 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 1 1
428.12 568.79 649.08 544.56 589.81 483.24 484.91 536.66 569.31 750.47 869.88 513.49 825.29 695.87 611.15 815.58 723.41 678.61 694.75 614.37 710.87 705.62 694.44 484.97 637.56 531.08
384.32 479.86 479.86 384.32 479.86 384.32 384.32 479.86 495.54 718.72 718.72 495.54 718.72 495.54 495.54 718.72 654.25 654.25 654.25 654.25 654.25 654.25 677.02 501.98 616.27 552.45
Data are mean of triplicates analyses.
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82% of the variability in the response could be explained by the model. The response surface described by the model Equation (3) to estimate xylanase activity over independent variables X4 and X5 is shown in Fig. 1. The maximum predicted value of xylanase activity Yb ¼ 721:27 U/ml was determined. It is achieved at the following coded values of the independent variables: X4 = 1.00, X5 = 0.73. From the Equation (3) it can be seen that the xylanase activity does not depend on the value of X1, X2 and X3 in the variation intervals. From Fig. 1 it can be seen that at a fixed value of corn cobs (X4 = 1.00) a clearly expressed optimum in regard to wheat bran (X5) is observed, and vice versa at X5 = 1.00 there is a clearly expressed optimum respectively of the concentration of corn cobs (X4). The strongly expressed interaction effect X4 Æ X5 replaces the optimum ratios in the higher values of the two factors, i.e. the increase in concentration of one of the factors requires a higher concentration from the other factor. In decoding the factors X4 and X5, the optimum levels in real values were determined, which are respectively 24.0 g/l corn cobs and 14.6 g/l wheat bran. Biosynthesis of multiple xylanases has been reported by numerous microorganisms (Colins et al., 2005; Wong et al., 1988). It is possibly due to the high heterogeneity of xylans from different sources. Xylan from investigated corn cobs is characterized with considerably higher degree of branching (Ara/Xyl = 0.75) than xylan from the investigated wheat bran (Ara/Xyl = 0.55). It appears that xylosidic linkages in lignocellulose are not all equivalent and equally accessible to xylanolytic enzymes. The accessibility of some
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linkages also changes during the course of hydrolysis. The production of a system of enzymes, each enzyme with specialized functions, is one strategy that a microorganism may use to achieve superior xylan hydrolysis (Wong et al., 1988). It is possible that different xylans from corn cobs and wheat bran induce multiple xylanases, which act synergistically in xylan hydrolysis. The insignificance of the factors X1, X2 and X3 supposes saturation of their concentrations that gives grounds to change their variation intervals in lower concentration values. For the purpose a new 23 OCD was carried out at fixed values of X4 and X5 at their optimum concentrations determined by the 251 OCD. The new values of variation intervals for the variables X1, X2 and X3 are presented in Table 4. The coding of the variables was done according to the formula (2). The 23 OCD is presented in Table 5. After analysis for significance of the regression model coefficient values, the mathematical model was simplified by the elimination of statistically insignificant coefficients X1 Æ X2 (p = 0.690) and X1 Æ X3 (p = 0.331). The linear coefficient X1 (p = 0.223) and quadratics coefficients were maintained in the model because of their magnitude. At the 1% probability level, the linear coefficient X2 and X3 were found to be significant. At the 5% probability level, the coefficient of interaction X2 Æ X3 was found to be significant. The equation of regression is:
Table 4 Values of independent variables at different levels of the 23 factorial design Independent variables (g/l)
Levels
X1-(NH4)2HPO4 X2-Urea X3-Malt sprout
1
0
1
0.4 0.3 0.4
1.5 0.6 5.2
2.6 0.9 10.0
Table 5 Experimental design and results of the 23 factorial design
Fig. 1. Effect of corn cobs and wheat bran concentration on xylanase activity.
Run number
Coded levels X1
X2
X3
Ya
ˆ Y
1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 1 1 1 1 1 1 1 1 1 0 0 0 0
1 1 1 1 1 1 1 1 0 0 1 1 0 0
1 1 1 1 1 1 1 1 0 0 0 0 1 1
413.45 395.83 764.69 770.09 666.32 791.83 815.09 850.41 720.35 823.81 656.61 864.53 378.05 719.73
367.28 417.70 724.98 775.39 721.08 771.49 819.69 870.11 746.87 797.29 646.49 874.65 436.76 661.02
a
Xylanase activity (U/ml)
Data are mean of triplicates analyses.
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Yb ¼ 699:04 þ 25:21 X 1 þ 114:08 X 2 þ 112:13 X 3 64:77 X 2 X 3 þ 73:04 X 21 þ 61:53 X 22 150:15 X 23
ð4Þ
The statistically significance of the model (4) was done by means of ANOVA program. The low value of significance F = 0.0012 (<0.01) shows that this regression (4) is statistically significant at the 99% confidence level. It is characterized by a high determination coefficient (R2 = 0.96) explaining 96% of the variability in the response. The response surface described by the model Equation (4) to estimate xylanase activity over independent variables X1, X2 and X3 is shown in Figs. 2–4. The maximum value of xylanase activity Yb ¼ 976:48 U/ml is obtained at the following values of the independent variables X1 = 1.00, X2 = 1.00, X3 = 0.19. The optimum values of X1 and X2 are at the upper boundary value of the 23 OCD. The influence of their higher concentrations upon xylanase biosynthesis has been investigated in the 251 OCD where they are insignificant. That is why, the values obtained for (NH4)2HPO4 (X1), urea (X2) and malt sprout (X3), respectively 2.6 g/l, 0.9 g/l and 6.0 g/l can be accepted as optimum despite the fact that X1 and X2 are in the upper boundary point of the plan. The optimum nutrient medium obtained has the following composition (g/l): (NH4)2HPO4 2.6, urea 0.9, malt sprout 6.0, corn cobs 24.0, wheat bran 14.6. With that nutrient medium five parallel experiments have been carried out, at which the following results have been obtained for xylanase activity: Y1 = 1049.35 U/ml, Y2 = 979.81 U/ml, Y3 = 980.41 U/ml, Y4 = 984.62 U/ml, Y5 = 987.31 U/ml. The experimental results approximate to a great
Fig. 3. Effect of malt sprout and (NH4)2PO4 concentration on xylanase activity (U/ml) at fixed optimal level of urea.
Fig. 4. Effect of malt sprout and urea concentration on xylanase activity (U/ml) at fixed optimal level of (NH4)2PO4.
Fig. 2. Effect of urea and (NH4)2PO4 concentration on xylanase activity (U/ml) at fixed optimal level of malt sprout.
degree the value predicted by the model. That is an experimental confirmation of the statistically significance of the models obtained. With the optimized nutrient medium 996.30 U/ml xylanase activity was obtained. This value is 33% higher in comparison to the activity obtained with the basic nutrient medium (750.37 U/ml). The xylanase biosynthesis from other fungal strains grown on agricultural wastes was compared (Table 6). Xylanase activity obtained by the investigated A. niger B03 after optimization of nutrient medium (996.30 U/ml)
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Table 6 Comparison of xylanase production from other fungal strains grown on agricultural wastes Microorganism
Substrate
Xylanase activity (U/ml)
Reference
Aspergillus niger B03
1.5% Wheat bran + 2.4% Corn cobs + 0.6% Malt sprout 3% Wheat bran 1% Rice straw 3% Wheat bran 1% Corn cobs 3% Corn cobs 3% Wheat bran 5% Wheat bran 2% Corn cobs 7% Wheat bran
996.3
This work
12 138 85 7.54 125.1 119.2 36 245 450
Poutanen et al. (1987) Kang et al. (1995) Christov et al. (1999) Coelho and Carmona (2003) Lenartovicz et al. (2002)
Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus
awamori VTT-D-75028 niger KKS oryzae NRRL1808 giganteus CCT3232 fumigatus
Cephalosporium sp. NCL 87.11.9 Fusarium oxysporum Pseudomonas sp. WLUN024
is significantly higher than these cited in the literature. A. niger B03 is a potential microorganism for the production of xylanase. No addition of expensive media is required and the use of inexpensive agro-industrial wastes has important economic advantages. 4. Conclusions The response surface methodology was applied for the optimization of xylanase biosynthesis by submerged cultivation on A. niger B03 . The existence of interactions between the independent variables with the response was observed and established. The optimum nutrient medium has the following composition (g/l): (NH4)2HPO4 2.6, urea 0.9, malt sprout 6.0, corn cobs 24.0, wheat bran 14.6. The xylanase activity obtained with the optimized nutrient medium was 996.30 U/ml, which is 33% higher in comparison to the activity obtained with the basic medium. Determination of the optimal concentrations of nutrient medium components leads to decrease expenses of raw materials and achievement of maximal xylanase activity. Acknowledgements The authors acknowledge Biovet Ltd. for giving the strain A. niger with which the investigations were carried out. The authors also thank Miss Boriana Zhekova and Mrs Ginka Delcheva for editing the manuscript. This work was supported by the Grant-in-Aid for Scientific research of Priority area (Agricultural science) from the Ministry of Education of Bulgaria. References Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257–270. Bansod, S., Dutta-Choudhury, M., Srinivasan, M., Rele, M., 1993. Xylanase active at high pH from an alkalotolerant Cephalosporium species. Biotechnol. Lett. 15, 965–970. Bidlack, J., Malone, M., Benson, R., 1992. Molecular structure and component integration of secondary cell walls plant. Proc. Oklahoma Academy of Science 72, 51–56.
Bansod et al. (1993) Christakopoulos et al. (1996) Xu et al. (2005)
Bocchini, D.A., Alves-Prado, L.C., Roberto, I.C., Gomes, E., Da Silva, R., 2002. Optimization of xylanase production by Bacillus circulans D1 in submerged fermentation using reponse surface methodology. Process Biochem. 38, 727–731. Bonnin, E., Brunel, M., Gouy, Y., Lesage-Meesen, L., Asther, M., Tribault, J.F., 2001. Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL 39533, selected for the biotranformation of ferulic acid to vanilin, are also able to produce cell wall polysaccharidedegrading enzymes and feruloyl esterases. Enzyme Microb. Technol. 28, 70–80. Christakopoulos, P., Nerinckx, W., Kekos, D., Macris, B., 1996. Claeyssens M. purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3. J. Biotechnol. 51, 181–189. Christov, L., Szakacs, G., Balakrishnan, H., 1999. Production partial characterization and use of fungal cellulase-free xylanases in pulp bleaching. Process Biochem. 64, 511–517. Coelho, G., Carmona, E., 2003. Xylanolytic complex from Aspergillus giganteus: production and characterization. J. Basic Microbiol. 43, 269–277. Colins, T., Gerday, C., Feller, G., 2005. Xylanases, xylanase families and extermophilic xylanases. FEMS Microbiol. Lett. 29, 3–23. Coughlan, M., Hazlewood, G., 1993. b-1,4-D-xylan-degrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol. Appl. Bioc. 17, 259–289. Courtin, C.M., Delcour, J.A., 2002. Arabinoxylans and endoxylanases in wheat flour bread-making. J. Cereal Sci. 35, 225–243. Davidov, E.R., Atev, A., 1996. Method for production of enzyme preparation. Patent number Ru 2057179 C1. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Dutron, A., Georis, J., Genot, B., Dauvrin, T., Collins, T., Hoyox, A., Feller, G., 2004. Use family 8 enzymes with xylanolytic activity in baking. PCT WO 2004023879 A1. Gawande, P., Kamat, M., 1999. Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application. J. Appl. Microbiol. 87, 511–519. Gupta, S., Bhushan, B., Hoondai, G.S., 2000. Isolation and characterization of xylanase from Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. J. Appl. Microbiol. 88, 325–334. Hespell, R., 1998. Extraction and characterization of hemicellulose from the corn fiber produced by corn wet-milling processes. J. Agr. Food Chem. 46, 2615–2619. ICC, International association for cereal science and technology, 1991. Standard methods. Vienna.. Izydorczyk, M., Biliaderis, C., 1995. Cereal arabinoxylans: advances in structure and physicochemical properties. Carbohyd. Polym. 28, 33– 48.
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Kang, S., Kim, S., Lee, J., 1995. Production of cellulase and xylanase in a bubble column using immobilized Aspergillus niger KKS. Appl. Microbiol. Biotechnol. 53, 101–106. Kulkarni, N., Shendye, A., Rao, M., 1999. Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Lett. 23, 411–456. Lenartovicz, V., de Souza, C., Moreira, F., Peralta, R., 2002. Temperature effect in the production of multiple xylanases by Aspergillus fumigatus. J. Basic Microbiol. 42, 388–395. Marinova, D.N., Atev, A.P., Polizoev, J.V., 1993. Method for production of enzyme preparation with predominant xylanase activity. Patent number BG51306 A. Megazyme, 1988. Mixed-linkage b-Glucan Assay Procedure (McClary Method). Megazyme International Ireland Ltd., Bray Business Park, Bray, Co., Wichkows, Ireland. Nakamura, S., 2003. Structure and function of a multiplidomain alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1. Catalysis Surveys from Asia 7, 157–164. Park, Y.S., Kang, S.W., Lee, J.S., Hong, S.I., Kim, S.W., 2002. Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental design. Appl. Microbiol. Biot. 58, 761–766. Poutanen, K., Ratto, M., Puls, J., Viikari, L., 1987. Evaluation of different microbial xylanolytic systems. J. Biotechnol. 6, 49–60. Ryan, S.E., Nolan, K., Thompson, R., Gubitz, G.M., Savage, A.V., Tuohy, M.G., 2003. Purification and characterization of a new low molecular weight endoxylanase from Penicillium capsulatum. Enzyme Microb. Technol. 33, 775–785.
Saha, B., 2003. Hemicellulose bioconversion. J. Ind. Microbiol. Biot. 30, 279–291. Subramanyan, S., Prema, P., 2002. Biotechnology of microbial xylanases: Enzymology, molecular biology and application. Cr. Rev. Biotechn. 22, 33–64. Techapun, C., Charoenrat, T., Watanabe, M., Sasaki, K., Poosaran, N., 2002. Optimization of thermostable and alcaline-tolerant cellulase-free xylanase production from agricultural waste by thermotolerant Streptomyces sp. Ab 106, using the central composite experimental design. Biochem. Eng. J. 12, 99–105. Techapun, C., Poosaran, N., Watanabe, M., Sasaki, K., 2003. Thermostable and alkaline-tolerant cellulose-free xylanases produced from agricultural wastes and the properties required for use in pulp bleaching bioprocesses a review. Process Biochem. 38, 1327–1340. Vuchkov, I., Stoyanov, S., 1980. Mathematical modelling and optimization of technological objects, Technics, Sofia, pp. 135–151. Wong, K., Tan, L., Saddler, J., 1988. Multiplicity of b-1,4-xylanase in microorganisms: functions and applications. Microbiol. Rev. 52, 305– 317. Xu, Z-H., Bai1, Y-L., Xu1, X., Shi1, J-S., Tao, W-Y., 2005. Production of alkali-tolerant cellulase-free xylanase by Pseudomonas sp. WLUN024 with wheat bran as the main substrate. World J. Microbiol. Biotechnol. 21, 575–581. Yin, L., Jian, L., Guoxian, G., Zhonping, S., Zhongui, M., 2005. Studies on water-extractable arabinoxylans during malting and brewing. Food Chem. 93, 33–38.
Optimization of nutrient medium containing agricultural wastes for xylanase production by Aspergillus niger B03 using optimal composite experimental design Georgi Todorov Dobrev a,*, Ivan Genov Pishtiyski a, Veselin Stanchev Stanchev b, Rositza Mircheva c b
a University of Food Technologies, Department of Biochemistry and Molecular Biology, 26 Maritza Boulevard, Plovdiv 4002, Bulgaria University of Food Technologies, Department of Automatics, Information and Control Equipment, 26 Maritza Boulevard, Plovdiv 4002, Bulgaria c Biovet Ltd., Research and Development Analytical Department, 39 Petar Rakov, Peshtera 4550, Bulgaria
Received 23 May 2006; received in revised form 31 July 2006; accepted 17 September 2006 Available online 7 November 2006
Abstract The xylanase biosynthesis is induced by its substrate – xylan. The high xylan content in some of the wastes like corn cobs and wheat bran makes them an accessible and cheap source of inducers. Nutrient medium for xylanase biosynthesis in submerged cultivation of Aspergillus niger B03 has been optimized. The optimization process was analyzed using optimal composite experimental design and response surface methodology. The predicted by the regression model optimum components of nutrient medium are as follows (g/l): (NH4)2HPO4 2.6, urea 0.9, corn cobs 24.0, wheat bran 14.6 and malt sprout 6.0. Five parallel experiments have been carried out, at definite, optimum components concentrations of the nutrient medium, and a mean value of the activity Y = 996.30 U/ml has been obtained. The xylanase activity, obtained with the optimized nutrient medium is 33% higher than the activity, achieved with the basic medium. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Xylanase; Optimization; Agricultural waste; Aspergillus
1. Introduction The hemicelluloses unite a big group of high molecular polysaccharides, insoluble in water but soluble in alkaline solutions. These polysaccharides are associated with cellulose and lignin and play an important structurally-supportive role in building up of plant cell walls (Bidlack et al., 1992; Nakamura, 2003). The composition of hemicelluloses, includes branched heteropolymers of pentoses (xylose, arabinose), hexoses (mannose, galactose, glucose), and uronic acids. Xylan is the main hemicellulolytic polysaccharide found in plant cell walls. Xylan is the second
*
Corresponding author. Fax: +359 32644102. E-mail address: [email protected] (G.T. Dobrev).
0960-8524/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.09.022
one in its distribution polysaccharide in nature after cellulose. It comprises 20–30% dry weight of the plants (Saha, 2003). The molecule of xylan consists of a b-1,4-linked D-xylose backbone and can be substituted by different side groups such as L-arabinose, D-galactose, acetyl, feruloyl, p-coumarouyl and glucuronic acid residues (Izydorczyk and Biliaderis, 1995; Kulkarni et al., 1999; Subramanyan and Prema, 2002). For the total hydrolysis of xylan the action of several enzymes is needed: xylanase (endo-1,4-b-D-xylanase EC 3.2.1.8), b-D-xylosidase (EC 3.2.1.37), a-L-arabinofuranosidase (EC 3.2.1.55), a-L-glucuronosidase (EC 3.2.1.139), acetylxylan esterase (EC 3.1.1.72), ferulic acidic esterase (EC 3.1.1.73) and q-coumaric acidic esterase (EC 3.1.1.-). Xylanase plays a key role in xylan hydrolysis to xylooligosaccharides (Colins et al., 2005; Coughlan and Hazlewood,
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1993; Kulkarni et al., 1999; Subramanyan and Prema, 2002). Xylotriose is the lowest molecular olygosaccharide that can be obtained under the action of the most studied by now xylanases (Ryan et al., 2003). The interest towards xylanase enzyme preparations has been dictated by their wide applications in various fields of industry. The enzyme hydrolysis of xylan lies in the basis of its utilization as an energy source in animal feed or in different biotechnological processes (Coughlan and Hazlewood, 1993; Kulkarni et al., 1999; Subramanyan and Prema, 2002). The partial enzyme hydrolysis of xylan changes its physical and chemical properties, which concerns the quality of different products of the food and flavour industry. Xylanase finds applications in fruit juices and wines clarifying (Colins et al., 2005; Coughlan and Hazlewood, 1993). In brewing, xylanase is applied in filtering improvement (Yin et al., 2005). The utilization of xylanase in bread-making significantly improves the desirable texture, loaf volume and shelf life of bread (Courtin and Delcour, 2002; Dutron et al., 2004). Xylanase enzyme preparations are also widely used in bio-bleaching in paper industry. They facilitate the delignification of the plant pulp in the production of high-quality paper. In that way, the chlorine-containing bleaching agents, that are a serious ecological problem, can be reduced (Gupta et al., 2000). The industrial production of xylanase enzyme preparations is based on a microbial biosynthesis. Most often industrial xylanase producing strains moulds of the species Aspergillus sp. and Trichoderma sp., as well as bacterial strains of the species Bacillus sp. are used. There are two possibilities for cultivation of microbial enzyme producing strains: solid-state and submerged cultivation (Gawande and Kamat, 1999). At the present moment, the submerged cultivation is used more widely, allowing a higher degree of processes intensification and a better level of automation. Xylanases from moulds are extra-cellular, inducible enzymes. That determines the great significance of nutrient medium selection. The xylanase biosynthesis is induced by its substrate – xylan (Subramanyan and Prema, 2002; Kulkarni et al., 1999). The high xylan content in some of the wastes like corn cobs and wheat bran makes them an accessible and cheap source of inducers. Annually about seven million tons of wastes are deposited from the member-countries of the EU, in the form of corn and wheat bran (Bonnin et al., 2001). The inclusion of these components in the nutrient media composition is the main strategy in microbial xylanase biosynthesis (Davidov and Atev, 1996; Marinova et al., 1993). In order to improve the nutrient medium for xylanase production, conventional methods based on the ‘‘changeone-factor-at-a-time’’ in which, one independent variable is studied while fixing all others at a specific level, may lead to unreliable results and inaccurate conclusion. This experimental procedure is also expensive and time consuming for large number of variables. The mathematical design finds wide application in nutrient media optimization for microbial enzyme production. The aim is to obtain mathematical
models showing the dependence of the enzyme activity on independent variables (the concentration of the separate components of the nutrient medium). The mathematical dependences obtained are used for prediction of the optimum values of the independent variables, ensuring the maximum enzyme activity (Bocchini et al., 2002; Park et al., 2002; Techapun et al., 2002). The aim of this study is the optimization of the nutrient medium for xylanase production in submerged cultivation of the strain Aspergillus niger BO3, by using the methods of mathematical modeling and statistical processing of the results. 2. Methods 2.1. Analytical methods Moisture, ash, fat, protein content of wheat bran, corn cob and malt sprout were determined by ICC standard methods No. 109/1, 104/1, 136 and 105/2 (ICC, 1991). The b-glucan content was determinate by a mixed-linkage b-glucan assay procedure (Megazyme, 1988). Total sugars were determined by the method of Dubois (Dubois et al., 1956). The amounts of individual sugars in agricultural wastes were quantified by gas chromatography. The agricultural wastes sample (3 mg) was hydrolyzed with 2.0 M trifluoroacetic acid (1.5 ml) for 2 h at 120 °C. The excess trifluoroacetic acid was evaporated at about 40 °C with a stream of nitrogen. After drying, the sample was dissolved in 1 ml solution of internal standard phenyl-b-D-glucopyranoside (1 mg/ml) in pyridine. The received methyl glycosides were derivatized with hexamethyldisilazane (0.5 ml) with trifluoroacetic acid (0.15 ml) as a catalyst at 80 °C for 5 min. After cooling, the solution was analyzed on GS/FID system (Hewlett-Packard 6890) with column HP-5 5% phenyl methyl siloxane (30 m; 0.32 mm; 0.25 lm). The following temperature conditions were used: the initial temperature 190 °C was raised to 280 °C at 10 °C/min. Derivatized methylglycosides (the standards) were used for the identification of sugars. 2.2. Microorganism The experiments were carried out with the strain A. niger BO3 provided from company Biovet Ltd. The preservation and sporulation of the strain were done on a solid nutrient medium with the following composition (g/l): glucose 4.0, yeasts extract 4.0, malt extract 10.0 and agar 20.0. The pH was corrected up to 6.20–6.40 and the medium was sterilized at 121 °C for 30 min. The inoculated test-tubes with slant agar were incubated at 29 °C for seven days. They are stored at 4 °C for 3–4 months. 2.3. Inoculum preparation For inoculum preparation the nutrient medium described by Davidov and Atev (1996) was used. The cul-
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tivation was done in 500 ml flasks, containing 100 ml nutrient medium, inoculated with 2.5 ml spore suspension, containing 3.107–3.108 spores/ml. The cultivation was performed at 29 °C on a shaker, at 180 rpm for 22 h. 2.4. Xylanase biosynthesis Xylanase biosynthesis was performed into 500 ml flasks, containing 50 ml nutrient medium with the following basic composition (g/l): corn cobs 20.0, wheat bran 10.0, malt sprout 10.0, (NH4)2HPO4 2.5 and urea 1.0. pH was corrected up to 6.70–6.80 and the medium was sterilized at 121 °C for 30 min. The flasks were inoculated with 10% inoculum. The strain was cultivated at 28 °C for 64 h at 180 rpm shaking. The xylanase activity, obtained with the basic nutrient medium is 750.37 U/ml. For the optimization experiments, the nutrient medium was composed of different concentrations of corn cobs, wheat bran, (NH4)2HPO4, urea, malt sprout (Table 1). 2.5. Xylanase activity It was determined by the method of Bailey et al. (1992). One unit (U) of xylanase activity is defined as the amount of enzyme releasing 1 lmol of xylose equivalent per minute under pH 5.0 and temperature 50 °C. The substrate of the enzyme under is 1% solution of oat spelt xylan (Sigma) in acetate buffer with pH 5.0. 2.6. Optimal composite design Response surface methodology and 251 fractional factorial design was used to optimize of nutrient medium for the production of xylanase by A. niger B03. Optimal composite design (OCD) was used to find the optimum concentration of the nutrients components for xylanase production. The OCD contained 251 fractional factorial matrix with ‘‘star points’’ around the center points. The distance from the center of design space to a factorial point is a = ±1. The quadratic regression models are one of the most widely used in practice. They allow description of the object in a comparatively wide area of the input variables change (Vuchkov and Stoyanov, 1980). They are expressed as follows: Table 1 Value of independent variables at different levels of the 251 factorial design Independent variables (g/l) X1-(NH4)2HPO4 X2-Urea X3-Malt sprout X4-Corn cobs X5-Wheat bran
Levels 1
0
1
2.6 0.9 6.0 12.0 6.0
4.0 1.5 12.0 18.0 11.0
5.4 2.1 18.0 24.0 16.0
Yb ¼ bo þ
m X
bi x i þ
i¼1
m X
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bij xi xj þ
i¼1;j¼iþ1
m X
bii x2i
ð1Þ
i¼1
where Yb is the response variable, b the regression coefficients of the model and x the coded levels of the independent variables. MATLAB v.5.2 software from MathsWorks Inc. was used for regression and graphical analysis. The independent variables participating in the 251 OCD and their values are presented in Table 1. The coding of the independent variables is done by using: xi ¼
ðxi xio Þ Dxi
ð2Þ
xi is the current value of the i-factor, xi0 is the current value of the i-factor in the center point of the plan, and Dxi is the step of variation of the i-factor. 3. Results and discussion Corn cobs and wheat bran are the agricultural wastes which are most often included in nutrient media for microbial xylanase production. They are characterized with indefinite composition, depending on the climate and agro technical conditions (Techapun et al., 2003). Wheat bran is a by-product of wheat–flour processing and accounts for 15–20% of the weight of the grain. It contains 20–30% arabinoxylan. Corn cobs do not vary greatly in chemical composition, except for starch content (Hespell, 1998). The composition of the agricultural wastes used for xylanase production by submerged fermentation of A. niger B03 is presented in Table 2.
Table 2 Chemical constituents of agricultural wastes Composition
Agricultural wastes Wheat bran
Corn cobs
Malt sprout
Water (%) Ash (%)a Fat (%)a Protein (%)a Total sugars (%)a Total (%)a
12.10 3.98 2.59 14.87 79.12 100
11.70 8.60 3.17 3.13 81.31 96.21
11.90 5.79 0.94 32.55 56.03 95.31
Sugars (%)a: Starch b-Glucans Arabinoxylansb
24.69 13.22 17.30
n.d. 9.57 35.56
n.d. 20.47 14.29
Neutral sugars (%)a: Glucose Xylose Arabinose Mannose Galactose
49.15 12.65 7.01 1.48 8.19
17.49 22.93 17.48 3.28 20.70
26.44 8.15 8.09 2.11 8.59
n.d. – Not detected Data are mean of duplicates analyses. a Expressed as percent of dry material-% (dm). b Arabinoxylans = 0.88(%Xyl + %Ara).
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The composition of the wheat bran used is: 79.12% (dm) total sugars, 14.87 % (dm) protein, 2.59 % (dm) fat and 3.98 % (dm) ash. The composition of the total carbohydrates includes 17.30 % (dm) arabinoxylan, 24.69 % (dm) starch and 13.22 % (dm) b-glucans (Table 2). The ratio arabinose/xylose (Ara/Xyl) is an important characteristic, determining degree of substitution in arabinoxylans macromolecule. The arabinoxylans in the investigated wheat bran are characterized with Ara/Xyl = 0.55. The corn cobs used have the following composition (Table 2): total sugars 81.31 % (dm), protein 3.13 % (dm), fat 3.17 % (dm) and ash 8.60 % (dm). In the composition of carbohydrates the absence of starch is noticed. However, the content of arabinoxylans is considerably high – 35.56 % (dm) and that of b-glucans is 9.57 % (dm). The arabinoxylans in the investigated corn cobs are characterized with a higher ratio Ara/Xyl = 0.75 and a higher degree of branching in comparison to arabinoxylans in wheat bran. Malt sprout, wastes of the brewing industry are also included in the nutrient medium for xylanase biosynthesis. They are characterized with high protein content – 32.55 % (dm). The content of total sugars is 56.03 % (dm), ash 5.79 % (dm) and fat 0.94 % (dm) (Table 2). Arabinoxylans – 14.29 % (dm) and b-glucans – 20.47 % (dm), are included in the composition of total carbohydrates. No starch was detected. The achievement of high xylanase activity in the inclusion of these plant wastes in nutrient medium composition
has an exceptionally important economical significance in xylanase production. The investigated agricultural wastes contain vastly different quantities of arabinoxylan which determine their inducing effect on xylanase biosynthesis. For optimization of nutrition medium 251 OCD has been carried out. The results at each point based on the experimental design are shown in Table 3. After the regression analysis for this experiment, the model was simplified by elimination of the statistically insignificant coefficients. At the 5% probability level, the linear coefficient X4 and the coefficient of interaction X4 Æ X5 were found to be significant. However, the coefficient X5 (p = 0.252) was maintained in the model because of its magnitude. The quadratic coefficients X42 (p = 0.377) and X52 (p = 0.339) were maintained in the model because they describe nonlinearity in the area of the maximum. The mathematical model was reduced to: Yb ¼ 654:25 þ 87:52 X 4 þ 31:91 X 5 þ 79:68 X 4 X 5 64:75 X 24 69:89 X 25
ð3Þ
The predicted values of xylanase activity Yb are presented in Table 3. The statistical significance of the second-order model Equation (3) was evaluated by the F-test analysis of variance (ANOVA), which showed that this regression is statistically significant at 99% confidence level (significance F = 1.14 Æ 106). The coefficient of determination of the model (R2 = 0.82) was calculated to be indicating that
Table 3 Experimental design and results of the 251 factorial design Run number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 a
Coded levels
Xylanase activity (U/ml)
X1
X2
X3
X4
X5
Ya
ˆ Y
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 0 0 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 1 1 0 0 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 1 1 0 0
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 1 1
428.12 568.79 649.08 544.56 589.81 483.24 484.91 536.66 569.31 750.47 869.88 513.49 825.29 695.87 611.15 815.58 723.41 678.61 694.75 614.37 710.87 705.62 694.44 484.97 637.56 531.08
384.32 479.86 479.86 384.32 479.86 384.32 384.32 479.86 495.54 718.72 718.72 495.54 718.72 495.54 495.54 718.72 654.25 654.25 654.25 654.25 654.25 654.25 677.02 501.98 616.27 552.45
Data are mean of triplicates analyses.
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82% of the variability in the response could be explained by the model. The response surface described by the model Equation (3) to estimate xylanase activity over independent variables X4 and X5 is shown in Fig. 1. The maximum predicted value of xylanase activity Yb ¼ 721:27 U/ml was determined. It is achieved at the following coded values of the independent variables: X4 = 1.00, X5 = 0.73. From the Equation (3) it can be seen that the xylanase activity does not depend on the value of X1, X2 and X3 in the variation intervals. From Fig. 1 it can be seen that at a fixed value of corn cobs (X4 = 1.00) a clearly expressed optimum in regard to wheat bran (X5) is observed, and vice versa at X5 = 1.00 there is a clearly expressed optimum respectively of the concentration of corn cobs (X4). The strongly expressed interaction effect X4 Æ X5 replaces the optimum ratios in the higher values of the two factors, i.e. the increase in concentration of one of the factors requires a higher concentration from the other factor. In decoding the factors X4 and X5, the optimum levels in real values were determined, which are respectively 24.0 g/l corn cobs and 14.6 g/l wheat bran. Biosynthesis of multiple xylanases has been reported by numerous microorganisms (Colins et al., 2005; Wong et al., 1988). It is possibly due to the high heterogeneity of xylans from different sources. Xylan from investigated corn cobs is characterized with considerably higher degree of branching (Ara/Xyl = 0.75) than xylan from the investigated wheat bran (Ara/Xyl = 0.55). It appears that xylosidic linkages in lignocellulose are not all equivalent and equally accessible to xylanolytic enzymes. The accessibility of some
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linkages also changes during the course of hydrolysis. The production of a system of enzymes, each enzyme with specialized functions, is one strategy that a microorganism may use to achieve superior xylan hydrolysis (Wong et al., 1988). It is possible that different xylans from corn cobs and wheat bran induce multiple xylanases, which act synergistically in xylan hydrolysis. The insignificance of the factors X1, X2 and X3 supposes saturation of their concentrations that gives grounds to change their variation intervals in lower concentration values. For the purpose a new 23 OCD was carried out at fixed values of X4 and X5 at their optimum concentrations determined by the 251 OCD. The new values of variation intervals for the variables X1, X2 and X3 are presented in Table 4. The coding of the variables was done according to the formula (2). The 23 OCD is presented in Table 5. After analysis for significance of the regression model coefficient values, the mathematical model was simplified by the elimination of statistically insignificant coefficients X1 Æ X2 (p = 0.690) and X1 Æ X3 (p = 0.331). The linear coefficient X1 (p = 0.223) and quadratics coefficients were maintained in the model because of their magnitude. At the 1% probability level, the linear coefficient X2 and X3 were found to be significant. At the 5% probability level, the coefficient of interaction X2 Æ X3 was found to be significant. The equation of regression is:
Table 4 Values of independent variables at different levels of the 23 factorial design Independent variables (g/l)
Levels
X1-(NH4)2HPO4 X2-Urea X3-Malt sprout
1
0
1
0.4 0.3 0.4
1.5 0.6 5.2
2.6 0.9 10.0
Table 5 Experimental design and results of the 23 factorial design
Fig. 1. Effect of corn cobs and wheat bran concentration on xylanase activity.
Run number
Coded levels X1
X2
X3
Ya
ˆ Y
1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 1 1 1 1 1 1 1 1 1 0 0 0 0
1 1 1 1 1 1 1 1 0 0 1 1 0 0
1 1 1 1 1 1 1 1 0 0 0 0 1 1
413.45 395.83 764.69 770.09 666.32 791.83 815.09 850.41 720.35 823.81 656.61 864.53 378.05 719.73
367.28 417.70 724.98 775.39 721.08 771.49 819.69 870.11 746.87 797.29 646.49 874.65 436.76 661.02
a
Xylanase activity (U/ml)
Data are mean of triplicates analyses.
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Yb ¼ 699:04 þ 25:21 X 1 þ 114:08 X 2 þ 112:13 X 3 64:77 X 2 X 3 þ 73:04 X 21 þ 61:53 X 22 150:15 X 23
ð4Þ
The statistically significance of the model (4) was done by means of ANOVA program. The low value of significance F = 0.0012 (<0.01) shows that this regression (4) is statistically significant at the 99% confidence level. It is characterized by a high determination coefficient (R2 = 0.96) explaining 96% of the variability in the response. The response surface described by the model Equation (4) to estimate xylanase activity over independent variables X1, X2 and X3 is shown in Figs. 2–4. The maximum value of xylanase activity Yb ¼ 976:48 U/ml is obtained at the following values of the independent variables X1 = 1.00, X2 = 1.00, X3 = 0.19. The optimum values of X1 and X2 are at the upper boundary value of the 23 OCD. The influence of their higher concentrations upon xylanase biosynthesis has been investigated in the 251 OCD where they are insignificant. That is why, the values obtained for (NH4)2HPO4 (X1), urea (X2) and malt sprout (X3), respectively 2.6 g/l, 0.9 g/l and 6.0 g/l can be accepted as optimum despite the fact that X1 and X2 are in the upper boundary point of the plan. The optimum nutrient medium obtained has the following composition (g/l): (NH4)2HPO4 2.6, urea 0.9, malt sprout 6.0, corn cobs 24.0, wheat bran 14.6. With that nutrient medium five parallel experiments have been carried out, at which the following results have been obtained for xylanase activity: Y1 = 1049.35 U/ml, Y2 = 979.81 U/ml, Y3 = 980.41 U/ml, Y4 = 984.62 U/ml, Y5 = 987.31 U/ml. The experimental results approximate to a great
Fig. 3. Effect of malt sprout and (NH4)2PO4 concentration on xylanase activity (U/ml) at fixed optimal level of urea.
Fig. 4. Effect of malt sprout and urea concentration on xylanase activity (U/ml) at fixed optimal level of (NH4)2PO4.
Fig. 2. Effect of urea and (NH4)2PO4 concentration on xylanase activity (U/ml) at fixed optimal level of malt sprout.
degree the value predicted by the model. That is an experimental confirmation of the statistically significance of the models obtained. With the optimized nutrient medium 996.30 U/ml xylanase activity was obtained. This value is 33% higher in comparison to the activity obtained with the basic nutrient medium (750.37 U/ml). The xylanase biosynthesis from other fungal strains grown on agricultural wastes was compared (Table 6). Xylanase activity obtained by the investigated A. niger B03 after optimization of nutrient medium (996.30 U/ml)
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Table 6 Comparison of xylanase production from other fungal strains grown on agricultural wastes Microorganism
Substrate
Xylanase activity (U/ml)
Reference
Aspergillus niger B03
1.5% Wheat bran + 2.4% Corn cobs + 0.6% Malt sprout 3% Wheat bran 1% Rice straw 3% Wheat bran 1% Corn cobs 3% Corn cobs 3% Wheat bran 5% Wheat bran 2% Corn cobs 7% Wheat bran
996.3
This work
12 138 85 7.54 125.1 119.2 36 245 450
Poutanen et al. (1987) Kang et al. (1995) Christov et al. (1999) Coelho and Carmona (2003) Lenartovicz et al. (2002)
Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus
awamori VTT-D-75028 niger KKS oryzae NRRL1808 giganteus CCT3232 fumigatus
Cephalosporium sp. NCL 87.11.9 Fusarium oxysporum Pseudomonas sp. WLUN024
is significantly higher than these cited in the literature. A. niger B03 is a potential microorganism for the production of xylanase. No addition of expensive media is required and the use of inexpensive agro-industrial wastes has important economic advantages. 4. Conclusions The response surface methodology was applied for the optimization of xylanase biosynthesis by submerged cultivation on A. niger B03 . The existence of interactions between the independent variables with the response was observed and established. The optimum nutrient medium has the following composition (g/l): (NH4)2HPO4 2.6, urea 0.9, malt sprout 6.0, corn cobs 24.0, wheat bran 14.6. The xylanase activity obtained with the optimized nutrient medium was 996.30 U/ml, which is 33% higher in comparison to the activity obtained with the basic medium. Determination of the optimal concentrations of nutrient medium components leads to decrease expenses of raw materials and achievement of maximal xylanase activity. Acknowledgements The authors acknowledge Biovet Ltd. for giving the strain A. niger with which the investigations were carried out. The authors also thank Miss Boriana Zhekova and Mrs Ginka Delcheva for editing the manuscript. This work was supported by the Grant-in-Aid for Scientific research of Priority area (Agricultural science) from the Ministry of Education of Bulgaria. References Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257–270. Bansod, S., Dutta-Choudhury, M., Srinivasan, M., Rele, M., 1993. Xylanase active at high pH from an alkalotolerant Cephalosporium species. Biotechnol. Lett. 15, 965–970. Bidlack, J., Malone, M., Benson, R., 1992. Molecular structure and component integration of secondary cell walls plant. Proc. Oklahoma Academy of Science 72, 51–56.
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