Isolation, Screening, and Evaluation of Cellulase-Producing …
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sterilization, the flasks were set to cool in room temperature and placed under laminar
flow keeping the UV light on. After few minutes, each flask was inoculated with 1%
standard inoculum (V /V ) of the SCB9 cellulolytic bacterial strain. The inoculated
flasks were then incubated on a rotary shaker (120 rpm) at specific temperatures (30,
37.5, and 45 °C) and for specific time (12, 42, and 72 h) as given in the experimental
design from the RSM calculation.
2.5.3 Quantitative Estimation of Cellulase Production
After incubation, according to the design experimental conditions, 1 mL of bacterial
from each flask was taken in the Eppendorf tube. In this study, cellulase activity
was measured in triplicate by preparing three Eppendorf tubes containing bacterial
cultures for each experimental condition. Each Eppendorf tube containing 1 mL
bacterial culture broth was centrifuged at 10,000 rpm for 10 min at 4 °C. After
centrifugation, the bacterial supplements were collected to perform the DNS assay
for the estimation of cellulase activity (Miller 1959). The predicted value of cellulase
activity obtained from each experimental condition was then used to generate threedimensional contour plots, regression analysis, and analysis of variance (ANOVA).
2.5.4 Statistical Analysis and Analysis of Variance (ANOVA)
Design Expert 12 (Stat Ease, Inc., Minneapolis, USA) was used for regression analysis of the experimental data to obtain working parameters and to generate polynomials and the surface contour plots. A second-order polynomial equation was
established based on the analysis of variance, and the optimum ratio of experimental
variable was found using the same software. Standard deviation, p value, F value,
and R
2 value were also analyzed.
2.5.5 Molecular Identification and Phylogeny Analysis
The most potent cellulolytic bacterial strain was identified using 16S rRNA
sequencing. The partial contig sequence obtained was further analyzed through
phylogeny tree in MEGA 7 (Kumar et al. 2016).
2.5.6 Model Validation
The mathematical generated during RSM implementation was validated by
conducting checkpoint studies. Experimentally obtained data were compared with
the predicted one and the prediction error was calculated.
461
sterilization, the flasks were set to cool in room temperature and placed under laminar
flow keeping the UV light on. After few minutes, each flask was inoculated with 1%
standard inoculum (V /V ) of the SCB9 cellulolytic bacterial strain. The inoculated
flasks were then incubated on a rotary shaker (120 rpm) at specific temperatures (30,
37.5, and 45 °C) and for specific time (12, 42, and 72 h) as given in the experimental
design from the RSM calculation.
2.5.3 Quantitative Estimation of Cellulase Production
After incubation, according to the design experimental conditions, 1 mL of bacterial
from each flask was taken in the Eppendorf tube. In this study, cellulase activity
was measured in triplicate by preparing three Eppendorf tubes containing bacterial
cultures for each experimental condition. Each Eppendorf tube containing 1 mL
bacterial culture broth was centrifuged at 10,000 rpm for 10 min at 4 °C. After
centrifugation, the bacterial supplements were collected to perform the DNS assay
for the estimation of cellulase activity (Miller 1959). The predicted value of cellulase
activity obtained from each experimental condition was then used to generate threedimensional contour plots, regression analysis, and analysis of variance (ANOVA).
2.5.4 Statistical Analysis and Analysis of Variance (ANOVA)
Design Expert 12 (Stat Ease, Inc., Minneapolis, USA) was used for regression analysis of the experimental data to obtain working parameters and to generate polynomials and the surface contour plots. A second-order polynomial equation was
established based on the analysis of variance, and the optimum ratio of experimental
variable was found using the same software. Standard deviation, p value, F value,
and R
2 value were also analyzed.
2.5.5 Molecular Identification and Phylogeny Analysis
The most potent cellulolytic bacterial strain was identified using 16S rRNA
sequencing. The partial contig sequence obtained was further analyzed through
phylogeny tree in MEGA 7 (Kumar et al. 2016).
2.5.6 Model Validation
The mathematical generated during RSM implementation was validated by
conducting checkpoint studies. Experimentally obtained data were compared with
the predicted one and the prediction error was calculated.