The simultaneous utilization of kinetic analysis and flow cytometry in the assessment of Lactobacillus rhamnosus ATCC 7469 physiological states produced by increasing oxygen limitation levels and lactic acid accumulation

Carbon limited continuous cultures of Lactobacillus rhamnosus ATCC 7469 were grown at dilution rates between 0.1 h−1 and 0.6 h−1. At 0.45 h−1, oxygen uptake decreases producing a deficiency in the production of cell energy, lowering the concentration of biomass and finally accumulating glucose in the broth. Under the lack of energy pressure, L. rhamnosus ATCC 7469 triggers the production of lactic acid from pyruvate freeing NAD+ and stimulates glycolysis to continue, producing extra ATP from substrate-level phosphorylation. The 12-fold growing concentration of lactic acid and the 2-fold increase of succinic acid are in parallel with the steep 4-fold decrease of acetic acid production and small concentration changes of formic and propionic acids.The way the cells balance the available energy between the growing dilution rate and detoxification produces a stress within the culture, detected and described by flow cytometry. As the dilution rate increased, the proportion of L. rhamnosus ATCC 7469 cells with depolarized membrane steadily increased (1% at D = 0.20 h−1, 8% at D = 0.30 h−1, 14% at D = 0.45 h−1 and 26% for D = 0.62 h−1, respectively). Only a low level of 3.7% of the population did not recover from the demanding growth rates in the acidic environment.

Density plot showing the distribution of Lactobacillus rhamnosus ATCC 7469 cells growing at a 0.62 h−1 steady-state in the presence of propidium iodide (PI) and 3,3′-dihexylocarbocyanine iodide (DiOC6(3)). Three main populations were distinguished. Healthy cells stained with DiOC6 holding polarized membranes. Cells with a depolarized cytoplasmic membrane (no staining). These cells are capable of recovering towards a fully growth state. Permeabilized cells with a disrupted cytoplasmic membrane stained with both DiOC6 and PI (dead cells).Figure optionsDownload as PowerPoint slideHighlights
► We describe the heterogenic nature of steady state microbial cultures.
► High dilution rates results in the change from glucose to oxygen limitation.
► Oxygen limitation leads to a change of organic acid profile produced by L. rhamnosus.
► Flow cytometry reveals that organic acids affect L. rhamnosus membrane potential.
► Dilution rate increased, the proportion of L. rhamnosus cells with depolarized membrane steadily increased.