# Development of Microbial Fuel Cell-Based Sensor for In-Situ Water Monitoring

Emna Hentati, Cyril Buttay, Naoufel Haddour

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Emna Hentati, Cyril Buttay, Naoufel Haddour. Development of Microbial Fuel Cell-Based Sensor for In-Situ Water Monitoring. International MEEP Symposium (Microbial/Enzymatic Electrochemistry Platform), Jul 2025, Lucerne, Switzerland. [hal-05525502](https://hal.science/hal-05525502v1)

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M0402

# Development of Microbial Fuel Cell-Based Sensor for In-Situ Water Monitoring

Emna Hentati, Cyril Buttay, Naoufel Haddour

INSA Lyon, Ecole Centrale de Lyon, Université Claude Bernard Lyon 1, CNRS,
Laboratoire Ampère, UMR 5005, 69621 Villeurbanne

Tel.: +33472186112

emna.hentati@insa-lyon.fr

## Abstract

Water pollution is a growing concern, with many possible causes such as discharges from hospitals, agriculture, industries, and saline intrusions in fresh water. Assessing water pollution requires reliable in-situ monitoring of water quality.

In the present study, a sensor based on microbial fuel cell (MFC) technology is developed to provide an inexpensive, long-term, and in-situ monitoring solution.

A novel MFC-based sensor architecture is designed to overcome cathode biofouling. The design incorporates a built-in chimney structure that supplies oxygen to the cathode even when the MFC is fully submerged. To further mitigate biofouling, a commercial air-cathode that carries out a two-electron reduction oxygen to generate hydrogen peroxide ($H_2O_2$) is employed. Iron-functionalized cedarwood biochar is used as the anode material. Performance tests across six wastewater batches collected from La Fessine wastewater treatment plant demonstrate good reproducibility and repeatability. A current of 0.312 ± 0.088 mA and a maximum power output of 0.019 ± 0.0045 mW are obtained. The durability of the MFCs is studied as well as the evolution of the cathode and the possibility of storage after active biofilm development. Vacuum storage tests confirm sustained biofilm electroactivity upon reactivation in synthetic wastewater environment, highlighting potential field deployment as an organic pollution biosensor.

<figure><img src="image_2.png" /><figcaption>Fig. 1 Newly developed MFC architecture</figcaption></figure>
Introduction

Microbial fuel cells (MFCs) are a promising and innovative electrochemical technology capable of converting chemical energy into electricity through the catalytic activities of microorganisms. MFCs have been developed originally to generate renewable energy (Potter 1911, [1]) and it has been reported that it is possible to produce electricity using any compound that can be metabolized by microorganisms [2]. In addition to electricity generation, MFCs offer multi-dimensional applications in various domains such as wastewater treatment, biosensors for environmental monitoring and in situ power supply [3]. Various MFCs architectures have been reported over the past few years by scientists aiming to improve their reliability and efficiency and reduce their manufacturing costs. In fact, the overall performance of an MFC in terms of electricity generation is governed by its design, the electrochemical properties of electrode material and the overall internal resistance of the cell. Electrodes play a fundamental role in facilitating electroactive biofilm growth and electrochemical reactions. Several metallic materials such as platinum, stainless steel, titanium, aluminum, copper, cobalt, nickel, gold and silver have also been explored primarily for their high conductivity which enhances the MFCs performance in terms of energy generation. However, some of these materials are costly and others are prone to corrosion [4]. Carbon which is available in many forms like graphite plates, cloth, paper, felt, foam and carbon nanotubes, is the most used electrode material due to its chemical, thermal and electrochemical stability [3], [4]. In addition, carbon-based electrodes offer a sizable surface area and a very good biocompatibility which facilitates bacterial adhesion on the electrode surface and enhances electroactive biofilm growth. Granular Activated carbon (GAC) and graphite granules (GG) have also been used, mainly in large-scale systems, due to GG's low cost and high conductivity and to GAC's high degree of micro-porosity and catalytic activities [5]. Biochar, derived from waste biomass, is emerging as a sustainable and cost-effective alternative to traditional electrode materials. It also offers high surface area, porosity and electrical conductivity which are key features for an efficient MFC. Since it is generally produced from available bio-waste, such as agricultural and forestry residues, costs associated with feedstock purchasing and transportation are highly reduced. It has been reported that biochar costs ten times less than GAC and GG [6]. It has also been reported that it is possible to enhance electron transfer capabilities and enhance microbial electroactivity (resulting in higher energy outputs) through the functionalization of biochar with metal oxides, particularly iron [7], [8].

Microbial fuel cells come in various architectural designs, the main ones being two-
compartment MFCs and single-compartment MFCs, each with its own advantages and
disadvantages. Two-compartment MFCs consist of an anodic and a cathodic chambers
separated by a proton exchange membrane (PEM). The anodic compartment is inhabited
by bacterial communities, usually containing carbon-based substances, enabling oxidation
as the main energy source. The purpose of incorporating a PEM is to allow proton transfer
from the anodic compartment to the cathodic chamber, while preventing the oxygen entry
from the cathode to the anode side [9]. The double-chambered architecture offers the ability
to control the cathode chamber, which improves the MFCs ability to generate power.
However, these MFCs can be extremely complex, expensive and difficult to sustain in the
long-term and often suffer from high internal resistance, making scale-up difficult This is why
they are mainly used for research and not a part of an in-situ solution [3], [9]. On the other
hand, single-compartment MFCs combine both electrodes in a single chamber, eliminating
the need for a membrane. It has been proven that MFCs without a membrane can produce
more power than the ones containing a membrane [10]. This may be because it leads to
lower internal resistance. This makes single-chambered MFCs easy to design, economical,
less costly as well as more efficient. However, research has shown that the cathode surface
of these MFCs is susceptible to biofouling, which is the development of a passive biofilm on the cathode surface, inhibiting its proper functioning and therefore decreasing the power output of the MFC. Several methods have been used to mitigate cathode biofouling in microbial fuel cells. Physical methods restore MFCs performance to a certain extent but deeply embedded biofilms cannot be reached and they are not suitable for large scale applications [11]. Chemical methods such as alkalis pre-treatment have also been used to mitigate cathode biofouling and proved to be more effective than physical ones but they may present certain environmental risks due to toxicity [12]. Many more methods seeking to overcome the persistent biofouling issue of the air cathodes have been developed but none proved to be 100% efficient making this matter an interesting subject of research. Hydrogen peroxide ($H_2O_2$) has proved to be an effective anti-biofouling agent due to its strong oxidative properties in different areas of research [13], [14], [15]. It acts by producing reactive oxygen species such as hydroxyl radicals which damage microbial cell membranes, proteins and DNA leading to cell death and disruption of biofilms. In MFCs, hydrogen peroxide production has been explored for several reasons such advanced wastewater treatment since it enables the breakdown of hard-to-degrade pollutants [16], [17] and cost and safety advantages [18]. However, to our knowledge, the anti-biofouling properties of hydrogen peroxide have not been studied in the context of MFCs.

Tubular microbial fuel cells have been a subject of discussion in the past few years. This architecture seeks to address one of the major limitations of MFCs, which is their high internal resistance due to ohmic, kinetic and mass transport losses [3]. It also aims to create a cost-effective, robust structure suitable for different in-situ applications such as scaling up wastewater treatment and energy generation [19]. This design often incorporates an up-flow (vertical) arrangement where the anolyte, generally wastewater, flows continuously from a bottom inlet, passing towards the anodic compartment, thus promoting better contact between the microorganisms and the electrode surfaces and exiting from the top [3], [9]. Some tubular MFCs have a cylindrical structure with concentric layers arranged around a central core [20], [21], [22], [23]. Another type of tubular design has been tested when components were stacked vertically rather than arranged concentrically. In this case, the anode is placed at the bottom where the anolyte flows and the air cathode at the top [21], [24], [25]. However, some limitations were observed such as high internal resistance [26] and a poor cathode reaction since it allowed the diffusion of a large quantity of oxygen towards the anode [25]. Plant-microbial fuel cells (PMFCs) have also been extensively studied in the past few years, and their designs have evolved from small-scale bottles or cube configurations toward tubular type reactors [27], [28], [29]. Suderjo et al. [27] developed a PMFC that can be completely submerged in paddy field soil all the while maintaining oxygen access towards the cathode through a silicone tube, generating continuous electricity throughout the rice growing season.

# 1. Scientific Approach

In this study, a systematic experimental approach was employed to develop and evaluate a novel one-chamber, tubular, up-flow MFC architecture to address the key limitations identified in current MFC technologies, specifically cathode biofouling, difficulties of in-situ usage and risks to the environment, with the ultimate goal of utilizing these improved MFCs as biosensors. Multiple batches were developed over the course of one year and a statistical analysis was performed to assess the MFCs reproducibility and repeatability based on different criteria. Furthermore, the durability of the MFCs has been studied as well as the evolution of the cathode and the possibility of storage after active biofilm development. Lastly, the potential of MFC deployment in an environment other than the original inoculum has been tested.
## 2. Experiments/Calculations/Simulations

**Preparation of the electrodes:** Different kinds of materials were used for the electrodes.

The anode was composed of two pieces of conductive biochar (dimensions: 30 x 20 x 10 cm), previously pyrolyzed under optimized conditions (Figure 1.A). To obtain this anode, cedar wood was cut into uniform pieces (20 mm × 70 mm × 3 mm), thoroughly washed with deionized water, and dried at 60 °C for a minimum duration of 24 hours. The wood pieces were subject to ferric iron pretreatment where they were submerged in a 4 g/L ferric chloride solution for 24 hours, followed by drying in an oven for an additional 24 hours at 80 °C. Subsequently, the dried wood pieces were placed onto ceramic combustion boats and inserted in a pyrolysis furnace (RSH 50/500/13, Nabertherm, Germany). The pyrolysis process was initiated by evacuating air from the furnace tube using a vacuum pump, followed by flushing with nitrogen gas at a flow rate of 100 L·h⁻¹ using a gas supply system (controller B 410 Nabertherm, Germany). The furnace temperature was increased gradually at a rate of 5 °C·min⁻¹ from room conditions to 900 °C. This targeted temperature was then maintained for 2 hours to achieve complete carbonization. After completion of the pyrolysis step, the furnace temperature cooled down to room temperature. Iron-doped biochar samples were then collected and sealed in aluminum foil.

The cathode was a commercially available air-breathing electrode (Gaskatel, Reference: 82061, Germany) composed of activated carbon loaded with hydrophobic PTFE backing, ensuring one-sided oxygen diffusing while minimizing water ingress (Figure 1.B). The electrode material was purchased as a roll and was cut into cylindrical pieces with a 34mm diameter using a hydraulic press (Unicraft WPP 10 HTE, Germany) and a die punching cutter.

<figure><img src="image_3.png" /><figcaption>Figure 1: (A) Schematic representation of the preparation of iron-doped biochar from cedar wood by pyrolysis; (B) The hydrogen peroxide air cathode</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

**Microbial fuel cell design:** The MFC biosensors were designed as compact, floating, single chamber systems incorporating the biochar-based anode and the commercial air cathode. The device was assembled using three PVC components (labeled as Pieces 1, 2, 3 in Figure 3.A) to facilitate structural stability, modularity and immersion in aqueous environments. The central piece served as the foundation of the cell and is the most critical to its function. The air cathode is placed inside this piece, clamped between two silicone gaskets with an outside diameter of 34 mm and an inside diameter of 30 mm, made with a die punching cutter and the hydraulic press. It is then pressed and fixed inside using pieces (b) and (c). Piece (c) is threaded and has two holes on its sides making it possible to turn it and fasten it using a screwdriver. Pieces 1 (diameter 24 mm) and 3 (diameter 34 mm) were assembled using PVC tubes softened and reshaped by heating with a hot air gun (RS PRO, Ref: 124-5159, France), then inserted into the central piece, one on top and one below. The first PVC tube serves as an extension of the system to prevent water entry into the second piece when the MFC is submerged in water while ensuring continuous air access to the cathode. The third piece is designed to protect the anode and the electroactive biofilm cultivated on its surface from any disturbances or mechanical shocks that might occur in the environment of the MFC. It is cut to a fixed length of 13.5 cm to ensure that the distance between both
electrodes for all developed MFCs is the same. 4.5 mm holes are drilled into this piece to
allow the medium to reach the anode evenly.

<figure><img src="image_5.png" /><figcaption>**Figure 3:** (A) Schematic representation of the MFC design; (B) Schematic representation of the central piece; (C) The produced MFC</figcaption></figure>

**Microbial fuel cell setup and operation:** The first step to build MFCs consists in assembling the electrodes inside the PVC pieces. The cathode was assembled in the central piece so it is positioned between two gaskets. A titanium wire (0.25 diameter, GoodFellow) was passed through one side of the cathode and out the other, serving as a current collector. The electrical contact was checked using an ohmmeter to ensure that it is sound (< 10Ω). A leakage test was then carried out to make sure that the cathode assembly is water-proof from below. The central pieces were immersed in an aquarium containing water for 24 hours. If any seepage is detected during this time period, the central piece assembly and test must be repeated. For the anode, two pieces of biochar were utilised as a single anode to optimise its surface area. Each biochar piece was slightly opened at the top using a scalpel to allow the insertion of the 0.25 mm titanium wire. The conductive wires of the two pieces are then intertwined and sealed with a sheath that is shrunk afterwards using the hot air gun. This ensures a good connexion and insulation of the wires from the surrounding environment. Finally, the electrical contact was checked with an ohmmeter to verify that it is consistent and reliable (< 10Ω). The complete MFC units were mounted vertically in a 60 L glass aquarium containing real wastewater collected from La Fessine municipal wastewater treatment plant (Lyon, France). Sludge was used at 1g/L. Sodium acetate was also added to the mixture at 1g/L as an additional degradable carbon source to facilitate initial microbial colonization and biofilm development on the anode surface. Each MFC was maintained in a floating configuration using a polyethylene foam support (made out of polystyrene, 12 x 12 cm², containing a 46 mm diameter hole) ensuring the cathode remained at the air-water interface and the anode fully submerged. Voltages across 1kΩ external resistances were measured every minute using a multi-channel data acquisition system (Agilent 34972A) monitored with a LabView platform. This system allows the progress of biofilm formation to be monitored in real time. The MFCs were operated continuously at room temperature (~22 °C) under batch mode. Six batches of MFCs (42 MFCs in total) have been developed in the past year allowing us to assess the reproducibility and repeatability of the designed system.

<figure><img src="image_6.png" /><figcaption>**Figure 4:** The launch of MFCs in wastewater</figcaption></figure>
**Evaluation of MFCs initial performances:** Once the biofilms were mature (the MFCs voltages were stable), continuous voltage logging was interrupted to avoid interference with subsequent electrical characterizations. Polarization curves were made to evaluate the electrical performances of the MFCs. Resistor boxes that allow the external resistance to be varied were used. 14 different external loads were applied (from 4 MΩ to 2 Ω) and the voltage between the anode and the cathode is measured with a multimeter after stabilisation which takes approximately 30 minutes. Anode and cathode potentials were measured versus an Ag/AgCl reference electrode (Origalys, France) at each load condition.

**Statistical analysis:** In this study, the reproducibility and repeatability of the MFCs were assessed using the coefficient of variation (CV) as a statistical metric. The experimental setup consisted of 6 batches with a total of 42 MFCs. The CV of four parameters (time taken for biofilm to reach maturity, open circuit potential, maximum power, and current measured on a 50 Ω load) was calculated using this formula: $CV(\%) = (\sigma/\mu) \times 100$, where $\sigma$ represents the standard deviation and $\mu$ represents the mean value of each parameter. For within-batch repeatability, the CV was determined by analysing the variability along MFCs in the same batch, while between-batch reproducibility was evaluated by comparing the values across all 42 MFCs. This approach provides a dimensionless measure of dispersion that allows direct comparison of variability.

**Linear sweep voltammetry analysis:** Linear sweep voltammetry (LSV) measurements were conducted to evaluate the electrochemical performance of cathodes used in four MFCs compared to new, unused cathodes. The electrochemical characterization was performed using a potentiostat (Origalys, France) in a three-electrode configuration. The cathode served as the working electrode while a platinum electrode was used as the counter electrode and an (Ag/AgCl) electrode as the reference electrode. All measurements were carried out in a 50 mM $Na_2SO_4$ electrolyte solution at ambient temperature. The potential was swept linearly from the open circuit potential (OCP) to -1V vs. Ag/AgCl at a scan rate of 10 mV/s to minimize capacitive effects. For each cathode, the current response was recorded as a function of the applied potential.

**Vacuum packaging method for MFCs preservation:** A vacuum packaging approach was employed to investigate the preservation of electroactive biofilms in MFCs for potential deployment in diverse environments. Five MFCs (F2-F6) with established electroactive biofilm were selected for this experiment. Each one was taken out of the wastewater inside a transportation bag filled with the same solution to ensure that the lower portion containing the anode remained submerged and was not exposed to air. They were then transferred to vacuum bags (25 x 40 cm), and sealed using a film sealing device (Severin Folio, Germany) after maintaining anaerobic conditions through a continuous nitrogen gas supply for approximately 10 minutes. Once the MFCs where properly positioned, the solution from the transportation bag was drained from a hole cut on the other side of the bag, which was then quickly sealed to maintain the oxygen free environment. The sealed devices were stored under vacuum at ambient temperature (~22 °C) for two weeks in the absence of light.

## 3. Results

**Study of biofilm growth of MFCs:** The development and electrochemical maturation of electroactive biofilms on biochar anodes were monitored during a period of about a week in six independent experimental batches (labeled A to G) conducted over one year (Figure 5). This temporal evolution is indicative of biofilm formation dynamics and microbial electroactivity. Biofilms maturity is reached generally between days 3 and 5, where Batch C had the fastest growth kinetics in comparison to the other batches. The coefficient of variation in percentage (CV) concerning the duration of biofilm maturity (in days) was calculated to assess the reproducibility of this parameter across all 42 MFCs (Table 1) and its repeatability across the 6 batches (Table 2). A CV of 20.2% was determined when comparing the duration of biofilm maturity across all MFCs indicating a moderate but
<figure><img src="image_7.png" /></figure>

acceptable discrepancy. Batch B presented the highest variance with a CV of 8.4%. Batches C and F presented slightly lower CVs of 6.8% and 4.9% respectively. MFCs of batches A, D and G presented identical growth kinetics with a CV of 0%. These statistics indicate that the designed MFC allows to have repeatable and reproducible results in terms of biofilm maturity.

<figure><img src="image_8.png" /><figcaption>Figure 5: Voltage outputs of MFCs of 6 batches recorded in function of time to monitor biofilm growth</figcaption></figure>

<table><caption>Table 1: Coefficient of variation (%) of 42 MFCs across different parameters</caption><thead><tr><td>Parameters</td><td>Biofilm maturity (days)</td><td>Open circuit potential (mV)</td><td>Maximum power (mW)</td><td>Current at 50Ω (mA)</td></tr></thead><tbody><tr><td>C.V (%)</td><td>20.2%</td><td>8.3%</td><td>13.9%</td><td>12.7%</td></tr></tbody></table>

<table><caption>Table 2: Coefficient of variation (%) of the 6 batches across different parameters</caption><thead><tr><td>Batches</td><td>Biofilm maturity (days)</td><td>Open circuit potential (mV)</td><td>Maximum power (mW)</td><td>Current at 50Ω (mA)</td></tr></thead><tbody><tr><td>Batch A</td><td>0%</td><td>2.5%</td><td>6.9%</td><td>7.2%</td></tr><tr><td>Batch B</td><td>8.4%</td><td>0.9%</td><td>8.7%</td><td>9%</td></tr><tr><td>Batch C</td><td>6.8%</td><td>3.4%</td><td>3.9%</td><td>2.8%</td></tr><tr><td>Batch D</td><td>0%</td><td>3.7%</td><td>8.7%</td><td>5.5%</td></tr><tr><td>Batch F</td><td>4.9%</td><td>11%</td><td>5.8%</td><td>15%</td></tr><tr><td>Batch G</td><td>0%</td><td>10.7%</td><td>9.9%</td><td>11.1%</td></tr></tbody></table>

**Study of electrical performances of MFCs:** MFCs were assessed through polarisation curves (Figure 6). Polarization and power density curves revealed consistent electrical behaviours across batches. As illustrated in Figures 6, each batch exhibited characteristic power-current profiles with peak power densities ranging from 14 to 23 µW and current outputs at 50Ω between 0.25 and 0.40 mA for most units. Open circuit potential (OCP/mV), maximum power output (Pmax/mW) and current at 50Ω (Imax/mA) were chosen to assess the MFCs repeatability and reproducibility where the coefficient of variation was calculated for each of these parameters (Table1, Table2). Comparison of the 42 MFCs shows CVs of 8.3% for OCP, and 13.9% for Pmax and 12.7% for Imax, indicating a few variances especially in terms of power output. The 7 MFCs of Batch C presented the least discrepancies with a CV not exceeding 4% for all parameters. The CVs associated to current values produced at 50Ω were the highest at 15% and 11.1% for Batches F and G respectively. These relatively high values may also be due to the larger number of MFCs in these batches in comparison to the
other ones. The CVs related to maximum power generation were mostly close for all batches between 3.9% and 9.9%. Batches A and B presented variations in terms of power and current (between 7 and 9%) but barely for open circuit potentials. These statistics indicate that the produced MFCs are globally repeatable and reproducible. These deviations might be due to the physicochemical parameters of the inoculum from one batch to the other (Table 3).

<figure><img src="image_9.png" /><figcaption>Figure 6: Polarization curves demonstrating MFCs electrical performances</figcaption></figure>

<table><caption>Table 3: Physicochemical parameters of the inoculum of the 6 MFCs batches; pH, Dissolved oxygen DO (mg/L), Electrical conductivity EC (µS/cm)</caption><thead><tr><th>P/C parameters</th><th>Batch A</th><th>Batch B</th><th>Batch C</th><th>Batch D</th><th>Batch F</th><th>Batch G</th></tr></thead><tbody><tr><td>pH</td><td>8.4</td><td>7.7</td><td>7.6</td><td>7.1</td><td>8</td><td>7.9</td></tr><tr><td>DO (mg/L)</td><td>0.28</td><td>0.16</td><td>0.05</td><td>0.19</td><td>0.07</td><td>0.16</td></tr><tr><td>EC (µS/cm)</td><td>1995</td><td>1389</td><td>1184</td><td>1200</td><td>1195</td><td>1325</td></tr></tbody></table>

**Study of the evolution of the cathode:** The electrochemical performance of cathodes is a critical factor influencing the overall efficiency and power output of MFCs. A series of LSV measurements comparing four used cathodes and four new ones was conducted to investigate the impact of operational use on cathode properties (Figure 7). The results revealed remarkable differences between the two groups, providing valuable insights into cathode evolution during MFC operation. Looking at the entire potential range, the used cathodes consistently generate higher current magnitudes than the new cathodes. At very negative potentials (-1000 mV to -800 mV), they produce approximately 1.5 times the current of the new cathodes. The new cathodes display extremely similar behaviour, indicating a good reproducibility in their initial properties. However, the used ones show notable variability, suggesting modification during MFC operation. When focusing on the potential range in which MFCs typically operate (0 mV to 500 mV), the used cathodes demonstrate significantly enhanced catalytic activity compared to the new cathodes. At 400 mV, they generate currents between -1 mA and -3.5 mA, while new cathodes produce only about -0.5 mA. This positive change suggests improved reaction kinetics and lower activation overpotential.
<figure><img src="image_10.png" /><figcaption>**Figure 7:** LSV curves of four used cathodes and four new cathodes</figcaption></figure>

**MFCs packaging:** In the interest of using the developed MFCs in different environments and facilitating their transport, the vacuum packaging of 5 MFCs was tested in order to determine whether the formed electroactive biofilm can be preserved for a period of time. The MFCs were sealed inside vacuum bags exempt of oxygen for 24 days after which they were put back in wastewater, along 1g/L of sodium acetate. Voltage outputs were recorded in function of time for a period of 8 days (Figure 8) and polarisation curves were made on two separate occasions, at days 9 and 12 to check if the MFCs regained their electrical performances (Figure 9). For all MFCs, the voltage is initially near zero, indicating that the cells were inactive immediately after being removed from vacuum storage. This is followed by a sigmoidal recovery pattern with a lag phase (1 – 3 days), then a rapid increase in voltage, finally reaching a stationary phase around days 6 to 8. MFC F4 possessed the earliest and most rapid recovery, beginning its voltage recovery around day 1.5 and reaching a plateau of approximately 160 mV around day 6. This suggests that the electroactive biofilm of this particular cell maintained better viability during vacuum storage. The remaining MFCs showed a slightly more delayed recovery with successful voltage increases.

<figure><img src="image_11.png" /><figcaption>**Figure 8:** Voltage outputs of 5 MFCs recorded in function of time after 24 days of vacuum sealing</figcaption></figure>

The “before_SV” curves (blue) in Figure 9 represent the baseline performance of the MFCs before vacuum storage. These demonstrate moderate power outputs with values typically between 0.03 mW and 0.04 mW. The “after_SV1” (orange) curves measured on day 9 after reintroduction to wastewater show significantly reduced performance compared to the baseline indicating limited electrochemical activity. The curves measured on day 12 (“after_SV2”, red) show substantial recovery and even enhancement compared to the original performances with a maximum power output of 0.04-0.05 mW. The current ranges also expanded by day 12, with most cells able to sustain current production up to 0.8-1 mA. This may be due to the changes of the wastewater’s physicochemical properties due to sodium acetate addition, where electrical conductivity has increased from 1.2 mS/cm to 3.9 mS/cm. This demonstrates the ability of the microbial communities in MFCs to enter a dormant state during oxygen-free storage without losing their electrochemical functionality. Such preservation capacity is particularly valuable for practical applications as it enables the transport the deployment of pre-colonized MFCs to various locations.
<figure><img src="image_12.png" /><figcaption>**Figure 9:** Polarization curves demonstrating MFCs electrical performances after 24 days of vacuum sealing</figcaption></figure>

**MFC deployment in a new environment:** Two MFCs, B1 and B5, have been transferred to podzol, which is a type of acidic soil found in various parts of the world, including France, Russia, Canada, and the Amazon river. This test aims to determine whether the previously formed electroactive biofilm can function in a new environment. The voltage outputs of both MFC have been measured (Figure 10). Initially, the measured voltages began declining but quickly increased when 1g/L of sodium acetate were added at the end of the first day (Figure 10.B), suggesting that the lack of carbon substances in this environment that are necessary for oxidation and therefore energy generation. This increase which attained values as high as 120 mV demonstrates that it is possible the already formed electroactive biofilm in a new environment. Voltages began declining around day 7, suggesting that all the organic matter had been consumed by the MFCs. In an interest to utilize these MFCs as biosensors, a pollution demonstration has been performed around day 15 after podzol became exempt of organic matter through the addition of newly collected wastewater (Figure 10.C). An increase in the MFCs output voltages was registered (25-30 mV) due to the addition of organic matter. This result indicates that it is possible to use the produced MFCs as biosensors for environmental pollution.

<figure><img src="image_13.png" /><figcaption>**Figure 10:** (A) MFCs transferred to podzol soil; (B-C) Voltage outputs monitored in function of time</figcaption></figure>
**Conclusion:** In this work, a novel one-chamber, tubular, up-flow microbial fuel cell architecture that addresses several key limitations of current MFC technologies have been developed. The innovative design, incorporating iron-doped biochar anodes, and hydrogen peroxide-producing cathodes, demonstrated remarkable consistency and reliability across different batches. Statistical analysis using the coefficient of variation (CV), confirmed good reproducibility and repeatability, with CV values mainly below 15% for critical performance parameters including biofilm maturity, open circuit potential, maximum generated power and current output on a 50Ω load. The electrochemical characterisation of cathodes revealed an interesting evolution after being used as part of the MFC, with used cathodes demonstrating significantly enhanced catalytic activity compared to new ones. The successful preservation of electroactive biofilms through vacuum packaging for extended periods has also been achieved, with all MFCs resuming electricity generation upon reintroduction to wastewater. The MFCs also showed adaptability when transferred to acidic podzol soil environment, confirming that established electroactive biofilms can function effectively in conditions different that their original growth environment when provided with appropriate organic sources. The observed voltage response to wastewater addition further validates that these MFCs are capable to function as biosensors for environmental pollution monitoring.

**Acknowledgement:** This file is distributed under a CC-BY 4.0 license.

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**Keywords: MEEP2025, Bio Fuel Cells, MFC-sensor architecture, Iron-doped biochar, anti-biofouling**

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