## 3 Results and Discussion
### Discussion  
#### 3.2.1 Analysis of Media Optimization with High-Throughput Bioscreen Study  
The effects of the four factors on the growth rates on each carbohydrate type are presented as response surface plots: glucose (Figure 3), glycerol (Figure 4), SHH (Figure 5), and MSH (Figure 6). For all four carbohydrate types, the carbohydrate concentration had a significant effect (\(p<0.05\)) on the growth rate. In Figures 3-6, the interaction plots involving carbohydrate concentration with other factors such as NE, DP, and Fe, show the same trend of a linear increase in the growth rate as carbohydrate concentration increased from 0 to 3% (\(w/v\)). The growth rates observed in this linear range, ranged from 0 to 0.2 for all sugar types; however, in the interaction plots not involving the sugar concentration, saddled curves were observed with the variance in growth rate being limited to the 0.15-0.2 log numbers in the OD h\({}^{-1}\) range. This indicates that the growth rates did not increase in media combinations with a low sugar concentration and high bioenhancer (150 \(\upmu\)M for NE and DP) and high Fe (0.03% \(w/v\)) concentrations. However, in all the response surface interaction plots, even at middle concentrations for carbohydrate for all sugar types (1.5-3% \(w/v\)), the growth rates increased linearly, regardless of the NE, DP, and Fe concentrations. The interaction effect between carbohydrate concentration and norepinephrine concentration and the singular effect of norepinephrine concentrations were the only two significant effects observed in the glucose sugar type. In all other sugar types in Figures 4-6, none of the interaction effects between factors nor the singular effects were significant, and no maxima on any of the factors were achieved for the predicted model; hence, a saddle point prediction was seen. Figure 4: Response surface plots showing the effects of varying carbohydrate, Fe, norepinephrine, and dopamine concentrations on growth rates in glycerol-based media. Color legend: The green-to-orange color differential denotes increasing magnitude. Axes: glycerol (0–3%), Fe concentration (0–0.05%), norepinephrine (0–150 \(\upmu\)M), dopamine (0–150 \(\upmu\)M). Figure 3: Response surface plots showing the effects of varying carbohydrate, Fe, norepinephrine, and dopamine concentrations on growth rates in glucose-based media. Color legend: The green-to-orange color differential denotes increasing magnitude. Axes: glucose (0–3%), Fe concentration (0–0.05%), norepinephrine (0–150 \(\upmu\)M), dopamine (0–150 \(\upmu\)M). Figure 5: Response surface plots showing the effects of varying carbohydrate, Fe, norepinephrine, and dopamine concentrations on growth rates in SHH-based media. Color legend: The green-to-orange color differential denotes increasing magnitude. Axes: SHH (0–3%), Fe concentration (0–0.05%), norepinephrine (0–150 \(\upmu\)M), dopamine (0–150 \(\upmu\)M). Figure 6: Response surface plots showing the effects of varying carbohydrate, Fe, norepinephrine, and dopamine concentrations on growth rates in MSH-based media. Color legend: The green-to-orange color differential denotes increasing magnitude. Axes: MSH (0–3%), Fe concentration (0–0.05%), norepinephrine (0–150 \(\upmu\)M), dopamine (0–150 \(\upmu\)M). Rhamnolipid synthesis is QS (quorum-sensing) regulated [15]. These systems are driven by regulatory pathways to build the backbone and chemical components of rhamnoids. In human bodies, these QS systems are also associated with the opportunistic pathogenicity of _Pseudomonas aeruginosa_ in iron- and oxygen-deprived conditions by sensing human neuro-endocrine hormones such as dopamine and norepinephrine, which are stress-triggered molecules [15]. Our study utilized this understanding to design a rhamnpid-generating growth media for _Pseudomonas aeruginosa_; however, the expected significant improvements in the growth rates were not observed.