Rice straw was collected from local paddy field of Hathazari, Chittagong to isolate actinomycetes using Starch Casein Agar medium. The isolates were tested for cellulolysis by Congo red overlay method using Winstead’s medium having 1.2% CMC as carbon source. For the Congo-Red method, plates were flooded with 0.1% Congo red (Sigma-Aldrich) for 10 - 15 min before destaining with 1M NaCl solution for 15 - 20 min for several times until the clear zones around the colonies were visualized. The use of Congo-Red as an indicator for cellulose degradation in an agar medium provides the basis for a rapid and sensitive screening test for cellulolytic microbes. Colonies showing discoloration of Congo-Red were taken as positive cellulose-degrading microbial colonies. Then the actinomycetes isolates were further screened (secondary screening) for their cellulolytic potentiality. It was done by using Winstead’s medium having 1.2% CMC in separate small conical flasks.
Among the numerous isolates SR1 was found to be promising cellulose degrader. For the characterization of the isolate, the basic routine laboratory investigation like morphological, cultural and different biochemical characteristics, which included Indole, methyl red, Voges–Proskauer, citrate utilization, catalase, urease, starch hydrolysis, gelatin hydrolysis, sugar fermentation, caseinase, hydrogen sulfide production and nitrate reduction tests were performed and compared with the standard description given in Bergey’s Manual of Determinative Bacteriology. Based on morphological, cultural and biochemical characteristics the isolate SR1 was provisionally identified as Thermomonospora viridis.
An attempt was made to determine the optimum culture conditions such as pH, temperature, carbon and nitrogen sources requirement for maximum cellulase production. The biomass yields, extracellular protein, reducing sugar level and cellulase production of the isolates were recorded. To be more precise, the effects of pH (4.5, 5.5, 6.5. 7.5, 8.5, 9.5 and 10.5) and temperature (20, 25, 30, 35, 40 and 45°C) on the growth and liquefaction were investigated and recorded. Similarly, the production of extracellular cellulase under different carbon and nitrogen sources was studied in the liquid Winstead’s culture medium using five carbon sources (Avicel, CMC, Rice bran, Rice straw and Saw dust of 1.2%) and six nitrogen sources (Asparagine, Beef extract, Ammonium sulphate, Yeast extract, Peptone and Urea of 0.2%). Furthermore, effects of these carbon and nitrogen sources on the production of extracellular protein, reducing sugar and biomass yield were recorded (Tables 3 and 4). Finally, the substrate concentration was optimized using different concentrations of Carboxymethyl cellulose (CMC) (1.0, 1.5, 2.0 and 2.5% of CMC). The optimum concentration was selected based on the maximum production of cellulase, extracellular protein, reducing sugar and biomass.
Physicochemical parameters such as different pH (3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5 and 10.5), temperature (20, 25, 30, 35, 40, 45, 50, 55 and 60°C), carbon sources (avicel, CMC, rice straw, rice bran, saw dust, filter paper, cotton and salicin) and nitrogen sources (asparagine, ammonium sulphate, beef extract, peptone, yeast extract and urea) on the activities of crude cellulase were determined. To determine cellulase activity, the reaction mixtures (2 ml filtrate + 2 ml of 1% substrate in citrate phosphate buffer + 1 ml phosphate buffer) were incubated at 35°C for 2 hrs in water bath followed by the determinations of reducing sugars released by Nelson’s modification of Somogyi method (Nelson 1944). Enzyme activity was expressed by the amount of glucose released in µg/ml of crude enzyme/hour (U/ml) (Mahadevan and Sridhar 1982) and soluble protein in culture filtrate was estimated following the Lowry method. The saccharification percentage was calculated by applying the following equation:
Saccharification % = mg of reducing sugar per ml / mg of substrate per ml x 100
The filter paper containing biomass residue was dried in an oven at 80°C for a constant weight and amount of biomass was calculated by subtracting the weight of filter paper. Yield was expressed as mg/g cellulose.
Effect of different metal ions, as well as reductant and inhibitors on the activities of crude cellulase, was investigated. Effects of ten different metal chlorides [viz. NaCl, KCl, NH4Cl, LiCl2, AgCl2, HgCl2, MnCl2, MgCl2, CaCl2, and FeCl3] and six different inhibitors and reductants [viz. Sodium azide, Sodium dodecyl sulphate (SDS), Urea, Cystein, β-Mercaptoethanol and Ethylene diamine tetraacetic acid (EDTA)] on cellulase activity were determined by adding 2 ml of culture filtrate to 2 ml of 1% CMC prepared in phosphate buffer followed by addition of 1% of metal and inhibitors and reductants solution, respectively. All tubes were incubated for 2 hrs in water bath at 350C and the enzyme activity was measured according to Nelson’s modification of Somogyi method.