Isolation, Screening, and Evaluation of Cellulase-Producing …
459
2 Materials and Methods
2.1 Collection of Soil Sample
The soil samples were collected from seven different locations of Sitakunda and
Lulung regions of Similipal Biosphere Reserve.
2.2 Isolation of Cellulolytic Bacteria
Bacterial isolates were isolated by serial dilution method (Aneja 2007). 1 gm of each
of the respective soil samples was homogenized in 9 mL of sterilized sterile distilled
H 2 O taken in test tube and the suspension was then serially diluted up to 10
–5 and
10
–6 , respectively.
2.3 Screening of Cellulolytic Bacteria
Each bacterial isolates were screened for cellulolytic activity using CMCase plate
assay (Muhammad et al. 2012). 10 µL of each bacterial broth was inoculated inside
4 mm pore made on the CMC agar plate. The CMC agar plates containing bacteria
inoculums were then incubated at 37 °C for 72 h. After incubation, CMC agar plates
were stained by 0.1% Congo Red solution for 1 h followed by distain using 1 N
NaCl solution for the determination of cellulose hydrolyzing zone surrounding the
bacterial growth on CMC agar plate.
2.4 Quantitative Assay of Bacterial Cellulase Activity
Quantitative assay of cellulase activity was estimated using DNS method (Miller
1959) by measuring the amount of reducing sugar hydrolyzed by individual bacterial isolates. Each selected bacterial strain was cultured in nutrient broth medium for
overnight incubation at 37 °C. Incubated bacterial broths were individually transferred to sterilized 1-mL Eppendorf tube. Each Eppendorf tube was centrifuged at
10,000 rpm for 10 min at 4 °C. Substrate solution was prepared by adding 1% of CMC
with phosphate buffer (0.1 M) at pH 6.8. 0.5 mL of substrate solution and 0.5 mL
of each bacterial supernatant (crude enzyme) were taken in assay tube to form 1 mL
reaction mixture. Assay tubes containing the reaction mixture were then incubated
at 55 °C for 15 min in water bath. After the incubation of reaction mixture, 1.5 mL of
DNS solution was added in each assay tube and boiled at 100 °C for 10 min in water
bath to stop the reaction. Amount of reducing sugar released in the hydrolysis by
459
2 Materials and Methods
2.1 Collection of Soil Sample
The soil samples were collected from seven different locations of Sitakunda and
Lulung regions of Similipal Biosphere Reserve.
2.2 Isolation of Cellulolytic Bacteria
Bacterial isolates were isolated by serial dilution method (Aneja 2007). 1 gm of each
of the respective soil samples was homogenized in 9 mL of sterilized sterile distilled
H 2 O taken in test tube and the suspension was then serially diluted up to 10
–5 and
10
–6 , respectively.
2.3 Screening of Cellulolytic Bacteria
Each bacterial isolates were screened for cellulolytic activity using CMCase plate
assay (Muhammad et al. 2012). 10 µL of each bacterial broth was inoculated inside
4 mm pore made on the CMC agar plate. The CMC agar plates containing bacteria
inoculums were then incubated at 37 °C for 72 h. After incubation, CMC agar plates
were stained by 0.1% Congo Red solution for 1 h followed by distain using 1 N
NaCl solution for the determination of cellulose hydrolyzing zone surrounding the
bacterial growth on CMC agar plate.
2.4 Quantitative Assay of Bacterial Cellulase Activity
Quantitative assay of cellulase activity was estimated using DNS method (Miller
1959) by measuring the amount of reducing sugar hydrolyzed by individual bacterial isolates. Each selected bacterial strain was cultured in nutrient broth medium for
overnight incubation at 37 °C. Incubated bacterial broths were individually transferred to sterilized 1-mL Eppendorf tube. Each Eppendorf tube was centrifuged at
10,000 rpm for 10 min at 4 °C. Substrate solution was prepared by adding 1% of CMC
with phosphate buffer (0.1 M) at pH 6.8. 0.5 mL of substrate solution and 0.5 mL
of each bacterial supernatant (crude enzyme) were taken in assay tube to form 1 mL
reaction mixture. Assay tubes containing the reaction mixture were then incubated
at 55 °C for 15 min in water bath. After the incubation of reaction mixture, 1.5 mL of
DNS solution was added in each assay tube and boiled at 100 °C for 10 min in water
bath to stop the reaction. Amount of reducing sugar released in the hydrolysis by