Isolation of Cellulose-Degrading Bacteria and to Use Their …
5
(28.7884° N, 77.0301° E). After collection, the soil samples were stored at 4 °C in
sterile containers.
2.2 Screening of Cellulose-Degrading Bacteria
For screening of cellulose-degrading bacteria (CDB), around 2 g of soil sample was
homogenized in 0.9% saline solution and serially diluted ranging from 10
−1 to 10
−3
under sterile condition. Around 100 µl of each serially diluted sample was spread
plate on carboxymethyl cellulose (CMC) agar media composed of KH 2 PO 4 , 0.5 g;
MgSO 4 , 0.25 g; cellulose, 2 g; agar, 15 g; gelatin, 2 g; distilled water, 1 L; pH, 6.8–
7.2. The plates were incubated at 37 °C for 24 h. Colonies obtained were maintained
on CMC agar plate and preserved at 4 °C.
2.3 Cellulolytic Ability Assay
Cellulose-degrading ability was analyzed by streaking the isolated bacterial strains on
Congo Red agar media composed of CMC agar media supplemented with Congo Red
0.2 g/l. The formation of a clear zone of hydrolysis indicated cellulose degradation
ability of the isolated bacterial strain. The colony showing maximum degradation
was selected and used for further study.
2.4 Estimation of Carboxymethyl Cellulase (CMCase)
Activity
Using the 3, 5-dinitrosalicylic acid (DNS) method, CMCase activity was determined
(Miller 1959). Supernatant obtained from overnight-grown culture after centrifugation at 5000 rpm for 10 min at 4 °C was used as crude enzyme source. Assay was
carried out by adding around 0.5 ml of crude enzyme to 0.5 ml of 1% CMC (solubilized in 0.05 M phosphate buffer, pH 8) followed by incubation at 50 °C in water
bath for 30 min. The reaction was stopped by the addition of 1.5 ml of DNS reagent
followed by boiling the reaction mixture for 10 min. Sugars liberated were estimated
by measuring absorbance at 540 nm. A unit of activity is defined as the amount of
enzyme required to liberate 1 mol of glucose per minute under the assay conditions.
2.5 Molecular Identification and Phylogenetic Analysis
The bacterial strain that showed maximum cellulose activity was used for molecular
characterization using 16S rRNA sequencing. The isolated bacterial strain streaked
5
(28.7884° N, 77.0301° E). After collection, the soil samples were stored at 4 °C in
sterile containers.
2.2 Screening of Cellulose-Degrading Bacteria
For screening of cellulose-degrading bacteria (CDB), around 2 g of soil sample was
homogenized in 0.9% saline solution and serially diluted ranging from 10
−1 to 10
−3
under sterile condition. Around 100 µl of each serially diluted sample was spread
plate on carboxymethyl cellulose (CMC) agar media composed of KH 2 PO 4 , 0.5 g;
MgSO 4 , 0.25 g; cellulose, 2 g; agar, 15 g; gelatin, 2 g; distilled water, 1 L; pH, 6.8–
7.2. The plates were incubated at 37 °C for 24 h. Colonies obtained were maintained
on CMC agar plate and preserved at 4 °C.
2.3 Cellulolytic Ability Assay
Cellulose-degrading ability was analyzed by streaking the isolated bacterial strains on
Congo Red agar media composed of CMC agar media supplemented with Congo Red
0.2 g/l. The formation of a clear zone of hydrolysis indicated cellulose degradation
ability of the isolated bacterial strain. The colony showing maximum degradation
was selected and used for further study.
2.4 Estimation of Carboxymethyl Cellulase (CMCase)
Activity
Using the 3, 5-dinitrosalicylic acid (DNS) method, CMCase activity was determined
(Miller 1959). Supernatant obtained from overnight-grown culture after centrifugation at 5000 rpm for 10 min at 4 °C was used as crude enzyme source. Assay was
carried out by adding around 0.5 ml of crude enzyme to 0.5 ml of 1% CMC (solubilized in 0.05 M phosphate buffer, pH 8) followed by incubation at 50 °C in water
bath for 30 min. The reaction was stopped by the addition of 1.5 ml of DNS reagent
followed by boiling the reaction mixture for 10 min. Sugars liberated were estimated
by measuring absorbance at 540 nm. A unit of activity is defined as the amount of
enzyme required to liberate 1 mol of glucose per minute under the assay conditions.
2.5 Molecular Identification and Phylogenetic Analysis
The bacterial strain that showed maximum cellulose activity was used for molecular
characterization using 16S rRNA sequencing. The isolated bacterial strain streaked