FIGURE 1.

Analysis pipeline of prolonged acidification in non-growing cells. L. lactis NCDO712 cells were pre-cultured in lactose 1%, harvested at mid-exponential growth and transferred to the assay medium with lactose 1%. (A) Fluorescent signal was measured for assay medium without cells at pH 4 and 6.5 (light gray), cells (2.5E+07/mL) in assay medium at an initial pH of 6.5 (light green) and an initial pH of 5.9 (dark green). (B) Based on a standard curve, the RFU were converted to pH. In samples with a low starting pH (dark green) acidification stopped due to product inhibition at pH 4 while for a high starting pH acidification was still ongoing after more than 300 h. (C) Based on a titration curve, the pH profile was converted to the amount of lactic acid produced. The noise threshold was determined from the non-biological fluctuation of signal based on a negative control at pH 6.5. (D) The lactate production rate is plotted as a function of the total amount of lactic acid produced. Decline in production could be calculated following first order (E) or second order (F) reaction kinetics. The experiment was carried out three times with four replicates with reproducible results. Four replicate curves are shown in this plot.