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Bioresource Technology 99 (2008) 56025609

Application of a statistical design to the optimization of parameters and culture medium for a-amylase production by Aspergillus oryzae CBS 819.72 grown on gruel (wheat grinding by-product)
Radhouane Kammoun *, Belgacem Naili, Samir Bejar
tabolites des Procaryotes, Route de sidi Mansour km 4, BPK 3038 Sfax, Tunisia Centre de Biotechnologie de Sfax, Laboratoire dEnzymes et Me Received 29 September 2006; received in revised form 22 October 2007; accepted 24 October 2007 Available online 3 January 2008

Abstract The production optimization of a-amylase (E.C.3.2.1.1) from Aspergillus oryzae CBS 819.72 fungus, using a by-product of wheat grinding (gruel) as sole carbon source, was performed with statistical methodology based on three experimental designs. The optimisation of temperature, agitation and inoculum size was attempted using a BoxBehnken design under the response surface methodology. The screening of nineteen nutrients for their inuence on a-amylase production was achieved using a PlackettBurman design. KH2PO4, urea, glycerol, (NH4)2SO4, CoCl2, casein hydrolysate, soybean meal hydrolysate, MgSO4 were selected based on their positive inuence on enzyme formation. The optimized nutrients concentration was obtained using a Taguchi experimental design and the analysis of the data predicts a theoretical increase in the a-amylase expression of 73.2% (from 40.1 to 151.1 U/ml). These conditions were validated experimentally and revealed an enhanced a-amylase yield of 72.7%. 2007 Elsevier Ltd. All rights reserved.
Keywords: Aspergillus oryzae; a-Amylase; BoxBehnken design; PlackettBurman design; Taguchi design

1. Introduction Several starch by-products, generated from many industrial processes, represent one of the most abundant carbon resources in nature. The use of such resources in the cultivation process is a promising way to reduce the cost price of produced molecules particularly those of high added value like antibiotics, enzymes, etc. Such resources are particularly attractive as they provide an inexpensive industrial substrate and contribute in solving pollution problems. Gruel (by-product of wheat grinding) is one such by-product that has gained attention as a substrate for the production of functional bioproducts like biopesticides from Bacillus thuringiensis (Zouari et al.,

Corresponding author. Tel.: +216 98418570; fax: +216 74440451. E-mail address: radhouan.kammoun@cbs.rnrt.tn (R. Kammoun).

2002) and special peptides for children diets (Kammoun et al., 2003). With the advent of new frontiers in biotechnology, the amylase family enzyme nds potential application in a number of industrial processes such as bread making, brewing, starch processing, pharmacy, textile, paper industries. a-Amylases (E.C.3.2.1.1.: endo a-1-4 glucan-4-glucanohydrolase) are extra-cellular enzymes that randomly cleave the 1, 4-a-D-glucosidic linkages between adjacent glucose units in the linear amylose chain. a-Amylase is secreted as a primary metabolite of microorganisms and its production is a growth related process (Spohr et al., 1998; Sudo et al., 1994). Aspergillus oryzae has been largely used in the production of food such as soy sauce (Manabe et al., 1984), organic acid such as citric and acetic acids and commercial enzymes including a-amylase (Ramachandran et al., 2004). For the production of this enzyme by culture of A. Oryzae, many by-products are tested like spent brewing grains (Francis

0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.10.045

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et al., 2003), coconut oil cake (Ramachandran et al., 2004) and orange waste water (Djekrif-Dakhmouche et al., 2006). Many works (Yabuki et al., 1977; Erratt et al., 1984; Tada et al., 1991; Lachmund et al., 1993) reported that the a-amylase production in A. oryzae is induced by carbohydrates having a-1,4 glucosidic bonds, e.g., maltose or starch. The study of the inuence of many carbon sources on alpha amylase production by this strain, realised by Carlsen and Nielsen (2001), showed that maltodextrins are the most inducer of alpha amylase expression. In addition, Arnesen et al. (1998) and Nirmala and Muralikrishna (2003) reported respectively that Tween-80 and calcium ions enhance a-amylase activity production. In the development of any fermentation process, the optimization of the process variables, particularly physical and chemical parameters, is of primary importance due to its impact on the economy and feasibility of the process. Classical optimization method involves single factor variation, keeping the other factors constant. This method is unsuited for multifactor optimisation not only because it is time-consuming but also because it is unable of detecting the true optimum due especially to the interactions between the factors (Liu and Tzeng, 1998; Weuster-Botz, 2000). The limitation of such method is avoided by using a statistical design (named also factorial experimental design): a collection of experimental strategies, mathematical methods and statistical inferences. Statistical designs allow explaining interactions between the dierent factors (Elibol, 2004; Liu and Tzeng, 1998). These designs have been applied to fermentation processes optimization precisely in the culture of bacteria (Ahuja et al., 2004; Weuster-Botz, 2000), animal cell (Ganne and Mignot, 1991) and fungus (Djekrif-Dakhmouche et al., 2006; Francis et al., 2003). The most popular choices are the PlackettBurman design (Djekrif-Dakhmouche et al., 2006; Viswanathan and Surlikar, 2001), the central composite design (Dey et al., 2001; Vohra and Satyanarayana, 2002), the BoxBehnken design (Sarra et al., 1993) and the GraecoLatin squares (Haltrich et al., 1994). Some works already reported; deal with the inuence of crucial operational parameters and media optimisation on alpha amylase production by A. oryzae strains. The eects of agitation on fragmentation of a recombinant strain of A. oryzae and its consequential eects on protein production have been investigated by Amanullah et al. (1999). Gigras et al. (2002) reported the optimization of production of an alpha-amylase from A. oryzae using a central composite design. However, in our knowledge, no works concerning the optimisation of both thermo and hydro dynamics parameters and media composition were already cited. In the present study, we reported the optimization of alpha amylase production by A. oryzae using gruel based culture media. Values of hydro and thermodynamics parameters (temperature and agitation) and inoculums size concentration were selected by BoxBehnken design with three levels and using response surface methodology (RSM). The nutrients composing the medium for amylase production

were selected using PlackettBurman design and their concentrations were optimized using Taguchi design. 2. Methods 2.1. Microorganism, maintenance of culture A fungal strain of A. oryzae CBS 819.72 (purchased from Centraalbureau voor Schimmelcultures, The Netherlands) was used in this study. It was propagated on Potato DextroseAgar (PDA) (Fluka, France) medium plate at 30 C. The plates were grown for ve to seven days then stored at 4 C. 2.2. Inoculum Preparation A. oryzae, seven days old, were harvested from plates by adding ten millilitres of distilled water containing 0.1% Tween-80. The spores were dislodged using the inoculation needle under aseptic conditions. After centrifugation at 9000g during 20 min at 4 C and elimination of supernatant, glycerol was added to the suspension. Then it was, appropriately diluted for the required density of spores and used as the master suspension. The number of viable spores in the inoculum was determined by the counting technique using the Thomas cell. 2.3. Substrate and culture media Gruel, obtained from a local semolina factory processing durum wheat, was used as substrate. It contains starch (60%), other carbohydrates (5%), cellulose (1.5%), gluten (12%) and total nitrogen (2.1%) (Bejar et al., 1999). Into 500 ml Erlenmeyer ask, two grams and half of Gruel were mixed with an enzyme production medium (100 ml), containing (g/l): peptone 10.0, yeast extract 5.0, NaCl 10, KH2PO4 0.5, MgSO4 0.5, CaCl2 0.5 and distilled water. The contents were thoroughly mixed and the initial pH was adjusted to 5.0. The cotton plugged asks were autoclaved at 121 C (15 psi) for 20 min. After cooling the medium, they were inoculated with an appropriate volume of the master spore suspension then kept on rotary shaker at permanent conditions for 72 h. 2.4. Enzyme assay a-Amylase was assayed by the addition of 50 ll enzyme of culture supernatant to 0.5 ml of 1% (w/v) soluble starch made in 0.1 M acetate buer (pH 5.6) and incubation of the reaction mixture at 60 C for 30 min. Reducing sugars released were measured by the 3, 5-dinitrosalicylic acid method (Miller, 1959). A separate blank was set up for each sample to correct the non-enzymatic release of sugars. One unit of a-amylase was dened as the amount of enzyme that released reducing sugars equivalent to 1 lmol glucose per minute under the standard assay conditions.

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R. Kammoun et al. / Bioresource Technology 99 (2008) 56025609 Table 2 Nutrient supplements for screening using PlackettBurman design Nutrient code Nutrient name (+) level (%) g/g base substrate 0.2 0.2 0.1 0.1 0.2 () level (%) g/g base substrate 0.1 0.1 0.05 0.05 0.1 Bibliographic reference

2.5. Experimental design and statistical analysis Optimization of the parameters (temperature, agitation and inoculum size) for overproduction of amylase was done by BoxBehnken design especially made to require three levels coded as (), (0) and (+) (N = 13) (13 experiments and three factors at three levels) under the response surface methodology. Table 1 shows the dierent levels of each of the parameters. The basic points for the design were selected from a preliminary study (data not shown). BoxBehnken design is a fractional factorial design obtained by combining two-level factorial designs with incomplete block designs. The response surface methodology (RSM) was used to analyse the experimental design data. In order to be correlated to the independent variables, the response variable was tted by a second order model. The general form of the second degree polynomial equation is X X X y i b0 bi xi bii x2 bij xi xj i where Yi is the predicted response, x i, xj are input variables which inuence the response variable Y; b0 is the oset term; bi is the ith linear coecient; bii the ith quadratic coecient and bij is the ijth interaction coecient. To select the nutrients to add to gruel, 19 components have been chosen to evaluate their eect on a-amylase production. These components have been used in some works for the production of a-amylase by Aspergillus species (see Table 2). The selected compounds were screened using PlakettBurman design (N = 20) [20 experiments, 19 nutriments and one error]. This design is a fractional factorial plan which allows the investigation of up to N 1 variables with N experiments (N multiple of 4 and less or equal to 100). The PlakettBurman design assumes that there are no interactions between the dierent media constituents, xi in the range of variables under consideration (Plackett and Burman, 1946). A linear approach is considered to be sufcient for screening. Y b0 bixii 1; . . . ; k where Y is the estimated target function and bi are the regression coecients. The contrast coecient, noted b, was calculated as the dierence between the average of
Table 1 Parameters values for BoxBehnken design Factor Temperature (C) Basic level 30 Variation interval 5 Value of the factor 25 30 35 150 200 250 6.5 7 7.5 Coded value 0 + 0 + 0 +

A B C D E

K2HPO4 KH2PO4 NaCl MgSO4 Yeast extract Corn steep liquor Peptone Urea Soybean meal hydrolysate Casein acid hydrolysate Glycerol (NH4)2SO4

F G H I

0.2 0.2 0.2 0.2

0.1 0.1 0.1 0.1

Francis et al. (2003) Spohr et al. (1998), Dey et al. (2001) Spohr et al. (1998) Djekrif-Dakhmouche et al. (2006) Djekrif-Dakhmouche et al. (2006), Dey et al. (2001) Djekrif-Dakhmouche et al. (2006) Ramachandran et al. (2004) Ramachandran et al. (2004) Spohr et al. (1998), Mulimani et al. (2000) Mulimani et al. (2000) Tanyildizi et al. (2005) Mulimani et al. (2000), Ramachandran et al. (2004) Francis et al. (2003) Francis et al. (2003) Nirmala and Muralikrishna (2003) Francis et al. (2003) Djekrif-Dakhmouche et al. (2006) Djekrif-Dakhmouche et al. (2006) Arnesen et al. (1998)

J K L

0.2 0.2 0.2

0.1 0.1 0.1

M N O P Q R S

CoCl2 NH3 CaCl2 ZnCl2 FeCl3 MnSO4 Tween 80

0.2 0.2 0.1 0.1 0.1 0.1 0.1

0.1 0.1 0.05 0.05 0.05 0.05 0.05

Agitation (rpm)

200

50

Log10 (Inoculum size) (Spore/ml)

0.5

measurements made at the high (+) and the low () levels of the factors. This coecient noties the main eect of the studied factor. Table 2 shows the bibliographic reference and the two levels (+) and () of each of the constituents used in the PlackettBurman design. For the optimization of nutrients, a Taguchi orthogonal array (OA) experimental design (DOE) methodology (N = 18) [18 experimental trails, seven factors at three levels and one factor (Mg SO4) at two levels] was used. This method implies the study of any given system by a set of independent variables (factors) above a specic area of interest (levels) (Krishna Prasad et al., 2005). The Taguchi design makes it possible to establish the performance at the optimum levels obtained with some well dened experimental sets (Krishna Prasad et al., 2005). In the present study, the three levels of factor variations coded as (1), (2) and (3) were considered (Table 3). The total degree of freedom is equal to the number of trails minus one i.e., 17. In the orthogonal array design, each column consists of a number of conditions depending on the

R. Kammoun et al. / Bioresource Technology 99 (2008) 56025609 Table 3 Levels of selected nutrient supplements using Taguchi design Nutrient code X1 X2 X3 X4 X5 X6 X7 X8 Nutrient name Level (1) g/g base substrate 0.2 0.5 0.5 0.5 0.5 0.1 0.1 Level (2) g/g base substrate 0.1 0.1 0.25 0.25 0.25 0.25 0.05 0.05 Level (3) g/g base substrate 0 0 0 0 0 0 0 0

5605

The regression model is determined by the SPSS procedure that considers initially all the factors and then eliminates step-by-step those that dont have any eect. So, insignicant model terms were excluded. The model equation tted by regression analysis is given by ActivityU=ml 999:942 3:643 T 0:108 N 302:697 S 22:618 S 2 0:00359 T :N 0:564 T :S where Y is the a-amylase activity (U/ml of fermentation supernatant), T the temperature (C), N the agitation (rpm) and S the log10 (spores/ml culture media). The results of the second order response surface model in the form of analysis of variance (ANOVA) are realised. The Fisher F-test [F(6, 6) = 10.85] with a very low probability value (Pmodel > F = 0.005269) demonstrate a high signicance for the regression model (Khuri and Cornell, 1987). The goodness of t of the model was checked by the determination coecient (R2). In this case, the value of the determination coecient (R2 = 0.957) indicates that only 4.43% of the total variations are not explained by the model. A higher value of the correlation coecient, R(=0.9782), justies an excellent correlation between the independent variables (Khuri and Cornell, 1987). At the same time a relatively lower value of the coecient of variation (CV = 2.22%) indicates a better precision and reliability of the experiments carried out (Khuri and Cornell, 1987). From the model equation, it can be seen that the variables with the largest eect were, the linear term of the size of inoculum (S) and to a less extent the quadratic term of the inoculum size (S2). The great importance of inoculum size on the production of a-alpha amylase was already reported. Francis et al. (2003) and Ji et al. (1998) reported that inoculum size was a parameter which aects greatly the production of a-amylase in A. oryzae. Similarly, Mulimani et al. (2000) reported that inoculums volume per gram of substrate was among the most eective parameters in stimulating amylase production by Gibberella fujikuroi. Otherwise, the temperature is the second signicant term. In fact, the temperature shows a negative eect on a-amylase production of A. oryzae. This is in agreement with the results reported by Francis et al. (2003), showing a decrease in production at temperatures outside the mesophilic range. The speed of agitation shows limited positive eect on the enzyme production. An increase of the agitation, on such limits, causes an enzyme production enhancement since the aeration is an important factor for the growth of aerobic strains. This result slightly contrast with the studies undertaken by Amanullah et al. (1999, 2000, 2001), and Bennamoun et al. (2004). Indeed, Amanullah et al. (2001), showed that protein production (a-amylase and amyloglucosidase) was independent of agitation speed although signicant changes in mycelial morphology.

MgSO4 KH2PO4 Urea Soybean meal hydrolysate Casein acid hydrolysate Glycerol (NH4)2SO4 CoCl2

levels assigned to each factor. Table 3 gives the variation levels of the components added to gruel. All experiences are carried out at pH 5.0 for 72 h in duplicates to reach an optimum combination of the parameters. 3. Results and discussion 3.1. Optimization of operational parameters Contrary to the pH generally employed in the range of 5 and 5.5, the temperature, the agitation and the size of inoculum were recognized as being the most controlling parameters on the production of a-amylase by A. oryzae. The experimental BoxBehnken design was applied to analyze the interactions between these parameters and to determine the optimum conditions for production. The design and results of experiments carried out are given in Table 4. The average of amylase activity of triplicate values obtained was taken as the dependent variable or response Y. The results were analyzed using SPSS (Version 11.0.1 2001, LEAD Technologies, Inc., USA) statistical software and the response surface was generated using EXCEL (Version 2003, Microsoft oce, Inc., USA) software.

Table 4 BoxBehnken experimental design used to optimize the physical parameters for the production of a-amylase on gruel by A. oryzae CBS 819.72 Fermentation Temperature Agitation Log10 a-Amylase Code (C) (rpm) (Inoculum/size) activity (U/ml) (spore/ml) 1 2 3 4 5 6 7 8 9 10 11 12 13 25 35 25 35 25 35 25 35 30 30 30 30 30 150 150 250 250 200 200 200 200 150 250 150 250 200 7 7 7 7 6.5 6.5 7.5 7.5 6.5 6.5 7.5 7.5 7 21,81 17,52 31,31 16,36 11,61 15,30 15,94 12,24 11,12 15,93 24,00 16,26 20,23

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These authors suggested that rapid transients of morphological parameters in response to a speed change from 1000 to 550 rpm probably resulted from aggregation and they conclude that mycelial morphology does not directly aect protein production. Bennamoun et al. (2004) indicate that limited changes in the speed of agitation have no eect on the production of proteins while its eect on biomass is clearly positive. The 3D response surfaces are the graphical representations of the regression equation for a-amylase yields. They depict the eect of pair wise interaction of the parameters, when the third parameter is kept constant Figs. 13 show the eect of interaction respectively of temperature and agitation, agitation and inoculum size and temperature and inoculum size on a-amylase production. From these plots, it is very easy and convenient to understand the interactions between two parameters and also to locate their optimum levels. In Figs. 1 and 3, it can be seen that the yield of a-amylase increased by decreasing the temperature of 3525 C. On the other hand, the a-amylase output increased with increasing the agitation from 150 to 250 rpm. The eect of the temperature is more pronounced with a higher agitation (Fig. 1). This result indicates that A. oryzae tolerate a high agitation and a mesophilic incubation temperature. The literature review reported that most amylase production studies have been done with mesophilic fungi having a temperature range of 2537 C (Gupta et al., 2003). These observations are similar to those done for A. Niger ATCC16404 (Djekrif-Dakhmouche et al., 2006). The literature reported also that agitation intensity inuences the mixing and oxygen transfer rates in many fungal fermentation and thus controls mycelial morphology and a-amylase formation (Gupta et al., 2003). As observed in Fig. 1, the

25 22.5 20

amylase -1 Activity (U.ml )

17.5 15 12.5 10 7.5 5 2.5 0

7. 0 17 6 7. 0

15

19

7.

25

.)

23

(r.

p.m

21

-1

Lo

gS

(S

re po

.m

l )
22.5-25 20-22.5 17.5-20 15-17.5

6. 8

6. 6 6. 4

Fig. 2. a-Amylase production (U/ml) observed as a response to the interaction of agitation (rpm) and inoculum size (Log S) (spore/ml) as variables and temperature (C) at central point.

25 22.5 20 17.5 15 12.5 10 7.5 5 2.5 0

26

amylase Activity (U.ml-1)

24

7. 6

28

7. 4

30

7. 2

T(
25 22.5 20 17.5 15 12.5 10 7.5 5 2.5 0
17 0 19 0 21 0 23 0 0 25

C )

32

-1

34

36

Lo

gS

(Sp

.m ore

l )

22.5-25 20-22.5 17.5-20 15-17.5 12.5-15

7 6. 8 6. 6 6. 4

Fig. 3. a-Amylase production (U/ml) observed as a response to the interaction of inoculum size (Log S) (spore/ml) and temperature (C) as variables and agitation (rpm) at central point.

-amylase -1 Activity (U.ml )

15

34 32 30 28 26 24

36

(r.

p.m

.)

C T(

22.5-25 20-22.5 17.5-20 15-17.5

Fig. 1. a-Amylase production (U/ml) observed as a response to the interaction of temperature (C) and agitation (rpm) as variables and inoculum size at central point.

agitation and the temperature can signicantly aect a-amylase production. The contours were slightly inclined to the horizontal showing that there was a signicant interaction between the two parameters in particular at higher agitation. The interaction between these two parameters, often studied separately, was overlooked by classical experimental strategy. From Fig. 2, we can observe that the contours were parallel to the two axes suggesting that the two parameters were quite independent of each-other. Age and size of the inoculum are usually found to relate with time-course of incubation rather than agitation (Francis et al., 2003). Fig. 3 shows a decrease in production at high temperatures.

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The contours were slightly inclined to the horizontal showing a signicant interaction between the two parameters. 3.2. Screening of nutrient supplements Several experimental designs have been considered for choosing media supplements, and we selected the PlakettBurman design proposed by Francis et al. (2003). The analysis showed that mediums 17 and 19 (45.32 and 45.14 U/ml) gave the maximum yield followed by mediums 13 and 18 (44.12 and 43.87 U/ml). (NH4)2SO4, CoCl2, and two nitrogen sources among urea, soybean meal hydrolysate and casein acid hydrolysate, were present in higher titres in these media. The analysis of the contrast coecient (b) showed that eight compounds: CoCl2, casein acid hydrolysate, (NH4)2SO4, glycerol, urea, soybean meal hydrolysate (b vary between 7.29 and 3.33) and at less extend, KH2PO4, MgSO4, MnSO4 and peptone had pronounced inuence on the a-amylase production (b vary between 1.78 and 0.5). According to bibliographic studies, the eects of P and Cl ions on the stability of a-amylase have been well recognized (Chessa et al., 1999; Pandey et al., 2000). Otherwise, the organic nitrogen source, like casein acid hydrolysate and soybean meal hydrolysate, have traditionally a positive eect over inorganic ones since they are also carbon source and contain trace of minerals and ions that could enhance the enzyme secretion. The optimum combinations of the eight compounds were further analyzed by a Taguchi design (we ignored the eect of MnSO4 and peptone). 3.3. Optimization of nutrients levels For this study, an L18 Taguchi design proposed by Krishna Prasad et al. (2005) was employed. Production levels were found to be very much dependent on the culture conditions (0.25106.7 U/ml). The aect of the level, the yield dierence between level 1 and 2, the contribution of each factor and the resulted optimal conditions (value and level) are shown in Table 5. Individually at level stage, cobalt source (CoCl2) has the highest aect in level 1 whereas urea and casein acid hydrolysate has higher aects,

in level 2 and 3 respectively, on the a-amylase yield. The dierence between level 2 and level 1 (L2-L1) of each factor indicates the relative inuence of the aect. The larger the dierence (L2-L1), the stronger is the inuence of the factor level. It can be seen from Table 5, that among the factors studied, urea showed stronger inuence, compared to other factors followed by (NH4)2SO4, soybean meal hydrolysate, glycerol, casein acid hydrolysate, CoCl2, MgSO4 and KH2PO4. Gupta et al. (2003) indicated that, a-amylase activity in fungal cultures can be increased by the addition to the media of dierent compounds as inducers. Our study revealed that using many nitrogen sources: urea, casein acid hydrolysate, soybean meal hydrolysate, (NH4)2SO4, seems to be a better way to produce a-amylase. On the other hand, the study showed that an increase of N leads to an enhancement of amylase expression. This result might indicate a kind of coupling between N and C supply. This corroborate the results of New and Stevens (2004) which indicate that enzyme yield could be increased by using more N-source in the submerged fermentation medium. Other wise, literature indicated that urea is the best nitrogen source tested for the production of many fungal bio products (Gupta et al., 2003). Nevertheless, there are no data for the production of amylase in the presence of enhanced supply of N-source. Increase in concentration of factors such as KH2PO4, casein acid hydrolysate and (NH4)2SO4 has resulted in increase in enzyme production. While increase in urea, soybean meal hydrolysate or glycerol concentration has resulted in higher a-amylase expression up to level 2 and subsequent increase resulted in decrease in the a-amylase yield. CoCl2 concentration at level 1 showed higher yield of a-amylase. Increased to level 2 then to level 3 concentrations, it showed progressive increase in yield. This may be reasoned due to the constitutive eect of the other culture media compounds. Phosphate and magnesium seem to play important roles on the expression of alpha amylase. This result conrm the literature purpose which indicate that phosphate has a main regulatory role in the synthesis of primary and secondary metabolites in microorganisms and likewise it aects the growth of the organism and production of

Table 5 Main eect of factors, dierence of level (2) and (1), contribution and optimum levels and values of factors Nutrient code X1 X2 X3 X4 X5 X6 X7 X8 Level (1)a 35.19 31.94 21.17 30.56 25.32 35.91 24.91 63.14 Level (2)a 45.62 38.73 61.65 50.60 35.93 53.90 47.80 23.18 Level (3)a 50.55 38.40 40.07 59.97 31.42 48.52 34.90 L2-L1ab 10.43 6.79 40.48 20.04 10.61 17.99 22.89 39.96 Optimum valuesc 0.1 0.2 0.5 0.25 0.5 0.25 0.1 0 Optimum level 0 + + 0 + 0 + Contribution (%) 5.21 10.14 21.24 10.19 19.57 13.49 8.11 22.73

Total contribution from all factors = 110.66 U/ml, current grand average performance = 40.41 U/ml, expected result at optimum condition = 151.1 U/ml. a The value is given in term of activity (U/ml). b Level (2)level (1). c g/g base substrate.

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Table 6 Comparison between the original and optimized media Media Original Nutrient name Peptone Yeast extract NaCl KH2PO4 MgSO4 CaCl2 0.5 Urea Casein acid hydrolysate Soybean meal hydrolysate Glycerol KH2PO4 (NH4)2SO4 MgSO4 Nutrient concentration (%) (g/g base substrate) 4.0 2.0 4.0 0.2 0.2 0.2 0.5 0.5 0.25 0.25 0.2 0.1 0.1 a-Amylase activity (U/ml) 31.3

Optimised

148.0

a-amylase by A. oryzae strains (Gupta et al., 2003). Literature indicates also that production was reduced to 50% when Mg2+ was omitted from the medium. On the other hand, we demonstrated that the CaCl2 is not important for the amylase production. This corroborates the results of Kundu et al. (1973). In fact these authors demonstrate that Ca2+ was inhibitory to amylase production by A. oryzae EI 212 although Ca2+ has been reported as essential for alpha amylase stability (Gupta et al., 2003). 3.4. Optimum validation The optimum combination was found to be (g/g dry base substrate): urea 0.5, casein acid hydrolysate 0.5, soybean meal hydrolysate 0.25, Glycerol 0.25, KH2PO4 0.2, (NH4)2SO4 0.1, MgSO4 0.1 and CoCl2 0.0. The model showed that CoCl2 was not essential for a-amylase production. This result may be explained by the fact that agro nitrogen source like casein acid hydrolysate and soybean meal hydrolysate can contain trace of ions like Co++ and Cl. Further, to validate the proposed experimental methodology, fermentation experiments were performed for aamylase production by employing the obtained optimized culture conditions. Table 6 gives the comparison between the yield of a-amylase from original and optimized media under the experimental conditions. The obtained yield 148.0 U/ml is very close to expected result 151.1 U/ml. The results of this study have clearly demonstrated the noteworthy increase in yield (148.0 U/ml). Use of Gruel has been beneciary, as the yield of a-amylase has been high in comparison to a-amylase production reported under submerged fermentation using other by-products, such as orange waste water (Djekrif-Dakhmouche et al., 2006). 4. Conclusions In the present work, we have demonstrated the optimization of A. oryzae culture conditions and media composi-

tion by a factorial experimental design leading to a substantial increase in a-amylase production yield. This strategy was proved to be useful and powerful tool for screening, optimisation and modelling of fermentation process. Our study showed that gruel (maltodextrins source) constitutes a good carbon source for the growth of A. oryzae and a best inducer for the production of alpha amylase. Statistical analysis showed that inoculum size is among the most important factors aecting alpha amylase release. The signicant achievement of the present work lies in the fact that using enhanced quantities of many nitrogen sources leads to an increase of alpha amylase expression. In this study, the experimental results clearly showed that phosphate and magnesium play an important role on the enzyme expression. These minerals might be essentials for the maintenance of mould, enzyme production and stability. Using the optimized conditions, the produced activity reaches 148 U/ml and 5920 U/g of gruel. The results show a close concordance between the expected and obtained activity level. This is the rst report about production of alpha amylase by A. oryzae CBS 819.72 using gruel based medium. This paper proposes a low cost medium formulation that could be of industrial value and could serve as a basal media for further optimization studies of this and similar strains. Furthermore, this study serves as another example for the application of the statistical methodology to biological systems. In addition, after further optimization is carried out using optimized medium for batch and fed batch systems and after optimizing the fermentation parameters (aeration, agitation speed, dilution rate, etc.), the enzyme activity is expected to increase. Acknowledgements This work was supported by funds from the Tunisian Government (Contract-Programme CBS-L.E.M.P.). We thank Mr. Moncef Aes for critically reading the manuscript. References
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