Project Summary The natural environment is intrinsically spatiotemporally heterogenous at both macroscopic and microscopic levels. What shapes such a heterogeneity includes the concentration gradients of biologically relevant chemical species in the extracellular medium including dioxygen (O2), reactive oxygen species (ROS), as well as essential redox-active transition metals. While a significant amount of effort has been devoted to spectroscopically image these chemical moieties, our capability to spatiotemporally control their concentration distributions in the extracellular medium remains limited. This is especially the case for biofilms and microbiota, in which the microorganisms’ small length scales pose significant challenges for concentration modulation. The inadequate control of concentration heterogeneity limits our capability of mimicking the natural environments in vitro and investigating how local concentration gradients affect microbial functionality. Therefore, there is a need for an advanced method of controlling chemical concentrations at microscopic level. Our proposed research aims to use electrochemical nano-/micro-electrodes to spatiotemporally control the concentration gradients in the extracellular medium. When an electrochemical reaction occurs on an electrode’s surface, a concentration gradient is established near the electrode. Taking advantages of this phenomena with the assistance of numerical simulation, we will employ an array of nano-/micro-electrodes with individually addressable electrochemical potentials to program any arbitrary spatiotemporal concentration profiles. We will fine-tune the surface chemistry and the electrochemical properties of these electrodes to ensure biocompatibility and reaction specificity. The developed system will be applied to biofilms and we aim to investigate how the microbial social behavior will be affected by a perturbation of local O2 concentration. Moreover, we will use this device to mimic the heterogenous environment in the gut and culture gut microbiota in vitro. An algorithm based on machine learning will be employed to actively adjust electrode potentials, maintaining a stable concentration profile despite the accumulation of gut microorganisms. Ultimately, our work will expand our capability of controlling the concentration heterogeneity in nature. The developed electrochemical system will serve an in vitro platform to culture microorganisms in their native environment, or as a tool to perturb the concentration profiles. Combining electrochemistry, inorganic chemistry, and nanomaterials the research will enable a deeper understanding of the spatial distribution and temporal response of microbial systems.