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

In our lab, we investigate extracellular electron transfer, or EET, in bacteria like lactiplantibacillus plantarum, which is a fermentative bacteria critical in food industries. In EET, the cell transfers electrons to an extracellular electronic scepter, like an electrode in a bioelectric chemical system. We want to understand and engineer EET for applications in biosensing, biocatalysis, and food fermentation.

We discovered that alpine tunnel can purchase current through EET in the presence of quinone such as DHNA. This process regenerates NAD plus, accelerate the overall loss of the primary fermentation pathways, and boosts cell growth. Moreover, we find that diverse quinone derivatives, other than DHNA, can also mediate EET in L plantarum.

Investigating mediated EET requires specialized, biochemical set ups. Specifically, we use a three electrode, two chamber bioelectrochemical system to prevent cross-talking between the anodic and the cathodic reactions. We also use a carbon field, working electrode that has a large surface area to enhance electron transfer from electron mediators.

Our work exploring EET pathways in L plantarum and how those pathways interact with fermentative metabolism could have useful applications in food industries. We know EET and L plantarum alters metabolic flux through fermentation, and that could be manipulated for useful applications and altering food flavors and producing valuable chemicals in electro fermentation. In the future, we will continue to increase our fundamental knowledge of EET and pursue novel EET applications in organisms like L plantarum.

We plan to engineer proteins in the EET pathway, which will allow us to further control EET for applications in electro fermentation and biosensing. To begin, sand precut one millimeter diameter titanium wires for the working in 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 square centimeter 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 encounter electrodes into GL 45 caps by piercing the rubber septum with the wire and pulling it through a few centimeters. To build paired reactors, assemble the O-ring and place a precut cation exchange membrane, pre-soaked 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 a ring with the membrane with a knuckle clamp. Drop a magnetic stir bar into each anodic chamber before closing all small openings at the top of each bottle with GL14 caps, fitted with rubber septa.

Fill each reactor bottle with 110 milliliters of deionized water. Insert the caps fitted with a carbon felt round working electrode and gently press on the top of the felt round to keep it secured on the hook. Close the bottles with the appropriate, electrode fitted GL45 cap.

Autoclave water-filled reactors and GL14 electrode caps. Under sterile conditions, scrape lactoplant abasilisk plantarum culture from the top of a glycerol stock, and inoculate in three milliliters of commercial demand rugosa sharp, or MRS medium. Incubate the culture overnight at 37 degrees Celsius, without shaking.

To begin, in a sterile biosafety cabinet, discard the autoclaved water from the bio electrochemical system reactors. Fill the cathodic chambers with 110 milliliters of autoclaved M9 medium and anodic chambers with 110 milliliters of freshly prepared MCDM. Replace one GL14 cap from the anodic chamber with an autoclave GL14 cap with a silicone ceiling ring.

Spray the prepared reference electrodes with 70%ethanol before placing it through the electrode cap into each anodic chamber. Tighten all caps and clamps to avoid leaking. To attach the reactors to the water pump system, first, place each reactor on the appropriate stir bar platform.

Then connect the water jacket spigots of each reactor to the next with rubber tubing, connecting the end reactors to the inflow and outflow tubes of the water pump. Fill the pump with water and add four to six drops of water conditioner. Turn the pump system on and set the temperature to 30 degrees Celsius.

Start the pump and observe the water flow through all reactor water jackets, confirming there are no leaks. Turn on the stir platforms and set them to continuous stirring at 220 RPM. To attach the reactors to the nitrogen sparing gas lines, first, attach an air filter to a 22 gauge needle and insert the needle through the top septum of a reactor anodic chamber into the media.

Insert an 18 gauge needle in the top septum of a reactor anodic chamber, then connect the gas line from a nitrogen source to the air filter and open the valve to allow gas to bubble gently through the reactor. To attach the bioreactors to the potentiostat leads, connect the working counter and reference electrode alligator clip leads from the potentiostat to their corresponding electrodes. After inputting all parameters, press the green start triangle to begin the run.

Observe the open circuit voltage traces for a few minutes to ensure all reactors read positively. and close together with a steady signal. Under sterile conditions, subculture the previously grown L plantarum culture one to 200 in 50 milliliters of MMRS.

Grow the cells overnight at 37 degrees Celsius, without shaking. To begin, remove the L plantarum culture grown in MMRS from the incubator. Transfer the culture to a 50 milliliter conical tube under sterile conditions and place the tube on ice.

Centrifuge the culture at 4, 000 G for five minutes at four degrees Celsius. Remove the supernatant before resuspending the pellet in 50 milliliters of PBS. After the second wash, resuspend the cells in cold PBS to optical density at 600 nanometers of 11.

Load two milliliters of resuspended cells into a three milliliter syringe, fitted with a needle. At the reactor station, decap a cell syringe and insert the needle into the top of a reactor anodic chamber. Once all syringes are in place, depress the plungers to inject the cells.

Record the time of injection from the chronoamperometry trace. Allow the current to stabilize on the trace for two to four hours. At the bioelectrochemical system, label the experimental reactors as plus DHNA, and the solvent control reactors as minus DHNA.

Decap a syringe loaded with 110 microliters of 20 milligrams per milliliter DHNA, and insert it into the top of the anodic chamber, designated as plus DHNA. Insert a syringe loaded with 110 microliters of DMSO into the anodic chamber, designated as minus DHNA. Using a three milliliter syringe fitted with a 21 gauge needle, remove two milliliters of media from each anodic chamber through the unused small cap septum.

Transfer the samples to a 24 deep well plate to measure pH for the zero hour time point. Depress the plungers of DHNA and DMSO syringes to inject into the reactors. Record the time of injection from the chronoamperometry trace.

Discard all syringes and needles. After 24 hours, remove two milliliters of media from each anodic chamber as described previously, and transfer it to a 24 deep well plate for 24 hour pH measurements. Run the cyclic voltammetry for the 24 hour time point.

Measure and record the pH for the 24 hour samples from each reactor. The current density due to extracellular electron transfer reached a peak of approximately 132 microamperes per square centimeter, eight hours after the DHNA injection. In contrast, the DMSO injection resulted in a negligible current density.

The cyclic voltammetry data showed a distinct increase in oxidative current at 50 millivolts in the presence of L plantarum with DHNA, compared to L plantarum with DMSO and a 256%increase in current density at 300 millivolts, compared to the abiotic DHNA trace. Extracellular electron transfer resulted in a notable drop in pH to an average of 3.33 over 24 hours in the samples with L plantarum and DHNA, while the samples with L plantarum and DMSO had an average pH of 6.50.