Bacterial Motility's Role in Aqueous Phase Dynamics

Abstract

We study the partitioning of motile bacteria in an aqueous two-phase mixture of dextran (DEX) and polyethylene glycol (PEG), which can phase separate into DEX-rich and PEG-rich phases. While nonmotile bacteria partition exclusively into the DEX-rich phase in all conditions tested, we observed that motile bacteria penetrate the soft DEX-PEG interface and partition variably among the two phases. For our model organism Bacillus subtilis, the fraction of motile bacteria in the DEX-rich phase increased from 0.58 to 1 as we increased the DEX composition within the two-phase region. We hypothesized that the chemical affinity between DEX and the bacteria cell wall acts to weakly confine the bacteria within the DEX-rich phase; however, motility can generate sufficient mechanical forces to overcome the soft confinement and propel the bacteria into the PEG-rich phase. Using optical tweezers to drag a bacterium across the DEX-PEG interface, we demonstrate that the overall bacteria partitioning is determined by a competition between the interfacial forces and bacterial propulsive forces. Our measurements are supported by a theoretical model of dilute active rods embedded within a periodic soft confinement potential.

A collaborative team of physicists and microbiologists from UNIST and Stanford University has, for the first time, uncovered the fundamental laws governing the distribution of self-propelled particles, such as bacteria. Published in the September 2025 edition of Physical Review Letters, this breakthrough has been jointly led by Professor Joonwoo Jeong in the UNIST Department of Physics, Professor Robert J. Mitchell in the UNIST Department of Biological Sciences, and Professor Sho C. Takatori at Standford University.

The study reveals that the distribution of living bacteria is governed by a delicate balance between their motility and their affinity for specific liquid environments. Interestingly, the findings highlight a phenomenon consistent with the like-attracts-like principle. Motile bacteria tend to aggregate with other bacteria exhibiting similar motility behaviors, influencing their spatial distribution within complex fluids. While forces that draw bacteria into certain liquids tend to confine them, the bacteria's ability to move allows them to escape these confinements and distribute more broadly-challenging traditional expectations based solely on energy preferences.

Using optical tweezers, the researchers precisely measured the forces bacteria exert to favor one liquid phase over another, finding that this attractive force is approximately 1 piconewton (pN)-a force about ten million times weaker than what is experienced by a human hair under gravity. Remarkably, the bacteria's propulsion force was measured at around 10 pN, sufficient to overcome these weak attractive forces and enable bacteria to traverse between phases, further illustrating how motility influences distribution in active matter systems.

Figure 1. Motile bacteria B. subtilis immersed in an ATPS of DEX and PEG partition variably across the two phases compared to nonmotile ones, which partition exclusively into the DEX-rich phase. (a) Top and (b) side views of our quasi-2D setup for measuring bacterial partitioning. The chamber thickness is 5  μ⁢m to prevent the bacteria (average body length ≈5  μ⁢m) from orienting vertically. (c) Nonmotile B. subtilis partition exclusively into the DEX-rich phase (yellow region), whereas (d) motile ones partition into both phases. Total concentrations of DEX and PEG are 3.2 and 2.5  wt/wt%, respectively. The scale bar is 20  μ⁢m.

The experiments involved injecting Bacillus subtilis (B. subtilis)-a bacterium commonly used in fermented soybean products-into a two-phase dextran/PEG system, which naturally separates into dextran-rich and PEG-rich phases. While non-motile bacteria remained confined to their preferred phase, motile bacteria were evenly distributed across both, a phenomenon that cannot be explained solely by thermal fluctuations. Instead, the motile bacteria exhibit a form of like-attracts-like behavior, where their self-propulsion and mutual attraction lead them to cluster with similar bacteria, influencing their phase partitioning.

Key contributors include Dr. Jiyong Cheon, a former doctoral student at UNIST, now working as a postdoctoral researcher at Georgia Tech, and Kyu Hwan Choi, a postdoctoral researcher at Stanford University. The team emphasized that this interdisciplinary work-spanning physics, chemical engineering, and microbiology-has successfully quantified the forces acting on active particles in non-equilibrium conditions. This model system offers new insights into how bacteria and other active particles behave in environments where energy is continuously supplied or consumed, reinforcing the idea that like behaviors tend to attract and influence collective dynamics.

Professor Jeong explained, "This research not only helps us understand how bacteria establish their niches within the body, but also has potential applications in protein purification, biosensor development, and the design of micro-robots."

The findings of this research have been published in the online version of *Physical Review Letters* on September 16, 2025. The study was supported by funding from the National Research Foundation of Korea (NRF), the U.S. National Institutes of Health (NIH), the U.S. National Science Foundation (NSF), the U.S. Air Force Research Laboratory (AFRL), and the Parker Fellowships for Science and Engineering.

Journal Reference

Jiyong Cheon, Kyu Hwan Choi, Kevin J. Modica, et al., "Motility Modulates the Partitioning of Bacteria in Aqueous Two-Phase System," Phys. Rev. Lett., (2025).