## 3 Results and Discussion  
### Determination of Process Parameters for Optimal Exoperoxidase Production  
The utility of peroxidase in different industrial sectors has led to an increased demand, which in the near future may not be met by horseradish peroxidase (HRP), the major commercially available peroxidase. Hence, the need for novel sources of peroxidase, particularly bacteria because they are more efficient, they grow faster, and can easily be modified genetically [14]. This study optimized extracellular peroxidase production by _Ensifer adherens_ NWODO-2 through determination of the process parameters (medium pH, incubation temperature and agitation speed), which are significant for optimal growth and metabolic activities of an organism and amending the composition of the production medium. Bacterial growth has been linked to constant production of extracellular enzymes [12; 31; 32]. Hence, factors capable of influencing bacteria growth such as pH, temperature and agitation as well as the composition of the medium will invariably play an important part in enzyme secretion by the bacteria. The pH of the cultivation environment significantly influences the electric charge of the microbial cell, consequently affecting the absorption of nutrients and intracellular enzymatic activities [33; 34]. The effect of initial medium pH on exoperoxidase production by _Ensifer adherens_ NWODO-2 is presented in Figure 1a. The results showed that peroxidase production by the test strain, _Ensifer adherens_ NWODO-2 within pH 5-9 was significantly different (F = 123.7, \(p\) value = \(p<0.0001\) for ANOVA). According to [PERSON]'s test, however, peroxidase production at pH 5 (2.92 \(\pm\) 0.14 U mL\({}^{-1}\)) and pH 8 (3.03 \(\pm\) 0.32 U mL\({}^{-1}\)) was not significantly different. In this study, maximum peroxidase production was observed at an initial pH 7 (5.83 \(\pm\) 0.00 U mL\({}^{-1}\)); peroxidase activity was not observed at pH 3, 4, 10 and 11 (Figure 1a). This finding agrees with the works of [PERSON] et al. [35] and [PERSON] et al. [36], in which maximum peroxidase production by _Streptomyces avermitilis_ UAH30 and _Streptomyces_ sp. K37 was achieved at pH 7 and 7.5, respectively. However, [PERSON] et al. [12] observedoptimum peroxidase production by _Streptomyces_ sp. BSII#1 at pH 8 while we recorded pH 5 as the optimal pH for peroxidase production by _Raoultella ornithinolytica_ OKOH-1 in a recent study [25]. It is worthy of note that the characteristic production of peroxidase by the test bacteria at a neutral pH augurs well for biotechnological applications as the large volume of acid and base required for pH adjustment would be saved [37], consequently, reducing the cost of peroxidase production. The effect of incubation temperature on exoperoxidase production by _Ensifer adherens_ NWODO-2 is shown in Figure 1b. The results revealed that peroxidase production differs significantly (F = 63.44, \(p\) value = \(p<\) 0.0001 for ANOVA) across the investigated temperatures: 20-45 \({}^{\circ}\)C, with optimum production recorded at 30 \({}^{\circ}\)C (5.83 \(\pm\) 0.00 U mL\({}^{-1}\)). Nonetheless, [PERSON]'s multiple comparison test showed there was no significant difference in the enzyme production at 20 \({}^{\circ}\)C (1.05 \(\pm\) 0.00 U mL\({}^{-1}\)), 35 \({}^{\circ}\)C (0.58 \(\pm\) 0.00 U mL\({}^{-1}\)), 40 \({}^{\circ}\)C (1.52 \(\pm\) 0.59 U mL\({}^{-1}\)) and 45 \({}^{\circ}\)C (0.94 \(\pm\) 0.12 U mL\({}^{-1}\)). Although there is no previous report on optimal temperature for exoperoxidase production by _Ensifer_ species, our finding agrees with the result obtained by [PERSON] et al. [38], where optimal peroxidase production by a _Bacillus_ sp. was also observed at 30 \({}^{\circ}\)C. Nevertheless, [PERSON] and [PERSON] [39] and [PERSON] et al. [12] recorded 37 \({}^{\circ}\)C as the temperature for optimum production of peroxidase by _Bacillus subtilis_ and _Streptomyces_ sp. BSII#1, respectively, whereas [PERSON] et al.