## 3 Results and Discussion
### Discussion
#### 3.2.1 Analysis of Media Optimization with High-Throughput Bioscreen Study
The lack of an increase in bacterial growth rate in our study, compared to non-growth enhancer media controls for all media combinations except glucose sugar type, can be attributed to the lack of human transferrin protein in the growth media. The presence of the human transferrin protein has been identified as critical to the generation of NEGF, an intermediary molecule for the auto-induction of the quorum-sensing-directed bulk growth of the _Pseudomonas_ strain, as described in the above-mentioned studies by [PERSON] et al. 1992 and [PERSON] et al. 2009 [16; 17]. The critical difference in the media formulation in our study compared to that in previous studies was the exclusion of human transferrin protein, which seems to have played a critical role in supplying iron in the media, which is then coupled with the generation of norepinephrine-induced growth factor, leading to quorum-sensing-driven higher growth rates. This lack of enhanced bioenbancer effect (NE and DP) is also consistent with our results in which varying the Fe concentration in the media did not have any effect on the growth rate, due to lack of the iron-transferring activity of the transferrin protein. 2.2 Analysis of Verification of Optimized Conditions in 100 mL Shake-Flask Study for Growth Performance and Rhamnolipid Titers  
As shown in Figure 7, the highest ODs were observed for the glucose- and glycerol-based media, with higher ODs when yeast with extract was added to the media. In a pairwise comparison of the peak OD values, no significant differences (\(p>0.05\)) were observed among the mean OD values of any of the sugar blocks except for the obvious significant differences between the sugar blocks and the \"no sugar\" controls. Figure 7: Growth (optical density at 600 nm) performance of _P. aeruginosa_ PAO1 on optimized media combinations in 100 mL shake-flask cultures for 48 h (composition of controls: 0% sugar, 160 uM Nor, 66 uM Dop, 0.03% Fe (n = 2 replicates each)). Experimental carbohydrate blocks: glucose (G), glucose + YE (G + YE), glycerol (GL), glycerol + YE (GL + YE), MSH, MSH + YE, SHH, SHH + YE. This could be attributed to higher total protein availability in the media for the strain to grow; however, no significant slope deflections were observed during the log phase of all the cultures, as observed by [PERSON] et al. 2009, where norepinephrine-treated media combinations for _Pseudomonas aeruginosa_ were on glycerol-based minimal media [16]. Figure 7 also shows that there was no significant difference (\(p>0.05\)) between the peak OD values of the SHH and MSH combinations, with or without the addition of yeast extract, although the glucose-based media and the glycerol-based media had higher peak OD values with the yeast extract addition in the Bioscreen studies. The media utilized for rhamnoid reduction in this study were optimized for minimal salt and protein requirements as the addition of a human transferrin protein to this media would not only make the process cost-ineffective due to the extremely high price of purified human transferrin protein, but it will also raise regulatory concerns when the process is scaled up to industrial production. Figure 8 compares the rhamnoid titers for each optimized media combination at the 100 mL volumes. The media combinations G + Y, GL + Y, and GL produced significantly higher rhamnoid titers (2.78 g/L, 2.65 g/L, and 2.72 g/L, respectively) compared to other media combinations; however, the difference among these three combinations was statistically insignificant.