Synthetic Media Composition for Adaptive Laboratory Evolution

The composition of the synthetic media (SM) used in this study is specified in Table 1. The chemicals used in the preparation of the SM exhibited a purity level of ≥ 99.9%.
Table 1

Composition of different synthetic media used in this study

	Concentration (g/L)
	SM 1 (1:1)*	SM 2 (1:2)*	SM 3 (1:4)*	SM 4 (1:6)
Acetic acid	3.57	2.60	1.48	1.02
Propionic acid	0.64	0.61	0.68	0.66
Butyric acid	5.93	5.47	5.78	5.84
Valeric acid	1.42	1.33	1.31	1.29
Hexanoic acid	3.52	5.19	5.90	6.24
Total	15.08	15.20	15.15	15.06

* Acetic acid:hexanoic acid ratio (A:H) are in brackets

SM 1 was prepared according to the SCFAs profile obtained by Greses et al., (2020) in the anaerobic fermentation of food wastes [15 ]. To carry out the ALE of Y. lipolytica, SM 2 and 3 were modified from SM 1 to have higher hexanoic acid concentrations. In order to maintain 15 g/L of SCFAs in all media, the concentration of acetic acid, the least inhibitory acid, was reduced while hexanoic acid was increased. Thus, four different A:H ratios (1:1, 1:2, 1:4 and 1:6) were established, corresponding to SM 1, 2, 3 and 4. Since the YNB composition was the same in all media (1.7 g/L of yeast nitrogen base and 7.5 g/L of ammonium sulfate), the initial C:N ratio was kept at 3.5.