Active motility and wetting cooperatively regulate liquid-liquid phase separation

Abstract

Liquid-liquid phase separation of aqueous two-phase system (ATPS) is fundamental across physical and biological sciences. While well understood for passive systems, how this process is regulated by active agents such as motile bacteria remains largely unexplored. By combining experiments on Pseudomonas aeruginosa in a prototypical dextran-polyethylene glycol ATPS with hydrodynamic simulations, we show that the interplay between bacterial activity and interfacial wetting gives rise to a robust sequence of nonequilibrium morphologies, including self-spinning droplets, elongated droplet chains, branched capillary-like clusters, and highly deformed droplets. We find that activity plays a dual role in coarsening kinetics: it suppresses coarsening through hydrodynamically driven, activity-induced droplet rotation, yet accelerates it when dextran is the minority phase, where wetting-mediated attraction governs bacterial aggregation. These findings reveal a generic physical mechanism through which motility and wetting cooperatively control phase-separation dynamics, offering new physical insight into activity-regulated LLPS and suggesting strategies for engineering ATPS morphology using active agents.

Figures (8)

• Figure 1: Interfacial wettability and confocal visualization. (a) Contact angles characterizing the wettability of P. aeruginosa across four interfaces: the DEX--PEG--bacteria interface and the water/PEG/DEX--air--bacteria interfaces. (b) Schematic of the spinning-disk confocal microscopy setup used to image the DEX--PEG system containing P. aeruginosa.
• Figure 2: Morphologies of phase-separated domains in an ATPS with a small amount of P. aeruginosa ($\phi_{\mathrm{bac}}=0.01$), showing a transition upon decreasing the DEX volume fraction $\phi_\text{dex}$. (a) Self-spinning droplets at $\phi_\text{dex}=0.05$. (b) Elongated, interconnected droplet chains at $\phi_\text{dex}\approx\phi_{\mathrm{bac}}$. (c) Branched, capillary-like bacterial clusters at $\phi_\text{dex}=0.001$. Upper panels: experimental images of green fluorescent protein (GFP)-tagged bacteria. Lower panels: corresponding simulation snapshots (black: head; white: tail; red: DEX phase; blue: PEG phase).
• Figure 3: Dynamic behavior and mechanism of coarsening in self-spinning droplets. (a) Experimental time evolution of the average droplet radius $R$ (time $t$ scaled by $\tau_\text{e}=\ell_\text{bac}/v_\text{bac}\approx0.1\,$s, with $\ell_\text{bac}\approx1.5\,\mu$m and $v_\text{bac}\approx15\,\mu$m/s) for $\phi_\text{dex}=0.05$ and $\phi_\text{bac}=0.01$. Inset: visual comparison of $R$ for active and non-motile systems at $t=2400\tau_\text{e}$ (left), and a magnified confocal image of a self-spinning droplet (right). (b) Simulated evolution of the domain size $\langle \ell \rangle = 2\pi/\langle q\rangle$, showing suppressed coarsening in the active case. Inset: time evolution of the average neighbor number $N_{\text{neigh}}$. (c) Heat map of the composition field $\psi$ with the velocity field $\bm{v}$, demonstrating spontaneous droplet rotation. (d) Simulated center-to-center distance $D$ between two rotating spheres for various rotation orientations, showing effective hydrodynamic repulsion. (e) Experimental time-lapse images showing two self-spinning droplets separating over time without coalescing.
• Figure 4: Dynamic behavior and mechanism of branched bacterial clusters. (a) Experimental time evolution of the DEX-phase cluster area $A$ for $\phi_\text{dex}=0.001$ and $\phi_\text{bac}=0.01$, showing accelerated phase-separation kinetics under activity. Inset: magnified confocal image of a branched bacterial cluster. (b) Simulated evolution of the $\psi$-based domain size $\langle \ell \rangle$, confirming faster coarsening for $f_\text{act}=1$ compared with $f_\text{act}=0$. Inset: time evolution of the average neighbor number $N_{\mathrm{neigh}}$. (c) Two-bacterium simulation showing that attractive interactions arise only when wetting affinity is present ($\gamma_1=-4$, $\gamma_2=2$); without affinity, no attraction is observed. (d) Heat map of the chemical potential $\mu$ overlaid with the velocity field $\bm{v}$, revealing solvent fluxes directed toward the inter-bacterial region that forms a capillary bridge.
• Figure A1: Phase separation under $\phi_{\text{dex}} = 0.6$ and $\phi_{\text{bac}} = 0.05$. (a) Brightfield microscopy image showing spherical PEG droplets that exhibit no rotation. (b) Corresponding simulation in which P. aeruginosa populate the DEX phase, producing similarly stationary PEG droplets.