monitoring single-cell bioenergetics via the coarsening ?· monitoring single-cell bioenergetics...

Download Monitoring single-cell bioenergetics via the coarsening ?· Monitoring single-cell bioenergetics via…

Post on 13-Aug-2018




0 download

Embed Size (px)


  • Monitoring single-cell bioenergetics via thecoarsening of emulsion dropletsL. Boitarda,1, D. Cottineta, C. Kleinschmitta, N. Bremonda, J. Baudrya, G. Yvertb, and J. Bibettea

    aEcole Superieure de Physique et de Chimie Industrielles, Paris Tech, Laboratoire de Collodes et Matriaux Diviss, Universit Pierre et Marie Curie, Paris06, Unite Mixte de Recherche 7195, 10 Rue Vauquelin, 75231 Paris, France; and bEcole Normale Superieur Lyon, Laboratoire de Biologie Molculaire de laCellule, Universit Lyon 1, Unit Mixte de Recherche 5239, 46 Alle dItalie, 69007 Lyon, France

    Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved February 28, 2012 (received for review January 17, 2012)

    Microorganisms are widely used to generate valuable products,and their efficiency is a major industrial focus. Bioreactors are ty-pically composed of billions of cells, and available measurementsonly reflect the overall performance of the population. However,cells do not equally contribute, and process optimization wouldtherefore benefit from monitoring this intrapopulation diversity.Such monitoring has so far remained difficult because of the inabil-ity to probe concentration changes at the single-cell level. Here,we unlock this limitation by taking advantage of the osmoticallydrivenwater flux between a droplet containing a living cell towardsurrounding empty droplets, within a concentrated inverse emul-sion. With proper formulation, excreted products are far moresoluble within the continuous hydrophobic phase compared to in-itial nutrients (carbohydrates and salts). Fast diffusion of productsinduces an osmotic mismatch, which further relaxes due to slowerdiffusion of water through hydrophobic interfaces. By measuringdroplet volume variations, we can deduce the metabolic activitydown to isolated single cells. As a proof of concept, we presentthe first direct measurement of the maintenance energy of indivi-dual yeast cells. This method does not require any added probesand can in principle apply to any osmotically sensitive bioactivity,opening new routes for screening, and sorting large libraries ofmicroorganisms and biomolecules.

    biosensors metabolism

    Microorganisms, including bacteria, yeast, fungi, and algae,have the potential to safely produce valuable moleculesfor various fields of applications, including sustainable energy,packaging, detergency, food, cosmetics, and therapeutics (13).In all cases, optimizing the yield of production by choosing thebest microorganism phenotypes remains a key advantage, raisingthe importance of monitoring parameters of individual cells. Thismonitoring implies measuring the rate of nutrient consumption,or the rate of metabolite production, for each single cell, whichhas so far remained impossible because of the difficulty in detect-ing small concentration changes around each isolated cell in alarge population. Inverse emulsion droplets (water droplets dis-persed in an oil phase) have been increasingly used over the pastdecade to compartmentalize biomolecules or cells for individualassays or amplification (48). Interestingly, when droplet compo-sitions change, droplets can exhibit composition ripening (9).Indeed, if two droplets have different concentrations of some so-lute, either water or the solute molecule will diffuse to equilibratechemical potentials; the relaxation is dominated by the fastestdiffusing species, which in the case of inverse emulsion is water.We therefore reasoned that if bioactivity within a drop lowersits overall solute concentration, this would decrease the waterchemical potential and induce a water flux outward. As a result,droplets with high bioactivity would progressively decrease insize, as already observed (10, 11). We detail the key criteria thatemulsions must satisfy to exhibit this active coarsening. We thendemonstrate that the sensitivity of this coarsening allows nutrientconsumption to be detected in very small exponentially growingmicrocolonies as well as for single nondividing cells. We then

    determine the statistical distribution of the maintenance energyof individual yeast cells at various ploidies, revealing at the micro-scopic level the relationship between size and glucose consumption.We finally illustrate the universality of this osmotic phenomenonin the cases of bacterial growth and a lytic enzymatic reaction.

    ResultsIn the presence of glucose, yeast metabolism is dominated byfermentation, where 1 mol of glucose provides 2 mol of ATP(12) from the following chemical equation: C6H12O6 2ADP2Pi 2C2H5OH 2CO2 2ATP. For ripening to occur andreflect glucose consumption, a number of criteria must be satisfiedby the emulsion formulation. First, waste products of fermenta-tion (carbon dioxide and ethanol) must diffuse out of the dropletfaster than water. This diffusion is essential to decrease the totalsolute concentration within the yeast-containing droplets (callednursing droplets hereafter) and thereby to create a chemicalpotential mismatch as a result of nutrient consumption. Secondly,solely water but not nutrients must diffuse out of the nursingdroplet so that the relaxation of this osmotic mismatch inducesa change in droplet size. Finally, because products remainingentrapped in cells (ATP) do not contribute to osmotic change,themeasurable droplet shrink rate quantitatively reflects nutrientsuptake.

    To fulfill these requirements, we chose an organic continuousphase consisting of a mixture of mineral oil and a partially fluori-nated alkane chain containing 0.75% of a surfactant blend. Thisparticular blend and the continuous phase composition produceda controlled adhesion (wetting) between droplet interfaces (13).Using this formulation, contacts between adjacent drops formeda bilayer structure associated with a contact angle (Fig. S1).Adhesion imposes the distance between all the adhesive dropletsand thus provides a homogeneous exchange rate overall theemulsion that is a least two times faster than for a nonadhesiveformulation (see SI Text, Formulation and Diffusion Model andParameters, Without Cells). To check if the above criteria wereobeyed by this formulation, we first brought into adhesive con-tacts droplets that contained different concentrations of glucosein water. As expected, the volume of droplets having a high glu-cose concentration increases to the detriment of the less concen-trated ones (Fig. 1A andMovie S1). This observation is consistentwith water diffusing across the hydrophobic membrane from theless concentrated drop toward the most concentrated one in theabsence of any glucose permeation. For dilute solutions ( c),water transport parameters can be estimated from water diffusionrelaxation curves obeying Ficks law:

    Author contributions: L.B., N.B., J. Baudry, G.Y., and J. Bibette designed research; L.B. andC.K. performed research; D.C., J. Baudry, and G.Y. contributed new reagents/analytic tools;L.B., D.C., and C.K. analyzed data; and L.B., N.B., G.Y., and J. Bibette wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail:

    This article contains supporting information online at PNAS Early Edition 1 of 6










  • dVdt

    Fvwc; [1]

    where V is the volume of the drop, vw is the molar volume ofwater, c is the time-dependent glucose concentration mismatchbetween the two adhesive droplets and F is a transport factorreflecting membrane properties, which can be written as F DAKd, where D is the diffusion coefficient into the oil mem-brane, A and d are the area and thickness of the adhesive bilayer,respectively, and K is the solubility of the diffusing species intothe oil membrane (14). We then performed the same experimentto measure the equilibration time from a concentration mismatchin ethanol. We brought into adhesive contact two droplets havingequal concentration of glucose and salt, adding 1 M of ethanolin only one of them. This experiment did not result in any obser-vable change in droplet sizes over time, consistent with ethanoldiffusing out much faster than water (Fig. S2 and Movie S2).Such phenomenon is in perfect agreement with the values of Kin olive oil (15), which change by more than three orders of mag-nitude between glucose (3 105), water (103), and ethanol(3 102). Finally, because it is highly soluble in the oil (16), car-bon dioxide diffuses faster than water or is totally dissolved intothe oil. Hence, it does not contribute to the osmotic mismatcheither. Altogether, these properties set the basis of our strategyto measure the rate of glucose consumption by living cells.