## 2 Materials and Methods
### Design of Response Surface Experiment and Statistical Analyses for Bioscreen Study  
After an initial assessment of the factors and media design, carbohydrate concentration (0-3% \(w/v\)), norepinephrine (0-150 uM), dopamine (0-150 uM), and Fe (0-0.03%) were selected as growth enhancement factors in the previously defined MSM. This MSM contained only a minimal amount (0.1 g/L) of protease peptone as a protein source. No other complex nutritional factors such as yeast extract or other undefined components were present in the media, as minimum interference of extraneous protein components was intended to observe the combination effects of the above-mentioned growth enhancement factors. Central composite design (CCD) is a commonly used response surface methodology to optimize independent variables concerning the measured response. Typically, CCD design consists of 2P factorial runs with 2p axial runs and P\({}_{\text{c}}\) center runs to optimize process parameters, where p is the number of factors or independent variables. In this study, four independent variables (\(p\) = 4), carbohydrate concentration for glycerol/glucose/SHH/MSH (X\({}_{1}\)), Fe concentration (X\({}_{2}\)), norepinephrine (X\({}_{3}\)) concentration, and dopamine (X\({}_{4}\)) concentration, were optimized, with growth rate as the response. Sixteen (24 = 16) factorial points, eight (2 \(\times\) 4 = 8) axial points, and three replicates at the center points resulted in 27 experimental conditions. \"Low\" and \"High\" levels of the independent variables were coded as \(-\)1 and +1 and the axial points were located at (\(\pm\alpha\), 0, 0), (0, \(\pm\alpha\), 0) and (0, 0, \(\pm\alpha\)), where \(\alpha\) is the distance of the axial point, which was fixed at 1. The center points were used to determine the experimental error and the reproducibility of the data. The experimental sequence was randomized to minimize the effects of uncontrolled errors. The cell growth rates (log numbers in OD h\({}^{-1}\) at 600 nm) for the fermentations were calculated by determining the slope of the logarithmic conversion of the optical density for each growth curve between 0 and 10 h (the log-phase) and were used as a response variable. The optical density (OD) was not used as a response variable due to the lack of dilution capability in the Bioscreen unit, as any OD value above 1.0 would not be an accurate numerical value; hence, the growth rate during the log phase (0-10 h) was selected as the response parameter. The effect of four independent variables was used to develop a second-degree polynomial model (Equation (1)) for the growth rate response variable (Y\({}_{1}\)). \[\hat{Y}=\beta_{0}+\sum_{i}^{n}\beta_{i}x_{i}+\sum_{ii}^{n}\beta_{ii}\chi_{i}^{2 }+\sum_{i=1}^{n-1}\sum_{j=i+1}^{n}\beta_{ij}x_{i}x_{j} \tag{1}\]  
where \(\hat{Y}\) is the predicted response, \(\beta_{0}\) the constant coefficient, \(\beta_{i}\) the linear coefficients, \(\beta_{ij}\) the interaction coefficients, \(\beta_{ii}\) the quadratic coefficients, and \(x_{i},\ x_{j}\) are the assigned independent variables. The design matrix generation and model fitting were performed using JMP 15 (SAS Institute, Inc., Cary, NC, USA). Experimental Design and Determination of Rlamnolipid Concentration in 100 mL Shake-Flask Experiments  
The seed culture for _Pseudomonas aeruginosa_ PAO1 for the 100 mL shake-flask study was also grown in LB media at 30 \({}^{\circ}\)C for 24 h with 150 rpm shake speed in a shaking incubator. A 0.1% \(v/v\) inoculation percentage was used for this study as well. Optimized media combinations from the Bioscreen study were grown for each sugar type (glucose, glycerol, SHH, and a mimic soy hull hydrolysate mix produced from pure sugars, MSH) with two variants: (a) mineral-salt-optimized media and (b) mineral-salt-optimized media with 1.5 g/L of yeast extract. MSH was a pure monomeric sugar mix control in the ratio of 3:2.5:0.6:2 for glucose, xylose, arabinose, and galactose, as found in soy hull hydrolysate (Chapter 3; [PERSON] et al. 2016) [13].