UCLA

Bacteria live in environments featuring various complex chemical concentration gradients. The formation of these gradients is a combined result of mass transfer and bacterial activities, creating a challenge in isolating the effects of specific gradients on bacterial activities. Since the size of a single microbe is 1 to 10 μm, the spatial profiles of the surrounding chemical concentration gradients are at a resolution similar to the size of a single microbe. Moreover, time-dependent bacterial activities could lead to temporal variation in these chemical concentration gradients. Both spatial chemical heterogeneity and temporal chemical variation are important in shaping bacterial activities. The critical roles of gradients on bacterial behavior poses a significant challenge: the coexistence of numerous gradients simultaneously complicates discerning the effects of a specific gradient on a particular bacterial activity. To address this challenge, I established a workflow using electrochemistry to create electrochemical gradients to simulate specific chemical gradients in bacterial environments. Additionally, I leveraged electrode design to control the generated gradients and minimize side effects where electrochemistry could introduce to the bacterial environment. In my first research project (Chapter 2), I fabricated a flow device equipped with a microwire electrode to generate and control oxygen (O2) and hydrogen peroxide (H2O2) gradient to mimic bacterial gradients. The first highlight of this research is that I generated electrochemical H2O2 gradients through an electrocatalyst design to simulate spatial distributions of reactive oxygen species in bacterial environments. By coating the electrodes with Au catalysts to facilitate the two-electron oxygen reduction reaction (2e-ORR), I achieved a spatial profile of H2O2 perpendicular to the electrode surface. The second highlight of this project is that I enabled spatial and temporal control of both electrochemical gradients via electrode morphology design and applying time-dependent external voltages, respectively. This spatiotemporal control strategy achieved spatial control at a precision within 10 μm and temporal control on a second scale, comparable to the spatiotemporal variation of bacterial environments. Moreover, the concentration ranges of O2 (0 to 250 μM) and H2O2 (0 to 40 μM) in the electrochemical gradient, aligned with the conditions of microbiological research. To advance this research, I, together with co-authors Jingyu Wang and Ben Hoar, developed a machine-learning-assisted design strategy for desired O2 and H2O2 gradients, offering a rapid and efficient solution for electrode optimization. I created another novel electrochemical flow device for my second research project (Chapter 3). I introduced two main electrode design strategies to enhance compatibility and convenience in gradient-bacteria research. Firstly, I deposited a layer of porous copolymer on the electrode surface to increase the oxygen reduction reaction (ORR) selectivity and create a “clean” O2 gradient for bacterial research. This innovation was particularly critical in mitigating the interference of external voltages with bacterial redox systems, thus increasing the integrity of experimental results. Based on size exclusion, the coated polymer maintained the ORR current while rejecting the access of larger bacterial redox molecules to the electrode surface. Using a redox molecule produced by Pseudomonas aeruginosa, pyocyanin, as an example, the coated polymer enabled the ORR current to remain around 70% compared to that on the current on a bare electrode while suppressing almost 100% of the redox current of pyocyanin. Secondly, I adopted planar electrode arrays instead of the vertical electrode arrays used in Chapter 2. The planar geometry enabled spatial O2 concentration profile gradient generation both perpendicular and parallel to the electrode surface. This innovation proved instrumental as it facilitated microscopic microbial research while also allowing the replication of gradients within three-dimensional bacterial structures. In summary, this porous polymer coating enabled the creation of a “clean” O2 gradient, allowing investigation of the effect of O2 on microbial behavior. In Chapter 4, I proposed a potential application using electrochemical gradients in microbiological research. I describe utilizing electrochemical O2 gradients to investigate the effect of O2 variation on bacterial adenosine triphosphate concentration ([ATP]) at the single-cell level. I reviewed the published literature on the relationship between O2 concentration and microbial ATP activity. Most previous research is focused on the average effect of O2 on adenosine energy charge (AEC) and [ATP] in a bulk bacteria culture. As such, the effect of O2 on single cells is missing. I proposed an experiment plan to utilize the electrochemical O2 gradient platform to study the effects of O2 on single microbes and explored the feasibility of this plan. I raised several potential concerns or questions that could arise from an electrochemistry-microbiology hybrid system. In summary, my thesis comprehensively explores electrochemical concentration gradients and their implications for microbial behavior. By employing innovative electrode design strategies, I explored the feasibility of using electrochemical concentration gradients for nuanced investigations into the intricate dynamics of bacterial ecosystems. Looking ahead, I believe the fusion of electrochemistry and microbiology holds promise for advancing our understanding of bacterial behavior, especially at the single-cell level and beyond.