## 2 Materials and Methods
### 2.2. _Rhamolipid Production of the Isolate Under Laboratory Conditions_  
#### 2.2.1 Optimization of Rhamolipid Production  
A central composite design (CCD) was applied to optimize the rhamnoid production of the selected bacterial strain ([PERSON] and [PERSON], 1951). The experiment was carried out in 500-mL Erlenmeyer flasks containing 200 mL of mineral salt medium (MSM), amended with crude oil as a sole carbon source. Thirty treatments factors (runs) with variable substrate concentrations, pH, temperature, and inoculation loads were the factors used in the experiment (Table S1). The coded values were-\(\alpha\),-1,0,1,+,\(\alpha\), with the corresponding values of parameters: for A (crude oil concentration) 0, 1, 2, 3, and 4%; B (inoculation load) 35 \(\times\) 10\({}^{2}\), 10 \(\times\) 10\({}^{4}\), 29 \(\times\) 10\({}^{5}\), 16 \(\times\) 10\({}^{7}\), and 33 \(\times\) 10\({}^{8}\) CFU mL-1; C (pH) 3.5, 5.0, 6.5, 8.0, and 9.5; D (temperature) 12.5, 20.0, 27.5, 35, and 42.5 \({}^{\circ}\)C. The agitation was maintained at 150 rpm, 120 h, during the experiment. Rhamolipid production was quantified as rhamne equivalents (RE) using the phenol-sulfuric acid method with a standard curve prepared using rhamnose [27]. The critical micelle concentration (CMC) of the biosurfactant was determined based on a plot of surface tension vs. surfactant concentration (expressed mg mL-1) [28]. The results of the experiments were analyzed using Design-Expert 9.0 software (Stat-Ease, Inc., Minneapolis, MN, USA), and the following second-order polynomial regression model equation was obtained:  
\[ \begin{split}\text{Y}=\beta_{\text{o}}+\beta_{1}\text{A}+\beta_{2 }\text{B}+\beta_{3}\text{C}+\beta_{4}\text{D}+\beta_{11}\text{A}+\beta_{22} \text{B}^{2}+\beta_{33}\text{C}^{2+}\beta_{44}\text{D}^{2+}\beta_{12}\text{ AB}+\beta_{13}\text{AC}\\ +\beta_{14}\text{AD}+\beta_{23}\text{BC}+\beta_{24}\text{BD}+\beta_ {34}\text{CD}\end{split}  \tag{3}\]  
where Y represents the predicted response (rhamolipid production in RE), b0 is an intercept, A is the crude oil concentration (%), B is the inoculation load (CFU mL-1), C is pH, and D is the temperature (\({}^{\circ}\)C). \(\beta\)1, \(\beta\)2, \(\beta\)3, and \(\beta\)4 are the linear coefficients; \(\beta\)11, \(\beta\)22, \(\beta\)33, and \(\beta\)44 are the squared coefficients; \(\beta\)12, \(\beta\)13, \(\beta\)14, \(\beta\)23, \(\beta\)24, \(\beta\)34 are the interaction coefficients; A\({}^{2}\), B\({}^{2}\), C\({}^{2}\), D\({}  
was extracted with chloroform-methanol (3:1, v:v). Finally, the precipitate was decanted, and the solvent was evaporated. Thin-layer chromatography (TLC) was used for the identification of the biosurfactants, according to the protocols of [PERSON] et al. [30]. Silica gel plates were prepared by adding 30 g of silica-G (200-245 mesh) to 60 mL distilled water followed by mixing. Microscopic slides were coated by the slurry and air-dried. Ten microliters of the purified biosurfactant fraction were applied at the point of origin (near the bottom) of each preparative silica gel plate. The silica gel plates containing biosurfactant were eluted using a solution mix of chloroform-methanol-acetic acid (65:15:2, v/v/v) and visualized using iodine vapors. The retention factor (\(R_{f}\)) value of BS was calculated according to the following formula:  
\[R_{f}=\frac{\text{Distance travelled by the substance}}{\text{Distance travelled by the solvent}} \tag{4}\]  
For partial rhamnpid purification, rhamnoids specifying TLC spots were dissolved in 1 mL methanol and centrifuged at 15,000\(\times\)\(g\) for 3 min at 4 \({}^{\circ}\)C to remove the silica gel. The supernatant was collected, air-dried for 24 h, followed by dissolving the dried fraction in 25 \(\upmu\)L methanol. Finally, the rhamnoids were separated by centrifugation at 1000\(\times\)\(g\) for 10 min and air-drying. The dried fraction was further purified on a silica gel (60-120 mesh) column eluted with a gradient of chloroform and methanol, as described by [PERSON] et al. [31].