Developing a Bacterial Monolayer Biofilm Using an Electrochemical Reactor

Begin with an electrochemical reactor equipped with an external water jacket, tightly sealed over a working electrode.

Next, insert the reference and counter electrodes to complete the electrical circuit.

Remove the reference electrode, add nutrient media, and place the electrode back.

Introduce filtered nitrogen gas to create anaerobic conditions.

Place the setup inside a Faraday cage to avoid external electrical interference.

Connect the water circulation system to maintain the reactor temperature during the voltage application.

Next, connect to a potentiostat and apply a constant electrical voltage to the working electrode relative to the reference electrode.

Inject the bacterial suspension into the reactor and close the cage. Continue applying electrical voltage.

The applied voltage stimulates extracellular electron transfer from the bacterial cells to the working electrode, promoting initial surface attachment.

Over time, bacteria form a monolayer biofilm on the working electrode.

To construct a three-electrode electrochemical reactor, put an ITO substrate as the working electrode at the bottom of the reactor.

Subsequently, insert a glass cylinder and a polytetrafluoroethylene cover and insert a platinum wire as the counter-electrode. Then, insert a silver chloride electrode into the reactor as the reference electrode. Next, add four milliliters of DM supplemented with 10 millimolar lactate and 0.5 grams per liter yeast extract into the electrochemical reactor.

After confirming that there is no leakage from the electrochemical reactor, flow the nitrogen gas into the electrochemical reactor over 20 minutes to maintain anaerobic conditions inside the electrochemical reactor. Connect the electrochemical reactor to a potentiostat and apply a positive 0.4 volts to the ITO electrode, keeping the temperature of the electrochemical reactor at 30 degrees Celsius using an external water circulation system.

Add 0.3 milliliters of the cell suspension into the electrochemical reactor through the injection port using a syringe. The OD₆₀₀ in the reactor changes to 0.1. Continue the potential application at positive 0.4 volts to the ITO electrode for 25 hours.