Wetting-coupled phase separation as an energetic mechanism for active bacterial adhesion

TL;DR

This work tackles the puzzle of rapid bacterial adhesion from dilute suspensions by showing that wetting-coupled LLPS in aqueous two-phase systems creates an energetic trap at solid–liquid interfaces, enabling stable confinement and lateral clustering. A combined experimental and coarse-grained particle--field modeling approach demonstrates that preferential partitioning into a surface-wetting phase, together with capillary interactions, drives surface accumulation even when electrostatic repulsion would hinder adhesion; bacterial motility further modulates this process by accelerating transport at low minority-phase volume and by generating hydrodynamic lift at high minority-phase volume. The non-monotonic adhesion dependence on the minority phase volume and the demonstrated generality across P. aeruginosa and S. aureus establish wetting-coupled LLPS as a generic framework for interfacial organization in active suspensions, with implications for biofilm initiation and control of interfacial transport in complex fluids. Collectively, the results provide a minimal, physics-based explanation for rapid surface colonization in phase-separated environments and suggest routes to tune adhesion via phase behavior and surface wettability.

Abstract

The rapid adhesion of motile bacteria from dilute suspensions poses a fundamental non-equilibrium problem: hydrodynamic interactions bias bacterial motion near surfaces without generating stable confinement, while electrostatic interactions are predominantly repulsive. Here, combining experiments on Pseudomonas aeruginosa and Staphylococcus aureus in a polyethylene glycol/dextran aqueous two-phase system with large-scale hydrodynamic simulations, we identify wetting-coupled liquid--liquid phase separation (LLPS) as an energetic trapping mechanism for bacterial adhesion. When bacteria partition into a phase that preferentially wets the substrate, interfacial free-energy minimization creates a deep energetic trap that stabilizes adhesion and induces lateral clustering via capillary interactions. Crucially, bacterial motility plays a dual role: at low phase volume fractions, activity enhances transport into the wetting layer and promotes accumulation, whereas at higher phase volumes it suppresses adhesion through the formation of self-spinning droplets that generate hydrodynamic lift opposing interfacial trapping. Our results establish wetting-coupled LLPS as a generic physical route governing interfacial organization in active suspensions. This provides a unified energetic framework for bacterial adhesion in complex fluids, with broad implications for deciphering bacterial-cell interactions and controlling biofilm formation.

Figures (12)

• Figure 1: Wetting-mediated interfacial adhesion induced by liquid--liquid phase separation. (a) Conceptual illustration of the adhesion mechanism. (i) In a homogeneous PEG solution, bacterial accumulation at the wall is weak due to the absence of an energetic trapping mechanism. (ii) Upon the introduction of a minority DEX phase, bacteria are preferentially wetted by the DEX-rich phase. (iii) When the DEX-rich phase also wets the solid surface, a thin wetting layer forms at the interface, energetically confining bacteria near the wall and thereby enhancing interfacial accumulation and in-plane clustering. (b) Contact angle measurements of the polymer phases on the glass substrate, demonstrating the preferential wetting of the DEX-rich phase relative to the PEG-rich phase.
• Figure 2: Phase-dependent bacterial accumulation. Confocal microscopy images of green fluorescent protein (GFP)-tagged P. aeruginosa in binary fluids with increasing volume fraction of the DEX-rich phase. Panels (a)--(c) correspond to $\phi_{\textnormal{dex}}=0$, $0.05$, and $0.2$, respectively, at a fixed bacterial volume fraction of $\phi_{\textnormal{bac}}=0.1$. (i) Vertical cross-sectional views ($xz$ plane) showing the three-dimensional distribution of bacteria above the substrate. (ii) Horizontal views ($xy$ plane) taken within a 2.5 $\mu$m-thick layer near the substrate. (iii) Corresponding simulation snapshots illustrating preferential wetting of bacteria (particles) by the DEX-rich phase (red) relative to the PEG-rich phase (blue). (a) In the absence of DEX, bacteria remain broadly dispersed in the bulk with minimal interfacial accumulation. (b) At $\phi_{\textnormal{dex}}=0.05$, a surface-associated DEX-rich wetting layer forms, energetically trapping bacteria and leading to enhanced interfacial accumulation. (c) At $\phi_{\textnormal{dex}}=0.2$, large self-spinning droplets form in the bulk, reducing bacterial accumulation at the solid surface.
• Figure 3: Motility enhances interfacial accumulation at low phase volumes. (a,b) Temporal evolution of the accumulated bacterial fraction $f_{\textnormal{ads}}$ obtained from (a) experiments and (b) simulations. Active bacteria (blue) exhibit faster accumulation kinetics and reach higher steady-state levels than non-active controls (red). The inset in (a) shows a representative confocal image of the non-active bacterial layer, while the inset in (b) displays the time evolution of the average number of neighboring bacteria $n_{\textnormal{neigh}}$. In this study, bacteria are defined as surface-bound if they reside within a proximal layer with a thickness of 2.5 $\mu$m, corresponding to the average bacterial length. (c,d) Radial distribution functions $g(r)$ characterizing the in-plane spatial organization of bacteria at the interface. Pronounced peaks for active bacteria in both experiments (c) and simulations (d) indicate enhanced lateral clustering relative to the non-active case. (e) Schematic illustration of the confinement mechanism. At $\phi_{\textnormal{dex}}=0.05$, the surface-associated DEX-rich wetting layer creates a deep energetic trap that confines bacteria near the interface, whereas in the single-phase control ($\phi_{\textnormal{dex}}=0$), bacteria readily escape into the bulk. (f) Probability distribution of the bacterial orientation angle $P(\theta)$. The peak near $\theta \approx 90^\circ$ indicates that confined bacteria predominantly undergo two-dimensional sliding parallel to the wall substrate.
• Figure 4: Active motility suppresses interfacial accumulation at high phase volumes. (a,b) Accumulation kinetics of active (blue) and non-active (red) bacteria. In contrast to the low-$\phi_{\textnormal{dex}}$ regime, bacterial motility suppresses interfacial accumulation at high phase volume fractions. The inset in (a) shows dense interfacial accumulation of non-active bacteria, whereas the inset in (b) quantifies the reduced local clustering of active bacteria through the neighbor number $n_{\textnormal{neigh}}$. (c,d) Hydrodynamic suppression mechanism revealed by simulations. Panel (c) shows the flow field generated by self-spinning droplets in the bulk, which emerge from collective bacterial motion. These rotating droplets produce hydrodynamic flows that oppose the chemical-potential gradient driving transport toward the surface. As a result, the wall-normal velocity $\langle \bm v_z \cdot \bm n \rangle$ is significantly reduced for active bacteria, as shown in panel (d), leading to suppressed accumulation.
• Figure 5: Simulation of phase-dependent bacterial accumulation near neutral walls ($\gamma_{\textnormal{wall}}=0$). Representative simulation snapshots for low ($\phi_{\textnormal{dex}}=0.05$; (a,b)) and high ($\phi_{\textnormal{dex}}=0.2$; (d,e)) volume fractions of the minority phase. (c) Accumulation kinetics at $\phi_{\textnormal{dex}}=0.05$, demonstrating that bacterial motility promotes interfacial accumulation by accelerating transport of the wetting phase toward the walls. (f) Accumulation kinetics at $\phi_{\textnormal{dex}}=0.2$, showing that motility suppresses accumulation in this high-phase-volume regime. These results are qualitatively similar to those obtained with wetting walls.