Fig. 5: Key physiological and metabolic characteristics of 1A01 and 3B05 monocultures under acetate stress.

3B05 was precultured alone in strongly buffered acetate medium. Then the culture was washed and transferred to a weakly buffered medium (2 mM NaHCO₃) with 4.5 mM acetic acid (with pH = 4.9), supplemented with (filled symbols) or without (open symbols) the addition of pyruvate, lactate, and glutamate (referred to collectively as a Supplement, or “Supp”). a OD, with the gray symbols showing results from the same experiment starting at a lower pH (4.6, by the addition of HCl). The measurement variability for the determination of OD₆₀₀ was ±0.002 on the basis of repeated measurements of the same culture sample. b Acetate (left axis) and the sum of pyruvate, lactate, and glutamate concentrations ([Supp], right axis) in the medium of the two cultures described in (a), and (c) pH of the medium. The measurement variability for the determination of acetate, pyruvate, lactate, and glutamate concentrations by HPLC was ~2% on the basis of repeated measurements of the same spent media sample. The pH measurement was accurate to ±0.02 pH unit on the basis of repeated measurements of pH standards. Exponentially growing 1A01 monoculture was initiated at an OD₆₀₀ of 0.2 and grew in steady-state in GlcNAc medium with the weak buffer (2 mM NaHCO₃) until acetate excretion dropped the pH to ~5 where OD₆₀₀ reached 0.45, corresponding to viable cell density of ~4 × 10⁸ CFU/mL. The pH was then maintained in a narrow pH range by manually titrating with 0.1 M NaHCO₃. d Viable cell count (black circles) and pH (orange circles); the arrows indicate times at which NaHCO₃ was added. e Concentrations of GlcNAc (blue triangles) and acetate (red squares) in the medium. f Concentrations of pyruvate, lactate, and glutamate in the medium.