MATERIALS AND METHODS

Fungus cultivation for spore production and inoculum preparation: Local isolate, Aspergillus terreus SUK-1 from School of Biosciences and biotechnology, The National University of Malaysia (UKM), were grown and maintained on Potato Dextrose Agar (PDA). Spore suspension of 10⁷ spore mL^-1 was prepared by harvesting from 7 days old culture of both molds with 15 mL sterile distilled water and 10% (w/v) of inoculum (of the tested ratios) was used in all experiments.

Sorghum production and medium preparation: Sorghum seeds were collected from Local market in Khartoum, Sudan. The seeds planted by drill in rows seeds in moist soil in National University (UKM) of Malaysia experimental plot Malaysia. The seeding depth was in one inch and after 70 days the sorghum were harvested (Kansas State University, 1998), the sorghum straw was firstly washed then dried and then milled into small particles using miller. The sorghum straw particles were sieved using 1 mm mesh sieve and utilized as the substrate for solid substrate fermentation. The medium used for SSF was developed by Mandels and Rees (Sternberg et al., 1977), the medium composition is shown in Table 1.

Enzyme production under SSF: Five gram of the sorghum straw was introduce into 250 mL Erlenmeyer flask, the Mandels medium was added to the 5 g of the sorghum straw to give different moisture ratio. Medium was sterilized for 30 min at 121°C and spore sustention was aseptically added to the flasks and mixed thoroughly, incubation temperature was set at 25-40°C for 7 days experimental run conducted based on the statistical design.

Enzyme extraction: The enzyme was extracted by a simple contact method distilled water was introduced into each fermented flask in 1:10 (v/w) ratio kept in shaker at 120 rpm for 30 min and then filtered via muslin cloth and centrifuge at 10,000 rpm for 15 min at 4°C. The clarified supernatant was used for further enzyme analysis.

Determination of enzyme activity: Filter paper activity (FPase) for total cellulase activity in the culture filtrate was determined according to the standard method (Hankin and Anagnostakis, 1975). Aliquots of appropriately diluted cultured filtrate as enzyme source was added to whatman No. 1 filter paper strip (1×6 cm; 50 mg) immersed in 1 mL of 0.05 M Sodium citrate buffer of pH 5.0. After incubation at 50±2°C for 1 h, the reducing sugar released was estimated by dinitrosalicylic acid (DNS) method (Miller, 1959). One unit of filter paper (FPU) activity was defined as the amount of enzyme releasing 1 μmole of reducing sugar from filter paper per mililiter per min.

Experimental design: The variables used were (a) Temperature, (b) pH and (c) moisture content and the coded value variable were -1, 0, 1 (low, basal low, high) (Table 2). Twenty experiments were performed for each microorganism at three level including five replicates at the centre points with three levels according second order face-centred composite design (Table 3).

Table 1:	Medium composition

Table 2:	Range of variable and their coded level for the central composite design uses for FPase activity from sorghum straw using Aspergillus terreus SUK-1
A: Temperature at low level (1-) of 25°C and high level (1+) of 40°C, B: pH at low level (1-) of 4 and high level (1+) of 7, C: Moisture content at low level (1-) of 40 %(w/v) and high level (1+) of 80% (w/v)

Table 3:	Experimental Central Composite Design (CCD) with experimental value and predicted value on FPase activity by Aspergillus terreus SUK-1

The experimental data was employed in statistical package, software Design Expert 6.0.0 (Stat Ease Inc. Minneapolis, USA) to fit a second order polynomial response surface methodology according to Eq. 1.

(1)

RESULTS

The effect of the three variables on FPase activity is illustrated in Table 4. Statistical analysis by ANOVA showed that the effect of temperature and moisture content had significant effect on FPase activity. However, the effect of pH was insignificant on FPase activity (Table 4).

The experimental design and result of experiment are presented in Table 3. The results for the second ordered response surface model for FPase activity in the forms of ANOVA is shown in Table 4.

The model F-value of 7.27 implies the model is significant. There is only a 0.23% chance that a model F-value could occur due to noise. The pure error was very low (0.005207), indicating good reproducibility of the experimental data. Values of probe >F-value less than 0.0023 indicates model terms are significant.

In this case A and C are significant terms. Values that are greater than 0.05 indicate the model terms to be not significant. The fit of the model checked by the co-efficient of determination R² was calculated to be 0.8674 indicating that 86.74% of variables can be explained by the model and only 13.3% of variability in the response cannot be explained by the model. The multiple coefficient of determination (R²) represent variability in the value formed response which can be explained by test factors and their interactions.

A regression model can be used to predict observation of the response (FPase) coresponding to the particular values of the regression variable (Table 5). The model equation for the individual parameters, interaction (as second order equation ) can be shown by using Eq. 2:

(2)

The three dimensional response surface graphs (3D) for response surfaces are displayed in Fig. 1a-c represented the combined effects of temperature and pH (Fig. 1a), temperature and moisture content (Fig. 1b), pH and moisture content (Fig. 1c). In all 3D graphs, two variables were varied while the other variable was kept constant at its central level. The central level of temperature was 32.5°C, pH was 5.5 and moisture content was 60% as shown in Table 3.

The response surfaces obtained suggested that optimum cellulase activity by Aspergillus terreus SUK-1 would be obtained at a temperature 25°C, pH and 40% moisture content. The quadratic model (Eq. 2) predicted an optimum FPase activity of 0.35 U mL^-1 under the following SSF parameters: Temperature of 25°C, pH of 4 and moisture content of 40%.

The interaction effect between temperature and pH on cellulase production was presented in Fig. 1a. The 3D graph shows that decreasing the temperature and pH around 5 leads to increase the cellulase activity. Figure 1b displays the interaction effect between temperature and moisture content on cellulase activity. The interaction effect between pH and moisture content on cellulase production was presented in Fig. 1c. It was found that pH 5 and moisture content 60% maximized the FPase activity.

Fig. 1(a-c):	Three-dimensional response surface plot for the effects of, (a) Incubation temperature and pH, (b) Moisture content and temperature and (c) pH and moisture content on FPase activity

Table 4:	Analysis of Variance (ANOVA) for regression model to optimize FPase activity
SS: Sum of squares, DF: Degree of freedom, MS: Mean of squares

Table 5:	Regression coefficient for optimization of FPase activity

DISCUSSION

Present results are in agreement with the results of Latifian et al. (2007), who have reported that moisture had a significant effect on cellulase production using T. reesei QM 9414 and T. reesei MCG 77. In addition they found that the temperature was in the range of 25-30°C for optimal cellulase production.

Acharya et al. (2008) reported that the optimum pH and temperature for cellulase production using A. niger were at pH 4.0 and temperature of 28°C, respectively which is in agreement with present results. Ahmed et al. (2009) reported that the optimum pH for Trichoderma viride growth for maximal exoglucanase (EXG), endoglucanase (EG) and β-glucosidase production was found to be 5.5 at 28°C. Optimum pH for fungal cellulases varies from species to species; though in most cases the optimum pH ranges from 3.0 to 6.0 (Niranjane et al., 2007; Karuppaiya et al., 2009).

It is reported in the literature that solutions with higher desirability gave optimum temperature of 30°C, pH of 5 and moisture content of 70% (Oberoi et al., 2011).

Moisture content in the solid state fermentation is a key factor for cellulase enzyme production. Ahmed et al. (2010) have optimized the moisture content by varying moisture content from 10-50% to lignocellulosic substrate wheat straw. The study indicates that maximum cellulase (301±1.16 μM mL^-1 min^-1) production was observed at 40% moisture level, while further increase in moisture influenced the enzyme production negatively. They concluded that there is a positive relationship between cellulase production and moisture content (Ahmed et al., 2010). Higher and lower water contents adversely affect the primary metabolic activities of microbes leading to slower enzyme formation (Wang et al., 2006; Fang et al., 2010).

Optimal water fractions in the solid substrate between 40-60% (by mass) under solid-state fermentation (Narasimha et al., 2006). Sohail et al. (2009) reported that optimization of incubation temperature and initial moisture content of the medium resulted in a 6.2 fold. Increase in production from 0.605 to 3.8 U gds^-1 of cellulase by Aspergillus niger MS82.

The effect of initial pH on enzyme production ranging from 3 to 7 was determined and maximum cellulase activity (348±1.29 μM mL^-1 min^-1) was recovered at pH 5.5. Furthermore, it was found that maximum induction of endoglucanase was achieved at pH 5.5 as there was a correlation between the initial pH of the medium and cellulase produced by Trichoderma reesei Rut C-30 (Khurshid et al., 2001; Xiong et al., 2004).

Model validation: The model was validated by applying the optimized conditions and the obtained cellulase activity was found to be 0.36 U mL^-1 which is slightly higher than the predicted value (0.35U mL^-1). This verifies the fitness of the model in predicting the combined interactions of the three independent variables (temperature, pH and moisture content) on the cellulase activity.

CONCLUSION

The optimization of FPase activity from sorghum straw under solid substrate fermentation by Aspergillus terreus SUK-1 was successfully conducted by using Response Surface Methodology (RSM). The temperature and moisture content was found to significant effect on cellulase activity. Optimum cellulose activity was established 0.35 U mL^-1 at the temperature of 25°C, pH 4 and 40% moisture content.

ACKNOWLEDGMENTS

To staff of laboratory assistants of the School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, UKM Bangi, 43600, Selangor, Malaysia. We also acknowledge grant from GUP 2012-074 from UKM.