## 3 Results and Discussion  
### Results  
#### 3.1.1 Results of Media Optimization with High-Throughput Bioscreen Study  
The results for the batch optimization and model fitting for growth rates on each control and supplemented carbohydrate media illustrated that the OD-based growth rates ranged between 0 and 0.5 (log numbers in OD h\({}^{-1}\)) for all the media combinations and carbohydrate types. Figure 2 shows the range of actual and predicted (Equation (1)) growth rates achieved for each media combination, where the highest growth rates were achieved for glucose (up to 0.5 log numbers in OD h\({}^{-1}\)); however, the data variability was higher with an R\({}^{2}\) value of 0.83. Glycerol had a slightly lower (up to 0.35 log numbers in OD h\({}^{-1}\)) peak growth rate than glucose, with lower data variability as indicated with an R\({}^{2}\) value of 0.96. The SHH and MSH sugar types showed smaller peak growth rates (0.25-0.3 log numbers in OD h\({}^{-1}\)) than the pure glucose and glycerol sugars. This phenomenon can be attributed to the metabolic preference of _Pseudomonas aeruginosa_ to readily uptake six-carbon sugars and utilize them as building blocks for growth and the generation of the sugar backbone of rhamnipid. [PERSON] et al. (2016) reported some biofilm formation inhibition when _Pseudomonas aeruginosa_ was grown on raffinose-rich media, although no significant growth-related inhibition was observed. The lower growth rates on SHH and MSH could be explained by the likely presence of raffinose in SHHs and MSHs, which is made up of galactose as one of its constituent sugars along with glucose and fructose. Galactose has been shown to inhibit biofilm formation in Pseudomonas strains in previous studies [20] and may have limited the production of rhamnoids [20]. Figure 2: Scatter plots for microbial growth rates (log numbers in OD h\({}^{-1}\)) on four carbohydrate media at 2.5% carbohydrate concentration: (**a**) glucose, (**b**) glycerol, (**c**) SHH, and (**d**) MSH. The plots show a scatter showing the range of the actual and predicted (Equation (1)) growth rates achieved for each media combination. 1.2 Results of Verification of Optimized Conditions in 100 mL Shake-Flask Study for Growth Performance and Rhamolipid Titers  
Hydrolysate sugars from the hydrolysis of soy hulls as carbohydrates in SHH-based media yielded 68.2% and 71.50% carbohydrate conversion, respectively, for the Bioscreen study and the 100 mL verification, which is consistent with the results that were achieved for the enzyme hydrolysis process for soy hulls in our previous study (Chapter 3; [PERSON] et al. 2016) [13]. As noted in the previous section, the only significant effect of any bioenhancer was observed for the norepinephrine concentration and the interaction effect of the carbohydrate concentration and the norepinephrine concentration of effect in the glucose sugar type. The best media combination for verification with the 100 mL study was selected from one of the solutions provided by the response surface model as, 2.5% sugar concentration, 160 \(\upmu\)M NE concentration, 66 \(\upmu\)M NE concentration, and 0.03% Fe concentration. This media combination (hitherto referred to as G, GL, SHH, and MSH for each sugar type at 2.5% concentration) was tested with the original MSM and with a variant with 1.5 g/L yeast extract added (hitherto referred to as G + Y, GL + Y, SHH2 + Y, and MSH2 + Y). The addition of 1.5 g/L yeast extract as a media variant in addition to the MSM was implemented to mimic the interaction of norepinephrine with much more expensive human transferrin protein to induce a combined effect of NE and DP binding to larger peptides present in the yeast extract, considering the similarity of the peptidomes of humans and yeast extract [21]. However, no significant improvement in rhamnipid titers was observed, although some improved optical density in the culture media containing the yeast extract was observed for all the different sugar types.