Isolation and selection of thermophilic cellulase-producing bacteria
A total of ten bacterial isolates were obtained on CMC-containing enrichment media plates from Tapovan soil samples. Two isolates (TP-1 and TP-3) showed comparatively higher cellulase activity based on the zone of hydrolysis (diameter in mm) on agar plates containing 1% Carboxyl Methyl Cellulose (CMC) medium. Of these two isolates, TP-3 was selected for further analysis due to the maximum zone of clearance during the plate diffusion assay (Figure 2A). Therefore, TP-3 was selected for further optimizing the cellulase production. The isolate exhibited optimum growth at 50 °C; hence, it is considered a thermophilic bacterium.

Morphological and growth characteristics
Bacterial colonies were grown on the nutrient agar plate and subjected to phenotypic characterization. It had a spherical colony, off-white, transparent, and slimy appearance with flat, slightly undulating margins (Figure 2B). Gram staining identified it as a Gram-positive bacterium. A compound light microscope at 100X magnification revealed long rod-shaped bacteria (Figure 2C), and SEM corroborated similar findings (Figure 2D). Gram-positive bacterial cells with terminal and sub-terminal endospores were observed. The TP-3 strain exhibited enzyme activity of 0.692 µmoles/min/mL using the DNSA assay of glucose estimation.

Identification of TP-3 cellulose-degrading bacterial strain
A ~1.5 kb fragment of the 16S rRNA gene was successfully amplified and sequenced to identify the cellulolytic bacterial isolate (Figure 3A). Phylogenetic analysis based on the sequence data confirmed that the isolate, designated as TP-3, belongs to the genus Geobacillus. The 16S rRNA gene of TP-3 exhibited the highest sequence similarity with multiple Geobacillus stearothermophilus strains, including G. stearothermophilus YE-6 (98.53%), G. subterraneus E-55 I (98.03%), G. stearothermophilus D6a (98.02%), Geobacillus sp. TC-S8 (97.64%), Geobacillus sp. G1 (97.57%), G. kaustophilus HTA426 (97.44%), and G. thermoleovorans SURF-48B (97.37%). The 16S rRNA gene sequence has been deposited in GenBank under accession number OP962434 (Figure 3B).

Using the Neighbor-Joining (NJ) method with the T-Coffee multiple sequence alignment tool, phylogenetic analysis revealed that TP-3 shares the closest evolutionary relationship with Geobacillus sp. H6a (Figure 3C). These findings suggest that TP-3 is a novel strain within the Geobacillus genus, exhibiting significant cellulolytic potential. Its close phylogenetic relationship with G. stearothermophilus and other thermophilic species underscores its adaptation to high-temperature environments, making it a promising candidate for industrial applications involving lignocellulose degradation.

Selection of media for cellulase production and OFAT analysis
The maximum cellulase production was observed in a medium reported by Sakthivel et al.²⁶ of the six different media. The selected medium was optimised using a One-Factor-at-a-Time (OFAT) approach to enhance cellulase production by Geobacillus stearothermophilus. This method systematically assessed the effect of individual parameters, with results presented in Figure 3.

Screening of various carbon sources, including glucose, carboxymethyl cellulose (CMC), xylose, and maltose, identified glucose and CMC as the most effective combination for maximizing cellulase activity (Figure 4A). Similarly, among the nitrogen sources tested, yeast extract yielded the highest cellulase activity (Figure 4B).

The effect of pH on enzyme production was evaluated by adjusting the pH of the production medium (M5) from 4 to 12. Optimal growth was observed within the pH range of 7-9, with peak cellulase production occurring at pH 8 (Figure 4C). Temperature optimization, conducted over a range of 20 °C to 60 °C, revealed that maximum cellulase production occurred at 50 °C (Figure 4D).

The impact of inoculum size was also assessed, with variations tested to determine the ideal concentration. A 6% inoculum size resulted in the highest cellulase activity (Figure 4E). Additionally, cellulase production was monitored at different incubation times (0-60 hours), showing a steady increase up to 24 hours, followed by a gradual decline (Figure 4F). Following OFAT optimization, enzyme activity significantly improved, increasing approximately 1.5-fold from 0.692 µmol/min/mL to 1.06 µmol/min/mL. This enhancement highlights the effectiveness of systematic parameter optimization in boosting cellulase production for potential industrial applications.

Plackett-Burman design for statistical optimization of cellulase production
Seven factors were examined to identify the crucial variables appropriate for cellulase production. The model produced a design for 12 trials (Table 2), where each column corresponds to a variable and each row to an experiment. Geobacillus sp. TP-3 produced cellulase in varying amounts, and these differences are shown in Table 2, where they highlight the significance of factor optimization. The eighth experimental run had the highest cellulase activity (0.925 µmoles/min/mL), whereas the seventh run showed the lowest (0.051 µmoles/min/mL). The data in Table 2 were subjected to multiple linear regression analysis based on the PBD to estimate the F-value and p-value of each component. The first-order linear model determines how independent variables affect cellulase production, and it provides these results in equations 2 (coded factors) and 3. (actual factors).

Table (2):
Plackett-Burman experimental trial table with cellulase activity response

Independent variables: High level +1 and low level -1. The mean of triplicate culture trials is used to calculate response values

Final Equation in Terms of Coded Factors

Y (cellulase activity) = +102.23*A-119.99*B-147.79*C-87.35*D+94.79*E-65.97*H+67.73*J-128.17*K

…(2)

Final Equation in Terms of Actual Factors
Y (cellulase activity) = 1022.29171* Glucose-479.95132*CMC-1477.88043*Yeast Extract-87.35206*pH+9.47902*Temperature-65.96677*dummy1+67.73334*dummy2-128.16932*dummy3

…(3)

Y = b₀ + Σb_i + x_i;

where Y represents the response of the cellulase enzyme activity, b₀ the model intercept, b_i the linear variable coefficient, i the variable number, and x_i the level independent variable. Design Expert 6.1.10 software.

The factors affecting the generation of cellulase by Geobacillus stearothermophilus TP-3 were examined using an analysis of variance (ANOVA), and the findings were presented in Table 3. The p-value of 0.0371 in the statistical design made it clear that the model is significant. Three factors (glucose, CMC, and yeast extract) were determined by p–value analysis to impact cellulase production significantly. However, the yeast extract was the most important factor, with a p–value of 0.0187. A p–value greater than 0.05 indicates that the studied component was not statistically significant; however, it did not play a specific function in enzyme synthesis. The F-value of 10.98 for the model suggests that it is significant. A “Model F-value” this large might happen due to noise with a mere 3.71% probability. Because the “Pred R-Squared” of 0.4717 is not as close to the “Adj R-Squared” of 0.8789, the coefficient (R²) revealed that the model has a block effect. By the criterion known as “Adeq Precision,” a measure of signal-to-noise ratio that can be used to explore the design space, our model’s ratio value of 9.885 shows that it has a satisfactory signal-to-noise ratio.

Table (3):
Plackett-Burman design ANOVA analysis using the partial sum of squares for Geobacillus sp. TP-3 cellulase production

Std Dev 110.05, mean 311.05, C.V. 35.38, Press 5.813E+005; R squared 0.9670; Adj R-Squared 0.8789; Pred R squared 0.4717; Adeq Precisior 9.885 [*p-value < 0.01 highly significant; 0.01 < P < 0.05 significant]

Low-cost wastes as the substrate for cellulase production
Various agricultural waste products were evaluated as carbon sources to assess their impact on cellulase enzyme induction over five days. Among the tested substrates, cane sugar molasses supported the highest cellulase production (~9.60 µmol/min/mL), reaching its peak on the third day before experiencing a slight decline on the fourth day. In contrast, wheat bran yielded approximately 2.4 times lower cellulase production, with maximum enzyme activity (~4 µmol/min/mL) observed on the fourth day, followed by a significant decline on the fifth.

Untreated rice bagasse, a more complex lignocellulosic substrate, exhibited initial resistance to degradation. However, cellulase activity gradually increased, reaching its peak (~0.37 µmol/min/mL) on the fifth day, suggesting a slower enzymatic response likely due to its recalcitrant structure. Similarly, corn cob demonstrated minimal cellulase production (~0.19 µmol/min/mL) until the fourth day, with enzyme activity dropping to half its initial value by the fifth day (Figure 5).

These findings indicate that readily available and less structurally complex carbon sources, such as cane sugar molasses and wheat bran, facilitate higher cellulase production in shorter timeframes. In contrast, complex lignocellulosic materials like rice bagasse and corn cob require extended incubation periods for enzyme induction, highlighting the necessity of pretreatment strategies for efficient bioconversion. This study underscores the potential of agro-waste valorization for sustainable cellulase production, particularly from substrates with high fermentable sugar content.

Characterization of cellulase enzyme
The cellulase activity of Geobacillus stearothermophilus TP-3 was measured at different pH (5-9) and temperatures (40-70 °C). Optimum cellulase activity was observed at pH 8.0 and 50 °C (Figure 6A, 6B). Moreover, the cellulase enzyme was active at 40-70 °C. Up to 15 minutes, a little increase in cellulase activity was seen at 50 °C and 60 °C. The cellulase enzyme retained ~68% of its original activity at different temperature ranges, 40-70 °C after 3 hours of incubation (Figure 6C). From these results, the cellulase seems to have considerably high thermostability at higher temperatures for a long incubation period.