Inoculating fluid a

We comparatively analyzed the growth capacity of E. coli MG1655 and the evolved strains on non-evolutionary carbon sources in Biolog PM1 MicroPlate^TM (Biolog Inc., Hayward, CA, United States), which contains 95 different carbon sources as a sole nutrient source. The frozen cell stocks were inoculated in 3 mL of 0.2% lactate or glycerol M9 minimal media, cultured overnight at 37°C with agitation. Two technical replicates were tested per sample. Cells were collected using a centrifuge, the cell pellets re-suspended in 6 mL of Inoculating Fluid A (Biolog Inc., Hayward, CA, United States) with OD_{600 nm} of approximately 0.4 (42% T cell). To make 85% T cell suspension, 4 mL of 42% T cell suspension was transferred to 20 mL of Inoculating Fluid A. 100 μL of 85% T cell suspension was loaded onto each well on the PM01 plate. To quantitatively determine cellular growth kinetics, cell density was measured on EPOCH 2 plate readers (BioTek, Winnoski, VT) at 30°C with shaking (807 rpm double orbital) for 24 h, using default 96-well configuration.

Cells were streaked on Biolog Universal Growth Agar (Biolog) plates and grown overnight at 37 °C. Then, cells were resuspended and diluted with Inoculating Fluid A (80% IF-0a GN/GP Base in sterile water; Biolog) to 42% transmittance (T) measured using a Turbidimeter (Biolog). A 42% T cell resuspension was diluted with Inoculating Fluid B (83.33% IF-0a GN/GP Base and 1.2% Biolog Redox Dye mix A in sterile water) to generate 85% T cell resuspension. For PM plate 3B and 4A, 19.8 mM of sodium succinate and 1.98 nM of ferric citrate were added to Inoculating Fluid B as carbon sources. Finally, 100 μl of the 85% T cell resuspension was inoculated onto PM plates and cellular respiration was measured using an Omnilog instrument (Biolog).