Here we present a protocol for characterizing mediated extracellular electron transfer (EET) in lactic acid bacteria using a three-electrode, two-chamber bioelectrochemical system. We illustrate this method with Lactiplantibacillus plantarum and the redox mediator 1,4-dihydroxy-2-naphthoic acid and provide a thorough description of the electrochemical techniques used to evaluate mediated EET.
Many bacteria perform extracellular electron transfer (EET), whereby electrons are transferred from the cell to an extracellular terminal electron acceptor. This electron acceptor can be an electrode and electrons can be delivered indirectly via a redox-active mediator molecule. Here, we present a protocol to study mediated EET in Lactiplantibacillus plantarum, a probiotic lactic acid bacterium widely used in the food industry, using a bioelectrochemical system. We detail how to assemble a three-electrode, two-chambered bioelectrochemical system and provide guidance on characterizing EET in the presence of a soluble mediator using chronoamperometry and cyclic voltammetry techniques. We use representative data from 1,4-dihydroxy-2-naphthoic acid (DHNA)-mediated EET experiments with L. plantarum to demonstrate data analysis and interpretation. The techniques described in this protocol can open new opportunities for electro-fermentation and bioelectrocatalysis. Recent applications of this electrochemical technique with L. plantarum demonstrated an acceleration of metabolic flux towards producing fermentation end-products, which are critical flavor components in food fermentation. As such, this system has the potential to be further developed to alter flavors in food production or produce valuable chemicals.
Bioelectrochemical systems interface microbes with electrodes, allowing investigation of extracellular electron transfer (EET) mechanisms and providing renewable approaches to bioelectrocatalysis1,2,3. Microbes that naturally perform EET are known as exoelectrogens, which transfer electrons derived from metabolism to extracellular terminal electron acceptors, e.g., iron (hydr)oxides and electrodes1. First characterized in Geobacter and Shewanella species4,5, EET pathways have since been identified in many bacteria. These exoelectrogens play a central role in several microbial electrochemical technologies, such as generating electrical energy from waste streams, fixing CO2, and producing valuable chemicals via electrosynthesis1,6,7,8,9,10,11,12.
One such exoelectrogen is Lactiplantibacillus plantarum, a gram-positive lactic acid bacterium13. L. plantarum is a nomadic, probiotic bacterium that resides in a wide range of environments, including the guts of humans and other vertebrates, as well as many types of food such as meat, cereals, vegetables, and fermented foods and drinks14,15,16,17. Its genome encodes a flexible, heterofermentative metabolism, allowing successful adaptation across these diverse environments. It is well-studied, widely used in the food and health industries, and generally recognized as safe by the Food and Drug Administration18,19. As such, L. plantarum has the potential to serve as a useful platform for EET-based technologies.
Recent research in L. plantarum has identified a multi-gene operon encoding a complex EET pathway originally characterized in Listeria monocytogenes13,20. In L. plantarum, the proteins synthesized from this operon facilitate EET in a bioelectrochemical system (BES) when provided the quinone 1,4-dihydroxy-2-naphthoic acid (DHNA) as an electron mediator13. The first essential protein in this pathway is a membrane-bound NADH-quinone oxidoreductase (Ndh2), which oxidizes NADH and reduces DHNA. DHNA either delivers electrons directly to an electrode or indirectly via the accessory protein PplA (Figure 1)13,21,22. Recent research indicates L. plantarum may also use other quinones that are structurally similar to DHNA as electron mediators; however, L. plantarum is incapable of producing DHNA or these alternative quinones, thus mediators must be exogenously present in the environment for EET to occur13,22,23.
Figure 1: Electron flow in Lactiplantibacillus plantarum EET. Ndh2 passes electrons from NADH to the quinone DHNA. Electrons are shuttled to the electrode to produce current, either directly by reduced quinone or indirectly through the accessory protein PplA. Abbreviations: FAD = Flavin adenine dinucleotide; FMN = Flavin mononucleotide; EET = extracellular electron transfer; NADH = reduced nicotinamide adenine dinucleotide; Ndh2 = NADH-quinone oxidoreductase; DHNA = 1,4-dihydroxy-2-naphthoic acid; PplA = phospholipase A. Please click here to view a larger version of this figure.
In this article, we provide a comprehensive protocol for using a BES-based method to characterize DHNA-mediated EET in L. plantarum. A three-electrode, two-chamber system confines bacteria to the working electrode, allowing precise control of the potential applied to the bacteria while preventing crosstalk between the working and counter electrode. We present a comprehensive protocol spanning 5 days, which covers pre experiment preparation, BES assembly, EET analysis utilizing chronoamperometry (CA) and cyclic voltammetry (CV), and post experiment sample analysis. This protocol can be applied to unravel the mechanisms of EET pathways and to build systems for electrofermentation and electrocatalysis.
NOTE: Two-chambered BES assemblies will be referred to as "reactors" in the following protocol.
1. Media preparation
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.
3. Day 2: Preparation of reference electrodes, preparation of reactors for experiment start, and L. plantarum sub-culturing
4. Day 3: Injection of cells and DHNA/DMSO
5. Day 4: Experiment completion and sample collection
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.
Chronoamperometry analysis
The EET of L. plantarum can be observed through the chronoamperometry (CA) data depicted in Figure 3, in which the current density trace visualizes electron transfer from L. plantarum to the working electrode. We monitored the current density (j) versus time while maintaining a constant potential of +200 mV versus Ag/AgCl for 24 h. Upon injecting 20 µg/mL DHNA into the stirring electrolyte solution, an abiotic DHNA oxidation spike was observed, followed by a rapid increase in biotic current density peaking at 132.0 ± 2.47 µA/cm2 at approximately the 8 h time point. Conversely, the injection of DMSO resulted in negligible current density. These results emphasize the significance of DHNA as a necessary and efficient mediator to facilitate electron transfer between L. plantarum and the electrode. Users can adjust the current output by adjusting the concentration of DHNA in the BES. Prior research also indicates L. plantarum responds to DHNA in a dose-dependent manner across a wide range of DHNA concentrations, producing significant current in the presence of DHNA concentrations as low as 0.01 µg/mL13,22.
Figure 3: Chronoamperometry analysis of Lactiplantibacillus plantarum EET mediated by DHNA. DHNA (20 µg/mL) or DMSO was injected into mCDM electrolytes (pH~ 6.5) with injection time identified as t = 0. j represents current density as a function of the working electrode area. Experiments were conducted at 200 mV versus Ag/AgCl with a carbon felt electrode (16 cm2) and stirring. Values are plotted as mean ± sd obtained in triplicate BES reactors. Abbreviations: EET = extracellular electron transfer; DHNA = 1,4-dihydroxy-2-naphthoic acid; DMSO = dimethyl sulfoxide; mCDM = Chemically Defined Medium with mannitol. Please click here to view a larger version of this figure.
Cyclic voltammetry analysis
To further evaluate DHNA-mediated EET in L. plantarum, we conducted cyclic voltammetry 24 h after DHNA injection. Here we show CV traces for three conditions: L. plantarum with 20 µg/mL DHNA, L. plantarum with DMSO, and media with 20 µg/mL DHNA. As shown in Figure 4A, the presence of 20 µg/mL DHNA in reactors containing L. plantarum resulted in a distinct increase in oxidative current at 50 mV that did not occur in the presence of DMSO alone. These data confirm that adding the redox mediator DHNA is necessary to facilitate electron transfer between L. plantarum and the anode. While we observed various smaller redox peaks in the L. plantarum + DMSO trace, these peaks were similar to the media control trace and are likely attributed to redox-active components in mCDM (Supplemental Figure S1). In Figure 4B, we compared traces of DHNA under biotic conditions (L. plantarum + DHNA) versus DHNA under abiotic conditions (Media + DHNA). While both traces exhibited a distinct DHNA oxidative peak around 50 mV, we observed a sustained increase in current beyond 50 mV only under biotic conditions. The catalytic peak reached a current density of 129 µA/cm2 at 300 mV, representing a 256% increase compared to the abiotic trace. This turnover CV profile is characteristic of microbial EET27, indicating re-reduction of DHNA by L. plantarum cells in the presence of an electron source (mannitol) after oxidation of DHNA at the anode. Additionally, the abiotic trace exhibited new oxidative peaks around -240 mV and -180 mV. Prior research indicates the appearance of these peaks may be due to the degradation of DHNA into ACNQ (2-amino-3-carboxy-1,4-naphthoquinone)21,28. We did not observe these peaks in the biotic trace, indicating that the interaction of L. plantarum cells with DHNA may stabilize DHNA and prevent degradation. A point to note is that the 24 h trace for media with 20 µg/mL DHNA was conducted separately according to this protocol without adding cells.
Figure 4: Representative cyclic voltammetry traces. All CV experiments were performed in mCDM using carbon felt (16 cm2) as the working electrode at a scan rate of 2 mV/s while stirring the solution. (A) CV traces for Lactiplantibacillus plantarum with either DHNA (20 µg/mL) or DMSO at t = 24 h. (B) CV traces of 20 µg/mL DHNA in either L. plantarum (biotic conditions) or mCDM only (abiotic conditions) at t = 24 h. Abbreviations: CV = cyclic voltammetry; mCDM = Chemically Defined Medium with mannitol; DHNA = 1,4-dihydroxy-2-naphthoic acid; DMSO = dimethyl sulfoxide. Please click here to view a larger version of this figure.
pH analysis
EET activity in L. plantarum resulted in a notable drop in pH over 24 h. As shown in Figure 5, the average sample pH of L. plantarum exposed to DHNA dropped to 3.33 ± 0.01 (p = 6.85 × 10-6, n = 3), while the average sample pH of L. plantarum exposed to DMSO dropped to 6.50 ± 0.06 (p = 0.0409, n = 3). As exhibited in prior research, this drop is attributed to an increase in fermentative metabolism that occurs when L. plantarum performs EET13. L. plantarum normally metabolizes mannitol through glycolysis and fermentative pathways, which produce acetate, lactate, and ethanol as end-fermentation products and generate ATP through substrate-level phosphorylation29. Under EET conditions, metabolic flux through fermentation increases, thereby increasing the production of end-fermentation products in the BES media13. This metabolic shift causes the media pH to drop more rapidly in the reactors with DHNA compared to the DMSO control reactors.
Figure 5: pH analysis of Lactiplantibacillus plantarum bioelectrochemical system. Samples were collected at t = 0 and t = 24 h during chronoamperometry. Values are plotted as mean ± sd obtained in triplicate BES reactors. Significance was determined by a one-tailed t-test. DHNA: P-value = 6.85 × 10-6. DMSO: P-value = 0.0409. Abbreviations: DHNA = 1,4-dihydroxy-2-naphthoic acid; DMSO = dimethyl sulfoxide. Please click here to view a larger version of this figure.
Table 1: Ingredients for preparation of mMRS media24. Please click here to download this Table.
Table 2: Ingredients for preparation of mCDM media. This table is taken from Tejedor-Sanz et al.13 and Aumiller et al.25. Please click here to download this Table.
Table 3: Ingredients for preparation of M9 media. Please click here to download this Table.
Table 4: EC-Lab parameter settings for OCV, CA, and CV techniques. Abbreviations: OCV = open circuit voltage; CA = chronoamperometry; CV = cyclic voltammetry. Please click here to download this Table.
Supplemental Figure S1: Representative cyclic voltammetry traces of Lactiplantibacillus plantarum with DMSO and mCDM alone. CV traces for L. plantarum with DMSO at t = 24 h and mCDM alone at t = 0 h. All CV experiments were performed using carbon felt (16 cm2) as the working electrode at a scan rate of 2 mV/s while stirring the solution. Abbreviations: CV = cyclic voltammetry; mCDM = Chemically Defined Medium with mannitol; DHNA = 1,4-dihydroxy-2-naphthoic acid; DMSO = dimethyl sulfoxide. Please click here to download this File.
Using the three-electrode, two-chamber bioelectrochemical system described here, we showed measurement of current generation from DHNA-mediated EET in L. plantarum. These BES experiments generate high-quality data; however, BESs are sensitive. Thus, protocol success depends on user precision, particularly in reactor and reference electrode assembly, positioning of needles and electrodes within the anodic chamber, and cation exchange membrane replacement. It is critical to assemble reactors carefully, ensuring no water/media leakage during autoclaving or experimentation. Water leakage can be resolved by ensuring cation exchange membranes are cut to precisely fit the O-ring and tightening the knuckle clamp to finger tight. It is also essential to fully submerge the carbon felt round fully in water during autoclaving to allow it to become hydrophilic for experimentation. We recommend new users allow newly assembled reactors filled with water to sit for 2 h prior to autoclaving, checking for signs of slow leaks below the main bottle junctions. Moreover, ensuring a proper reference electrode assembly guarantees consistent data replication across reactors. If the Teflon frit inside the glass housing becomes discolored, cracked, or dried, this can cause a high resistance of the reference electrode. Users can replace the glass housing to restore reference electrode performance.
Proper orientation of all needles and electrodes within the anodic chamber during experimentation is critical to experiment success. The reference electrode must not directly contact any part of the carbon-felt working electrode. Users can adjust the carbon felt position by gently rotating the working electrode titanium wire from above the reactor. Additionally, needle placement for nitrogen sparging should not make direct contact with electrodes within the chamber or any electrode/potentiostat connections above the chamber. The nitrogen stream should be adjusted to not flow into either electrode. Finally, users should make sure the stir bar does not contact the working electrode by positioning the working electrode 1-2 cm above the stir bar. If an erratic signal is observed in OCV, this can usually be resolved by ensuring proper placement of electrodes and the nitrogen stream within the reactor, and by checking that connections between the potentiostat leads and the reactor electrodes are correct and secure. Lastly, our experience shows that electron mediators like DHNA can be retained within the cation exchange membrane and cause a high background current if reused too many times. We recommend replacing the cation exchange membrane after two to three uses, especially when investigating mediated EET, to guarantee reliable experimental results.
Unlike direct EET, where direct microbial attachment to the electrode facilitates electron transfer, mediated EET necessitates consistent diffusion of electron shuttles across the cell membrane and the electrode, resulting in the unique BES settings described here. First, we chose a double-chamber BES over the single-chamber counterpart in our protocol to separate anodic and cathodic reactions with a cation exchange membrane. This separation prevents the freely diffusing electron mediators (DHNA) and microbes from cross-interacting with the cathode, ensuring microbial EET is the major electron source to reduce the electron mediators and the anode. The separation also allows precise control over parameters like mediator concentration/distribution and the potential poised to the anode. Additionally, we chose carbon felt as the anode material amongst other options such as graphite rods, metal electrodes, glassy carbon, or indium tin oxide (ITO). This is because the 3D porous structure of carbon felt provides a much larger surface area than those electrodes30, allowing efficient utilization of mediators even at high concentrations. Our three-electrode, two-chamber BES settings provide a reliable and reproducible readout of mediated EET even over long-term monitoring; however, this process is relatively low throughput. This protocol is suitable for bench-scale understanding of EET mechanisms or to test prototype EET applications. Alternative BES architectures such as portable or printed BESs31,32, complementary metal oxide semiconductor (CMOS) arrays33, or up-scaled BESs34 can be considered by researchers for different fundamental or application purposes.
In this protocol, we provide detailed instructions for the most commonly used electrochemical techniques: chronoamperometry (CA) and cyclic voltammetry (CV). It is worth noting that other electrochemical techniques, such as Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV), can provide deeper insights into the BES by analyzing charge transfer resistance and double-layer capacitance35,36,37. While this BES protocol enables EET measurements, complementing electrochemical data with metabolic activity and cell biomass measurements can also be essential for a comprehensive analysis. Microbes like L. plantarum engage EET as one of the electron sinks alongside other fermentation byproducts such as lactate and ethanol. Moreover, it is noteworthy that cell biomass growth also serves as an electron sink13. Therefore, quantifying consumed electron donors (e.g., mannitol), assessing cell biomass growth, and monitoring fermentation byproducts offer deeper insights into the efficiency and physiological ramifications of EET. Cellular metabolites are commonly quantified using chromatography and enzymatic assays, while cell viability and growth are assessed by counting colony-forming units and measuring the optical density of spent media at 600 nm, respectively13. It is also important to note that EET measurements are sensitive to small perturbations in experimental conditions. This includes but is not limited to pH, temperature, stirring speed, and nitrogen gas sparging rate38. Therefore, normalizing measured EET levels with bioanalytical measurements acts as an internal control, facilitating consistent assessment across experiments conducted on different days.
Combining electrochemical techniques with other bioanalytical measurements, mediated EET creates new opportunities for electro-fermentation and bioelectrocatalysis. Conventional use of organic, inorganic, or enzymatic electrocatalysts poses challenges due to their high cost and are prone to degradation. Alternatively, using microbes as living electrocatalysts offers a less expensive and more scalable solution due to microbes' self-repair and self-replication abilities39. L. plantarum, generally recognized as a safe lactic acid bacterium, is a particularly intriguing chassis. Using identical electrochemical setups described in this protocol, we have previously shown that L. plantarum can ferment kale juice under EET conditions and accelerate the metabolic flux toward producing more fermentation end-products such as lactate, acetate, and succinate13; these organic acids are essential flavor compounds in food fermentation. This implies that, by using electrochemical techniques, mediated EET in L. plantarum can be potentially hijacked to manipulate metabolic flux, alter food flavors, or produce valuable chemicals. It is worth noting that the electrochemical techniques presented in this protocol can not only be applied to L. plantarum but can also be generically applied to other native or engineered microbes that perform mediated EET40,41. Different electron mediators, such as flavin, ferrocene, neutral red, ferricyanide, lawsone, and menadione can be selected based on the electron transfer mechanism of the specific microbe being used22,42. Moreover, the BES protocol established in this work can be extended to exoelectrogens that perform mediatorless EET as previously demonstrated with Shewanella and Geobacter species43,44. An optimized growth medium should be used to support the cellular activity of the particular microbe to facilitate its EET performance. This protocol fine-tunes parameters for DHNA-mediated EET in L. plantarum, but modifications are expected when a different microbe and electron mediators are applied.
The authors have nothing to disclose.
We thank members of the Ajo-Franklin lab for insightful discussions on BES assembly, maintenance, critical steps, and troubleshooting. The research was sponsored by the Army Research Office and was accomplished under Grant Number W911NF-22-1-0239 (to C. M. A-F, supporting R. A.) and by the Cancer Prevention and Research Institute of Texas, grant # RR190063 (to C. M. A-F, supporting R. C., S. L., and B. B. K.). Figure 1 was created with BioRender.com.
1,4-Dihydroxy-2-naphthoic acid (DHNA) | Sigma-Aldrich | 281255-25G | |
1.0 mm diameter titanium wire | Thermo Fisher Scientific | 045485.BY | Cut to size for working and counter electrodes |
120-C Aluminum Oxide Sheets 9" x 11" | Johnson Abrasives | 10108-15 | |
3 mL plastic syringes | Thermo Fisher Scientific | 14955457 | |
3M KCl solution saturated with silver chloride | Millipore Sigma | 60137-250ML | |
6.35-mm-thick carbon felt | Thermo Fisher Scientific | 043200.RF | Cut into 16 cm2 rounds |
Ag/AgCl reference electrode | CH Instruments | CH111 | |
Air-Tite Premium Hypodermic Needles | Thermo Fisher Scientific | 14-817-102 | |
AlK(SO)4 * 12H2O | Sigma-Aldrich | 237086-100G | |
Ammonium citrate tribasic | Millipore Sigma | A1332 | |
Avanti J-15R Centrifuge | Beckman Coulter | B99517 | |
BD Precision Glide Needle, 18 G x 1 inch | Thermo Fisher Scientific | 14-826-5G | |
BD Precision Glide Needle, 21 G x 2 inch | Thermo Fisher Scientific | 14-821-13N | |
Bel-Art SP Scienceware Cleanware Aqua-Clear Water Condtioner | Thermo Fisher Scientific | 23-278339 | |
Biotin | Millipore Sigma | B4639 | |
CaCl2 | Millipore Sigma | C4901 | |
Calcium D-(+)-pantothenate | Millipore Sigma | 1087009 | |
Casamino acids | Millipore Sigma | 2240-OP | |
cation exchange membrane | Membranes International | CMI-7000 | Cut into rounds fit to the BES O-ring |
CoCl2 * 6H2O | Millipore Sigma | C8661 | |
CuSO4 * 5H2O | Millipore Sigma | C8027 | |
Cysteine-HCl * H2O | Millipore Sigma | 30129 | |
DMSO | Millipore Sigma | 5439001000 | |
DS-11+ Spectrophotometer | Denovix | N/A | |
EC-Lab Software | BioLogic | N/A | |
ECO E 4 S heating circulator | Lauda-Brinkmann | Cat. No. 115 V; 60 Hz : L001191 | |
FeSO4 * 7H2O | Millipore Sigma | 215422 | |
Folic acid | Millipore Sigma | F8758 | |
H3BO3 | Millipore Sigma | B6768 | |
Insulin syringes with BD Micro-Fine IV Needle | Thermo Fisher Scientific | 14-829-1A | |
Lactiplantibacillus plantarum NCIMB8826 | N/A | N/A | Reference: Tejedor-Sanz et al., 2022 |
Lactobacillus MRS Broth | HiMedia | M369 | |
M9 Broth | Milliport Sigma | 63011 | |
Magnesium sulfate anhydrous | Millipore Sigma | 208094 | |
Manganese sulfate monohydrate | Millipore Sigma | 221287 | |
mannitol | Millipore Sigma | M1902-1KG | |
Mettler Toledo FiveEasy Benchtop pH Meter | Hogentogler | F20-KIT | |
MgCl2 * 6H2O | Millipore Sigma | M9272 | |
MgSO4 * 7H2O | Millipore Sigma | M2773 | |
Millex – GV 0.22 µm PVDF Membrane Filter Unit | Millipore Sigma | SLGV004SL | |
MnCl2 * 4H2O | Millipore Sigma | 203734 | |
MnSO4 * H2O | Millipore Sigma | 221287 | |
MOPS | Millipore Sigma | M1442 | |
N2 gas | Airgas | NI UHP300 | Filter before use |
Na2MoO4 * 2H2O | Millipore Sigma | 331058 | |
Na2SO4 | Millipore Sigma | 238597 | |
NaCl | Millipore Sigma | S9888 | |
NH4Cl | Millipore Sigma | A9434 | |
Nicotinic acid | Millipore Sigma | N-0761 | |
Nitrilotriacetic acid (NTA) | Millipore Sigma | 72560 | |
p-Aminobenzoic acid | Millipore Sigma | P9879 | |
Phosphate buffered saline, 10x solution | Thermo Fisher Scientific | BP399-1 | |
Potassium phosphate dibasic | Millipore Sigma | P8281 | |
potentiostat | BioLogic | VMP-300 | |
Protease peptone #3 | Bacto | 211693 | |
Pyridoxine HCl | Millipore Sigma | P6280 | |
Riboflavin | Millipore Sigma | 555682 | |
RO10 magnetic stir bar platform | IKA | 3691000 | |
Sodium acetate trihydrate | Millipore Sigma | 935700 | |
Stir bar, egg-shaped | Thermo Fisher Scientific | 14-512-121 | Place in anodic chamber of BES |
Thiamine HCl | Millipore Sigma | V-014 | |
Thioctic acid (α-Lipoic acid) | Millipore Sigma | T-1395 | |
Tryptophan | Millipore Sigma | 9136 | |
Tween80 | Millipore Sigma | P4780 | |
Vitamin B12 | Millipore Sigma | V6629 | |
Jacketed MCF set, 100 ml, NW25, 2 x GL14 port | Adams & Chittenden Scientific Glass | NA | Customized |
Yeast extract | Millipore Sigma | Y1625 | |
ZnSO4 * 7H2O | Millipore Sigma | Z0251 |
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