Effect of Minerals and Moisture Level on the Solid Cultures of A. niger and T. viride for Extracellular Cellulolytic and Xylanolytic Enzymes Production

MATERIALS AND METHODS

Microorganisms and Cultural Conditions
Aspergillus niger and Trichoderma viride were obtained from the culture bank of Institute of Industrial Biotechnology, GC University, Lahore. These strains were re-cultured at 30±1°C in culture tubes with 4.0% Potato Dextrose Agar (PDA) medium. In the tubes containing five day old fungal growth, 5.0 mL sterilized distilled water was added to obtain conidial suspension. One milliliter this suspension (containing 3.75x10⁷ conidia) was transferred to Erlenmeyer conical flasks containing 20 g Solid Medium (SM). The SM were prepared by mixing the 20 mL distilled water/E and P medium [supplemented with (g L^-1); KH₂PO₄ (1.0), KCl (0.5), (NH₄)₂SO₄ (0.5), MgSO₄.7H₂O (0.2), L-asparginine (0.5), CaCl₂ (0.1), yeast extract (0.5) with 5.0 pH] (Eggins and Pugh, 1962) with 20 g agricultural wastes (wheat bran, sugarcane bagasse, corncob, dried grass and rice bran), separately following a manual homogenization (all the wastes were washed, dried and powdered to a diameter of 2.0 mm). After 72 h of incubation, 100 mL distilled water was added to each flask. These flasks were maintained under occasional shaking for about 1 h at room temperature. The contents in flasks were centrifuged at 8,000 rpm for 15 min and supernatant was used for analytical purposes.

Analytical Methods
Endo-β-glucanase activity determination was based on the degradation of carboxymethyl cellulose. The method used was after Wood and Bhat (1988) using glucose as standard. Avicel was used as substrate for the analysis of avicelase in the supernatant. β-Glucosidase was estimated according to the method used by Rajoka and Malik (1996). p-Nitrophenyl-β-D-glucopyranoside was used as substrate and the results were compared with the standard curve of p-nitrophenol. Xylanase in the samples was determined using xylan as substrate. The xylose was used as standard for comparison. In the estimation of all the enzymes except β-glucosidase, reducing sugars were measured according to the method as described by Miller (1959). Finally the units of all the estimated enzymes were calculated and data was further processed to statistical analysis. One unit of enzyme activity is defined as the amount of enzyme required to liberate one micromole of substrate in one minute under standard conditions.

Statistical Analysis
Treatment effects were compared after Snedecor and Cochrane (1980) using computer software Costat, cs6204W.exe by applying two-way analysis of variance (two-way ANOVA). Significance difference among replicates has been presented as Duncan’s multiple range tests in the form of probability <p> values.

RESULTS AND DISCUSSION

The present study describes the utilization of agricultural wastes for the production of extracellular hydrolytic enzymes by Aspergillus niger and Trichoderma viride under solid-state fermentation. Agricultural wastes like wheat bran, wheat straw, rice bran, rice straw, sugar cane bagasse, corncob and dried grass were used in this investigation. Distilled water and E and P medium were used as moistening agents.

The results were presented after statistical analysis by applying two-way ANOVA. Standard deviation and least significance difference among replicates were determined. After one-way ANOVA, results indicated that A. niger and T. viride secretes both cellulolytic and xylanolytic enzymes, but their rates are varied. Mitchell (1992) also stated that filamentous fungal strains like A. niger and T. viride are the best adapted micro-organisms for solid-state fermentation owing to their physiological, enzymological and bio-chemical properties. Moreover, hyphal mode of fungal growth gives them a power to penetrate into the solid substrates, which gives them major advantage over unicellular micro-organisms for the colonization of the substrate and the utilization of available nutrients. Haq et al. (2006) also reported that A. niger and T. viride are best source of cellulolytic and hemicellulolytic enzymes. Gawande and Kamat (1999) observed that crude culture filtrate of Aspergillus strains hydrolyzed various lignocellulosic materials. Results indicated that A. niger and T. viride used agricultural wastes as carbon sources in the presence of distilled water (1:1 ratio). This is because these substrates are heterogeneous water insoluble materials, which have lignocellulosic nature with different percentages (Raimbault, 1998). However, higher rate of enzymes production (CMC-ase, avicelase, β-glucosidase and xylanase) was obtained with corncob. It is because that corncob is a complete substrate with all required micro- and macro-nutrients for the growth of microorganisms. Corncob contains 90% dry matter, 9.0% net energy for growth maintenance (NEg), 3.0% crude protein (CP), 50% bypass, 36% crude fiber (CF), 39% acid detergent fiber (ADF), 88% neutral detergent fiber (NDF), 56% effective neutral detergent fiber (eNDF), 0.5% ether extract (EE), 2.0% Ash, 0.12% calcium (CA), 0.04% phosphorous (P), 0.8% potassium (K), 0.40% sulphur (S) and 5.0% Zinc (Zn).

Using corncob as a substrate, A. niger produced 0.586 U mL^-1 min^-1 CMC-ase (Table 1), 0.46 U mL^-1 min^-1 avicelase (Table 3), 0.71 U mL^-1 min^-1 β-glucosidase (Table 5) and 18.7 U mL^-1 min^-1 xylanase (Table 7) on corncob at 5.0% level of significance with 0.011, 0.0078, 0.0065 and 0.0391 LSD, respectively.

Table 1:	Effect of distilled water and different ratios (v/w) of E and P medium on the production of carboxymethyl cellulase (U mL-1 min-1) by A. niger under solid-state fermentation conditions
Superscript alphabets show significant ranges among distilled water and different ratios of E and P medium. Subscript alphabets indicate significant ranges among different agricultural by-products (Significance level increases with ascending order of alphabets and vive versa)

Table 2:	Effect of distilled water and different ratios (v/w) of E and P medium on the production of carboxymethyl cellulase (U mL-1 min-1) by T. viride under solid-state fermentation conditions
Superscript alphabets show significant ranges among distilled water and different ratios of E and P medium. Subscript alphabets indicate significant ranges among different agricultural by-products (Significance level increases with ascending order of alphabets and vive versa)

Table 3:	Effect of distilled water and different ratios (v/w) of E and P medium on the production of avicelase (U mL-1 min-1) by A. niger under solid-state fermentation conditions
Superscript alphabets show significant ranges among distilled water and different ratios of E and P medium. Subscript alphabets indicate significant ranges among different agricultural by-products (Significance level increases with ascending order of alphabets and vive versa)

Table 4:	Effect of distilled water and different ratios (v/w) of E and P medium on the production of avicelase (U mL-1 min-1) by T. viride under solid-state fermentation conditions
Superscript alphabets show significant ranges among distilled water and different ratios of E and P medium. Subscript alphabets indicate significant ranges among different agricultural by-products (Significance level increases with ascending order of alphabets and vive versa)

On the other hand, T. viride produced 0.90 U mL^-1 min^-1 CMC-ase (Table 2), 0.77 U mL^-1 min^-1 avicelase (Table 4), 0.59 U mL^-1 min^-1 β-glucosidase (Table 6) and 13.4 U mL^-1 min^-1 xylanase (Table 8), respectively with 0.012, 0.0140, 0.005 and 0.0345 LSD. da Silva et al. (2005) also worked on xylanase and CMC-ase by Thermoascus aurantiacus in solid-state fermentation and reported that best production of these two enzymes were achieved, when corncob was used as substrate.

Table 5:	Effect of distilled water and different ratios (v/w) of E and P medium on the production of β-glucosidase (U mL-1 min-1) by A. niger under solid-state fermentation conditions
Superscript alphabets show significant ranges among distilled water and different ratios of E and P medium. Subscript alphabets indicate significant ranges among different agricultural by-products (Significance level increases with ascending order of alphabets and vive versa)

Table 6:	Effect of distilled water and different ratios (v/w) of E and P medium on the production of β-glucosidase (U mL-1 min-1) by T. viride under solid-state fermentation conditions
Superscript alphabets show significant ranges among distilled water and different ratios of E and P medium. Subscript alphabets indicate significant ranges among different agricultural by-products (Significance level increases with ascending order of alphabets and vive versa)

Table 7:	Effect of distilled water and different ratios (v/w) of E and P medium on the production of xylanase (U mL-1 min-1) by A. niger under solid-state fermentation conditions
Superscript alphabets show significant ranges among distilled water and different ratios of E and P medium. Subscript alphabets indicate significant ranges among different agricultural by-products (Significance level increases with ascending order of alphabets and vive versa)

Orgaz et al. (2006) reported that different plant polysaccharides are best substrates for the production of hydrolytic enzymes.

A series of experiments was performed to compare between the efficiency of distilled water and E and P medium as moistening agent. Different ratios of E and P medium were used (1:1, 2:1 and 3:1).

Table 8:	Effect of distilled water and different ratios (v/w) of E and P medium on the production of xylanase (U mL-1 min-1) by T. viride under solid-state fermentation conditions
Superscript alphabets show significant ranges among distilled water and different ratios of E and P medium. Subscript alphabets indicate significant ranges among different agricultural by-products (Significance level increases with ascending order of alphabets and vive versa)

Data obtained was compared statistically applying two-way ANOVA. With E and P medium (1:1 ratio), A. niger and T. viride produced 0.678 and 1.110 U mL^-1 min^-1 CMC-ase (Table 1 and 2), 0.60 and 1.00 U mL^-1 min^-1 avicelase (Table 3 and 4), 0.87 and 0.72 U mL^-1 min^-1 β-glucosidase (Table 5 and 6) and 23.4 and 17.1 U mL^-1 min^-1 xylanase (Table 7 and 8). These results revealed that E and P mineral salts medium is the best moistening agent for the production of hydrolytic proteins by both A. niger and T. viride as it contains the minerals for best growth of fungal strains. These minerals provide and fulfill the basic requirements for fungal growth like phosphate, sulphate, potassium, chloride, ammonium, magnesium, calcium and nitrogen. All these are considered to be essential in fermentation process for the growth of microbial strains and ultimate production and secretion of various hydrolytic proteins. After the optimization of E and P medium as best moistening agent, moisture level analysis was made and it was observed that 2:1 ratio of E and P medium was best as higher rates of enzymes were achieved, when A. niger and T. viride were cultured with E and P medium (2:1). Whereas; lower moisture content causes reduction in solubility of nutrients of the substrate, low degree of swelling and high water tension. On the other hand, higher moisture levels can cause a reduction in enzyme yield due to stearic hindrance of the growth of the producer strain by reduction in porosity of the solid matrix, thus interfering oxygen transfer (Lonsane et al., 1985).

CONCLUSIONS

Cellulosic and hemi-cellulosic compounds are mainly presented in agricultural by products as these compounds are an important part. These are being replenished constantly in nature by photosynthesis, goes waste in a lion’s share in the form of pre-harvest and post-harvest agricultural losses and wastes of food processing industry. These cellulosic and hemi-cellulosic wastes have an immense potential to be utilized by filamentous fungi in the presence of limited amount of moisture and some essential ions. By the utilization of these wastes, production and recovery of several products and ingredients can be achieved as these are rich materials with all essential substances. These products mainly include hydrolytic enzymes, which possess a wide spectrum of researches in industrial area like food, paper, sugar and textile industries.