3.5.1. Inoculum size Enhancing the cellulolytic potential of Bacillus sp. RL-07 involved optimizing various factors, among which inoculum size played a crucial role in increasing enzyme activity. Insufficient inoculum size can lead to delayed microbial growth, consequently prolonging the overall time required for enzyme production. The maximum cellulase activity, 4.94 ± 0.11 U/ml, was observed at an inoculum size of 5% (v/v), while the lowest cellulase activity, 2.68 ± 0.07 U/ml, was recorded at an inoculum size of 1% (v/v), as depicted in Fig. 2a. Bacillus sp. RL-07 exhibited an increase in cellulase activity up to an inoculum size of 5% (v/v), followed by a decline in activity. However, Behl et al. (2023) utilized a 3% inoculum size of 24-hr-grown seed culture for the production of cellulase enzyme from Bacillus sp. PHS-05.
3.5.2. Carbon source and its concentration The selection of a carbon source in a growth medium plays a crucial role in influencing the growth, metabolism, and behavior of microorganisms, as it serves as their primary energy source. In this study, glucose, lactose, xylose, maltose, and sucrose were investigated as carbon sources. Among these, glucose exhibited the highest activity, with a value of 5.21 ± 0.13 U/ml, while lactose showed the lowest activity at 3.04 ± 0.06 U/ml, as illustrated in Fig. 2b. Similar results were reported by Shanmugapriya (2012) in the case of cellulase activity with CMC as a substrate. When the substrate concentration exceeds the required level, enzyme units may become saturated and fail to function efficiently. The maximum cellulase activity was achieved at a glucose concentration of 1.5% (w/v), reaching 5.69 ± 0.09 U/ml, whereas the lowest activity (3.75 ± 0.14 U/ml) was observed at a glucose concentration of 0.5% (w/v), as depicted in Fig. 2c. A sharp decline in activity was noted after a certain increase in concentration, which could be attributed to elevated viscosity leading to reduced nutrient circulation and disrupted oxygen transfer (Saini et al., 2023).
3.5.3 Nitrogen sources and its concentration
Nitrogen plays a vital role in the physiology and metabolism of enzyme production in bacteria. Different nitrogen sources can significantly influence enzyme production rates. Beef extract, as the primary nitrogen source, exhibited a positive impact on cellulase activity, reaching 5.94 ± 0.09 U/ml. Conversely, the lowest cellulase activity of 3.48 ± 0.11 U/ml was observed with ammonium nitrate, as depicted in Fig. 2d. A study by Iram (2012) reported a similar trend in cellulase activity during the optimization of various nitrogen sources for cellulase production by A. niger. Beef extract as a nitrogen source demonstrated maximum cellulase activity of 6.08 ± 0.06 U/ml at a concentration of 1.6% (w/v). Conversely, the lowest cellulase activity was recorded at 0.4% (w/v), yielding 2.91 ± 0.24 U/ml, as illustrated in Fig. 2e. At lower nitrogen source concentrations, enzyme activity remained low as the media failed to meet microbial demands for vitamins and minerals. However, beyond a optimum concentration, activity levels began to decline, possibly due to reduced nutrient consumption and accumulation, resulting in a constrained environment for microbial growth and the production of essential enzymes, including cellulase (Behl et al., 2023).
3.5.4 Optimization of temperature, pH and incubation time These various factors play a crucial role in the growth, production, and activity of enzymes by stabilizing the protein structure and accelerating kinetic energy. Cellulase exhibited maximum activity at 40°C, reaching 6.25 ± 0.15 U/ml. Initially, as temperature increased, the rate of reaction also rose due to increased kinetic energy. However, beyond optimum temperature, both activity and reaction rate began to decline, likely due to bond breakage and changes in enzyme structure. The lowest cellulase activity was recorded at 50°C, with a value of 2.83 ± 0.09 U/ml (Fig. 2f). This finding aligns with a study by Pramanik et al. (2021), which reported maximum cellulase activity at 40°C for B. pseudomycoides.
Enzyme activity is highly dependent on pH, as changes in hydrogen ion concentration can affect intra- and intermolecular bonds, altering the enzyme's shape and catalytic site effectiveness. In our study, maximum cellulase activity of 6.45 ± 0.16 U/ml was observed at pH 7.0. However, activity declined beyond the optimum pH, with the lowest cellulase activity recorded at pH 11.0, reaching 3.05 ± 0.08 U/ml (Fig. 2g). Acharya and Chaudhary (2012) reported similar findings for Bacillus licheniformis MVS1 and Bacillus sp. MVS3, with maximum cellulase activity observed at pH 6.5 and 7.0, respectively.
Incubation time also significantly influences enzyme production, with protein denaturation occurring with prolonged incubation, leading to loss of catalytic activity over time. In our study, maximum cellulase activity of 6.98 ± 0.36 U/ml was achieved after 36 hr, whereas the lowest activity was observed after 12 hr incubation with a value of 2.80 ± 0.31 U/ml (Fig. 2h). This underscores the importance of optimizing incubation time to maximize enzyme productivity while mitigating the risk of denaturation and reduced activity over time (Nargorta et al., 2016).