During storage and ripening of fermented foods, Lactococcus cremoris is predominantly in a non-growing state. L. cremoris can become stationary due to starvation or acidification, and its metabolism in these non-growing states affects the fermented product. Available studies on the response of L. cremoris to acid and starvation stress are based on population level data. We here characterized the energetic state and the protein synthesis capacity of stationary L. cremoris cultures at the single cell level. We show that glucose starved stationary cells are energy-depleted, while acid-induced stationary cells are energized and can maintain a pH gradient over their membrane. In the absence of glucose and arginine, a small pH gradient can still be maintained. Subpopulations of stationary cells can synthesize protein without a nitrogen source, and the subpopulation size decreases with increasing stationary phase length. Protein synthesis capacity during starvation only benefits culturability after 6 days. These results highlight significant differences between glucose starved stationary and acid-induced stationary cells. Furthermore, they show that the physiology of stationary phase L. cremoris cells is multi-facetted and heterogeneous, and the presence of an energy source during stationary phase impacts the cells capacity to adapt to their environment.

Metabolic interactions between cells affect microbial community compositions and hence their function in ecosystems. It is well-known that under competition for the exchanged metabolite, concentration gradients constrain the distances over which interactions can occur. However, interaction distances are typically quantified in two-dimensional systems or without accounting for competition or other metabolite-removal, conditions which may not very often match natural ecosystems. We here analyze the impact of cell-to-cell distance on unidirectional cross-feeding in a three-dimensional aqueous system with competition for the exchanged metabolite. Effective interaction distances were computed with a reaction-diffusion model and experimentally verified by growing a synthetic consortium of 1 µm-sized metabolite producer, receiver, and competitor cells in different spatial structures. We show that receivers cannot interact with producers located on average 15 µm away from them, as product concentration gradients flatten close to producer cells. We developed an aggregation protocol and varied the receiver cells’ product affinity, to show that within producer–receiver aggregates even low-affinity receiver cells could interact with producers. These results show that competition or other metabolite-removal of a public good in a three-dimensional system reduces metabolic interaction distances to the low µm-range, highlighting the importance of concentration gradients as physical constraint for cellular interactions.