Microbial stir bars: light-activated rotation of tethered bacterial cells to enhance mixing in stagnant fluids

Microfluidics devices are gaining significant interest in biomedical applications. However, in a micron-scale device, reaction speed is often limited by the slow rate of diffusion of the reagents. Several active and passive micro-mixers have been fabricated to enhance mixing in microfluidic devices. Here, we demonstrate external control of mixing by rotating a rodshaped bacterial cell. This rotation is driven by ion transit across the bacterial flagellar stator complex. We first measured the flow fields generated by rotating a single bacterial cell rotationally locked to rotate either clockwise (CW) or counterclockwise (CCW). Micro-Particle Image Velocimetry (μPIV) and Particle Tracking Velocimetry results showed that a bacterial cell of ~ 2.75 μm long, rotating at 5.75 ± 0.39 Hz in a counterclockwise direction could generate distinct micro-vortices with circular flow fields with a mean velocity of 4.72 ± 1.67 μm/s and maximum velocity of 7.90 μm/s in aqueous solution. We verified our experimental data with a numerical simulation at matched flow conditions which revealed vortices of similar dimensions and speed. We observed that the flow-field diminished with increasing z-height above the plane of the rotating cell. Lastly, we showed we could activate and tune rotational mixing remotely using strains engineered with Proteorhodopsin (PR), where rotation could be activated by controlled external illumination using green laser light (561 nm).


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Magnetic stir bars are routinely used for mixing static solutions, that is, with no flow  all PIV pixels for the CCW (5.75 ± 0.39 Hz) and CW (3.54 ± 0.58 Hz) rotating cell was 1 5 8 greater in magnitude in the region with rotating cell than in the region without any rotating  Flow fields generated at different z-heights.

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After confirming generation of vortices as a measure of circular flow pattern for both CCW 1 6 9 and CW rotating cells, we measured azimuthal velocity (or tangential velocity) to determine 1 7 0 the magnitude of flow velocity generated across the plane of cell rotation i.e., x-y plane. Azimuthal velocity was measured for angles between 0 and 2π in the x-y plane vs radius, where radius was defined as a distance from the center of rotation (SI Figure 4) with the increasing distance away from the center of rotation ( Figure 4a). Velocity decreased 1 7 5 exponentially with distance from the center of rotation for both CCW and CW cell types (R 2 1 7 6 value of 0.99 and 0.98 respectively; SI Figure 2b).   After analyzing the velocity profile in the x-y spatial plane, we investigated the flow field velocity, which exponentially decreased with increasing distance away from the center of the 1 9 5 rotation in the x-y plane, we also observed an exponential decrease of velocity with 1 9 6 increasing z-height along the z plane for both CCW and CW cell types (R 2 value of 0.98 and 1 9 7 0.97 respectively (SI zure 2b). across the x-y plane (16 by 16 µm) at 0, 2, 4, and 6 µm in z-height (left to right).   rotating cells, exponential decay of velocity on increasing z-heights from 0 µm to 6 µm was 2 1 7 observed with R 2 value of 0.99 (SI Figure 5). In the x-y plane of the cell rotation, velocity  rotational velocity of 56.11 µm/s from the experiment at the z-height of 0 µm (SI Figure 7).

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To test for possible confounding surface effects, we also simulated the CCW rotating cell 2 2 9 (1.13 Hz) in the middle of a region of interest 5 um from the bottom surface and top of the 2 3 0 chamber. In these simulations the velocity decayed by 70% within 1 um from the rotating 2 3 1 plane of the cell symmetrically in both directions (SI Figure 8). Flow fields generated by multiple cell rotation.

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To examine the potential interaction between adjacent cells' flow fields, we examined the beyond a z-height of 4 µm (SI Figure 10). Furthermore, we examined the flow field for the 2 5 2 multiple cells (more than two cells) rotating adjacent to each other. We observed multiple cells (SI Figure 11).  Light activated cell rotation.

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We used CCW rotating bacteria cell where we expressed proteorhodopsin (PR) from a 2 6 4 plasmid to control the proton-motive force in response to green light. Initial cell rotation was nm) at intensity of 55 W/cm 2 , the cell took 30 seconds to start the rotation. Cell speed 2 6 8 gradually increased and reached a constant speed of ~ 3.5 Hz after 1-2 minutes ( Figure 8).

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When the green laser was switched off, the cell stopped rotating within 1 millisecond. When  tethering could also potentially affect the penetration depth because it is known that rod-2 9 6 shaped bacteria tethered at the center rotate faster than bacteria tethered at the edge (Soman et   We cannot exclude that surface effects from the glass coverslip, from physical contact 3 0 5 between the 0.2 µm tracer particle and the 2 µm long cell, or from the shadow of the cell 3 0 6 obscuring tracer particle fluorescence might affect the accuracy of our velocity field 3 0 7 calculation. We note, however, that the azimuthal velocity 4.6 µm from the centre is 9% of 3 0 8 the value 0.8 µm from the centre, that is, the induced vortex persists at least 3 µm beyond the 3 0 9 path traced by the arc of the cell (1.66 µm radius). This gives evidence that the velocity effect 3 1 0 is observed beyond the boundary of the cell and its direct path. It is not easy to interrogate the 3 1 1 space between the cell and the surface, nor is it straightforward to measure the distance 3 1 2 between cell and surface for each cell, which can vary. The primary limitation is that it is 3 1 3 difficult to integrate tracer particles into this small volume due to particle size and steric  Similarly, it is challenging to simulate boundary effects from the bottom surface since the and the surface roughly are 0.5 µm apart). We were able to execute simulations with cells in the flow velocity had decayed to 10% of the velocity in the plane of the cell, and, as above, to 3 2 7 9% at 4.6 µm radially distant from the centre of rotation. Thus we are able to quantify fluid 3 2 8 flow around the cell in our tethered cell experiments including potential effects from the near 3 2 9 surface.
3 3 0 We note that the average velocity from numerical simulation did not match exactly to our rotational speed which is normally distributed (Fig. 1d). This fluctuation in rotational speed rotating out of plane, or a tether that is non-centered. We are limited to a cross-sectional slice When mixing solutions using a rotating object, control over the direction of rotation and the control over cell rotation through the genomic engineering of bacteria to rotate in CCW or 3 5 0 CW direction. We showed that rotation of single cell generated flow with localized micro-3 5 1 vortices and that these vortices' directionality corresponded to the rotation direction. The 3 5 2 rotation of multiple cells also caused distinct local micro-vortices for each cell that was 3 5 3 rotating. We saw no collective behavior due to hydrodynamic interaction in the region 3 5 4 between two vortices generated by unsynchronized cells separated by ~10 µm. We cannot 3 5 5 rule out vortex interaction at closer spacing, as we did not record data where the cells were 3 5 6 more densely packed. While we can easily generate very sparse cell densities as we can findings agree with previous work which held the separation distance between two rotating 3 6 3 microspheres at ten times the diameter of the microspheres and observed independent