Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules.biosensors | metabolism M icroorganisms, including bacteria, yeast, fungi, and algae, have the potential to safely produce valuable molecules for various fields of applications, including sustainable energy, packaging, detergency, food, cosmetics, and therapeutics (1-3). In all cases, optimizing the yield of production by choosing the best microorganism phenotypes remains a key advantage, raising the importance of monitoring parameters of individual cells. This monitoring implies measuring the rate of nutrient consumption, or the rate of metabolite production, for each single cell, which has so far remained impossible because of the difficulty in detecting small concentration changes around each isolated cell in a large population. Inverse emulsion droplets (water droplets dispersed in an oil phase) have been increasingly used over the past decade to compartmentalize biomolecules or cells for individual assays or amplification (4-8). Interestingly, when droplet compositions change, droplets can exhibit composition ripening (9). Indeed, if two droplets have different concentrations of some solute, either water or the solute molecule will diffuse to equilibrate chemical potentials; the relaxation is dominated by the fastest diffusing species, which in the case of inverse emulsion is water. We therefore reasoned that if bioactivity within a drop lowers its overall solute concentration, this would decrease the water chemical potential and induce a water flux outward. As a result, droplets with high bioactivity would progressively decrease in size, as already observed (10, 11). W...

To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.