Assay of acetate concentration in spent medium

Extracellular acetate concentration was determined in samples collected from cultures of the different strains under different conditions using a commercial enzymatic acetate assay (Megazyme). Cells were removed from 0.5 mL culture sample by centrifugation for 5 min at 13,000 rpm at 4 °C. Supernatant samples were stored at − 20 °C for later use. For the enzymatic acetate assay, approximately 200 µL of a supernatant was used according to the manufacturer’s instructions. During the assay, the conversion of the acetate present in the sample to acetyl phosphate is stoichiometrically coupled to the conversion of NADH to NAD+. The latter conversion was quantitated via the absorption (340 nm) of the sample, which was performed in 96-well plates (Greiner), using a plate reader (BMG FLUOstar OPTIMA Microplate Reader), operated at room temperature. For quantification, the assay was calibrated with a standard curve obtained under the same conditions.