A Custom Soil Electrochemical Profiling System for Detecting Electrochemical Activity Changes in Soil

A Custom Soil Electrochemical Profiling System for Detecting Electrochemical Activity Changes in Soil | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Custom Soil Electrochemical Profiling System for Detecting Electrochemical Activity Changes in Soil Won-Jun Kim, Ibrahim Bozyel, Suat Ay, Maren Friesen, Haluk Beyenal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8981935/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 19 You are reading this latest preprint version Abstract Physicochemical soil parameters are key drivers of biogeochemical processes, controlling microbial activity and the mobility, solubility, and bioavailability of organic and inorganic matter. These parameters vary with depth, yet tools for their quantification are limited. Microelectrodes, among the most common tools, are primarily designed for biofilms only a few micrometers to millimeters thick and thus unsuitable for centimeter-scale soil gradients. To address this gap, we developed a custom-built automated manipulator system for soil depth profiling. Using off-the-shelf components, we constructed an instrument called Soil Electrochemical Profiling System (SEPS) capable of operating to depths of up to 27 cm with a minimum step interval of 25 µm. SEPS integrates directly with a potentiostat, enabling electrochemical techniques such as redox potential measurements, cyclic voltammetry, and chronoamperometry experiments. We validated the system by generating depth-resolved redox profiles and cyclic voltammograms in soil and sediment reactors. Additionally, we demonstrated its utility for microbial enrichment by polarizing a microelectrode at a fixed depth over 40 days. This system uniquely combines fine-scale automated positioning with electrochemical instrumentation functionality, providing a versatile and affordable platform for soil depth profiling and advancing the study of biogeochemical and microbial processes in terrestrial environments. Physical sciences/Chemistry Earth and environmental sciences/Environmental sciences manipulator depth profiling redox soil Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. INTRODUCTION Soil can be viewed as a natural bioelectrochemical reactor, driven by microbial interactions with minerals, organic matter, and other constituents that generate redox gradients 1 . Nearly half of the soil volume consists of inorganic materials such as sand, silt, and clay 2 , while approximately 5% is composed of organic matter derived from decaying organisms, microbial biomass, or humic substances. Microbes utilize the diverse chemical species present within both inorganic and organic fractions of the soil. The remaining soil volume comprises pore spaces that hold air and water, enabling the transport of dissolved chemical species used for nutrient cycling. The metabolic activities of soil microorganisms govern the biogeochemical processes that occur in terrestrial environments and cause redox gradients controlling electrochemical activities 3 . Redox potential indicates the balance between oxidized and reduced compounds, determining the oxidative or reductive state 4 , 5 . The redox potential of soil increases with the abundance of oxidizing components, and oxic conditions typically prevail near the surface where oxygen is readily available. 6 . As depth increases, oxygen becomes depleted through aerobic microbial respiration, creating anoxic and reducing zones 7 . These redox gradients shape key biogeochemical processes. For example, in the nitrogen cycle, oxic regions drive nitrification by oxidizing ammonium to nitrate, while anoxic regions favor denitrification, the reduction of nitrate to atmospheric nitrogen 8 . Under anoxic conditions, certain microorganisms also reduce ferric oxides, releasing bound phosphates and enhancing their bioavailability in soil 9 . Hypoxic microenvironments can produce nitrous oxide, a potent greenhouse gas, as an intermediate during denitrification 10 , whereas strictly anoxic conditions promote methanogenesis and the release of methane 11 . Thus, soil redox potential not only represents the overall balance between oxidized and reduced species but also indicates the types of redox reactions likely to be favored by microbial activity within different soil zones. Therefore, to understand these microbial activities, redox potential in the soil must be measured by depth. In most well-drained soils, an oxic layer [75–100% of air saturation] typically occupies the upper ~ 0–5 cm, a suboxic transition zone [10–75% of air saturation] extends roughly ~ 5–15 cm, and more strongly reducing (often anoxic) conditions [< 5% of air saturation] prevail below ~ 15 cm 12 . Soil texture, moisture, available alternative electron donors, and dissolved organic matter loading can shift these boundaries 13 – 16 . In wet, high organic loaded or flooded soils, the oxic zone may shrink to just a few millimeters at the surface, with persistent anoxia throughout the profile beneath. On the other hand, coarse, dry soils or root-oxygenated rhizospheres can maintain oxic zone down to ~ 20–30 cm. These depth-structured redox gradients govern the onset of denitrification, Fe/Mn reduction, sulfate reduction, and methanogenesis with depth 17 , 18 . Redox potential reflects the equilibrium ratio between oxidized and reduced species according to the Nernst equation. In parallel with redox potential measurements, cyclic voltammetry (CV) can provide complementary electrochemical information about soil systems. In addition, CV allows the characterization of active redox processes rather than equilibrium states provided by the Nernst equation. By sweeping the potential over a defined range and measuring the resulting current response, CV can identify redox-active species, quantify their electrochemical reversibility, and provide insight into electron-transfer kinetics at soil–electrode interfaces. From the shape and position of anodic and cathodic peaks, one can infer parameters such as formal redox potential, peak current, and reaction reversibility. Additionally, CV can help distinguish between diffusion-controlled and surface-confined reactions, detect catalytic redox cycles mediated by microbes, and reveal electron shuttle behavior of humic substances or mineral phases in soil. Thus, CV offers a dynamic and mechanistic understanding of redox-active components that complement static redox potential measurements. In addition to cyclic voltammetry, chronoamperometry (CA) can be employed to monitor the activity of soil biofilms grown on electrode surfaces. Chronoamperometry is an electrochemical characterization technique that measures current by applying a constant potential over time. In this technique, biofilms are cultivated directly on microelectrode tips by maintaining a constant applied potential, allowing electroactive microorganisms to colonize the surface. These electrode-grown biofilms have been used to quantify metabolic substrates such as acetate and fumarate concentrations 19 , 20 . Building on this approach, Babauta et al. demonstrated that such biofilms could also be used to measure local current as a function of depth, revealing microscale electrochemical activity gradients 21 . Electrode-grown, electrochemically active biofilms thus offer a means to infer local nutrient availability and redox dynamics in soil microenvironments. However, to date, such measurements have not been realized in soil systems due to the absence of appropriate instrumentation capable of high-resolution electrochemical profiling in situ . As detailed above, measuring redox gradients and electrochemical behavior (CV and CA) in soil is critical for understanding microbial respiration, nutrient cycling, and greenhouse gas production, yet in situ quantification remains limited by limited sensing technology and the lack of integrated instrumentation. Accurate measurement of redox potential and CV in soil requires a sensor, an electrochemical control unit to apply potential sweeps and record current responses (potentiostat), a positioning system to move the sensor to defined depths, and integration of these elements into a unified automated platform. Traditionally, soil redox potential is measured with platinum (Pt) electrodes calibrated using Zobell’s solution containing a known ferri/ferrocyanide redox couple; the potential is measured against a known reference electrode (e.g., Ag/AgCl) as an open-circuit potential representing the redox potential in the soil. While such measurements indicate the oxidative/reductive balance, they reveal little about the identity or kinetics of the redox-active species involved. CV complements this by resolving oxidation and reduction peaks corresponding to specific electroactive components, providing additional information about electron-transfer kinetics, reaction reversibility, and microbially mediated redox processes. Chronoamperometry is another tool that can measure current by applying a constant potential over time and enrich microbes in soil while doing so. Despite their potential, existing commercial systems (e.g., Unisense, Mettler Toledo) are limited to shallow depths, manual operation, and single-function measurements, preventing comprehensive depth-resolved electrochemical profiling. To overcome above limitations, our goal is to develop a custom-built automated manipulator capable of acquiring redox potential and CV data across soil depths up to 27 cm. Our system integrates a Pt working electrode, a custom-made Ag/AgCl reference, and a counter electrode with a programmable potentiostat and automated custom-made vertical positioning, enabling low-cost, high-resolution characterization of soil redox dynamics and microbially driven electron-transfer processes. Using off-the-shelf components, we constructed a Soil Electrochemical Profiling System (SEPS) capable of automated operation to depths of up to 27 cm with a minimum positioning resolution of 25 µm. The SEPS integrates directly with a potentiostat, allowing the use of multiple electrochemical techniques including redox potential measurement, CV, and chronoamperometry. We validated SEPS by generating depth-resolved redox potential profiles and CVs in control samples as well as in soil and sediment reactors. To further demonstrate its CA capability for biological studies, we polarized a microelectrode at a fixed depth over a 40 days enrichment period, which revealed clear evidence of microbial electrochemical activity. Using biofilms developed on this microelectrode, we subsequently measured current depth profiles in soil - to our knowledge, the first such measurement reported in the literature. 2. METHODS 2.1 Construction of Soil Electrochemical Profiling System The Soil Electrochemical Profiling System (SEPS) was assembled using commercially available components integrated via the RAMPS 1.4 kit (Hilitand, Wuhan, China, ASIN B07DFKC3RF, Amazon), which connects to an Arduino Mega 2560 microcontroller to enable a compact motion-control system for driving a stepper motor and reading limit switches (Fig. 1 ). The system includes a linear stage actuator with a stepper motor, RAMPS 1.4 motor driver shield, A4988 stepper driver, mechanical limit switches, a 12 V DC power supply, and all necessary wiring and connectors. Custom 3D-printed mounts and acrylic holders were fabricated in-house to secure the electrodes and potentiostat leads during operation (Fig. 1 ). The core components of the system consist of 1) a motor assembly for precise microelectrode positioning, 2) a Gamry 1000E potentiostat for electrochemical measurements such as open-circuit potential which we called as redox potential in this manuscript, cyclic voltammetry (CV), and chronoamperometry (CA), and 3) custom software that integrates motion control, data acquisition, and potentiostat operation into a single automated platform. A schematic overview and instrumentation workflow are shown in Fig. 1 . All components used for constructing the SEPS were obtained from commercial suppliers and are listed in the Supporting Information (Table SI1). The motor driver board (RAMPS 1.4) was mounted directly onto the Arduino Mega 2560 controller board. Wiring and configuration followed the official RAMPS 1.4 documentation 22 . Two “ endstop ” mechanical switches were installed on the y-min and y-max ports of the RAMPS 1.4 board to limit the linear stage actuator (Fig. 1 ). The y-min switch was positioned on the motor-side (top) end of the stage, and the y-max switch was mounted on the opposite end (bottom), ensuring that actuator motion automatically ceased at both limits. Motion control was achieved using an A4988 stepper-motor driver installed on the x-axis header of the RAMPS board (Fig. 1 ). Prior to mounting, three jumpers were inserted to enable microstepping with 1/16-step, providing the precise positioning resolution. The linear actuator’s stepper motor was wired using male-to-female extensions soldered to the four motor leads. Power was supplied by a 12 V DC regulated unit. After completing the wiring, the Arduino IDE software 23 was installed. The StepperDriver v1.4.1 library (Laurentiu Badea) was added by using the Arduino IDE Library Manager. The RAMPS library was downloaded from github.com/momostein/Ramps and installed using Sketch → Include Library → Add .ZIP Library, selecting the file Ramps-master.zip. The control code that manages control signal between Arduino and the motor driver, “Demo_Stepper.ino,” was opened. After compilation and verification, the board type (Arduino Mega 2560) and correct COM port were selected under Tools, and the code was uploaded. Once programmed, the SEPS was ready for control through custom Python software for automated electrochemical profiling. 2.1.1 Custom Python Software for System Control and Integration A custom Python-based software package was developed to coordinate all operations of the SEPS, integrating motion control of the microelectrode manipulator with electrochemical measurements performed by a Gamry potentiostat. The software functions as a unified control interface, providing synchronized automation of electrode positioning and data acquisition across varying soil depths. The program establishes serial communication with the Arduino Mega 2560 microcontroller, which manages the stepper motor via a RAMPS 1.4 shield and A4988 driver module. Through user-defined parameters, the software sends G-code-style commands to move the microelectrode vertically in user-determined increments. A homing routine is included to reference the initial position using mechanical limit switches before each profiling sequence begins. In parallel, the software interfaces with the Gamry potentiostat using the GamryCOM API. It supports two electrochemical techniques: OCP and CA (Fig. 2 B and 2 C). The user selects the desired method, and the software automatically configures and initiates the corresponding measurement protocol. Collected data is saved locally in structured files for post-analysis. CV is measured separately using Gamry’s stock program because depth-profile CV measurements do not require synchronized electrode positioning during data acquisition. For OCP and CA depth profiles, the response time between measurement points is critical for obtaining accurate profiles. In contrast, CV has a relatively long and fixed acquisition time determined by the initial parameter settings. We also chose to run CV manually because, after each scan, the user may need to adjust parameters such as the scan rate, initial and final potentials, and scan limits. The workflow is fully automated. The software initiates a measurement at the current depth, waits for completion, logs the data, moves the electrode to the next depth, and repeats the cycle until the profiling sequence is complete (Fig. 2 and Figure SI1A). Timing synchronization, device status monitoring, and error handling are all managed within the Python environment. A graphical user interface (GUI) built with PyQT allows users to set parameters (step size, number of steps, delay time, technique type), monitor real-time progress, and manually trigger actions such as homing, start, and abort (Figure SI1). The interface is designed to be intuitive and adaptable for future extensions, such as multi-electrode support or remote operation. To measure redox potential, the user selects the OCP mode and specifies the starting position, ending position, total number of steps, step size, and delay time between measurements. The software acquires OCP readings from Gamry at each depth and reports it as a plot in the bottom left portion of the GUI (Fig. 2 B). In manual mode, the user adjusts the microelectrode position using the “ Move Up ” or “ Move Down ” button based on the step size the user has set. It can also move to a specific location by setting the target position along the length of the manipulator (0-270 mm) and pressing the “ Go to Position ” button. For automated operation, the user defines the starting and ending depths, step size, and delay parameters; the software then controls the microelectrode movement and data acquisition accordingly. The same workflow applies to CA measurements, with the additional requirement that the user enters an applied potential. CA data can also be collected manually or automatically. In contrast, CV measurements are supported only in manual mode because it does not require synchronized electrode positioning during data acquisition and require frequent parameter adjustments, where the user sets the parameter and initiates each CV scan on Gamry’s stock program (Gamry Framework) at the desired depth. 2.2 Microelectrodes 2.2.1 Pt-Tipped Microelectrodes We followed the fabrication procedures described by Atci et al. 24 to construct Pt-tipped microelectrodes, introducing several modifications to improve their mechanical durability for use in soil environments. The original design was intended for biofilms, which are significantly softer than soil; therefore, adjustments were necessary to prevent breakage during insertion and profiling. A glass Pasteur pipette was used to achieve a longer (~ 10cm of length available for soil application insertion) and sturdier electrode. The reference electrode’s outer casing was fabricated from the widened section of the pipette and positioned concentrically around the Pt microelectrode shaft. An Ag/AgCl wire was inserted at the junction between the inner and outer casings and sealed using 5-minute epoxy, which was cured for 24 hours 24 . The outer casing was then filled with saturated KCl solution (Potassium Chloride, 4 M, saturated with Silver Chloride, Electrode filling Solution, RICCA Chemical Company, Arlington, TX, USA, Cat. No. 5920-16), and its open end was sealed with a molecular sieve disc (Molecular Sieve 4 Å, Consolidated Chemical & Solvents LLC, CAS 70955-01-0). This modification prevented soil particles from penetrating into and contaminating the internal chamber during depth-profiling while maintaining ionic conductivity with the surrounding medium. A 100 µm platinum wire (California Fine Wire Company, Grover Beach, CA, USA) was affixed to the outer casing using heat-shrink tubing as counter electrode for CV or CA measurements. After the final sealing, the assembly was cured for another 24 hours and stored in saturated KCl solution inside 15 mL Falcon tubes. Prior to each experiment, the electrodes were validated in Zobell’s solution by performing OCP or CV measurements to confirm functionality and assess the electroactive surface area. 2.2.2 Carbon-Tipped Microelectrodes In addition to the Pt-tipped microelectrodes, a second type of microelectrode was fabricated using a graphite rod as the sensing element. The construction process followed the same protocol described by Atci et al. 24 , and Babauta et al 25 with the only modification being the substitution of the platinum wire with a graphite rod of 500 µm diameter. The graphite was pulled into a glass Pasteur pipette, identical to the method used for the Pt version. This carbon-based microelectrode was specifically designed for experiments involving biofilm growth on the tip, where CA and depth-resolved CA were the primary objectives. Due to its biocompatibility for biofilm growth and electron transfer properties for microbial systems, carbon was selected as a more suitable material than platinum. 2.3 Soil Electrochemical Profiling System Verification and Sample Types To validate the operation of the SEPS, two types of samples were used. The first was a control sample consisting of a hydrogel matrix made from xanthan gum, designed to simulate the soft materials. This matrix was in contact with a bulk Zobell solution containing the ferri/ferrocyanide redox couple. Over time, the redox species diffused from the bulk solution into the hydrogel, allowing us to monitor changes in redox chemistry both in the bulk solution and within the matrix using OCP and CV. The second sample type consisted of soil mesocosm prepared with garden soil (GPS: 46.7211° N, 117.1722° W) and DI water. Using these two distinct systems, one controlled and synthetic, the other representative of real-world conditions, we verified the SEPS's capability to perform automated electrochemical measurements, including OCP, CV, and CA, in both idealized and complex environments. 2.3.1 Xanthan Gum Hydrogel System for Monitoring Redox Potentials and Cyclic Voltammograms Over Time and Depth To evaluate the SEPS system's ability to measure redox gradients, we designed an experiment using a xanthan gum hydrogel matrix and Zobell solution to simulate time- and depth-dependent electrochemical changes. The concentration of xanthan gum (Namaste Foods, LLC, Coeur d’Alene, ID, USA) was optimized to achieve a consistency that was neither too fluid (which would allow mixing with the overlying solution) nor too viscous (which would trap air bubbles and prevent the formation of a flat surface). A concentration of 15 g/L in 0.1 M KCl was found to meet these conditions. One liter of hydrogel was prepared by slowly adding the xanthan gum powder to the heated and stirred KCl solution. The mixture was then autoclaved at 121 o C for 15 minutes to improve homogeneity and degassed under vacuum at − 25 psi to eliminate residual air bubbles. While still hot, the hydrogel was poured into a 600 mL beaker up to the 500 mL mark and was allowed to cool overnight, forming a smooth, bubble-free surface. Zobell solution, containing 3.33 × 10 − 3 M potassium ferrocyanide (K₄Fe(CN) 6 ·3H 2 O), 3.33 × 10 − 3 M potassium ferricyanide (K 3 Fe(CN) 6 ), and 0.1 M potassium chloride (KCl), was prepared by dissolving the components in deionized water. The hydrogel was topped with Zobell solution carefully poured along the beaker wall using a 50 mL Falcon tube to avoid mixing; the liquid layer was maintained at approximately 3 cm above the hydrogel surface. Right after adding the Zobell solution, depth-resolved redox potential profiles were recorded using the SEPS. The microelectrode was initially positioned manually so that its tip was ~ 2 cm above the hydrogel surface. The system was then programmed to automatically measure OCP from the top of the solution down to 55 mm into the hydrogel, with 1 mm step intervals and a 5-second delay between each measurement. These profiling measurements were conducted at 0, 6, 12, and 24 hours to capture the progression of redox couple diffusion over time. After completing the OCP scan, the microelectrode was retracted and repositioned at a new location on the hydrogel surface, at least 1 cm laterally from the previous measurement site to minimize disturbance. To verify the long-distance measurement capability of the SEPS, a column consisting of 15 cm of Zobell solution over 25 cm of xanthan gum hydrogel was prepared in a graduated cylinder. Redox potentials were then measured over the 27 cm depth, its maximum range, at 1 cm intervals. CV was then conducted at depths of + 2 cm, + 0.5 cm, -1 cm, -2.5 cm, -4 cm, and − 5.5 cm (relative to the hydrogel surface level) using a scan rate of 10 mV/s over a potential range of − 300 mV to + 600 mV Ag/AgCl , with three consecutive cycles performed at each depth. 2.3.2 Soil Mesocosm and Measurements Garden soil was homogenized for at least 1 minute using a food processor (Spectrum Brands / Black+Decker, 120 V AC, 450 W, model FP1600B) and then sieved through a 1 mm mesh. The sieved soil was added to a 600 mL beaker until it reached the 450 mL mark. Deionized water was gradually added while mixing the soil with a spatula to ensure full saturation. As the soil settled, additional soil was added to restore the height to the 450 mL mark. Once the soil level was stable, deionized water was added to create a 3 cm water column above the soil surface. This soil mesocosm was allowed to equilibrate before beginning measurements. The same procedures used in the hydrogel experiments were followed for OCP and CV measurements. However, in this case, measurements were taken at time points of 0, 7, 14, and 21 days. Additionally, the cyclic voltammetry scan range was extended to a scan range of -600 mV Ag/AgCl to + 600 mV Ag/AgCl , compared to the scan range of -300mV Ag/AgCl to + 600 mV Ag/AgCl used in the hydrogel setup. The carbon-tipped microelectrode was used to enrich biofilm at its tip over a period of 40 days by polarizing it to + 300 mV Ag/AgCl inside a soil mesocosm. Following biofilm formation, the same microelectrode was employed to measure depth-resolved current profiles by maintaining the polarization at + 300 mV Ag/AgCl and using CA to capture the electrochemical activity associated with the biofilm at the electrode tip. 2.3.3 Declaration of generative AI and AI-assisted technologies in the manuscript preparation process During the preparation of this manuscript, the authors used generative artificial intelligence tools solely to assist with editing and improving the clarity and readability of the text. No figures (except part of Fig. 1 ), images, or data were generated using AI tools. All content was reviewed and edited by the authors, who take full responsibility for the accuracy, integrity, and originality of the published work. 3. RESULTS AND DISCUSSION Using the SEPS, we quantified redox potential profiles in a hydrogel-Zobell solution system as a function of both depth and time. This was followed by CV measurements to monitor electrochemical changes over time and at different depths. To validate the full profiling capability of the system, measurements were extended across the entire operational length (27 cm), confirming functionality of the SEPS over the intended depth range. After completing tests in these well-controlled systems, we used the SEPS for soil mesocosms. First, we measured redox profiles. Following this, biofilms were first enriched on the tip of a carbon microelectrode using CA. The same microelectrode grown biofilm was then used to perform depth-resolved current measurements to assess the electrochemical activity of the surface-associated biofilms in soil. 3.1 Monitoring Redox Potentials Over Time and Depth of a Controlled Hydrogel System in the Presence and Absence of Redox Couple 3.1.1 Absence of redox-active compounds yields uniform redox profiles with time-dependent shifts In the absence of redox-active compounds, redox potentials remained uniform with depth while changing over time (Fig. 3A). At the start of the experiment, the redox potential was approximately 280 mV Ag/AgCl in both the bulk solution and throughout the hydrogel, resulting in a flat depth profile, as expected under non-reactive conditions. After 24 hrs, the redox potential increased to ~ 420 mV Ag/AgCl , while the profile remained linear and depth-independent. The time-dependent increase in redox potential is attributed to changes in ambient conditions, including a decrease in room temperature and re-equilibration between dissolved oxygen in the liquid phase and oxygen from the air. The persistence of a flat redox profile across the entire depth confirms that, in the absence of redox-active species, the SEPS system reliably measures uniform redox potentials without introducing spatial artifacts. 3.1.2. Redox-active compounds generate time- and depth-dependent redox gradients In the presence of the ferricyanide/ferrocyanide redox couple, pronounced redox potential gradients developed with both depth and time (Fig. 3B). Zobell solution, which has a redox potential of ~ 223 mV Ag/AgCl , was initially confined to the bulk phase. At time zero, the bulk solution exhibited a nearly uniform redox potential of ~ 237 mV Ag/AgCl , while the hydrogel showed a pre-existing oxygen-driven gradient, with a maximum of ~ 296 mV Ag/AgCl at 15 mm depth that decreased to ~ 273 mV Ag/AgCl at the bottom. After 6 hrs, the bulk redox potential increased to ~ 253 mV Ag/AgCl , indicating diffusion of Zobell into the system. Concurrently, the hydrogel redox potential decreased to ~ 232 mV Ag/AgCl at ~ 24 mm depth, consistent with penetration of the ferricyanide/ferrocyanide couple. By 12 hrs, the bulk potential further increased to ~ 256 mV Ag/AgCl , and the redox minimum within the hydrogel shifted deeper to ~ 32 mm with a potential of ~ 236 mV Ag/AgCl . After 24 hrs, the bulk redox potential reached ~ 261 mV Ag/AgCl . The redox trough within the hydrogel progressed to ~ 44 mm depth and stabilized near ~ 237 mV Ag/AgCl . The progressive deepening of this trough, coupled with its convergence toward the Zobell redox potential, reflects continued diffusion and dilution of the ferricyanide/ferrocyanide redox couple within the hydrogel over time. Figure 3. Redox potential profiles in the hydrogel–Zobell system. A) In the absence of redox-active compounds, redox potentials remain uniform with depth but vary over time. B) Addition of Zobell solution induces time- and depth-dependent redox gradients in the hydrogel, measured at 0, 6, 12, and 24 hrs. C) A full-depth (27 cm) profile measured immediately after Zobell addition captures large-scale redox changes but lacks the spatial resolution shown in panel B. 3.1.3 Large step sizes capture broad redox trends but obscure fine spatial gradients To evaluate the maximum depth-profiling capability of the SEPS system, redox potential was measured across a 27 cm depth at 1 cm intervals (Fig. 3C). Within the Zobell solution layer, the redox potential remained relatively constant at ~ 227 mV Ag/AgCl . Upon entering the hydrogel, the potential increased abruptly to ~ 238 mV Ag/AgCl and then gradually rose to ~ 257 mV Ag/AgCl toward the bottom of the hydrogel, consistent with oxygen diffusion into the matrix. Because this profile was acquired immediately after Zobell solution addition, diffusion of ferricyanide/ferrocyanide into the hydrogel was minimal. Consequently, the measurement captures large-scale redox transitions across phases but lacks the spatial resolution required to resolve fine redox gradients within the hydrogel, in contrast to the higher-resolution profiles shown in Fig. 3B. 3.2 Monitoring Cyclic Voltammograms over Time and Depth 3.2.1 Hydrogel-water system oxygen reduction kinetics In the hydrogel–water system, oxygen was the only electrochemically active species present. Accordingly, the cyclic voltammograms primarily reflect oxygen reduction kinetics, with minor background contributions likely originating from impurities associated with xanthan gum used (Figure SI3). The voltammograms remained qualitatively consistent over time, indicating stable electrochemical conditions in the absence of added redox-active compounds 3.2.2 Ferricyanide/ferrocyanide system reveals diffusion-driven spatial and temporal electrochemical gradients Cyclic voltammetry of the hydrogel–Zobell system is shown in Fig. 4, with additional datasets provided in Figures SI1 and SI2. In the absence of Zobell solution, voltammograms remained unchanged over time, confirming the lack of electrochemically active species (Figure SI1A–F). Following Zobell addition, distinct spatial and temporal changes in electrochemical behavior were observed at 0, 3, 6, 12, and 24 hrs (Figure SI2G–L), consistent with diffusion of the ferricyanide/ferrocyanide redox couple into the hydrogel. To illustrate the depth-profiling capability of cyclic voltammetry, representative voltammograms collected at three depths (+ 2 cm, -1.0 cm, and − 5.5 cm) after 24 hrs are shown in Fig. 4A. The shallowest position (+ 2 cm) exhibited the largest and most well-defined redox waves, indicating higher local availability of redox-active species near the hydrogel surface. At -1.0 cm, peak currents decreased and the voltammogram narrowed, suggesting a transition zone where mass transport increasingly limits the electrochemical response. At -5.5 cm, the voltammogram showed minimal peak structure and reduced currents, reflecting limited penetration of redox species and slower diffusion at depth. The systematic attenuation of current magnitude and peak definition with depth demonstrates the presence of sharp vertical redox gradients that can be directly resolved using cyclic voltammetry. Temporal evolution of the electrochemical environment is illustrated by voltammograms collected at 1 cm depth at 0, 12, and 24 hrs (Fig. 4B). At 0 h, the voltammogram exhibited modest currents, consistent with background electrochemical activity prior to significant diffusion of the redox couple. After 12 hrs, both anodic and cathodic peak currents increased and the response broadened, indicating the combined influence of oxygen and partially diffused ferricyanide/ferrocyanide. By 24 hrs, the voltammogram displayed more defined redox features, albeit with reduced peak magnitude, consistent with dilution of the redox couple as diffusion progressed. These results demonstrate that cyclic voltammetry sensitively captures time-dependent restructuring of the local electrochemical environment and provides mechanistic processes into redox species transport and distribution within the hydrogel matrix. Figure 4. Cyclic voltammograms measured in the hydrogel–Zobell system. A) Depth-dependent voltammograms collected 24 hrs after Zobell solution addition at 2 cm above the hydrogel surface (bulk solution) and at 1 cm and 5.5 cm below the surface, illustrating attenuation of electrochemical activity with depth. B) Time-dependent voltammograms collected at 1 cm depth at 0, 12, and 24 hrs, showing the evolution of local redox behavior following diffusion of the ferricyanide/ferrocyanide couple. 3.3 Monitoring Redox Gradients by Depth and Time in Soil Mesocosm Reactors 3.3.1 Redox potential profiles Redox potential profiles in the soil mesocosm reactors exhibited a progressive shift toward lower values over time, indicating a transition to increasingly reducing conditions (Fig. 5 ). Such temporal and depth-dependent redox gradients are characteristic of saturated soils, where microbial respiration sequentially consumes available electron acceptors as oxygen diffusion becomes limited. In the standing water above the soil surface, redox potential decreased gradually from approximately + 215 mV Ag/AgCl at week 0 to + 200, +190, and + 105 mV Ag/AgCl at weeks 1, 2, and 3, respectively. This decline reflects reduced oxygen availability in the overlying water column as microbial and chemical oxygen demand increased, consistent with observations reported for flooded soils and wetland systems. At week 0, the redox profile within the soil remained relatively uniform at approximately + 350 mV Ag/AgCl down to ~ 27 mm below the surface, indicating well-oxidized conditions shortly after reactor setup. Increased signal variability below this depth is likely attributable to trapped air pockets within the soil matrix following water addition, a common artifact during early saturation stages. This setup also simulates post-tillage soil aeration, during which oxygen is temporarily introduced into the soil profile. By week 1, a pronounced redox gradient had developed. Redox potential decreased sharply from ~ + 200 mV Ag/AgCl at the soil surface to ~ + 95 mV Ag/AgCl within the upper 7 mm and remained near this value at greater depths. This rapid downward shift is consistent with oxygen depletion driven by aerobic microbial respiration, a process widely reported as the first stage in the establishment of reducing conditions in saturated soils. By weeks 2 and 3, the profiles converged toward substantially lower redox potentials, reaching approximately + 30 mV Ag/AgCl at ~ 11 mm depth. The week 2 profile remained relatively constant below this depth, whereas the week 3 profile exhibited an additional gradual decrease of ~ 20 mV with depth, suggesting continued progression toward more reduced conditions. These trends are consistent with the onset of anaerobic processes such as nitrate, manganese, and iron reduction, which typically occur after oxygen has been depleted and redox potentials fall below ~ + 200 mV Ag/AgCl . Overall, the measured profiles capture the expected spatiotemporal evolution of redox conditions in water-saturated soils and demonstrate the capability of the SEPS system to resolve dynamic redox gradients at millimeter-scale resolution over extended time periods. 3.4 Monitoring Cyclic Voltammograms by Depth and Time in Soil Mesocosm Reactors 3.4.1 Depth- and time-dependent evolution of soil redox activity revealed by cyclic voltammetry Cyclic voltammetry revealed pronounced depth- and time-dependent evolution of electrochemical activity within the soil mesocosm reactors (Fig. 6 and Figure SI3), consistent with the redox potential gradients observed by redox potential measurements (Fig. 5 ). Unlike redox profiles, which report the dominant redox state at a given depth, cyclic voltammetry provides dynamic information on electron-transfer processes, including the presence, intensity, and reversibility of redox reactions occurring within the soil matrix. At positions above the soil surface (+ 2 cm and + 0.5 cm), the voltammograms were dominated by oxygen reduction, characterized by strong cathodic currents at negative potentials and the absence of corresponding anodic peaks (Fig. 6A). This behavior indicates an oxygen-rich environment where electrochemical activity is controlled primarily by dissolved oxygen rather than reversible redox couples. This observation is consistent with the relatively high redox potentials measured in the overlying water layer in Fig. 5 . In contrast, voltammograms collected below the soil surface (− 1 cm, − 2.5 cm, − 4 cm, and − 5.5 cm) exhibited a progressive emergence of paired anodic and cathodic features over time, reflecting the development of active redox processes within the soil. At week 0, voltammograms at these depths showed low current magnitudes and weak structure, indicating limited faradaic activity immediately after reactor setup. By week 1, distinct anodic-cathodic responses became apparent, coinciding with the sharp decline in redox potential observed in Fig. 5 and consistent with rapid oxygen depletion and the onset of anaerobic conditions. As incubation progressed through weeks 2 and 3, the redox features increased in magnitude and definition (Fig. 6B), indicating enhanced electron-transfer activity and a growing contribution from reduced soil constituents. Unlike the well-defined peaks associated with single reversible redox couples in homogeneous electrolytes, the broadened and asymmetric voltametric features observed here reflect the superposition of multiple overlapping redox processes, including microbially mediated and mineral-associated reactions. Such behavior is characteristic of natural soils, where redox chemistry is governed by a complex mixture of organic matter, metal oxides, and microbial electron-transfer pathways. The depth dependence of the CV response closely mirrors the vertical redox stratification measured by redox profiling (Fig. 5 ), with stronger and more complex voltametric signatures observed at greater depths where redox potentials were lowest. Together, these results demonstrate that cyclic voltammetry complements redox potential measurements by resolving not only where redox gradients exist, but also how actively electrons are exchanged within each soil layer. This combined approach enables sensitive, in situ tracking of dynamic biogeochemical processes and demonstrate the utility of SEPS for characterizing spatially and temporally evolving soil redox environments. Figure 6. Cyclic voltammograms measured in the soil mesocosm reactor. A) Depth-dependent voltammograms collected at week 1, showing oxygen-dominated electrochemical behavior above the soil surface and the emergence of redox activity within the soil. B) Time-resolved voltammograms collected at 5.5 cm depth over four weeks, illustrating the progressive development of soil redox processes under increasingly reducing conditions. Figures 3–6 together define a coherent model of redox evolution driven by diffusion, stratification, and biological activity. In controlled hydrogels, uniform redox profiles persist in the absence of redox-active species, while introduction of a defined redox couple produces predictable, diffusion-controlled gradients that deepen over time. In soil mesocosms, similar principles apply, but redox gradients emerge from oxygen depletion and microbial respiration, leading to vertically stratified and increasingly reducing environments. Redox potential profiling captures the spatial structure of these gradients, whereas cyclic voltammetry reveals their dynamic electrochemical activity. Combined, these measurements demonstrate that complex soil redox behavior can be interpreted through the same mechanistic framework established in controlled systems. By integrating these complementary measurements across depth and time, SEPS uniquely enables a mechanistic interpretation of complex redox environments that cannot be obtained using conventional, single-point redox measurements. 3.5 Biofilm Enrichment at the Microelectrode Tip and Chronoamperometric Measurements 3.5.1 Chronoamperometry reveals electroactive biofilm enrichment on polarized microelectrodes To enable long-term and stable enrichment of electroactive biofilms, the soil mesocosm was operated as a wick system rather than a fully flooded system, allowing improved control of soil moisture over extended incubation periods. Carbon-tipped microelectrodes were inserted 7 cm below the soil surface and continuously polarized at + 300 mV Ag/AgCl for 40 days (Fig. 7A). Under these conditions, the measured current reflects electron transfer from microorganisms capable of donating electrons to the electrode and, thus, serves as a proxy for electroactive biofilm growth 26 , 27 . The chronoamperometric response exhibited a characteristic rise, plateau decline pattern consistent with microbial enrichment and nutrient depletion dynamics. During the initial several days, current remained low, indicating adaptation, minimal electroactive biomass and limited extracellular electron transfer. Beginning around Day 5, current increased steadily, exceeding 300 nA by approximately Day 20. This increase corresponds to the enrichment of microorganisms able to utilize the polarized microelectrode tip as a terminal electron acceptor, supported by the availability of electron donors in the surrounding soil, such as reduced organic compounds, fermentation products, or reduced metal species. Between Days 20 and 30, the current remained elevated, indicating the establishment of a stable and metabolically active electroactive biofilm. This sustained anodic current suggests that the local soil environment was sufficiently reduced to favor electrode respiration, a condition commonly associated with diffusion-limited soils. After approximately Day 30, the current declined sharply, likely reflecting changes in soil biogeochemical conditions, including depletion of readily available electron donors, accumulation of inhibitory metabolites, or shifts in microbial community composition 26 . Such temporal patterns are consistent with redox cycling and substrate turnover in natural soil systems. Overall, these results demonstrate that chronoamperometry provides a sensitive, real-time measure of electroactive biofilm development and reflects evolving redox conditions in soil. 3.5.2 Microelectrode-grown biofilms detect current fluctuations in soil and respond to distant electrochemical changes Depth-resolved chronoamperometric measurements performed with the 40-day-enriched microelectrode biofilms revealed vertical structuring of electrochemical activity within the soil (Fig. 7B). Under constant polarization, the measured anodic current reflects the rate of microbial electron transfer to the electrode and is therefore governed by the local availability of electron donors and their transport to the biofilm-electrode interface 28 . Immediately above the soil surface, measured currents were relatively high (~ 8–10 nA), consistent with conditions that support biofilm respiration due to greater availability of diffusible electron donors. Upon entry into the soil, the current decreased sharply, indicating a rapid transition in redox conditions and electron-donor availability near the microelectrode tip. Between approximately 5 and 25 mm below the soil surface, the current exhibited substantial variability (∼1–10 nA), reflecting fine-scale heterogeneity in microbial activity, substrate distribution, and local redox microenvironments. Below ~ 25–30 mm, currents stabilized at lower values (< 3 nA), consistent with a deeper, more strongly reduced zone where electron-donor supply is limited by slow diffusion and depleted electron donor pools. This depth-dependent pattern-high current near the surface, a sharp transition zone, and a relatively uniform reduced region at depth-is consistent with established models of redox stratification in saturated soils 17 . Importantly, the persistence of anodic current throughout the depth profile confirms that the enriched biofilm on the microelectrode remained metabolically active and responsive to spatial variations in soil redox conditions. Notably, anodic current was detected even near the soil surface, where competing soluble electron acceptors would typically expect to suppress electrode respiration 25 . The relatively higher anodic currents observed across all soil depths suggest a system dominated by electron-donor availability (e.g., organic carbon) with limited availability of alternative terminal electron acceptors 29 , 30 . Together, these results demonstrate that biofilm-functionalized microelectrodes function as sensitive, living sensors capable of resolving fine-scale vertical transitions in soil biogeochemistry and detecting electrochemical responses to subsurface redox heterogeneity 20 , 21 . Figure 7. Chronoamperometric characterization of microelectrode biofilm enrichment and depth-resolved current measurements in soil. A) Current response of a carbon microelectrode polarized at + 300 mV Ag/AgCl and placed 7 cm below the soil surface over 40 days, illustrating electroactive biofilm enrichment. B) Depth-resolved chronoamperometric measurements using the biofilm-enriched microelectrode at the same applied potential, revealing vertical structuring of electrochemical activity within the soil. 3.6 Integrated Interpretation of Soil Redox Dynamics Using Redox Potential, Cyclic Voltammetry, and Chronoamperometry Sections 3.3 – 3.5 together demonstrate how combining redox potential, cyclic voltammetry, and chronoamperometry provides a multi-modal view of soil redox dynamics that cannot be obtained from any single technique. Redox potential measurements resolve the spatial and temporal structure of redox gradients, revealing the progressive development of vertically stratified and increasingly reducing conditions 26 . Cyclic voltammetry complements this by identifying the emergence and evolution of faradaic redox activity within these gradients, reflecting the onset and intensification of coupled microbial and geochemical electron-transfer processes. Chronoamperometry extends this framework by directly tracking sustained electron flux to a polarized microelectrode, linking redox structure and activity to biofilm-mediated respiration. Together, these measurements connect redox state, redox activity, and electron flux, providing an integrated and mechanistically grounded picture of soil redox processes. 4. CONCLUSIONS We demonstrated development, validation, and application of a custom Soil Electrochemical Profiling System (SEPS) designed to resolve depth-dependent electrochemical behavior in soils, hydrogels, and controlled redox systems. By integrating automated motion control with multi-modal electrochemical measurements, SEPS enables high-resolution interrogation of spatially and temporally evolving redox environments that are difficult to capture using conventional approaches. We have concluded that: The SEPS successfully performed automated electrochemical depth profiling to 27 cm with micrometer-scale positional resolution (25 µm), demonstrating reliable integration of mechanical positioning and potentiostat control. Depth-resolved redox potential measurements in hydrogel–Zobell systems confirmed that SEPS can capture fine-scale redox gradients and track the diffusion of well-defined redox couples (ferricyanide/ferrocyanide) over time, providing a controlled benchmark for interpreting transport-limited redox systems. Cyclic voltammetry measurements in hydrogels further demonstrated that SEPS resolves spatial and temporal changes in redox-active species, revealing systematic depth and time dependent evolution of faradaic electrochemical behavior. In soil mesocosms, SEPS detected strong vertical redox stratification, capturing the transition from oxic surface conditions to progressively reduced subsurface zones as incubation proceeded, consistent with established models of soil redox evolution. Time-resolved cyclic voltammetry in soils revealed the gradual emergence and intensification of redox activity, reflecting the development of coupled microbial and geochemical electron-transfer processes within the soil matrix. Chronoamperometry enabled the enrichment of electroactive biofilms on microelectrode tips; when used for depth profiling, these biofilm-functionalized electrodes resolved clear vertical patterns in microbial electron-transfer activity, providing indirect but mechanistically meaningful evidence of electron donor and acceptor availability and biogeochemical heterogeneity in soils. Collectively, these results demonstrate that SEPS uniquely integrates redox state (redox potential), redox activity (cyclic voltammetry), and electron flux (chronoamperometry) into a single, automated, depth-resolved platform. This multi-modal capability enables mechanistically grounded, in situ investigation of soil redox dynamics and microbially driven processes that cannot be obtained from single-point or single-technique measurements. Beyond soil systems, the flexibility and scalability of SEPS make it broadly applicable to studies of sediments, wetlands, biofilms, and engineered porous media, offering new opportunities to link redox structure, activity, and function across complex environmental systems. Declarations 5. FUNDING DECLARATION This work was supported by the National Science Foundation (NSF), USA under Grant No. 2226680. 6. AUTHOR CONTRIBUTIONS Haluk Beyenal conceptualized and designed the research. Ibrahim Bozyel developed the device and its operation software. Won-Jun Kim performed the data curation. Both Won-Jun Kim and Haluk Beyenal conducted data analysis and wrote the original draft. Won-Jun Kim, Ibrahim Bozyel, Suat Ay, Maren Friesen, and Haluk Beyenal contributed to the manuscript editing. All authors approved the final version of the paper. 7. DATA AVAILABILITY STATEMENT The Python code for operating the manipulator system will be available upon request. All collected data is included in this published article and its supplementary materials. 8. COMPETING INTERESTS STATEMENT The authors declare that they have no competing interests. References Webster, C. F., Kim, W. J., Reguera, G., Friesen, M. L. & Beyenal, H. Review: can bioelectrochemical sensors be used to monitor soil microbiome activity and fertility? Curr. Opin. Biotechnol. 90 , 103222 (2024). An Introduction to Soil Concepts and the Role of Soils in. Watershed Management - Schoonover – 2015 - Journal of Contemporary Water Research & Education - Wiley Online Library. https://onlinelibrary.wiley.com/doi/full/ 10.1111/j.1936-704X.2015.03186.x Borch, T. et al. Biogeochemical Redox Processes and their Impact on Contaminant Dynamics. Environ. Sci. Technol. 44 , 15–23 (2010). Markelova, E. Redox potential and mobility of contaminant oxyanions (As, Sb, Cr) in argillaceous rock subjected to oxic and anoxic cycles (Université Grenoble Alpes; University of Waterloo (Canada), 2016). Schüring, J., Schulz, H. D., Fischer, W. R., Böttcher, J. & Duijnisveld, W. H. M. Redox: Fundamentals, Processes and Applications (Springer Science & Business Media, 2000). Sojka, R. E. & Scott, H. D. Aeration Measurement. in Encyclopedia of Soil Science - Two-Volume Set (CRC, (2005). Lacroix, E. M. et al. Consider the Anoxic Microsite: Acknowledging and Appreciating Spatiotemporal Redox Heterogeneity in Soils and Sediments. ACS Earth Space Chem. 7 , 1592–1609 (2023). Ward, B. B. Nitrification. in Encyclopedia of Ecology 351–358Elsevier, (2013). 10.1016/B978-0-12-409548-9.00697-7 Gross, A., Lin, Y., Weber, P. K., Pett-Ridge, J. & Silver, W. L. The role of soil redox conditions in microbial phosphorus cycling in humid tropical forests. Ecology 101 , e02928 (2020). Khalil, K., Mary, B. & Renault, P. Nitrous oxide production by nitrification and denitrification in soil aggregates as affected by O2 concentration. Soil. Biol. Biochem. 36 , 687–699 (2004). Wagner, D., Pfeiffer, E. M. & Bock, E. Methane production in aerated marshland and model soils: effects of microflora and soil texture. Soil. Biol. Biochem. 31 , 999–1006 (1999). Dorau, K. et al. Redoxtrons – An experimental system to study redox processes within the capillary fringe. Eur. J. Soil. Sci. 74 , e13347 (2023). Slimani, I., Zhu-Barker, X., Lazicki, P. & Horwath, W. Reviews and syntheses: Iron – a driver of nitrogen bioavailability in soils? Biogeosciences 20 , 3873–3894 (2023). Li, Y. et al. Oxygen availability regulates the quality of soil dissolved organic matter by mediating microbial metabolism and iron oxidation. Glob Change Biol. 28 , 7410–7427 (2022). Zausig, J., Stepniewski, W. & Horn, R. Oxygen Concentration and Redox Potential Gradients in Unsaturated Model Soil Aggregates. Soil. Sci. Soc. Am. J. 57 , 908–916 (1993). Keiluweit, M., Gee, K., Denney, A. & Fendorf, S. Anoxic microsites in upland soils dominantly controlled by clay content. Soil. Biol. Biochem. 118 , 42–50 (2018). Ponnamperuma, F. N. The Chemistry of Submerged Soils. in Advances in Agronomy vol. 24 29–96 (Elsevier, (1972). Reddy, K. R. & DeLaune, R. D. Biogeochemistry of Wetlands: Science and Applications (CRC, 2008). 10.1201/9780203491454 Atci, E., Babauta, J. T., Sultana, S. T. & Beyenal, H. Microbiosensor for the detection of acetate in electrode-respiring biofilms. Biosens. Bioelectron. 81 , 517–523 (2016). Atci, E., Babauta, J., Ha, P. & Beyenal, H. A Fumarate Microbiosensor for Use in Biofilms. J. Electrochem. Soc. 164 , H3058–H3064 (2016). Atci, E., Babauta, J. T., Sultana, S. T. & Beyenal, H. Microbiosensor for the detection of acetate in electrode-respiring biofilms. Biosens. Bioelectron. 81 , 517–523 (2016). RAMPS 1. 4 - RepRap. https://reprap.org/wiki/RAMPS_1.4 arduino.cc/en/software. https://www.arduino.cc/en/software/ Atci, E., Babauta, J. T. & Beyenal, H. A hydrogen peroxide microelectrode to use in bioelectrochemical systems. Sens. Actuators B Chem. 226 , 429–435 (2016). Babauta, J. & Beyenal, H. Local Current Variation by Depth in Geobacter Sulfurreducens Biofilms. J Electrochem. Soc 161 , (2014). Beyenal, H. & Babauta, J. Biofilms in Bioelectrochemical Systems: From Laboratory Practice to Data Interpretation | Wiley. https://www.wiley.com/en-us/Biofilms+in+Bioelectrochemical+Systems%3A+From+Laboratory+Practice+to+Data+Interpretation-p-9781118413494 Lewandowski, Z. & Beyenal, H. Fundamentals of Biofilm Research (CRC, 2013). 10.1201/b16291 Bond, D. R. & Lovley, D. R. Electricity Production by Geobacter sulfurreducens Attached to Electrodes. Appl. Environ. Microbiol. 69 , 1548–1555 (2003). Tender, L. M. et al. Harnessing microbially generated power on the seafloor. Nat. Biotechnol. 20 , 821–825 (2002). Lovley, D. R. Bug juice: harvesting electricity with microorganisms. Nat. Rev. Microbiol. 4 , 497–508 (2006). Additional Declarations No competing interests reported. Supplementary Files SIJunpaperDec23Final.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Mar, 2026 Reviews received at journal 21 Mar, 2026 Reviews received at journal 21 Mar, 2026 Reviews received at journal 20 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviews received at journal 15 Mar, 2026 Reviews received at journal 14 Mar, 2026 Reviewers agreed at journal 13 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers invited by journal 11 Mar, 2026 Editor invited by journal 11 Mar, 2026 Editor assigned by journal 07 Mar, 2026 Submission checks completed at journal 07 Mar, 2026 First submitted to journal 26 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8981935","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":606321169,"identity":"292b75bf-6fc9-4bf5-a98e-300b56c338a1","order_by":0,"name":"Won-Jun Kim","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Won-Jun","middleName":"","lastName":"Kim","suffix":""},{"id":606321170,"identity":"f61bd931-b793-4170-83eb-560071db9e48","order_by":1,"name":"Ibrahim Bozyel","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Bozyel","suffix":""},{"id":606321171,"identity":"1078b1c1-269b-405d-8766-f0262b959ff6","order_by":2,"name":"Suat Ay","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Suat","middleName":"","lastName":"Ay","suffix":""},{"id":606321172,"identity":"acd8291a-9460-44c8-847d-030352da83b2","order_by":3,"name":"Maren Friesen","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Maren","middleName":"","lastName":"Friesen","suffix":""},{"id":606321173,"identity":"07a62dd8-a6ec-47cb-8e1b-96733116f4d6","order_by":4,"name":"Haluk Beyenal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYBACAwhlIQeh2YjXImFMupbEBqK1mLP3GD4uqJFI33D+jAHDh7LDhLVY9pwxNp5xTCJ3w40cA8YZ54jQYnAjx0yah00id9sNHgNm3jZitNx/Y/6b559EuhnQYcx/idJyg8cMaLhEgtmBHANmRmK0WPakFUvz9kkY7r+RVnCw51w6YS3m7Ic3fub5ZiMv2X9444MfZdaEtTAwcBjAmQeIUQ8E7A+IVDgKRsEoGAUjFgAAj4k4GtacA7wAAAAASUVORK5CYII=","orcid":"","institution":"Washington State University","correspondingAuthor":true,"prefix":"","firstName":"Haluk","middleName":"","lastName":"Beyenal","suffix":""}],"badges":[],"createdAt":"2026-02-26 23:23:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8981935/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8981935/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104702440,"identity":"f656c91a-282c-4bdd-a7ee-20fbbe7cfc25","added_by":"auto","created_at":"2026-03-16 08:43:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":180112,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic overview and instrumentation workflow of the Soil Electrochemical Profiling System (SEPS).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/a5e4c722243dca9a0738b7e5.jpg"},{"id":104702438,"identity":"cb60aaa8-2ed9-424a-b03c-65828680940b","added_by":"auto","created_at":"2026-03-16 08:43:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":117255,"visible":true,"origin":"","legend":"\u003cp\u003eA) Software logic flow diagram of the custom Python interface developed for the SEPS. The software initializes connections with both the Arduino-based manipulator and the Gamry potentiostat, performs automated microelectrode positioning, executes electrochemical measurements (OCP or CA), logs the data, and repeats the cycle until profiling is complete.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/adbbb5e9a7d0764ed782ed0a.jpg"},{"id":104702536,"identity":"3f5c5e91-0e0d-4746-a831-606a75053883","added_by":"auto","created_at":"2026-03-16 08:43:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71127,"visible":true,"origin":"","legend":"\u003cp\u003eRedox potential profiles in the hydrogel–Zobell system. A) In the absence of redox-active compounds, redox potentials remain uniform with depth but vary over time. B) Addition of Zobell solution induces time- and depth-dependent redox gradients in the hydrogel, measured at 0, 6, 12, and 24 hrs. C) A full-depth (27 cm) profile measured immediately after Zobell addition captures large-scale redox changes but lacks the spatial resolution shown in panel B.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/9ec876e04c00aff2e7b98fe3.jpg"},{"id":104702538,"identity":"e16946d0-cd7c-48d5-9036-cc27c015e68a","added_by":"auto","created_at":"2026-03-16 08:43:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":83240,"visible":true,"origin":"","legend":"\u003cp\u003eCyclic voltammograms measured in the hydrogel–Zobell system. A) Depth-dependent voltammograms collected 24 hrs after Zobell solution addition at 2 cm above the hydrogel surface (bulk solution) and at 1 cm and 5.5 cm below the surface, illustrating attenuation of electrochemical activity with depth. B) Time-dependent voltammograms collected at 1 cm depth at 0, 12, and 24 hrs, showing the evolution of local redox behavior following diffusion of the ferricyanide/ferrocyanide couple.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/36a4dd721dfcabe1781d94d1.jpg"},{"id":104702549,"identity":"a3b79f64-cab7-4baa-89fd-459b5dbda637","added_by":"auto","created_at":"2026-03-16 08:43:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16429,"visible":true,"origin":"","legend":"\u003cp\u003eRedox potential profiles measured in the soil mesocosm reactor over three weeks, illustrating the temporal development of depth-dependent redox gradients under water-saturated conditions.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/f48b2f4a2a2cd6c9622a231c.jpg"},{"id":104702557,"identity":"22686701-8238-47e5-b679-72e3e0cb4c84","added_by":"auto","created_at":"2026-03-16 08:43:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":75355,"visible":true,"origin":"","legend":"\u003cp\u003eCyclic voltammograms measured in the soil mesocosm reactor. A) Depth-dependent voltammograms collected at week 1, showing oxygen-dominated electrochemical behavior above the soil surface and the emergence of redox activity within the soil. B) Time-resolved voltammograms collected at 5.5 cm depth over four weeks, illustrating the progressive development of soil redox processes under increasingly reducing conditions.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/f0891b9faa422b12b92ef6dd.jpg"},{"id":104702556,"identity":"d0985160-bd1f-4059-b36f-afaeae337bef","added_by":"auto","created_at":"2026-03-16 08:43:49","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":61128,"visible":true,"origin":"","legend":"\u003cp\u003eChronoamperometric characterization of microelectrode biofilm enrichment and depth-resolved current measurements in soil. A) Current response of a carbon microelectrode polarized at +300 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e and placed 7 cm below the soil surface over 40 days, illustrating electroactive biofilm enrichment. B) Depth-resolved chronoamperometric measurements using the biofilm-enriched microelectrode at the same applied potential, revealing vertical structuring of electrochemical activity within the soil.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/a55b63a9e9baaedc5e16fb40.jpg"},{"id":104702606,"identity":"eb3f0936-a251-430a-8e2a-c44e506436b0","added_by":"auto","created_at":"2026-03-16 08:44:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1805446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/73e8c629-20ab-49d6-ae9d-d078cb3529c9.pdf"},{"id":104702589,"identity":"2559d265-1896-4dc9-84de-bd44c78aa633","added_by":"auto","created_at":"2026-03-16 08:44:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":928719,"visible":true,"origin":"","legend":"","description":"","filename":"SIJunpaperDec23Final.docx","url":"https://assets-eu.researchsquare.com/files/rs-8981935/v1/5aa2f2841242177958117a6a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Custom Soil Electrochemical Profiling System for Detecting Electrochemical Activity Changes in Soil","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eSoil can be viewed as a natural bioelectrochemical reactor, driven by microbial interactions with minerals, organic matter, and other constituents that generate redox gradients \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Nearly half of the soil volume consists of inorganic materials such as sand, silt, and clay\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, while approximately 5% is composed of organic matter derived from decaying organisms, microbial biomass, or humic substances. Microbes utilize the diverse chemical species present within both inorganic and organic fractions of the soil. The remaining soil volume comprises pore spaces that hold air and water, enabling the transport of dissolved chemical species used for nutrient cycling. The metabolic activities of soil microorganisms govern the biogeochemical processes that occur in terrestrial environments and cause redox gradients controlling electrochemical activities \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRedox potential indicates the balance between oxidized and reduced compounds, determining the oxidative or reductive state \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The redox potential of soil increases with the abundance of oxidizing components, and oxic conditions typically prevail near the surface where oxygen is readily available. \u003csup\u003e6\u003c/sup\u003e. As depth increases, oxygen becomes depleted through aerobic microbial respiration, creating anoxic and reducing zones \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These redox gradients shape key biogeochemical processes. For example, in the nitrogen cycle, oxic regions drive nitrification by oxidizing ammonium to nitrate, while anoxic regions favor denitrification, the reduction of nitrate to atmospheric nitrogen \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Under anoxic conditions, certain microorganisms also reduce ferric oxides, releasing bound phosphates and enhancing their bioavailability in soil \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Hypoxic microenvironments can produce nitrous oxide, a potent greenhouse gas, as an intermediate during denitrification \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, whereas strictly anoxic conditions promote methanogenesis and the release of methane \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Thus, soil redox potential not only represents the overall balance between oxidized and reduced species but also indicates the types of redox reactions likely to be favored by microbial activity within different soil zones. Therefore, to understand these microbial activities, redox potential in the soil must be measured by depth.\u003c/p\u003e \u003cp\u003eIn most well-drained soils, an oxic layer [75\u0026ndash;100% of air saturation] typically occupies the upper\u0026thinsp;~\u0026thinsp;0\u0026ndash;5 cm, a suboxic transition zone [10\u0026ndash;75% of air saturation] extends roughly\u0026thinsp;~\u0026thinsp;5\u0026ndash;15 cm, and more strongly reducing (often anoxic) conditions [\u0026lt;\u0026thinsp;5% of air saturation] prevail below ~\u0026thinsp;15 cm \u003csup\u003e12\u003c/sup\u003e. Soil texture, moisture, available alternative electron donors, and dissolved organic matter loading can shift these boundaries \u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In wet, high organic loaded or flooded soils, the oxic zone may shrink to just a few millimeters at the surface, with persistent anoxia throughout the profile beneath. On the other hand, coarse, dry soils or root-oxygenated rhizospheres can maintain oxic zone down to ~\u0026thinsp;20\u0026ndash;30 cm. These depth-structured redox gradients govern the onset of denitrification, Fe/Mn reduction, sulfate reduction, and methanogenesis with depth \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRedox potential reflects the equilibrium ratio between oxidized and reduced species according to the Nernst equation. In parallel with redox potential measurements, cyclic voltammetry (CV) can provide complementary electrochemical information about soil systems. In addition, CV allows the characterization of active redox processes rather than equilibrium states provided by the Nernst equation. By sweeping the potential over a defined range and measuring the resulting current response, CV can identify redox-active species, quantify their electrochemical reversibility, and provide insight into electron-transfer kinetics at soil\u0026ndash;electrode interfaces. From the shape and position of anodic and cathodic peaks, one can infer parameters such as formal redox potential, peak current, and reaction reversibility. Additionally, CV can help distinguish between diffusion-controlled and surface-confined reactions, detect catalytic redox cycles mediated by microbes, and reveal electron shuttle behavior of humic substances or mineral phases in soil. Thus, CV offers a dynamic and mechanistic understanding of redox-active components that complement static redox potential measurements.\u003c/p\u003e \u003cp\u003eIn addition to cyclic voltammetry, chronoamperometry (CA) can be employed to monitor the activity of soil biofilms grown on electrode surfaces. Chronoamperometry is an electrochemical characterization technique that measures current by applying a constant potential over time. In this technique, biofilms are cultivated directly on microelectrode tips by maintaining a constant applied potential, allowing electroactive microorganisms to colonize the surface. These electrode-grown biofilms have been used to quantify metabolic substrates such as acetate and fumarate concentrations \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Building on this approach, Babauta et al. demonstrated that such biofilms could also be used to measure local current as a function of depth, revealing microscale electrochemical activity gradients \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Electrode-grown, electrochemically active biofilms thus offer a means to infer local nutrient availability and redox dynamics in soil microenvironments. However, to date, such measurements have not been realized in soil systems due to the absence of appropriate instrumentation capable of high-resolution electrochemical profiling \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAs detailed above, measuring redox gradients and electrochemical behavior (CV and CA) in soil is critical for understanding microbial respiration, nutrient cycling, and greenhouse gas production, yet \u003cem\u003ein situ\u003c/em\u003e quantification remains limited by limited sensing technology and the lack of integrated instrumentation. Accurate measurement of redox potential and CV in soil requires a sensor, an electrochemical control unit to apply potential sweeps and record current responses (potentiostat), a positioning system to move the sensor to defined depths, and integration of these elements into a unified automated platform. Traditionally, soil redox potential is measured with platinum (Pt) electrodes calibrated using Zobell\u0026rsquo;s solution containing a known ferri/ferrocyanide redox couple; the potential is measured against a known reference electrode (e.g., Ag/AgCl) as an open-circuit potential representing the redox potential in the soil. While such measurements indicate the oxidative/reductive balance, they reveal little about the identity or kinetics of the redox-active species involved. CV complements this by resolving oxidation and reduction peaks corresponding to specific electroactive components, providing additional information about electron-transfer kinetics, reaction reversibility, and microbially mediated redox processes. Chronoamperometry is another tool that can measure current by applying a constant potential over time and enrich microbes in soil while doing so. Despite their potential, existing commercial systems (e.g., Unisense, Mettler Toledo) are limited to shallow depths, manual operation, and single-function measurements, preventing comprehensive depth-resolved electrochemical profiling.\u003c/p\u003e \u003cp\u003eTo overcome above limitations, our goal is to develop a custom-built automated manipulator capable of acquiring redox potential and CV data across soil depths up to 27 cm. Our system integrates a Pt working electrode, a custom-made Ag/AgCl reference, and a counter electrode with a programmable potentiostat and automated custom-made vertical positioning, enabling low-cost, high-resolution characterization of soil redox dynamics and microbially driven electron-transfer processes. Using off-the-shelf components, we constructed a Soil Electrochemical Profiling System (SEPS) capable of automated operation to depths of up to 27 cm with a minimum positioning resolution of 25 \u0026micro;m. The SEPS integrates directly with a potentiostat, allowing the use of multiple electrochemical techniques including redox potential measurement, CV, and chronoamperometry. We validated SEPS by generating depth-resolved redox potential profiles and CVs in control samples as well as in soil and sediment reactors. To further demonstrate its CA capability for biological studies, we polarized a microelectrode at a fixed depth over a 40 days enrichment period, which revealed clear evidence of microbial electrochemical activity. Using biofilms developed on this microelectrode, we subsequently measured current depth profiles in soil - to our knowledge, the first such measurement reported in the literature.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Construction of Soil Electrochemical Profiling System\u003c/h2\u003e \u003cp\u003eThe Soil Electrochemical Profiling System (SEPS) was assembled using commercially available components integrated via the RAMPS 1.4 kit (Hilitand, Wuhan, China, ASIN B07DFKC3RF, Amazon), which connects to an Arduino Mega 2560 microcontroller to enable a compact motion-control system for driving a stepper motor and reading limit switches (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The system includes a linear stage actuator with a stepper motor, RAMPS 1.4 motor driver shield, A4988 stepper driver, mechanical limit switches, a 12 V DC power supply, and all necessary wiring and connectors. Custom 3D-printed mounts and acrylic holders were fabricated in-house to secure the electrodes and potentiostat leads during operation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The core components of the system consist of 1) a motor assembly for precise microelectrode positioning, 2) a Gamry 1000E potentiostat for electrochemical measurements such as open-circuit potential which we called as redox potential in this manuscript, cyclic voltammetry (CV), and chronoamperometry (CA), and 3) custom software that integrates motion control, data acquisition, and potentiostat operation into a single automated platform. A schematic overview and instrumentation workflow are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All components used for constructing the SEPS were obtained from commercial suppliers and are listed in the Supporting Information (Table SI1).\u003c/p\u003e \u003cp\u003eThe motor driver board (RAMPS 1.4) was mounted directly onto the Arduino Mega 2560 controller board. Wiring and configuration followed the official RAMPS 1.4 documentation \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Two \u0026ldquo;\u003cem\u003eendstop\u003c/em\u003e\u0026rdquo; mechanical switches were installed on the y-min and y-max ports of the RAMPS 1.4 board to limit the linear stage actuator (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The y-min switch was positioned on the motor-side (top) end of the stage, and the y-max switch was mounted on the opposite end (bottom), ensuring that actuator motion automatically ceased at both limits. Motion control was achieved using an A4988 stepper-motor driver installed on the x-axis header of the RAMPS board (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Prior to mounting, three jumpers were inserted to enable microstepping with 1/16-step, providing the precise positioning resolution. The linear actuator\u0026rsquo;s stepper motor was wired using male-to-female extensions soldered to the four motor leads. Power was supplied by a 12 V DC regulated unit. After completing the wiring, the Arduino IDE software \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e was installed. The StepperDriver v1.4.1 library (Laurentiu Badea) was added by using the Arduino IDE Library Manager. The RAMPS library was downloaded from github.com/momostein/Ramps and installed using Sketch \u0026rarr; Include Library \u0026rarr; Add .ZIP Library, selecting the file Ramps-master.zip. The control code that manages control signal between Arduino and the motor driver, \u0026ldquo;Demo_Stepper.ino,\u0026rdquo; was opened. After compilation and verification, the board type (Arduino Mega 2560) and correct COM port were selected under Tools, and the code was uploaded. Once programmed, the SEPS was ready for control through custom Python software for automated electrochemical profiling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Custom Python Software for System Control and Integration\u003c/h2\u003e \u003cp\u003eA custom Python-based software package was developed to coordinate all operations of the SEPS, integrating motion control of the microelectrode manipulator with electrochemical measurements performed by a Gamry potentiostat. The software functions as a unified control interface, providing synchronized automation of electrode positioning and data acquisition across varying soil depths. The program establishes serial communication with the Arduino Mega 2560 microcontroller, which manages the stepper motor via a RAMPS 1.4 shield and A4988 driver module. Through user-defined parameters, the software sends G-code-style commands to move the microelectrode vertically in user-determined increments. A homing routine is included to reference the initial position using mechanical limit switches before each profiling sequence begins.\u003c/p\u003e \u003cp\u003eIn parallel, the software interfaces with the Gamry potentiostat using the GamryCOM API. It supports two electrochemical techniques: OCP and CA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The user selects the desired method, and the software automatically configures and initiates the corresponding measurement protocol. Collected data is saved locally in structured files for post-analysis. CV is measured separately using Gamry\u0026rsquo;s stock program because depth-profile CV measurements do not require synchronized electrode positioning during data acquisition. For OCP and CA depth profiles, the response time between measurement points is critical for obtaining accurate profiles. In contrast, CV has a relatively long and fixed acquisition time determined by the initial parameter settings. We also chose to run CV manually because, after each scan, the user may need to adjust parameters such as the scan rate, initial and final potentials, and scan limits.\u003c/p\u003e \u003cp\u003eThe workflow is fully automated. The software initiates a measurement at the current depth, waits for completion, logs the data, moves the electrode to the next depth, and repeats the cycle until the profiling sequence is complete (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figure SI1A). Timing synchronization, device status monitoring, and error handling are all managed within the Python environment. A graphical user interface (GUI) built with PyQT allows users to set parameters (step size, number of steps, delay time, technique type), monitor real-time progress, and manually trigger actions such as homing, start, and abort (Figure SI1). The interface is designed to be intuitive and adaptable for future extensions, such as multi-electrode support or remote operation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo measure redox potential, the user selects the OCP mode and specifies the starting position, ending position, total number of steps, step size, and delay time between measurements. The software acquires OCP readings from Gamry at each depth and reports it as a plot in the bottom left portion of the GUI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In manual mode, the user adjusts the microelectrode position using the \u0026ldquo;\u003cem\u003eMove Up\u003c/em\u003e\u0026rdquo; or \u0026ldquo;\u003cem\u003eMove Down\u003c/em\u003e\u0026rdquo; button based on the step size the user has set. It can also move to a specific location by setting the target position along the length of the manipulator (0-270 mm) and pressing the \u0026ldquo;\u003cem\u003eGo to Position\u003c/em\u003e\u0026rdquo; button. For automated operation, the user defines the starting and ending depths, step size, and delay parameters; the software then controls the microelectrode movement and data acquisition accordingly. The same workflow applies to CA measurements, with the additional requirement that the user enters an applied potential. CA data can also be collected manually or automatically. In contrast, CV measurements are supported only in manual mode because it does not require synchronized electrode positioning during data acquisition and require frequent parameter adjustments, where the user sets the parameter and initiates each CV scan on Gamry\u0026rsquo;s stock program (Gamry Framework) at the desired depth.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microelectrodes\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Pt-Tipped Microelectrodes\u003c/h2\u003e \u003cp\u003eWe followed the fabrication procedures described by Atci et al. \u003csup\u003e24\u003c/sup\u003e to construct Pt-tipped microelectrodes, introducing several modifications to improve their mechanical durability for use in soil environments. The original design was intended for biofilms, which are significantly softer than soil; therefore, adjustments were necessary to prevent breakage during insertion and profiling. A glass Pasteur pipette was used to achieve a longer (~\u0026thinsp;10cm of length available for soil application insertion) and sturdier electrode. The reference electrode\u0026rsquo;s outer casing was fabricated from the widened section of the pipette and positioned concentrically around the Pt microelectrode shaft. An Ag/AgCl wire was inserted at the junction between the inner and outer casings and sealed using 5-minute epoxy, which was cured for 24 hours \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The outer casing was then filled with saturated KCl solution (Potassium Chloride, 4 M, saturated with Silver Chloride, Electrode filling Solution, RICCA Chemical Company, Arlington, TX, USA, Cat. No. 5920-16), and its open end was sealed with a molecular sieve disc (Molecular Sieve 4 \u0026Aring;, Consolidated Chemical \u0026amp; Solvents LLC, CAS 70955-01-0). This modification prevented soil particles from penetrating into and contaminating the internal chamber during depth-profiling while maintaining ionic conductivity with the surrounding medium. A 100 \u0026micro;m platinum wire (California Fine Wire Company, Grover Beach, CA, USA) was affixed to the outer casing using heat-shrink tubing as counter electrode for CV or CA measurements. After the final sealing, the assembly was cured for another 24 hours and stored in saturated KCl solution inside 15 mL Falcon tubes. Prior to each experiment, the electrodes were validated in Zobell\u0026rsquo;s solution by performing OCP or CV measurements to confirm functionality and assess the electroactive surface area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Carbon-Tipped Microelectrodes\u003c/h2\u003e \u003cp\u003eIn addition to the Pt-tipped microelectrodes, a second type of microelectrode was fabricated using a graphite rod as the sensing element. The construction process followed the same protocol described by Atci et al.\u003csup\u003e24\u003c/sup\u003e, and Babauta et al \u003csup\u003e25\u003c/sup\u003e with the only modification being the substitution of the platinum wire with a graphite rod of 500 \u0026micro;m diameter. The graphite was pulled into a glass Pasteur pipette, identical to the method used for the Pt version. This carbon-based microelectrode was specifically designed for experiments involving biofilm growth on the tip, where CA and depth-resolved CA were the primary objectives. Due to its biocompatibility for biofilm growth and electron transfer properties for microbial systems, carbon was selected as a more suitable material than platinum.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Soil Electrochemical Profiling System Verification and Sample Types\u003c/h2\u003e \u003cp\u003eTo validate the operation of the SEPS, two types of samples were used. The first was a control sample consisting of a hydrogel matrix made from xanthan gum, designed to simulate the soft materials. This matrix was in contact with a bulk Zobell solution containing the ferri/ferrocyanide redox couple. Over time, the redox species diffused from the bulk solution into the hydrogel, allowing us to monitor changes in redox chemistry both in the bulk solution and within the matrix using OCP and CV. The second sample type consisted of soil mesocosm prepared with garden soil (GPS: 46.7211\u0026deg; N, 117.1722\u0026deg; W) and DI water. Using these two distinct systems, one controlled and synthetic, the other representative of real-world conditions, we verified the SEPS's capability to perform automated electrochemical measurements, including OCP, CV, and CA, in both idealized and complex environments.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Xanthan Gum Hydrogel System for Monitoring Redox Potentials and Cyclic Voltammograms Over Time and Depth\u003c/h2\u003e \u003cp\u003eTo evaluate the SEPS system's ability to measure redox gradients, we designed an experiment using a xanthan gum hydrogel matrix and Zobell solution to simulate time- and depth-dependent electrochemical changes. The concentration of xanthan gum (Namaste Foods, LLC, Coeur d\u0026rsquo;Alene, ID, USA) was optimized to achieve a consistency that was neither too fluid (which would allow mixing with the overlying solution) nor too viscous (which would trap air bubbles and prevent the formation of a flat surface). A concentration of 15 g/L in 0.1 M KCl was found to meet these conditions. One liter of hydrogel was prepared by slowly adding the xanthan gum powder to the heated and stirred KCl solution. The mixture was then autoclaved at 121 \u003csup\u003eo\u003c/sup\u003eC for 15 minutes to improve homogeneity and degassed under vacuum at \u0026minus;\u0026thinsp;25 psi to eliminate residual air bubbles. While still hot, the hydrogel was poured into a 600 mL beaker up to the 500 mL mark and was allowed to cool overnight, forming a smooth, bubble-free surface. Zobell solution, containing 3.33 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M potassium ferrocyanide (K₄Fe(CN)\u003csub\u003e6\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO), 3.33 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M potassium ferricyanide (K\u003csub\u003e3\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e), and 0.1 M potassium chloride (KCl), was prepared by dissolving the components in deionized water. The hydrogel was topped with Zobell solution carefully poured along the beaker wall using a 50 mL Falcon tube to avoid mixing; the liquid layer was maintained at approximately 3 cm above the hydrogel surface. Right after adding the Zobell solution, depth-resolved redox potential profiles were recorded using the SEPS. The microelectrode was initially positioned manually so that its tip was ~\u0026thinsp;2 cm above the hydrogel surface. The system was then programmed to automatically measure OCP from the top of the solution down to 55 mm into the hydrogel, with 1 mm step intervals and a 5-second delay between each measurement. These profiling measurements were conducted at 0, 6, 12, and 24 hours to capture the progression of redox couple diffusion over time. After completing the OCP scan, the microelectrode was retracted and repositioned at a new location on the hydrogel surface, at least 1 cm laterally from the previous measurement site to minimize disturbance. To verify the long-distance measurement capability of the SEPS, a column consisting of 15 cm of Zobell solution over 25 cm of xanthan gum hydrogel was prepared in a graduated cylinder. Redox potentials were then measured over the 27 cm depth, its maximum range, at 1 cm intervals. CV was then conducted at depths of +\u0026thinsp;2 cm, +\u0026thinsp;0.5 cm, -1 cm, -2.5 cm, -4 cm, and \u0026minus;\u0026thinsp;5.5 cm (relative to the hydrogel surface level) using a scan rate of 10 mV/s over a potential range of \u0026minus;\u0026thinsp;300 mV to +\u0026thinsp;600 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e, with three consecutive cycles performed at each depth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Soil Mesocosm and Measurements\u003c/h2\u003e \u003cp\u003eGarden soil was homogenized for at least 1 minute using a food processor (Spectrum Brands / Black+Decker, 120 V AC, 450 W, model FP1600B) and then sieved through a 1 mm mesh. The sieved soil was added to a 600 mL beaker until it reached the 450 mL mark. Deionized water was gradually added while mixing the soil with a spatula to ensure full saturation. As the soil settled, additional soil was added to restore the height to the 450 mL mark. Once the soil level was stable, deionized water was added to create a 3 cm water column above the soil surface. This soil mesocosm was allowed to equilibrate before beginning measurements. The same procedures used in the hydrogel experiments were followed for OCP and CV measurements. However, in this case, measurements were taken at time points of 0, 7, 14, and 21 days. Additionally, the cyclic voltammetry scan range was extended to a scan range of -600 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e to +\u0026thinsp;600 mV \u003csub\u003eAg/AgCl\u003c/sub\u003e, compared to the scan range of -300mV\u003csub\u003eAg/AgCl\u003c/sub\u003e to +\u0026thinsp;600 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e used in the hydrogel setup.\u003c/p\u003e \u003cp\u003eThe carbon-tipped microelectrode was used to enrich biofilm at its tip over a period of 40 days by polarizing it to +\u0026thinsp;300 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e inside a soil mesocosm. Following biofilm formation, the same microelectrode was employed to measure depth-resolved current profiles by maintaining the polarization at +\u0026thinsp;300 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e and using CA to capture the electrochemical activity associated with the biofilm at the electrode tip.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Declaration of generative AI and AI-assisted technologies in the manuscript preparation process\u003c/h2\u003e \u003cp\u003eDuring the preparation of this manuscript, the authors used generative artificial intelligence tools solely to assist with editing and improving the clarity and readability of the text. No figures (except part of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), images, or data were generated using AI tools. All content was reviewed and edited by the authors, who take full responsibility for the accuracy, integrity, and originality of the published work.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cp\u003eUsing the SEPS, we quantified redox potential profiles in a hydrogel-Zobell solution system as a function of both depth and time. This was followed by CV measurements to monitor electrochemical changes over time and at different depths. To validate the full profiling capability of the system, measurements were extended across the entire operational length (27 cm), confirming functionality of the SEPS over the intended depth range. After completing tests in these well-controlled systems, we used the SEPS for soil mesocosms. First, we measured redox profiles. Following this, biofilms were first enriched on the tip of a carbon microelectrode using CA. The same microelectrode grown biofilm was then used to perform depth-resolved current measurements to assess the electrochemical activity of the surface-associated biofilms in soil.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.1 Monitoring Redox Potentials Over Time and Depth of a Controlled Hydrogel System in the Presence and Absence of Redox Couple\u003c/b\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003cdiv class=\"Heading\"\u003e3.1.1 Absence of redox-active compounds yields uniform redox profiles with time-dependent shifts\u003c/div\u003e \u003cp\u003eIn the absence of redox-active compounds, redox potentials remained uniform with depth while changing over time (Fig.\u0026nbsp;3A). At the start of the experiment, the redox potential was approximately 280 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e in both the bulk solution and throughout the hydrogel, resulting in a flat depth profile, as expected under non-reactive conditions. After 24 hrs, the redox potential increased to ~\u0026thinsp;420 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e, while the profile remained linear and depth-independent. The time-dependent increase in redox potential is attributed to changes in ambient conditions, including a decrease in room temperature and re-equilibration between dissolved oxygen in the liquid phase and oxygen from the air. The persistence of a flat redox profile across the entire depth confirms that, in the absence of redox-active species, the SEPS system reliably measures uniform redox potentials without introducing spatial artifacts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003cdiv class=\"Heading\"\u003e3.1.2. Redox-active compounds generate time- and depth-dependent redox gradients\u003c/div\u003e \u003cp\u003eIn the presence of the ferricyanide/ferrocyanide redox couple, pronounced redox potential gradients developed with both depth and time (Fig.\u0026nbsp;3B). Zobell solution, which has a redox potential of ~\u0026thinsp;223 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e, was initially confined to the bulk phase. At time zero, the bulk solution exhibited a nearly uniform redox potential of ~\u0026thinsp;237 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e, while the hydrogel showed a pre-existing oxygen-driven gradient, with a maximum of ~\u0026thinsp;296 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e at 15 mm depth that decreased to ~\u0026thinsp;273 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e at the bottom. After 6 hrs, the bulk redox potential increased to ~\u0026thinsp;253 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e, indicating diffusion of Zobell into the system. Concurrently, the hydrogel redox potential decreased to ~\u0026thinsp;232 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e at ~\u0026thinsp;24 mm depth, consistent with penetration of the ferricyanide/ferrocyanide couple. By 12 hrs, the bulk potential further increased to ~\u0026thinsp;256 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e, and the redox minimum within the hydrogel shifted deeper to ~\u0026thinsp;32 mm with a potential of ~\u0026thinsp;236 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e. After 24 hrs, the bulk redox potential reached\u0026thinsp;~\u0026thinsp;261 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e. The redox trough within the hydrogel progressed to ~\u0026thinsp;44 mm depth and stabilized near ~\u0026thinsp;237 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e. The progressive deepening of this trough, coupled with its convergence toward the Zobell redox potential, reflects continued diffusion and dilution of the ferricyanide/ferrocyanide redox couple within the hydrogel over time.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3.\u003c/b\u003e Redox potential profiles in the hydrogel\u0026ndash;Zobell system. A) In the absence of redox-active compounds, redox potentials remain uniform with depth but vary over time. B) Addition of Zobell solution induces time- and depth-dependent redox gradients in the hydrogel, measured at 0, 6, 12, and 24 hrs. C) A full-depth (27 cm) profile measured immediately after Zobell addition captures large-scale redox changes but lacks the spatial resolution shown in panel B.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003cdiv class=\"Heading\"\u003e3.1.3 Large step sizes capture broad redox trends but obscure fine spatial gradients\u003c/div\u003e \u003cp\u003eTo evaluate the maximum depth-profiling capability of the SEPS system, redox potential was measured across a 27 cm depth at 1 cm intervals (Fig.\u0026nbsp;3C). Within the Zobell solution layer, the redox potential remained relatively constant at ~\u0026thinsp;227 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e. Upon entering the hydrogel, the potential increased abruptly to ~\u0026thinsp;238 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e and then gradually rose to ~\u0026thinsp;257 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e toward the bottom of the hydrogel, consistent with oxygen diffusion into the matrix. Because this profile was acquired immediately after Zobell solution addition, diffusion of ferricyanide/ferrocyanide into the hydrogel was minimal. Consequently, the measurement captures large-scale redox transitions across phases but lacks the spatial resolution required to resolve fine redox gradients within the hydrogel, in contrast to the higher-resolution profiles shown in Fig.\u0026nbsp;3B.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Monitoring Cyclic Voltammograms over Time and Depth\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Hydrogel-water system oxygen reduction kinetics\u003c/h2\u003e \u003cp\u003eIn the hydrogel\u0026ndash;water system, oxygen was the only electrochemically active species present. Accordingly, the cyclic voltammograms primarily reflect oxygen reduction kinetics, with minor background contributions likely originating from impurities associated with xanthan gum used (Figure SI3). The voltammograms remained qualitatively consistent over time, indicating stable electrochemical conditions in the absence of added redox-active compounds\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Ferricyanide/ferrocyanide system reveals diffusion-driven spatial and temporal electrochemical gradients\u003c/h2\u003e \u003cp\u003eCyclic voltammetry of the hydrogel\u0026ndash;Zobell system is shown in Fig.\u0026nbsp;4, with additional datasets provided in Figures SI1 and SI2. In the absence of Zobell solution, voltammograms remained unchanged over time, confirming the lack of electrochemically active species (Figure SI1A\u0026ndash;F). Following Zobell addition, distinct spatial and temporal changes in electrochemical behavior were observed at 0, 3, 6, 12, and 24 hrs (Figure SI2G\u0026ndash;L), consistent with diffusion of the ferricyanide/ferrocyanide redox couple into the hydrogel.\u003c/p\u003e \u003cp\u003eTo illustrate the depth-profiling capability of cyclic voltammetry, representative voltammograms collected at three depths (+\u0026thinsp;2 cm, -1.0 cm, and \u0026minus;\u0026thinsp;5.5 cm) after 24 hrs are shown in Fig.\u0026nbsp;4A. The shallowest position (+\u0026thinsp;2 cm) exhibited the largest and most well-defined redox waves, indicating higher local availability of redox-active species near the hydrogel surface. At -1.0 cm, peak currents decreased and the voltammogram narrowed, suggesting a transition zone where mass transport increasingly limits the electrochemical response. At -5.5 cm, the voltammogram showed minimal peak structure and reduced currents, reflecting limited penetration of redox species and slower diffusion at depth. The systematic attenuation of current magnitude and peak definition with depth demonstrates the presence of sharp vertical redox gradients that can be directly resolved using cyclic voltammetry.\u003c/p\u003e \u003cp\u003eTemporal evolution of the electrochemical environment is illustrated by voltammograms collected at 1 cm depth at 0, 12, and 24 hrs (Fig.\u0026nbsp;4B). At 0 h, the voltammogram exhibited modest currents, consistent with background electrochemical activity prior to significant diffusion of the redox couple. After 12 hrs, both anodic and cathodic peak currents increased and the response broadened, indicating the combined influence of oxygen and partially diffused ferricyanide/ferrocyanide. By 24 hrs, the voltammogram displayed more defined redox features, albeit with reduced peak magnitude, consistent with dilution of the redox couple as diffusion progressed. These results demonstrate that cyclic voltammetry sensitively captures time-dependent restructuring of the local electrochemical environment and provides mechanistic processes into redox species transport and distribution within the hydrogel matrix.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4.\u003c/b\u003e Cyclic voltammograms measured in the hydrogel\u0026ndash;Zobell system. A) Depth-dependent voltammograms collected 24 hrs after Zobell solution addition at 2 cm above the hydrogel surface (bulk solution) and at 1 cm and 5.5 cm below the surface, illustrating attenuation of electrochemical activity with depth. B) Time-dependent voltammograms collected at 1 cm depth at 0, 12, and 24 hrs, showing the evolution of local redox behavior following diffusion of the ferricyanide/ferrocyanide couple.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.3 Monitoring Redox Gradients by Depth and Time in Soil Mesocosm Reactors\u003c/b\u003e\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Redox potential profiles\u003c/h2\u003e \u003cp\u003eRedox potential profiles in the soil mesocosm reactors exhibited a progressive shift toward lower values over time, indicating a transition to increasingly reducing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Such temporal and depth-dependent redox gradients are characteristic of saturated soils, where microbial respiration sequentially consumes available electron acceptors as oxygen diffusion becomes limited. In the standing water above the soil surface, redox potential decreased gradually from approximately\u0026thinsp;+\u0026thinsp;215 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e at week 0 to +\u0026thinsp;200, +190, and +\u0026thinsp;105 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e at weeks 1, 2, and 3, respectively. This decline reflects reduced oxygen availability in the overlying water column as microbial and chemical oxygen demand increased, consistent with observations reported for flooded soils and wetland systems.\u003c/p\u003e \u003cp\u003eAt week 0, the redox profile within the soil remained relatively uniform at approximately\u0026thinsp;+\u0026thinsp;350 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e down to ~\u0026thinsp;27 mm below the surface, indicating well-oxidized conditions shortly after reactor setup. Increased signal variability below this depth is likely attributable to trapped air pockets within the soil matrix following water addition, a common artifact during early saturation stages. This setup also simulates post-tillage soil aeration, during which oxygen is temporarily introduced into the soil profile.\u003c/p\u003e \u003cp\u003eBy week 1, a pronounced redox gradient had developed. Redox potential decreased sharply from ~\u0026thinsp;+\u0026thinsp;200 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e at the soil surface to ~\u0026thinsp;+\u0026thinsp;95 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e within the upper 7 mm and remained near this value at greater depths. This rapid downward shift is consistent with oxygen depletion driven by aerobic microbial respiration, a process widely reported as the first stage in the establishment of reducing conditions in saturated soils.\u003c/p\u003e \u003cp\u003eBy weeks 2 and 3, the profiles converged toward substantially lower redox potentials, reaching approximately\u0026thinsp;+\u0026thinsp;30 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e at ~\u0026thinsp;11 mm depth. The week 2 profile remained relatively constant below this depth, whereas the week 3 profile exhibited an additional gradual decrease of ~\u0026thinsp;20 mV with depth, suggesting continued progression toward more reduced conditions. These trends are consistent with the onset of anaerobic processes such as nitrate, manganese, and iron reduction, which typically occur after oxygen has been depleted and redox potentials fall below ~\u0026thinsp;+\u0026thinsp;200 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eOverall, the measured profiles capture the expected spatiotemporal evolution of redox conditions in water-saturated soils and demonstrate the capability of the SEPS system to resolve dynamic redox gradients at millimeter-scale resolution over extended time periods.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Monitoring Cyclic Voltammograms by Depth and Time in Soil Mesocosm Reactors\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Depth- and time-dependent evolution of soil redox activity revealed by cyclic voltammetry\u003c/h2\u003e \u003cp\u003eCyclic voltammetry revealed pronounced depth- and time-dependent evolution of electrochemical activity within the soil mesocosm reactors (Fig.\u0026nbsp;6 and Figure SI3), consistent with the redox potential gradients observed by redox potential measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Unlike redox profiles, which report the dominant redox state at a given depth, cyclic voltammetry provides dynamic information on electron-transfer processes, including the presence, intensity, and reversibility of redox reactions occurring within the soil matrix.\u003c/p\u003e \u003cp\u003eAt positions above the soil surface (+\u0026thinsp;2 cm and +\u0026thinsp;0.5 cm), the voltammograms were dominated by oxygen reduction, characterized by strong cathodic currents at negative potentials and the absence of corresponding anodic peaks (Fig.\u0026nbsp;6A). This behavior indicates an oxygen-rich environment where electrochemical activity is controlled primarily by dissolved oxygen rather than reversible redox couples. This observation is consistent with the relatively high redox potentials measured in the overlying water layer in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, voltammograms collected below the soil surface (\u0026minus;\u0026thinsp;1 cm, \u0026minus;\u0026thinsp;2.5 cm, \u0026minus;\u0026thinsp;4 cm, and \u0026minus;\u0026thinsp;5.5 cm) exhibited a progressive emergence of paired anodic and cathodic features over time, reflecting the development of active redox processes within the soil. At week 0, voltammograms at these depths showed low current magnitudes and weak structure, indicating limited faradaic activity immediately after reactor setup. By week 1, distinct anodic-cathodic responses became apparent, coinciding with the sharp decline in redox potential observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e and consistent with rapid oxygen depletion and the onset of anaerobic conditions.\u003c/p\u003e \u003cp\u003eAs incubation progressed through weeks 2 and 3, the redox features increased in magnitude and definition (Fig.\u0026nbsp;6B), indicating enhanced electron-transfer activity and a growing contribution from reduced soil constituents. Unlike the well-defined peaks associated with single reversible redox couples in homogeneous electrolytes, the broadened and asymmetric voltametric features observed here reflect the superposition of multiple overlapping redox processes, including microbially mediated and mineral-associated reactions. Such behavior is characteristic of natural soils, where redox chemistry is governed by a complex mixture of organic matter, metal oxides, and microbial electron-transfer pathways.\u003c/p\u003e \u003cp\u003eThe depth dependence of the CV response closely mirrors the vertical redox stratification measured by redox profiling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e), with stronger and more complex voltametric signatures observed at greater depths where redox potentials were lowest. Together, these results demonstrate that cyclic voltammetry complements redox potential measurements by resolving not only where redox gradients exist, but also how actively electrons are exchanged within each soil layer. This combined approach enables sensitive, \u003cem\u003ein situ\u003c/em\u003e tracking of dynamic biogeochemical processes and demonstrate the utility of SEPS for characterizing spatially and temporally evolving soil redox environments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 6.\u003c/b\u003e Cyclic voltammograms measured in the soil mesocosm reactor. A) Depth-dependent voltammograms collected at week 1, showing oxygen-dominated electrochemical behavior above the soil surface and the emergence of redox activity within the soil. B) Time-resolved voltammograms collected at 5.5 cm depth over four weeks, illustrating the progressive development of soil redox processes under increasingly reducing conditions.\u003c/p\u003e \u003cp\u003eFigures 3\u0026ndash;6 together define a coherent model of redox evolution driven by diffusion, stratification, and biological activity. In controlled hydrogels, uniform redox profiles persist in the absence of redox-active species, while introduction of a defined redox couple produces predictable, diffusion-controlled gradients that deepen over time. In soil mesocosms, similar principles apply, but redox gradients emerge from oxygen depletion and microbial respiration, leading to vertically stratified and increasingly reducing environments. Redox potential profiling captures the spatial structure of these gradients, whereas cyclic voltammetry reveals their dynamic electrochemical activity. Combined, these measurements demonstrate that complex soil redox behavior can be interpreted through the same mechanistic framework established in controlled systems. By integrating these complementary measurements across depth and time, SEPS uniquely enables a mechanistic interpretation of complex redox environments that cannot be obtained using conventional, single-point redox measurements.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Biofilm Enrichment at the Microelectrode Tip and Chronoamperometric Measurements\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Chronoamperometry reveals electroactive biofilm enrichment on polarized microelectrodes\u003c/h2\u003e \u003cp\u003eTo enable long-term and stable enrichment of electroactive biofilms, the soil mesocosm was operated as a wick system rather than a fully flooded system, allowing improved control of soil moisture over extended incubation periods. Carbon-tipped microelectrodes were inserted 7 cm below the soil surface and continuously polarized at +\u0026thinsp;300 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e for 40 days (Fig.\u0026nbsp;7A). Under these conditions, the measured current reflects electron transfer from microorganisms capable of donating electrons to the electrode and, thus, serves as a proxy for electroactive biofilm growth \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The chronoamperometric response exhibited a characteristic rise, plateau decline pattern consistent with microbial enrichment and nutrient depletion dynamics. During the initial several days, current remained low, indicating adaptation, minimal electroactive biomass and limited extracellular electron transfer. Beginning around Day 5, current increased steadily, exceeding 300 nA by approximately Day 20. This increase corresponds to the enrichment of microorganisms able to utilize the polarized microelectrode tip as a terminal electron acceptor, supported by the availability of electron donors in the surrounding soil, such as reduced organic compounds, fermentation products, or reduced metal species.\u003c/p\u003e \u003cp\u003eBetween Days 20 and 30, the current remained elevated, indicating the establishment of a stable and metabolically active electroactive biofilm. This sustained anodic current suggests that the local soil environment was sufficiently reduced to favor electrode respiration, a condition commonly associated with diffusion-limited soils. After approximately Day 30, the current declined sharply, likely reflecting changes in soil biogeochemical conditions, including depletion of readily available electron donors, accumulation of inhibitory metabolites, or shifts in microbial community composition \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Such temporal patterns are consistent with redox cycling and substrate turnover in natural soil systems. Overall, these results demonstrate that chronoamperometry provides a sensitive, real-time measure of electroactive biofilm development and reflects evolving redox conditions in soil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 Microelectrode-grown biofilms detect current fluctuations in soil and respond to distant electrochemical changes\u003c/h2\u003e \u003cp\u003eDepth-resolved chronoamperometric measurements performed with the 40-day-enriched microelectrode biofilms revealed vertical structuring of electrochemical activity within the soil (Fig.\u0026nbsp;7B). Under constant polarization, the measured anodic current reflects the rate of microbial electron transfer to the electrode and is therefore governed by the local availability of electron donors and their transport to the biofilm-electrode interface \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Immediately above the soil surface, measured currents were relatively high (~\u0026thinsp;8\u0026ndash;10 nA), consistent with conditions that support biofilm respiration due to greater availability of diffusible electron donors. Upon entry into the soil, the current decreased sharply, indicating a rapid transition in redox conditions and electron-donor availability near the microelectrode tip.\u003c/p\u003e \u003cp\u003eBetween approximately 5 and 25 mm below the soil surface, the current exhibited substantial variability (\u0026sim;1\u0026ndash;10 nA), reflecting fine-scale heterogeneity in microbial activity, substrate distribution, and local redox microenvironments. Below ~\u0026thinsp;25\u0026ndash;30 mm, currents stabilized at lower values (\u0026lt;\u0026thinsp;3 nA), consistent with a deeper, more strongly reduced zone where electron-donor supply is limited by slow diffusion and depleted electron donor pools. This depth-dependent pattern-high current near the surface, a sharp transition zone, and a relatively uniform reduced region at depth-is consistent with established models of redox stratification in saturated soils \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImportantly, the persistence of anodic current throughout the depth profile confirms that the enriched biofilm on the microelectrode remained metabolically active and responsive to spatial variations in soil redox conditions. Notably, anodic current was detected even near the soil surface, where competing soluble electron acceptors would typically expect to suppress electrode respiration \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The relatively higher anodic currents observed across all soil depths suggest a system dominated by electron-donor availability (e.g., organic carbon) with limited availability of alternative terminal electron acceptors \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Together, these results demonstrate that biofilm-functionalized microelectrodes function as sensitive, living sensors capable of resolving fine-scale vertical transitions in soil biogeochemistry and detecting electrochemical responses to subsurface redox heterogeneity\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 7.\u003c/b\u003e Chronoamperometric characterization of microelectrode biofilm enrichment and depth-resolved current measurements in soil. A) Current response of a carbon microelectrode polarized at +\u0026thinsp;300 mV\u003csub\u003eAg/AgCl\u003c/sub\u003e and placed 7 cm below the soil surface over 40 days, illustrating electroactive biofilm enrichment. B) Depth-resolved chronoamperometric measurements using the biofilm-enriched microelectrode at the same applied potential, revealing vertical structuring of electrochemical activity within the soil.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Integrated Interpretation of Soil Redox Dynamics Using Redox Potential, Cyclic Voltammetry, and Chronoamperometry\u003c/h2\u003e \u003cp\u003eSections \u003cspan refid=\"Sec19\" class=\"InternalRef\"\u003e3.3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Sec23\" class=\"InternalRef\"\u003e3.5\u003c/span\u003e together demonstrate how combining redox potential, cyclic voltammetry, and chronoamperometry provides a multi-modal view of soil redox dynamics that cannot be obtained from any single technique. Redox potential measurements resolve the spatial and temporal structure of redox gradients, revealing the progressive development of vertically stratified and increasingly reducing conditions \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Cyclic voltammetry complements this by identifying the emergence and evolution of faradaic redox activity within these gradients, reflecting the onset and intensification of coupled microbial and geochemical electron-transfer processes. Chronoamperometry extends this framework by directly tracking sustained electron flux to a polarized microelectrode, linking redox structure and activity to biofilm-mediated respiration. Together, these measurements connect redox state, redox activity, and electron flux, providing an integrated and mechanistically grounded picture of soil redox processes.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eWe demonstrated development, validation, and application of a custom Soil Electrochemical Profiling System (SEPS) designed to resolve depth-dependent electrochemical behavior in soils, hydrogels, and controlled redox systems. By integrating automated motion control with multi-modal electrochemical measurements, SEPS enables high-resolution interrogation of spatially and temporally evolving redox environments that are difficult to capture using conventional approaches. We have concluded that:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe SEPS successfully performed automated electrochemical depth profiling to 27 cm with micrometer-scale positional resolution (25 \u0026micro;m), demonstrating reliable integration of mechanical positioning and potentiostat control.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eDepth-resolved redox potential measurements in hydrogel\u0026ndash;Zobell systems confirmed that SEPS can capture fine-scale redox gradients and track the diffusion of well-defined redox couples (ferricyanide/ferrocyanide) over time, providing a controlled benchmark for interpreting transport-limited redox systems.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eCyclic voltammetry measurements in hydrogels further demonstrated that SEPS resolves spatial and temporal changes in redox-active species, revealing systematic depth and time dependent evolution of faradaic electrochemical behavior.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eIn soil mesocosms, SEPS detected strong vertical redox stratification, capturing the transition from oxic surface conditions to progressively reduced subsurface zones as incubation proceeded, consistent with established models of soil redox evolution.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eTime-resolved cyclic voltammetry in soils revealed the gradual emergence and intensification of redox activity, reflecting the development of coupled microbial and geochemical electron-transfer processes within the soil matrix.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eChronoamperometry enabled the enrichment of electroactive biofilms on microelectrode tips; when used for depth profiling, these biofilm-functionalized electrodes resolved clear vertical patterns in microbial electron-transfer activity, providing indirect but mechanistically meaningful evidence of electron donor and acceptor availability and biogeochemical heterogeneity in soils.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that SEPS uniquely integrates redox state (redox potential), redox activity (cyclic voltammetry), and electron flux (chronoamperometry) into a single, automated, depth-resolved platform. This multi-modal capability enables mechanistically grounded, \u003cem\u003ein situ\u003c/em\u003e investigation of soil redox dynamics and microbially driven processes that cannot be obtained from single-point or single-technique measurements. Beyond soil systems, the flexibility and scalability of SEPS make it broadly applicable to studies of sediments, wetlands, biofilms, and engineered porous media, offering new opportunities to link redox structure, activity, and function across complex environmental systems.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e5. FUNDING DECLARATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science Foundation (NSF), USA under Grant No. 2226680.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. AUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaluk Beyenal conceptualized and designed the research. Ibrahim Bozyel developed the device and its operation software. Won-Jun Kim performed the data curation. Both Won-Jun Kim and Haluk Beyenal conducted data analysis and wrote the original draft. Won-Jun Kim, Ibrahim Bozyel, Suat Ay, Maren Friesen, and Haluk Beyenal contributed to the manuscript editing. All authors approved the final version of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. DATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Python code for operating the manipulator system will be available upon request. All collected data is included in this published article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. COMPETING INTERESTS STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWebster, C. F., Kim, W. J., Reguera, G., Friesen, M. L. \u0026amp; Beyenal, H. Review: can bioelectrochemical sensors be used to monitor soil microbiome activity and fertility? \u003cem\u003eCurr. Opin. Biotechnol.\u003c/em\u003e \u003cb\u003e90\u003c/b\u003e, 103222 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn Introduction to Soil Concepts and the Role of Soils in. Watershed Management - Schoonover\u0026thinsp;\u0026ndash;\u0026thinsp;2015 - Journal of Contemporary Water Research \u0026amp; Education - Wiley Online Library. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://onlinelibrary.wiley.com/doi/full/\u003c/span\u003e\u003cspan address=\"https://onlinelibrary.wiley.com/doi/full/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1936-704X.2015.03186.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1936-704X.2015.03186.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorch, T. et al. Biogeochemical Redox Processes and their Impact on Contaminant Dynamics. \u003cem\u003eEnviron. Sci. Technol.\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e, 15\u0026ndash;23 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarkelova, E. \u003cem\u003eRedox potential and mobility of contaminant oxyanions (As, Sb, Cr) in argillaceous rock subjected to oxic and anoxic cycles\u003c/em\u003e (Universit\u0026eacute; Grenoble Alpes; University of Waterloo (Canada), 2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSch\u0026uuml;ring, J., Schulz, H. D., Fischer, W. R., B\u0026ouml;ttcher, J. \u0026amp; Duijnisveld, W. H. M. \u003cem\u003eRedox: Fundamentals, Processes and Applications\u003c/em\u003e (Springer Science \u0026amp; Business Media, 2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSojka, R. E. \u0026amp; Scott, H. D. Aeration Measurement. in Encyclopedia of Soil Science - Two-Volume Set (CRC, (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLacroix, E. M. et al. Consider the Anoxic Microsite: Acknowledging and Appreciating Spatiotemporal Redox Heterogeneity in Soils and Sediments. \u003cem\u003eACS Earth Space Chem.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 1592\u0026ndash;1609 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWard, B. B. Nitrification. in \u003cem\u003eEncyclopedia of Ecology\u003c/em\u003e 351\u0026ndash;358Elsevier, (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/B978-0-12-409548-9.00697-7\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-12-409548-9.00697-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGross, A., Lin, Y., Weber, P. K., Pett-Ridge, J. \u0026amp; Silver, W. L. The role of soil redox conditions in microbial phosphorus cycling in humid tropical forests. \u003cem\u003eEcology\u003c/em\u003e \u003cb\u003e101\u003c/b\u003e, e02928 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalil, K., Mary, B. \u0026amp; Renault, P. Nitrous oxide production by nitrification and denitrification in soil aggregates as affected by O2 concentration. \u003cem\u003eSoil. Biol. Biochem.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, 687\u0026ndash;699 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWagner, D., Pfeiffer, E. M. \u0026amp; Bock, E. Methane production in aerated marshland and model soils: effects of microflora and soil texture. \u003cem\u003eSoil. Biol. Biochem.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 999\u0026ndash;1006 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDorau, K. et al. Redoxtrons \u0026ndash; An experimental system to study redox processes within the capillary fringe. \u003cem\u003eEur. J. Soil. Sci.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e, e13347 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlimani, I., Zhu-Barker, X., Lazicki, P. \u0026amp; Horwath, W. Reviews and syntheses: Iron \u0026ndash; a driver of nitrogen bioavailability in soils? \u003cem\u003eBiogeosciences\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 3873\u0026ndash;3894 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y. et al. Oxygen availability regulates the quality of soil dissolved organic matter by mediating microbial metabolism and iron oxidation. \u003cem\u003eGlob Change Biol.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 7410\u0026ndash;7427 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZausig, J., Stepniewski, W. \u0026amp; Horn, R. Oxygen Concentration and Redox Potential Gradients in Unsaturated Model Soil Aggregates. \u003cem\u003eSoil. Sci. Soc. Am. J.\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e, 908\u0026ndash;916 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeiluweit, M., Gee, K., Denney, A. \u0026amp; Fendorf, S. Anoxic microsites in upland soils dominantly controlled by clay content. \u003cem\u003eSoil. Biol. Biochem.\u003c/em\u003e \u003cb\u003e118\u003c/b\u003e, 42\u0026ndash;50 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePonnamperuma, F. N. The Chemistry of Submerged Soils. in Advances in Agronomy vol. 24 29\u0026ndash;96 (Elsevier, (1972).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy, K. R. \u0026amp; DeLaune, R. D. \u003cem\u003eBiogeochemistry of Wetlands: Science and Applications\u003c/em\u003e (CRC, 2008). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1201/9780203491454\u003c/span\u003e\u003cspan address=\"10.1201/9780203491454\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtci, E., Babauta, J. T., Sultana, S. T. \u0026amp; Beyenal, H. Microbiosensor for the detection of acetate in electrode-respiring biofilms. \u003cem\u003eBiosens. Bioelectron.\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e, 517\u0026ndash;523 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtci, E., Babauta, J., Ha, P. \u0026amp; Beyenal, H. A Fumarate Microbiosensor for Use in Biofilms. \u003cem\u003eJ. Electrochem. Soc.\u003c/em\u003e \u003cb\u003e164\u003c/b\u003e, H3058\u0026ndash;H3064 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtci, E., Babauta, J. T., Sultana, S. T. \u0026amp; Beyenal, H. Microbiosensor for the detection of acetate in electrode-respiring biofilms. \u003cem\u003eBiosens. Bioelectron.\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e, 517\u0026ndash;523 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRAMPS 1. 4 - RepRap. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://reprap.org/wiki/RAMPS_1.4\u003c/span\u003e\u003cspan address=\"https://reprap.org/wiki/RAMPS_1.4\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003earduino.cc/en/software. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arduino.cc/en/software/\u003c/span\u003e\u003cspan address=\"https://www.arduino.cc/en/software/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtci, E., Babauta, J. T. \u0026amp; Beyenal, H. A hydrogen peroxide microelectrode to use in bioelectrochemical systems. \u003cem\u003eSens. Actuators B Chem.\u003c/em\u003e \u003cb\u003e226\u003c/b\u003e, 429\u0026ndash;435 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabauta, J. \u0026amp; Beyenal, H. Local Current Variation by Depth in Geobacter Sulfurreducens Biofilms. \u003cem\u003eJ Electrochem. Soc\u003c/em\u003e \u003cb\u003e161\u003c/b\u003e, (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeyenal, H. \u0026amp; Babauta, J. Biofilms in Bioelectrochemical Systems: From Laboratory Practice to Data Interpretation | Wiley. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.wiley.com/en-us/Biofilms+in+Bioelectrochemical+Systems%3A+From+Laboratory+Practice+to+Data+Interpretation-p-9781118413494\u003c/span\u003e\u003cspan address=\"https://www.wiley.com/en-us/Biofilms+in+Bioelectrochemical+Systems%3A+From+Laboratory+Practice+to+Data+Interpretation-p-9781118413494\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewandowski, Z. \u0026amp; Beyenal, H. \u003cem\u003eFundamentals of Biofilm Research\u003c/em\u003e (CRC, 2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1201/b16291\u003c/span\u003e\u003cspan address=\"10.1201/b16291\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBond, D. R. \u0026amp; Lovley, D. R. Electricity Production by Geobacter sulfurreducens Attached to Electrodes. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e, 1548\u0026ndash;1555 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTender, L. M. et al. Harnessing microbially generated power on the seafloor. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 821\u0026ndash;825 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLovley, D. R. Bug juice: harvesting electricity with microorganisms. \u003cem\u003eNat. Rev. Microbiol.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 497\u0026ndash;508 (2006).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"manipulator, depth profiling, redox, soil","lastPublishedDoi":"10.21203/rs.3.rs-8981935/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8981935/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhysicochemical soil parameters are key drivers of biogeochemical processes, controlling microbial activity and the mobility, solubility, and bioavailability of organic and inorganic matter. These parameters vary with depth, yet tools for their quantification are limited. Microelectrodes, among the most common tools, are primarily designed for biofilms only a few micrometers to millimeters thick and thus unsuitable for centimeter-scale soil gradients. To address this gap, we developed a custom-built automated manipulator system for soil depth profiling. Using off-the-shelf components, we constructed an instrument called Soil Electrochemical Profiling System (SEPS) capable of operating to depths of up to 27 cm with a minimum step interval of 25 \u0026micro;m. SEPS integrates directly with a potentiostat, enabling electrochemical techniques such as redox potential measurements, cyclic voltammetry, and chronoamperometry experiments. We validated the system by generating depth-resolved redox profiles and cyclic voltammograms in soil and sediment reactors. Additionally, we demonstrated its utility for microbial enrichment by polarizing a microelectrode at a fixed depth over 40 days. This system uniquely combines fine-scale automated positioning with electrochemical instrumentation functionality, providing a versatile and affordable platform for soil depth profiling and advancing the study of biogeochemical and microbial processes in terrestrial environments.\u003c/p\u003e","manuscriptTitle":"A Custom Soil Electrochemical Profiling System for Detecting Electrochemical Activity Changes in Soil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 08:41:26","doi":"10.21203/rs.3.rs-8981935/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-23T05:36:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-22T00:10:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-21T15:35:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T11:22:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310148354281818627590527461840916039161","date":"2026-03-17T14:02:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322973779367381931553888059338322108152","date":"2026-03-17T02:25:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40751897944282645826806235998415013560","date":"2026-03-16T07:40:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-15T10:27:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-14T11:39:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129680370520233495125669029542455591731","date":"2026-03-13T15:42:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39414632438193990360713881268642021580","date":"2026-03-12T08:17:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120863953577207222333361142468988527569","date":"2026-03-11T16:54:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207734816686304511157737504417518742809","date":"2026-03-11T16:38:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210823078034829512759328068115361747752","date":"2026-03-11T14:19:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-11T13:56:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-11T09:50:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-07T07:20:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-07T07:20:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-26T23:18:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7affd9d0-7c6e-4d97-8515-269088401f81","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64523185,"name":"Physical sciences/Chemistry"},{"id":64523186,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2026-04-29T18:39:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 08:41:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8981935","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8981935","identity":"rs-8981935","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}