Characterizing Mediated Extracellular Electron Transfer in Lactic Acid Bacteria with a Three-Electrode, Two-Chamber Bioelectrochemical System

NOTE: Two-chambered BES assemblies will be referred to as "reactors" in the following protocol.

1. Media preparation

1. Prepare L. plantarum culturing media.
   1. Prepare commercial MRS (de Man Rogosa Sharpe) media as instructed and mMRS media²⁴ as described in Table 1. Adjust the pH of mMRS to 6.5. Filter-sterilize both media by passing them through a 0.22 µm filter and store both at 4 ˚C until use.
   2. Prepare a Chemically Defined Medium with mannitol (mCDM)^13,25 as described in Table 2, and adjust the pH to 6.5. Prepare enough medium to fill the anodic chamber of each reactor with 110 mL of medium. Filter-sterilize mCDM through a 0.22 µm filter.
      NOTE: mCDM should be prepared fresh on the day of intended use. The component solutions may be prepared in advance. Filter-sterilize all component solutions through a 0.22 µm filter and store at 4 ˚C. When preparing Wolfe's vitamins, adjust the pH to 11 before sterilizing and store in darkness or wrapped in foil. When preparing Wolfe's minerals, adjust the pH to 8 after adding nitrilotriacetic acid (NTA). Then, add the remaining components, sterilize, and store in darkness or wrapped in foil.
2. Prepare 1x PBS and autoclave to sterilize. Store at 4 ˚C for use in this protocol.
3. Prepare commercial M9 media as instructed (Table 3) and autoclave to sterilize. Prepare enough media to fill the cathodic chamber of each reactor with 110 mL. Store at room temperature.

2. Day 1: BES reactor assembly and initial  L. plantarum culturing

NOTE: Reference Figure 2 for a schematic of a BES reactor and a diagram detailing the assembly pieces indicated in the protocol.

Figure 2: BES components and diagram for assembly. (A) A schematic of a two-chamber BES reactor. Bacteria (green) in the anodic chamber transfer electrons to a working electrode (black circle) in the presence of a quinone mediator. Electrons flow through the circuit to the cathodic chamber, allowing current measurements to be taken between the anode and cathode by a potentiostat. (B) An image depicting a fully assembled BES reactor, including N₂ inlet and outlet needles in the anodic chamber. (C) An image depicting all parts of a disassembled reactor. Abbreviation: BES = bioelectrochemical system. Please click here to view a larger version of this figure.

1. Assemble and sterilize reactors.
   1. Prepare the working and counter electrodes. Sand precut 1.0 mm diameter titanium wires for the working and counter electrodes with aluminum oxide sandpaper until evenly shiny. Using pliers, bend one end of each working electrode wire into a small hook. Slide a 16 cm² carbon felt round onto each working electrode wire, weaving the wire in and out of the carbon felt round once and pulling the round down the wire until secured on the hook. Secure the working and counter electrodes into GL45 caps by piercing the rubber septum with the wire and pulling it through a few centimeters.
   2. To assemble paired reactors, first assemble the O-ring and place a precut cation exchange membrane that has been presoaked in water into the assembled O-ring. Place the O-ring with the membrane between the large bottom openings of two paired reactor bottles. Secure the reactor pair bottles and O-ring with the membrane with a knuckle clamp to finger tight. Drop a magnetic stir bar into each anodic chamber, and then close all small openings at the top of each bottle with GL14 caps fitted with rubber septa.
      NOTE: Do not overtighten the knuckle clamps or any caps as this can lead to cracks or breaks in the bottles.
   3. Fill each reactor bottle with 110 mL of deionized (DI) water, then close each bottle with the appropriate electrode-fitted GL45 cap. Insert caps fitted with a carbon felt round working electrode by gently pressing down the top of the felt round so the round remains on the hook.
      NOTE: Fresh carbon felt is hydrophobic and repels water in the reactor in the initial assembly. Autoclaving will cause the carbon felt to become appropriately hydrophilic for experimentation.
   4. Gather GL14 electrode caps fitted with silicone sealing rings.
   5. Gently loosen all GL45 caps prior to autoclaving. Autoclave water-filled reactors and electrode caps to sterilize. After autoclaving, allow the reactors to cool to room temperature.
2. Culture L. plantarum NCIMB8826. Under sterile conditions, scrape some bacteria from the top of a glycerol stock and inoculate into 3 mL of commercial MRS media. Grow the culture overnight at 37 ˚C without shaking.
   NOTE: L. plantarum does not use oxygen as a terminal electron acceptor; therefore, it is not necessary to oxygenate the media by shaking²⁶. However, culturing conditions will vary if other microbes are used.

3. Day 2: Preparation of reference electrodes, preparation of reactors for experiment start, and L. plantarum sub-culturing

1. Prepare Ag/AgCl reference electrodes.
   NOTE: These steps describe the preparation of the Ag/AgCl reference electrodes indicated in the Table of Materials. Users should follow the manufacturer's instructions if using a different reference electrode.
   1. Disassemble Ag/AgCl reference electrodes and sand the wires. Gently pull the electrode from its glass housing and flick the glass housing to empty the old KCl solution. Gently sand the electrode wires with aluminum oxide sandpaper until evenly shiny to remove oxidized material.
   2. Collect all sanded reference electrode wires in a small beaker with a small stir bar and fill the beaker with 100% bleach until the wires are fully submerged. Chloride the wires by allowing the wires to bleach on a stirring platform for 30 min until the electrodes turn darker gray. After bleaching, rinse the electrode wires thoroughly with DI water.
   3. To re-assemble a reference electrode, use a syringe to fill the glass housing fully with 3 M KCl solution saturated with silver chloride, gently tapping the side to dislodge any air bubbles. Using the same syringe, fill the electrode wire cap with KCl solution, then insert the wire into the housing. Place the bottom of the glass housing against a paper towel on the bench top, insert the electrode into the glass housing, then press firmly down on the cap to close the electrode. Store the reference electrodes in a beaker shallowly filled with KCl solution until needed and repeat with all remaining reference electrodes.
   4. Use a digital multimeter to measure the voltage of homemade Ag/AgCl reference electrodes.
      1. Submerge the ends of the Ag/AgCl reference electrodes (estimated at 197 mV versus the standard hydrogen electrode, SHE) shallowly in a beaker filled with 3 M KCl. Securely connect the multimeter to a commercially purchased saturated calomel electrode (SCE, 241 mV versus SHE) also submerged in the same KCl electrolytes.
      2. Measure the potential difference between each reference electrode and the SCE. Confirm that the reference electrodes differ from the SCE by 44 ± 10 mV. Disassemble and reassemble any reference electrode that falls outside this range.
   5. Seal the seam where the cap meets the glass housing with parafilm.
2. Prepare the reactors for experimentation.
   1. In a sterile biosafety cabinet, exchange the autoclaved water in the reactors with the appropriate media. Pour out the autoclaved water. Fill the cathodic chambers with 110 mL of autoclaved M9 medium. Fill the anodic chambers with 110 mL of freshly prepared mCDM.
   2. Install the reference electrodes. Remove one GL14 cap from each anodic chamber and replace it with an autoclaved electrode cap (GL14 cap with a silicone sealing ring). Spray the reference electrodes with 70% ethanol to sterilize, then place one reference electrode through the electrode cap into each anodic chamber.
      NOTE: Ensure the reference electrode does not make direct contact with the carbon felt round.
   3. Before removing reactors from the biosafety cabinet, tighten all caps and clamps to be finger-tight to avoid leaking.
   4. Attach the reactors to the water pump system. Place each reactor on the appropriate stir bar platform. Connect the water jacket spigots of each reactor to the next with rubber tubing, connecting end reactors to the inflow and outflow tubes of the water pump.
      NOTE: Ensure all connections are tight without leaks, using zip ties to secure tubing if necessary.
   5. Fill the pump with water and add 4-6 drops of water conditioner. Turn the pump system on and set the temperature to 30 ˚C. Start the pump and observe the water flow through all reactor water jackets, ensuring there are no leaks at any connections.
   6. Turn on the stir platforms and set them to continuous stirring at 220 RPM.
   7. Attach the reactors to the nitrogen-sparging gas lines. Attach an air filter to a 4 inch, 22 G needle and insert the needle through the top septum of a reactor anodic chamber into the media to serve as the nitrogen inlet. Insert another 1 inch, 18 G needle into the top septum of the anodic chamber to serve as the nitrogen outlet. Connect gas lines from a nitrogen source to the air filter and open the valve to allow gas to bubble gently through the reactor. Ensure that nitrogen bubbles through all anodic chambers continuously throughout the experiment to maintain anaerobic conditions.
      NOTE: Ensure the inlet needle is set away from the carbon felt round. The bubble stream should not make contact with either the carbon felt or reference electrodes.
   8. Attach the bioreactors to the potentiostat leads. Connect the working, counter, and reference electrode alligator clip leads from the potentiostat to their corresponding electrodes.
      NOTE: Use a multimeter to check the resistance between the wire/current collector and the electrodes to ensure proper electrical connections and minimize potential measurement errors.
3. Input potentiostat parameters for prerun.
   NOTE: Critical technique settings are given below. See Table 4 for an extended list of software settings for each technique.
   1. Turn on the potentiostat and initialize the EC-lab software on the computer. Connect the potentiostat to the computer by clicking the potentiostat symbol button in the top left panel under Devices. Once connected, the device name will appear with a green circle in the text box below.
   2. Synchronize all channels connected to the bioreactors into one group by clicking the Edit tab, then selecting synchronize. Click the appropriate channel number boxes | ok.
   3. Add the technique open circuit voltage (OCV) to the potentiostat by clicking the + button in the Parameters Settings panel on the left. In the blue settings box; set it to measure working electrode potential (E_WE) against the reference electrode (RE), recording time in interval d_t every 36 s for a total of 3 h.
   4. Next, add the technique cyclic voltammetry (CV) to the potentiostat. Set the initial potential of the E_WE to E_i of 0 V versus RE with a scan rate of 2 mV/s. Sweep to a vertex potential (E1) of 0.4 V versus RE reversing to vertex potential (E2) of -0.7 V versus RE. Repeat the scan for a total of two sweeps.
      NOTE: In microbial electrochemical systems, microbial inoculation and biofilm formation induce high capacities compared to metal materials or inorganic molecules. During cyclic voltammetry scanning, the potential varies, and a charging current acts as a background. To achieve a high signal-to-noise ratio, lower scan rates are necessary, though this increases the duration of the scan. Since we scan two cycles covering a wide electrochemical window of 1.1 V, a scan rate of 2 mV/s results in a half cycle taking up to 9.1 min. Consequently, two cycles take a total of 36.4 min. Reducing the scan rate further would be excessively time-consuming.
   5. Add the technique chronoamperometry (CA). Apply a constant potential E_WE of 0.2 V versus RE for a time of t = 200 h, recording time in intervals d_t every 25-40 s. Adjust the time interval based on potentiostat software to obtain the desired level of detail.
      NOTE: The midpoint potential of DHNA is approximately -0.093 V vs. Ag/AgCl; thus, an applied potential of 0.2 V versus RE to the working electrode is sufficient to allow electron transfer from DHNA to the electrode.
   6. After inputting all parameters, begin the run by pressing the green start triangle. Save the file as desired as directed by the software, then click Save. The software will begin technique 1, "OCV." Observe the OCV traces for a few minutes to ensure all reactors read positively and close together with a steady signal. Leave the experiment to run overnight to complete OCV and initial CV (see Supplemental Figure S1 for the media control CV) and allow CA to run until stabilized.
4. Under sterile conditions, subculture the MRS culture of L. plantarum 1:200 in 50 mL of mMRS. Grow the cells overnight at 37 ˚C without shaking.
   NOTE: A 50 mL overnight culture typically produces more than enough cells for six reactors, assuming a final OD₆₀₀ of 0.2 in the reactor. Adjust culture volumes accordingly for larger or smaller experiments.

4. Day 3: Injection of cells and DHNA/DMSO

1. Wash the cells and inject them into reactors.
   1. Remove the mMRS L. plantarum culture from the incubator in the morning. Under sterile conditions, transfer the culture to a 50 mL conical tube and place the culture on ice.
   2. Wash the cells 2x in sterile, cold 1x PBS. To do so, centrifuge the culture at 4,000 × g for 5 min in a 4 ˚C centrifuge to pellet the cells. Under sterile conditions, gently but thoroughly resuspend the cells in 50 mL of PBS, then centrifuge again as before; repeat for a second washing. After the final centrifuge, resuspend the cells in cold PBS to OD₆₀₀ = 11.
   3. Under sterile conditions, load 2 mL of resuspended cells into a 3 mL syringe fitted with a needle for each reactor.
   4. At the reactor station, decap a cell syringe and insert the needle into the top of a reactor anodic chamber. Do not depress the syringe plunger at this time; repeat for all reactors. Once all syringes are in place, depress the plungers to inject the cells into the reactors and record the time of injection from the CA trace. This volume of cells produces a final OD₆₀₀ of 0.2 in the reactors. Dispose of all syringes in biohazard boxes and needles in the designated sharps biohazard container. Allow the current to stabilize to flat on the CA trace for 2-4 h.
      NOTE: Upon injection, current fluctuations can be observed on the CA trace. After 2-4 h, these fluctuations will stabilize to a flat current (<2 µA change in current over the course of an hour), at which point DHNA can be injected.
2. Measure initial pH and inject DHNA.
   1. Prepare a DHNA solution. In a 1.5 mL tube, prepare 500 µL of a 20 mg/mL DHNA solution by dissolving powdered DHNA in 100% DMSO. Fill 3 insulin syringes with 110 µL of DHNA solution and 3 insulin syringes with 110 µL of DMSO only.
      NOTE: While DHNA is mildly soluble in water, DMSO is a better solvent for DHNA stocks at this concentration. Mediator solvent may vary if other mediators are used.
   2. At the BES reactor station, label the experimental reactors as + DHNA and the solvent control reactors as – DHNA. Decap a DHNA syringe and insert the syringe into the top of each anodic chamber designated as + DHNA. Insert a DMSO-only syringe into each anodic chamber designated as – DHNA. Do not depress the syringe plungers at this time.
   3. Take 0 h time point samples for sample analysis. Using a 3 mL syringe fitted with a 2 inch, 21 G needle, take a 2 mL media sample from each anodic chamber through the unused small cap septum and transfer the samples to a 24 deep-well plate to take pH measurements for the 0 h time point (DHNA injection point). If desired, take 1 mL of spent media from each anodic chamber and filter through a 0.2 µm filter into clean labeled tubes for quantifying metabolites using HPLC or other assays. Store spent media samples at -80 ˚C.
   4. Depress the plungers of all DHNA and DMSO syringes to inject into the reactors. Record the time of injection from the CA trace. Discard all syringes and needles appropriately.
   5. Measure and record the 0 h pH samples for each reactor.

5. Day 4: Experiment completion and sample collection

1. Conduct electrochemical analysis 24 h after DHNA injection and take final samples.
   1. End the CA run 24 h after DHNA injection.
   2. Take 24 h time point samples for sample analysis according to step 4.2.3.
   3. Run CV again for the 24 h time point according to the parameters described in step 3.3.4.
   4. Measure and record the pH for the 24 h samples from each reactor.
2. Disassemble and clean the reactors.
   1. Turn off the potentiostat. Then, disconnect the working, counter, and reference leads from each reactor. Wipe any moisture from the potentiostat lead alligator clips.
   2. Turn off the nitrogen gas flow. Disconnect the gas leads, and then remove the inflow and outflow needles. Dispose of all needles appropriately in a sharps container.
   3. Turn off the water pump. Disconnect inflow and outflow tubing from the pump and allow water to drain into a bucket. When disconnecting, keep the tubing ends elevated above the water line in the pump to prevent water from siphoning onto the floor. One by one, disconnect each reactor from the water jacket tubing, working from the final outflow reactor to the initial inflow reactor.
   4. Empty all media from the reactors into a large biohazard container. Follow normal culture bleaching methods for disposal.
   5. Once emptied, disassemble and clean all reactor pieces. Dispose of cation exchange membranes and carbon felt rounds into proper biohazard waste. Clean reference electrodes and titanium wires with 70% ethanol and store the cleaned reference electrodes in a beaker shallowly filled with water. Gently clean reactor bottles, caps, O-rings, and clamps in warm water with laboratory detergent, rinse thoroughly with DI water, and air dry all parts before storing.

6. Day 5: Electrochemical analysis

NOTE: Below is a general description of data plotting for this protocol. More detailed descriptions concerning analysis and data interpretation will be provided in the Representative Results section.

1. For CA analysis: Set the time of DHNA injection as the 0 h time point. Plot the average and standard deviation of the measured current density (j in µA/cm²) taken every 36 s for all replicates from the 0 h time point as a function of time (h). Calculate current density as a function of the working electrode area (16 cm²).
2. For CV analysis: Plot a representative CV trace for each experimental condition (media only, DMSO, or DHNA) depicting current density (j in µA/cm²) as a function of the working electrode potential (E_WE in V). Plot cycle two of the selected CV run.