Fungal strains and culture conditions
A total of 40 strains of Aspergillus niger used in this work were isolated from date by-products and identified in previous work . One millimeter of each diluent was plated on the solidified PDA plates (Biokar, France) by streaking. The plates were incubated at 25 °C for 3–5 days to ensure maximum fungal growth. Characteristic growth of A. niger (initially white, rapidly turning black) was subjected to microscopic observation. For microscopic identification, a drop of lactophenol blue was placed on a clean slide, with a fragment of the fungal growth, and observed under the microscope using a ×10 then ×40 objectives. The isolates obtained were given a number preceded by ASP. In this way, 40 isolates were purified and stored on inclined tubes of the PDA medium at 4 °C until they were used. These strains were routinely reactivated and cultured in PDA at 25 °C for 7 days before use.
Screening extracellular enzymes from A. niger
The cellulase activity of A. niger strains was realized in Czapek-Dox agar medium containing sucrose 30 g/L, sodium nitrate 3 g/L, magnesium sulfate 0.5 g/L, potassium chloride 0.5 g/L, iron(III) sulfate 0.01 g/L, dipotassium hydrogen phosphate 1 g/L, agar 12 g/L, and carboxymethylcellulose (CMC) 1% (w/v) (Sigma-Aldrich, USA). A. niger strains were inoculated with 5 mm of the mycelium at the center of the plate and incubated for 5 days at 25 °C. After incubation, the cultures were flooded with a Congo red solution (0.2%) and then bleached with 1M NaCl for 15 min. Clear zones obtained around the fungal colony indicated cellulolytic activity. All experiments were realized in triplicate.
The amylase activity was evaluated by measuring the ability of A. niger strains to hydrolyze starch in the agar medium. 5 mm of mycelia from strains was placed in the YPD medium containing dextrose 1g/L, yeast extract 0.1g/L, peptone 0.5 g/L, and agar 16 g/L, and supplemented with 1% (w/v) of soluble starch (Sigma-Aldrich, USA). After incubation at 25 °C for 5 days, the plates were flooded with a solution containing 1% iodine solution in 2% potassium iodide. Zone of clearance around the colony indicated amylase activity and was measured. The test was performed in triplicate.
The lipase activity was detected in a medium containing peptone 10 g/L, NaCl 5 g/L, CaCl2·2H2O 0.1 g/L, and agar 16 g/L and autoclaved at 121 °C for 20 min. Ten milliliters of Tween-20 was separately autoclaved and added into the medium and inoculated with 5 mm of mycelia from A. niger. After incubation at 25 °C for 5 days, the lipolytic activity was indicated by the appearance of a visible precipitate. All the assays were performed in triplicate.
Protease activity of A. niger strains was evaluated on YPD agar medium supplemented with 0.4% (w/v) of gelatin (Sigma-Aldrich, USA), and the plates were inoculated with 5 mm of mycelia from strains. After incubation at 25 °C for 5 days, the plates were flooded with saturated aqueous ammonium sulfate. The clear zone around the fungal colony indicated the hydrolysis of gelatin. All tests were realized in triplicate.
Semiquantification of extracellular enzymes from A. niger
Five A. niger strains (ASP2, ASP6, ASP28, ASP31, and ASP32), selected for their high enzyme production, were initially cultured on PDA medium for 5 days at 25 °C. After incubation, a spore suspension was prepared by flooding the grown fungal cultures with 10 mL sterile distilled water. The spore concentration was adjusted to 5×106 spores/mL using the Thoma cell counting chamber.
Semiquantification of extracellular enzyme production
An aliquot of 65 μL of the spore suspension of A. niger strains was then delivered into the API-ZYM cupules (BioMerieux, France) and incubated at 37 °C for 12 h. One drop of ZYM A (25 g Tris-hydroxymethyl-aminomethane, 11 mL 37% HCl, 10 g sodium lauryl sulfate,100 mL H2O) and ZYM B (0.12 g Fast Blue BB, 50 mL methanol, 50 mL dimethylsulfoxide) reagents was added to the cupules, which were placed under white light for 10 min. The API-ZYM test can detect 19 different enzymes and score their concentrations on a rating scale of 0–5. Scoring was done using the API-ZYM® color scale, in which 0 = no enzyme, 1 = 5 nmol, 2 = 10 nmol, 3 = 20 nmol, 4 = 30 nmol, and 5 = 40 nmol or more.
Quantification of amylase and cellulase activities of A. niger strains
Measurement of amylase and cellulase activities
The quantitative evaluation of amylase activity was studied in 50-mL flasks containing 25 mL of culture medium. The culture medium composition was (g/L) NaNO3 3 g, MgSO4·7 H2O 0.5 g, KCl 5 g, KH2PO4 1 g, FeSO4·7 H2O 0.01 g, and CaCl2 0.1 g, supplemented with 1% starch. On the other hand, the cellulase activity was studied on the liquid culture medium described by Hultin and Nordstr6m , supplemented with 1% (w/v) carboxymethylcellulose CMC (Sigma Aldrich Co, Germany). These flasks were then autoclaved at 121 °C for 15 min and cooled at room temperature. After sterilization, the flasks were inoculated with a spore suspension of 2×105 spores/mL and incubated for 3 days in an orbital shaker (KS 4000 I control) at 150 rpm. An uninoculated flask was used as a control.
The culture broth was filtered using Whatman filter paper N°1 (Indiamart, India), and then, the filtrate was centrifuged at 8000g for 10 min at 10 °C. The culture supernatant (1 mL) was added to 1% (w/v) starch for amylase activity and 1% (w/v) CMC for cellulase activity measurement in 0.05 M sodium acetate buffer (pH 5.6, 8 mL), and incubated at 50 °C for 30 min.
Reducing sugars were determined based on the DNS method . One unit of amylase and cellulase activity (U) is defined as the amount of enzyme that liberated 1 μmol of d-glucose from starch and CMC in a 1 μL reaction mixture under the assay conditions.
The fungal biomass was collected, after 7 days of incubation at 25 °C, on Whatman grade 1 filter paper (Indiamart, India), and dried in an oven at 100 °C for 18 h. The biomass of fungal culture was expressed as dry weight (g/L). All the tests were made in triplicate.
Factors influencing the production of amylase and cellulase enzymes
Effect of initial pH on production of amylase and cellulase activities
The effect of initial pH on the production of amylase and cellulase enzymes was evaluated on liquid culture. The medium broth of amylase and cellulase activities was adjusted of the initial pH of 3, 4, 5, 6, 7, and 8 with hydrochloric acid (4 M) and inoculated with 1% (v/v) of A. niger ASP2. After inoculation, the cultures were incubated at 25 °C for 7 days. Amylase and cellulase activities were measured. All tests were performed in triplicate.
Effect of temperature on production of amylase and cellulase activities
The effect of different temperatures on the production of amylase and cellulase enzymes was evaluated on liquid culture. The medium broth of amylase and cellulase activities was adjusted of the initial pH 7 with hydrochloric acid (4 M) and inoculated with 1% (v/v) of A. niger ASP2. After inoculation, the cultures were incubated at different temperatures (25, 28, 30, 35, 40, and 45°C) for 7 days. Amylase and cellulase activities were measured. All tests were performed in triplicate.
Effect of incubation period on the production of amylase and cellulase activities
The dynamic of production of amylase and cellulase enzymes by A. niger ASP2 strains was evaluated by the measurement of enzymatic activity in different incubation time. A. niger ASP2 strains were inoculated with 1% (v/v) in medium broth adjusted in pH 7 with hydrochloric acid (4 M) and cultivated at 25 °C. After 24, 48, 72, 96, 120, 144, and 168 h of incubation, the amylase and cellulase were determined. All assays were performed in triplicate.
Means were based on three replications. The values of different parameters were expressed as the mean ± standard deviation. Student-Newman-Keuls test was performed using the statistical analysis package SPSS 10 for Windows (SPSS Inc., Chicago, USA) at p<0.05, to evaluate the significance of differences between mean values.