Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations

In this study, we investigated the influence of three extracellular pH (pH(ex)) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pH(i)) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pH(i)) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pH(ex) was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pH(ex), the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pH(i) determination demonstrated an increase of the averaged pH(i) level for higher pH set points, while the pH gradient (pH(i)-pH(ex)) and the extent of pH(i) heterogeneity decreased. Cells maintained positive pH gradients at a low pH(ex) of 5.5 and even during substrate limitation at the more widely used pH(ex) 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pH(ex) of 7.5. The larger pH(i) heterogeneity at pH(ex) 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pH(i) heterogeneity at pH(ex) 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points.