## 3 Results and Discussion
### The Characteristics of Isolated Rhamolipid-Producing Bacterial Strain
#### 3.1.2 Production of Biosurfactant by the Isolate under Optimized Conditions  
In the present study, four parameters, namely, oil concentration, inoculation load, temperature, and pH, were evaluated for their influence on rhamn production by the isolated bacterial strain. Second-order polynomial equation fitted using multiple regression analysis was applied for the optimization of rhamnoid production. A second-order response model obtained after regression analysis for rhamnoid production was described by the following equation:  
\[\begin{split}\text{R1}&=+2.02+0.52\times\text{A}+0.24 \text{ }^{*}\text{B}+0.054\text{ }^{*}\text{C}+0.021\text{ }^{*}\text{D}-0.17\text{ }^{*}\text{AB}+0.019\text{ }^{*}\text{ AC}+0.019\text{ }^{*}\text{ AD}\\ &-0.031\text{ }^{*}\text{BC}-6.25\text{ }^{*}\text{BD}+0.031\text{ }^{*}\text{CD}-0.18\text{ }^{*}\text{A}^{2}-0.028\text{ }^{*}\text{B}^{2}-0.12\text{ }^{*}\text{C}^{2}-0.12\text{ }^{*}\text{D}^{2}\end{split} \tag{5}\]  
where A is the oil concentration, B the inoculation load, C the temperature, and D the soil pH. The parameters of the experimental Central Composite Design (CCD) runs, and the corresponding results are provided in Supplementary Table S1.  
The predicted R\({}^{2}\) value (0.827) was in reasonable agreement with the adjusted R\({}^{2}\) value (0.935) since the difference was less than 0.2 (Table 1). Based on the results obtained from the experiment designed according to the CCD model, the optimum concentration of crude oil was 2.9%, the inoculation load was \(54\times 10^{6}\) CFU mL\({}^{-1}\), the temperature was 30.5 \({}^{\circ}\)C, and the pH value was 6.7. The results of the experiment showed that an increase in crude oil concentration and inoculation load increased rhamnoid production at a fixed pH and temperature (6.5 and 27.5 \({}^{\circ}\)C, Figure 2a). The predicted and experimental values for rhamnoid production were close to a straight line (Figure 2b).  
Figure 1: Phylogenetic tree showing the position of _Shewanella_ spp. BS4 and its closest neighbors with a bootstrap value of 1000.  
When bacterial growth and rhamnpid production were studied in CCD-optimized growth medium, the highest bacterial abundance, \(58\times 10^{6}\) CFU mL\({}^{-1}\), was attained after 96 h of incubation, and the rhamnpid production was observed to be 2.5 mg cell dry weight (CDW) (Figure 2c). The CMC value of the biosurfactant was 27.4 (Figure 2d).  
Several studies have utilized CCD for the successful optimization of rhamnpid production in the case of _P. aeruginosa_ strains, such as AT10 [46], ATCC9027 [46], and _P. aeruginosa_ SG [47], but no study on rhamnpid production using _Shewanella_ species has been carried out previously. We adopted the response surface methodology for maximization of rhamnpid production using the optimization of operational factors [48]. [PERSON] [49] reported that _Shewanella putrefaciens_ strain LH4:18 could grow on crude oil under anaerobic conditions without the addition of any other carbon source. However, _Shewanella_ species are shown to be facultative aerobes using either oxygen under aerobic conditions or oxidized metals under anaerobic conditions as the terminal electron acceptor [50].