Hardware

Microbial fuel cells (MFCs) are bioelectrochemical systems that generate electricity using microorganisms. The concept was first described in 1911 by Michael Potter, who observed that Saccharomyces cerevisiae could produce an electrical current. Since then, research has focused on improving electron transfer mechanisms, optimizing hardware design and electrode material, to enhance MFC performance.

In the anode chamber, microorganisms such as yeast release electrons during metabolism. These electrons are transferred to mediators (e.g., methylene blue), which shuttle them to the anode. The electrons then flow through an external circuit to the cathode, where a terminal electron acceptor (such as potassium hexacyanoferrate) completes the reaction by accepting the electrons.

MFCs in iGEM: A Brief History

Over the past decade, several iGEM teams have explored microbial fuel cells (MFCs), applying diverse biological and engineering strategies to improve their performance and expand their applications.

iGEM Bielefeld 2013 pioneered synthetic biology approaches for MFCs by engineering Escherichia coli with porins, cytochromes, conductive nanowires, and electron-transferring mediators such as riboflavins and phenazines. They also developed two functional battery prototypes: one constructed with acrylic boards for performance testing, and another 3D-printed for flexible design. Their detailed hardware documentation remains a valuable reference for later teams.

iGEM Trento 2015 enhanced proton pumping by expressing proteorhodopsin under light conditions. They also designed a unique MFC architecture: a cylindrical anodic chamber connected to six independent cathodic chambers.

iGEM Hong Kong HKUST 2018 combined Escherichia coli and Shewanella oneidensis to convert degraded polyethylene into electricity. Their project included the development of a 3D-printed MFC prototype.

iGEM Harvard 2016 focused on plastic degradation. By expressing PETase on Escherichia coli, they demonstrated how degradation products could power MFCs while also enabling PET detection.

iGEM Westminster 2019 integrated plastic degradation with energy production by co-culturing engineered bacteria and algae that expressed PET-degrading enzymes and cytochromes.

iGEM Nanjing 2022 improved electron harvesting efficiency by using silver-binding proteins in the presence of silver nanoparticles.

Material Selection

Overview:
We surveyed several off-the-shelf options and the literature before finalizing our MFC hardware and materials. The University of Reading NCBE educational MFC kit (here) is a widely used testing platform (mediator-based, methylene blue) and provides a baseline for small-scale MFC experiments; however, the kit uses fixed chamber parts and a small, pre-cut membrane that limit reconfiguration for alternative cathode chemistry or electrode geometries. DMFC Adapta-Stack sold by Fuel Cell Store (here) is a PEM/MEA stack for direct methanol fuel cell work and proven to be functional as microbial fuel cells by iGEM Harward 2016, however, its thin MEA geometry and defined active area make it unsuitable for thick, porous carbon-felt electrodes intended for yeast colonization. Given these constraints, and following consultations with our engineering partner Renno Raudmäe, we designed an acrylic-board modular cell inspired by the Reading kit. We have improved the setup for reconfigurability and minimization of leakage.

Our design improvements:
We developed a custom acrylic-based MFC, inspired by the Reading kit but optimized for robustness and flexibility:

  • Transparent acrylic body - allows visual monitoring of chambers during operation.
  • Two anaerobic chambers - with custom inlet and outlet holes for electrodes and wiring.
  • Proton exchange membrane (Nafion 212) - supported by two rubber gaskets.
  • Carbon felt electrodes - thick (2 * 2 * 1.5 cm) porous electrodes provide high surface area for microbial colonization.
  • Flexible chamber volume - thick acrylic walls and modular design allow quick adjustment of chamber dimensions for future experiments.
  • Reduced leak risk - simplified construction with fewer parts, gaskets between chambers, and sealed joints (epoxy + silicone).
  • Improved electrical performance - the proximity of electrodes reduces internal ohmic resistance, improving current output.

Electrode:
Most iGEM teams and modern studies use carbonaceous electrodes because they are cost-effective and deliver competitive performance relative to precious metal electrodes Santoro et al., 2017 [3]. Carbon felts were selected to be used as material for our anode due to their high porosity, internal surface area and mechanical robustness - properties that facilitate colonization and increase the effective electrode area for extracellular electron transfer. Our electrode implementation uses a thick carbon felt block (dimensions and packing density given in hardware) that allows yeast to inhabit internal pores and provide stable current over time.

Cation exchange membrane:
We selected Nafion 212 ((Jenani et al., 2024 [4])) as the membrane for our prototyping work. Nafion remains widely used in MFC literature for its high proton conductivity and chemical stability. We understand that Nafion is relatively expensive and is often cited in the literature as a commercialization bottleneck. For the 2025 prototype, Nafion 212 offers reliable, reproducible performance that facilitates comparison with published studies.

Making Microbial Fuel Cells

Electrodes and hardware

  • Carbon felt electrodes (2 × 2 × 1.5 cm, two pieces)
  • Stainless steel wire (30 cm)
  • Nafion 212 membrane (6 × 6 cm)
  • Two acrylic chambers (see Hardware files)
  • Two rubber gaskets (see Hardware files)
  • Two cloths (2 × 2 cm, for membrane support)
  • Four M6 bolts
  • Eight M6 washers
  • Eight M6 nuts (wing or standard)

The total cost of the production is approximately 3 euro for a single cell.

Adhesives and surface treatment

Preparation of proton exchange membrane (PEM)

To ensure good proton conductivity, Nafion membranes must be pre-treated to remove metal cations and activate the surface.

Materials

  • Nafion 212 membrane (remove protective plastic covers)
  • Weak to moderate acid (0.1–1 M HCl, HNO₃, or H₂SO₄)
  • Millipore-quality (MQ) water

Steps

  1. Cut Nafion 212 to size. Remove protective covers.
  2. Place the membrane in a beaker with acid solution (10–20 mL per cm² of membrane).
  3. Stir gently on a stir plate for 2–24 h. Temperature can be room temperature or elevated (60–80 °C) to enhance proton exchange.
  4. Decant the acid. Rinse with fresh MQ water, stir, and repeat 3–5 times.
  5. Continue rinsing until the wash water is neutral (pH ~7).
  6. Store the cleaned membrane in MQ water until use.

Assembling

  1. Sterilize all parts with ethanol
  2. Roughen acrylic bonding surfaces with sandpaper.
  3. Glue acrylic plates to form two chambers; cure with epoxy and seal edges with silicone.
  4. Place one carbon felt electrode in each chamber. Connect to stainless steel wires.
  5. Insert a cloth layer and gasket.
  6. Repeat steps for the opposite chamber.
  7. Place pretreated Nafion 212 membrane between the gaskets.
  8. Align chambers, insert bolts with washers, and tighten nuts gradually in a cross pattern.
  9. Inspect for leaks with sterile water before use.

Measurements

To evaluate the performance of our engineered strains, we first measured the open-circuit voltage (OCV) of our microbial fuel cells (MFC) with a multimeter. OCV provides a simple indication of electrochemical activity between the electrodes, but it has limitations: because MFC currents are extremely small, multimeters cannot directly provide stable current readings to calculate power output.

A more reliable method is to connect the cell to an external resistor and measure the resulting voltage drop. Current is then calculated using Ohm’s law (I = V/R). Many iGEM teams use resistance boxes for this purpose, since they allow stable and reproducible measurements across a wide range of loads. From these data, two key performance curves can be obtained:

1. Polarization curves:

A polarization curve is generated by applying different resistances to the MFC and recording the steady-state voltage at each step. Plotting current (x-axis, calculated as I = V/R) against voltage (y-axis) shows how voltage decreases as current demand increases. This curve highlights the internal resistance of the system and the range where the cell performs most efficiently.

2. Power curves:

From the same measurements, power is calculated as P = V × I. Plotting current (x-axis) against power (y-axis) identifies the point where the cell delivers maximum power output, providing a straightforward metric for comparing performance across designs or experimental conditions.

Equipment:

  • Resistance box (x1, x10, x100, x1k, x10k Ω)
  • Multimeter
  • Wires
  • Syringes
  • Crocodile clips

Chemicals:

  • 1 M glucose solution (substrate)
  • 0.02 M potassium hexacyanoferrate solution (cathodic electron acceptor; store in dark)
  • 10 mM methylene blue solution (mediator)
  • 0.1 M potassium phosphate buffer, pH 7.0

Procedure:

  1. Prepare the anode chamber by mixing:
    • 10 mL glucose solution
    • 10 mL potassium phosphate buffer
    • 10 mL methylene blue solution together with the yeast culture. Inject this mixture into the anode chamber.
  2. Fill the cathode chamber with potassium hexacyanoferrate solution.
  3. Seal all inlets to maintain anaerobic conditions.
  4. Connect a multimeter to the electrodes and record the OCV at a fixed time point.
  5. Once the OCV has stabilized, generate polarization and power curves:
    • Connect the resistance box to the circuit.
    • Gradually decrease resistance step by step.
    • At each resistance, wait until the voltage stabilizes and record the value.
  6. Calculate current (I = V/R) and power (P = V × I) for each resistance and plot the results to obtain the polarization curve (voltage vs. current) and the power curve (power vs. current).
  7. Connect a multimeter to the electrodes and record the open-circuit voltage (OCV) at a fixed time point.

Results

In our custom-built hardware, we measured the electrochemical performance of our engineered Δgpd1 deletion strains and the DOM90 control. In the polarization curve (Figure 1), Δgpd1 displayed a less steep slope than DOM90, indicating stronger electrochemical potential and lower internal resistance.

Figure 1. Polarization curve of gpd1 and DOM90 strains. gpd1 exhibits a gentler slope compared to DOM90, indicating both stronger electrochemical potential and lower internal resistance. These results suggest that gpd1 has slightly better performance under the tested conditions.

The power curve (Figure 2) further showed that Δgpd1 achieved a higher maximum power output, confirming improved energy conversion. These results confirm that suppressing glycerol biosynthesis following GPD1 deletion increases the cytosolic NADH pool, thereby enhancing electron transfer to the electrodes, and validate our hypothesis that boosting NADH levels is an effective strategy for engineering yeast as a high-performance cell factory.

Thus, the measurement results obtained from our hardware directly validate our genetic design by linking the metabolic modifications in yeast to measurable improvements in MFC performance.

Figure 2. Power curve of gpd1 and DOM90 strains. The gpd1 strain reaches a higher maximum power output than DOM90, confirming the advantage suggested by the polarization data.

Our experiments showed that the Δgpd1 strain performs slightly better than the DOM90 control, achieving lower internal resistance, and greater maximum power output. Building on these results, we aim to further improve microbial fuel cell performance by exploring how parameters such as glucose concentration, chamber size, and potassium hexacyanoferrate levels influence voltage stability and power generation.