Lactiplantibacillus plantarum uses extracellular electron transfer to increase its NAD+/NADH ratio, generate more ATP through substrate-level phosphorylation, and accumulate biomass more rapidly. This novel, hybrid metabolism is dependent on a type-II NADH dehydrogenase (Ndh2) and conditionally requires a flavin-binding extracellular lipoprotein (PplA) under laboratory conditions. It confers increased fermentation product yield, metabolic flux, and environmental acidification in laboratory media and during kale juice fermentation. The discovery of a single pathway that simultaneously blends features of fermentation and respiration in a primarily fermentative microorganism expands our knowledge of energy conservation and provides immediate biotechnology applications.Energy conservation in microorganisms is classically categorized into respiration and fermentation; however, recent work shows some species can use mixed or alternative bioenergetic strategies. We explored the use of extracellular electron transfer for energy conservation in diverse lactic acid bacteria (LAB), microorganisms that mainly rely on fermentative metabolism and are important in food fermentations. The LAB  Bacteria produce the energy they need to live through two processes, respiration and fermentation. While respiration is often more energetically efficient, many bacteria rely on fermentation as their sole means of energy production. Respiration normally depends on the presence of small soluble molecules, such as oxygen, that can diffuse inside the cell, but some bacteria can use metals or other insoluble compounds found outside the cell to perform ‘extracellular electron transfer’.Lactic acid bacteria are a large group of bacteria that have several industrial uses and live in many natural environments. These bacteria survive using fermentation, but they also carry a group of genes needed for extracellular electron transfer. It is unclear whether they use these genes for respiration or if they have a different purpose.Lactiplantibacillus plantarum to study whether and how this group of bacteria use extracellular electron transfer. Analysis of L. plantarum and its effect on its surroundings showed that these bacteria use a hybrid process to produce energy: the cells use aspects of extracellular respiration to increase the yield and efficiency of fermentation. Combining these two approaches may allow L. plantarum to adapt to different environments and grow faster, allowing it to compete against other species.Tejedor-Sanz, Stevens et al. used a lactic acid bacterium called Tejedor-Sanz, Stevens et al. provide new information on a widespread group of bacteria that are often used in food production and industry. The next step will be to understand how the hybrid system is controlled and how it varies among species. Understanding this process could result in new biotechnologies and foods that are healthier, produce less waste, or have different tastes and textures. The ways in which microorganisms extract energy to maintain cellular functions are directly linked to their environment, including the availability of nutrients and cooperative or antagonistic interactions with other organisms . MicroorGeobacter spp., can anaerobically respire using electron acceptors outside the cell, such as iron (III) oxides or an electrode  are a diverse group of aerotolerant, saccharolytic microorganisms in the Firmicutes phylum which mainly use fermentation for energy conservation. LAB are essential for many food fermentations, including fermented milk and meats, fruits and vegetables, and grains . Strains citrate . Althoug citrate . Even th citrate . TherefoClostridium spp.  locus, that was identified in many Gram-positive species in the Firmicutes phylum, including LAB. Studies in individual species of LAB such as Lactococcus lactis  oxyhydroxide). Incubation of the model strain L. plantarum NCIMB8826 in the presence of ferrihydrite showed that this strain reduces Fe3+ to Fe2+  medium or chemically defined medium (CDM) (L. plantarum was incubated in mannitol-containing MRS (mMRS) with both DHNA and ferric ammonium citrate present  . Iron reum (CDM) . However present . The addlantarum , potentilantarum . Thus, LL. plantarum EET activity was confirmed in a bioelectrochemical reactor by quantifying electron output as current (L. plantarum reduced a carbon electrode (anode) polarized to +200 mVAg/AgCl in the presence of both DHNA and an electron donor (mannitol) (L. plantarum (L. plantarum produced a maximum current of 129 ± 19 µA/cm2 in mCDM (2 in mMRS (L. plantarum biomass was 2.7 mg (dry cell mass). Assuming 50% of the dry cell mass was protein, the specific electron transfer rate was 57 µmol electrons/mg- protein/hr and the current production was 1.5 mA/mg-protein. This value is lower than that reported for Geobacter sulfurrenducens (4–8 mA/mg-protein) (Shewanella oneidensis (0.67 mA/mg-protein)  . No currlantarum , indicat in mCDM  and 225  in mMRS . Under Eprotein) , the modprotein) , the modth media , and curth media . BecauseL. plantarum is relevant under these physiological concentrations, we probed whether L. plantarum can perform EET with a sub-physiological DHNA concentration of 0.01 μg/mL. Indeed, L. plantarum can reduce iron and produce significant current density  pathway  in MRS containing mannitol, DHNA, and ferric ammonium citrate  of mMRS. ORP is defined as the ratio of all oxidative to reductive components in an environment   , and at on assay . This dion assay . These ontations .L. plantarum Δndh2 produced a significantly lower current density . There was no significant difference in intracellular iron concentrations between L. plantarum growth in mMRS supplemented with DHNA and iron compared to growth in mMRS alone  produced by L. plantarum in mannitol CDM typically occurred within 24 hr after inoculation into the bioreactor (L. plantarum incubated in open circuit (OC) conditions  used in aerobic respiration, nor deletion of the respiratory nitrate reductase (ΔnarGHJI) significantly altered current production  cycle. e branch . To probe branch . MoreoveL. plantarum uses glycolysis to convert mannitol to two molecules of pyruvate which are then converted mainly to lactate or ethanol via NADH-consuming steps, or acetate via an ATP-generating reaction using substrate-level phosphorylation   . A simile the pH . When mue the pH . Overall600nm and dry cell weight were not significantly different  was re-oxidized to NAD+  were estimated using the calculated ATP and the measured dry weight. Under OC conditions, the YATP obtained (8.06 ± 0.8 g dw/mol ATP) for L. plantarum was similar to that observed previously (10.9 g-dw/mol ATP) (ATP was reached for L. plantarum performing EET (3.07 ± 0.35 g dw/mol ATP) (mannitol). Consequently, molar biomass yields (g-dw/mol-mannitol) under EET conditions were significantly lower  . Hence, mol ATP) . This obmol ATP) . EET conly lower , in agrely lower . These cL. plantarum as a starter culture  as compared to abiotic and biological non-EET promoting conditions (no DHNA)  . This cuno DHNA) , indicatL. plantarum growth and metabolism in the kale juice fermentation. Significant changes in the pH and fermentation products were detected under EET conditions  and conditionally requires an extracellular, flavin-binding reductase (PplA). EET in L. plantarum generates a high NAD+/NADH ratio, increases fermentation yield and flux, shortens lag phase, and increases ATP production. Thus, EET in L. plantarum is a hybrid energy metabolism that contains metabolic features of fermentation, redox features of anaerobic respiration, and predominately uses substrate-level phosphorylation to conserve energy. This pathway is active in L. plantarum with physiologically relevant DHNA concentrations and in a food fermentation and results in an increased metabolic flux and acidification rate.Increases in fermentation and energy conservation from EET have important bioenergetic implications for the mainly fermentative LAB. We showed that L. plantarum, a primarily fermentative bacterial species, is fundamentally different from EET-driven, anaerobic respiration of metal-reducing bacteria. Although aspects of EET in L. plantarum and metal-reducing Geobacter spp. are similar, such as the upregulation of NADH dehydrogenase, the reduction rate of extracellular electron acceptors, and the high NAD+/NADH ratio, other aspects of energy metabolism during EET in these two organisms are starkly different  and the shuttling of electrons outside of the cell. In addition, the reduction of the oxygen and organic compounds for cofactor regeneration by LAB leads to a metabolic shift toward acetate production and altered metabolic end-product ratios  and respiratory metabolisms  , but an overall ~1.5 fold weaker coupling between anabolism and catabolism (lower YATP). Additionally, the increased NAD+/NADH ratio arises from using an ~2:1 ratio of endogenous to extracellular electron acceptors. Thus, to our knowledge, the hybrid strategy that L. plantarum uses to generate ATP performing EET constitutes a novel mode of energy conservation in a primarily fermentative microorganism.In contrast to and expanding upon these studies, our work elucidates a qualitatively and quantitatively different blending of fermentation and respiration. equired) . QuantitL. plantarum, we showed that L. lactis is able to generate current despite lacking pplA. Current generation by L. lactis was observed previously, found to be riboflavin dependent, and resulted in a small metabolic shift (yet to be defined) in which the flux through NADH-oxidizing pathways was reduced and ATP generating pathways were increased   in Enterococcus rivorum  . E. faecquinones  and a tyquinones  for Fe3+lar iron . Moreoveeduction . The con rivorum . Thus, tL. plantarum and other LAB with FLEET systems such as L. casei are genetically and metabolically diverse and grow in a variety of nutrient rich environments including dairy and plant foods and the digestive tract   . This exsucrose) . BecauseL. plantarum EET will be key to unravel physiological and ecological questions and to develop other biotechnological applications.We expect that our study will improve the current understanding of energy conservation in primarily fermentative microorganisms and contribute to establishing the ecological relevance of EET in lactic acid bacteria. This work will ultimately allow the use of EET to electronically modulate the flavor and textural profiles of fermented foods and expand the use of lactic acid bacteria in bioelectronics, biomedicine, and bioenergy . The ideLactiplantibacillus spp., Lacticaseibacillus spp., Levilactobacillus brevis, Ligilactobacillus murinus, and Pediococcus pentosaceus, MRS ; Lactococcus lactis and Streptococcus agalactiae, M17 (BD) with 2% w/v glucose; Enterococcus faecalis, and Enterococcus faecium, BHI (BD); and Escherichia coli, LB . Bacterial strains were incubated without shaking except for E. coli (250 RPM) and at either 30 or 37 °C. Where indicated, strains were grown in filter-sterilized MRS (6H8FeNO7) (1.25 mM) , riboflavin , or 5 μg/mL erythromycin (VWR).All strains and plasmids used in this study are listed in ized MRS  lacking ized MRS  with 125ized MRS . RiboflaL. plantarum NCIMB8826 was identified using NCBI basic local alignment search tool (BLAST) (L. monocytogenes 10403S FLEET genes (lmo2634 to lmo2641) as a reference. L. plantarum genes were annotated based on predicted functions within the FLEET pathwa .The FLEET gene locus of  (BLAST)  using thT pathwa . FLEET las <10–3 . Heatmap complex  identifi complex  with cluhttp://cshprotocols.cshlp.org), and adjusted to an optical density (OD) at 600 nm (OD600nm) of 2 in the presence of 2.2 mM ferrihydrite (4 (Sigma-Aldrich) (0.25 mM to 0.016 mM) and 2 mM ferrozine. The FeSO4 was dissolved in 10 mM cysteine-HCl  to prevent environmental re-oxidation of Fe2+ to Fe3+ in the standard curve. For testing iron reduction activity of cells with a DHNA concentration of 0.01 μg/mL in the medium, iron(III) oxide nanoparticles < 50 nm (Sigma-Aldrich) were used as insoluble iron form , pH 7.2 .d pRV300 . For mutd pRV300 . PCR proL. plantarum NCIMB8826 strains were grown overnight (approximately 16–18 hr) from glycerol stocks in MRS. Cells were harvested by centrifugation  and washed twice in PBS. When L. plantarum wild-type EET activity versus the Δndh2 (MLES100) and ΔpplA (MLES101) deletion mutants was compared, cells were grown as described and the number of cells was normalized across the three strains prior to inoculation in the BES. The bioreactors consisted of double-chamber electrochemical cells   (BD) for the counter electrode chamber. Both media of the working electrode chamber were supplemented with 20 μg/mL DHNA or 0.01 μg/mL diluted 1:1 in DMSO:ddH2O where appropriate. To test the role of bd-cytochrome, heme was added in a final concentration of 10 μg/mL (diluted 1:1 in DMSO: ddH2O). The medium in the working electrode chamber was continuously mixed with a magnetic stir bar and N2 gas was purged to maintain anaerobic conditions for the course of the experiment. The applied potential to the working electrode was of +0.2 V versus Ag/AgCl (sat. KCl) . Reactors run under OC conditions were similarly assembled but kept at open circuit and used as control for non-current circulating conditions. Once the current stabilized, the electrochemical cells were inoculated to a final OD600 of 0.12–0.15 with the cell suspensions prepared in PBS. Current densities are reported as a function of the geometric surface area of the electrode (16 cm2). The bioreactors were sampled by taking samples under sterilized conditions at different time points for subsequent analysis. The samples for organic acids analyses were centrifuged  and the supernatant was separated for High-Performance Liquid Chromatography (HPLC) assessments. Samples for ATP and NAD+/NADH analyses were flash frozen in a dry ice/ethanol bath.ley, CA)  with a c–1. We used a refractive index detector 1260 Infinity II RID. A five-point calibration curve based on peak area was generated and used to calculate concentrations in the unknown samples. The following standards were included in the HPLC measurements: acetate, formate, pyruvate, malate, lactate, succinate, oxalacetate, fumarate, ethanol, acetoin, butanediol, mannitol, and glucose. No gaseous products were measured.Organic acids, ethanol, and sugar concentrations were measured by HPLC , using a standard analytical system  equipped with an Aminex Organic Acid Analysis column  heated at 60 °C. The eluent was 5 mM of sulfuric acid, used at a flow rate of 0.6 mL min2O. Afterwards cells were resuspended in 1 mL of ddH2O and transferred to microfuge tubes (previously weighted). Cells were harvested by centrifugation , and the tubes were then transferred to an evaporator to remove humidity. The microfuge tubes were then cooled in a desiccator for 30 min and the weight of each tube was measured to determine cell weight. The difference between the weight of each tube with the pellet and before containing it allowed us to determine the dry weight/mL.Bioreactors were shaken to remove the cells attached to the working electrode and afterwards sampled to measure viable cells (colony forming units [CFUs]) and total biomass (dry weight). Samples for CFU enumeration were collected under sterile conditions at the time of inoculation and at the time of approximately maximum current density. Samples were serially diluted (1:1000 to 1:1000000) in sterile PBS and plated on MRS for CFUs enumeration after overnight incubation at 30 °C. Dry weight was determined using a 25 mL sample collected at approximately maximum current density. Cells were harvested by centrifugation  and washed twice in 50 mL ddHL. plantarum NCIMB8826 was grown in triplicate to exponential phase (OD600 1.0) at 37 °C in mMRS with or without the supplementation of 20 μg/mL DHNA and 1.25 mM ferric ammonium citrate. Cells were collected by centrifugation at 10,000 g for 3 min at 4 °C, flash frozen in liquid N2 and stored at –80 °C prior to RNA extraction as previously described (rpoB as a control (escribed . Brieflyescribed . RNA was control .http://dnatech.genomecenter.ucdavis.edu/). An average of 36,468,428 raw paired-end reads per sample was collected  >0.5. Clusters of Orthologous Groups (COGs) were assigned to genes based on matches from the eggNOG (ver. 5.0) database  was depleted from 4 μg RNA using the RiboMinus Eukaryote Kit v2 with specific probes for prokaryotic rRNA (ThermoFisher) following the manufacturer’s instructions. RNA was then fragmented to approximately 200 bp, converted to cDNA, and barcoded using the NEBnext Ultra-directional RNA Library Kit for Illumina  with NEBnext Multiplex Oligos for Illumina (Primer Set 1) (New England Biolabs) following the manufacturer’s protocols. cDNA libraries containing pooled barcoded samples was run across two lanes of a HiSeq400  on two separate runs for 150 bp paired-end reads , or ΔpplA (MLES101) were inoculated at an OD600 of 0.10. Two uninoculated controls were used to measure baseline ORP over time. The ORP data was collected via Modbus TCP/IP protocol  into a database  and analyzed in MATLAB . pH was measured using a Mettler Toledo SevenEasy pH meter . Cells were collected at either 24 hr or at the greatest ORP difference between the wild-type and mutant strains (ΔmVmax) by centrifugation at 10,000 g for 3 min and used for ferrihydrite reduction analyses.Hamilton oxidation-reduction potential (ORP) probes  were inserted into air-tight Pyrex  bottles containing mMRS supplemented with 20 μg/mL DHNA and/or 1.25 mM ferric ammonium citrate and incubated in a water bath at 37 °C. A custom cap for the Pyrex bottles was 3D printed with polylactic acid filament (2.85 mm diameter) such that the ORP probe threads into the cap and an o-ring seal can be used to provide an air-tight seal between the probe and the cap. The ORP was allowed to equilibrate over 40 min before + and NADH in the supernatants were then quantified with the Molecular Probes ATP Quantification Kit (ThermoFisher) and the Promega NAD/NADH-Glo Kit , respectively according to the manufacturers’ instructions.Frozen cell pellets were suspended in PBS and lysed by mechanical agitation in a FastPrep 24  at 6.5 m/s for 1 min. The cell lysates were then centrifuged at 20,000 g for 3 min at 4 °C. ATP and NADL. plantarum was inoculated in mMRS with or without 20 μg/mL DHNA and 1.25 mM ferric ammonium citrate at an OD600 of 0.10 for 3.5 hr. Cells were then collected by centrifugation at 10,000 x g for 3 min and washed twice in PBS to remove cell-surface-associated metals. Viable cell numbers were enumerated by plating serial dilutions on MRS laboratory culture medium and the resulting cell materials were digested by incubating at 95 °C for 45 min in a 60% concentrated trace metal grade HNO3, allowed to cool, then diluted with MilliQ water to a final concentration of 6% HNO3. The contents were quantified with internal standards with an Agilent 7,500Ce ICP-MS  for simultaneous determination of select metals  at the UC Davis Interdisciplinary Center for Plasma Mass Spectrometry (http://icpms.ucdavis.edu/).L. plantarum NCIMB8826-R  (EET conditions) or kept at open circuit . Viable cells were measured by plating 10-fold serial dilutions in MRS agar plates with 50 μg/mL of Rif.Green organic kale purchased from a market (Whole Foods) was washed with tap water and air dried for 1 hr as previously recommended . A totalMB8826-R  .The total electrons harvested on the anode were estimated by integrating the area (charge) under the chronoamperometric curve (current response (A) over time (s)), which was corrected by subtracting the current baseline obtained before L. plantarum RNA-seq data are available in the NCBI Sequence Read Archive (SRA) under BioProject accession no. PRJNA717240. A list of the completed Lactobacillales genomes used in the DNA sequence analysis is available in the Harvard Dataverse repository at https://doi.org/107910/DVN/IHKI0C. Lactiplantibacillus plantarum. The ability to shift and/or accelerate metabolism of lactic acid bacteria capable of extracellular electron transfer may have interesting biotechnological applications.In this study, the authors describe unique metabolic strategies, including extracellular electron transfer, utilized by the lactic acid bacterium  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gisela Storz as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Extracellular electron transfer increases fermentation in lactic acid bacteria via a hybrid metabolism" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) The methods and results of metabolite analyses are not sufficiently described. Further explanation is required if conclusions are to be made about yield, fermentation, and physiology.2) Some claims are overstated and need to be revised or, better yet, backed up with more experimental data.Reviewer #1 (Recommendations for the authors):1) The only weakness: The methods and results of metabolite analyses are not sufficiently described. This requires explanation if conclusions are to be made about yield, fermentation, and physiology.The paper provides strong evidence that electron diversion to external acceptors is an electron sink. However, while quantitative conclusions are made about fermentation, only ~30% of the mannitol can be accounted for in the EET condition that is the focus of this paper. Put another way, the paper is about where electrons go, but the majority of the electrons are missing.Clarify if this strain can excrete products such as sugar polymers, sugars, pyruvate, formate, glycerol, acetoin, or butanediol. State if standards for these were included in the product analysis . Explain why formate isn't present (was it not measured?), as this would allow determination of flux through PFL vs. a pyruvate excretion, a carbon sink of acetoin, or butanediol. Could missing carbon be due to excretion of a partially oxidized sugar like fructose , or accumulation of storage polysaccharide?A) Address these questions in the section around line 299; where fermentation products are mentioned as being present or absent. What was looked for, but not found , vs which were not looked for, . This makes it clear that carbon is not missing, just that the methods were unable to detect it.B) Table 1 and all other tables making calculations need significant qualifiers-- since NADH flux is guessed from mannitol disappearance , and NADH fates + ATP yields are based on a small subset of fermentation products detected. Statements like line 347 "EET allowed cells to produce more fermentation products per mol mannitol" can't be made. Nor can one conclusively say what % of electrons went to electrodes, nor what the YATP is. If anything, the fact that the mix of lactate, acetate and ethanol do not shift dramatically to acetate production , is surprising and indicates something unexplained is going on.Again, the lack of a carbon balance takes nothing away from qualitative conclusion that mannitol electrons are diverted to EET, or the strength of the EET evidence. The electrons at the electrode or in the iron are real and countable, this just limits the quantitative statements that can be made.2) L136; Reported Geobacter current densities easily reach 1000 µA/cm2 for flat surfaces, not 100-200.3) Figure 3B: Many questions about this experiment. It is the only time it appears, yet is used as a quantitative way to compare mutants.a) State if the iron(III) in the medium was reduced by mutants during this assay-for redox potential to drop equally suggests that it was (which contradicts 3A).b) Provide the final pH during these experiments-- pH can affect E{degree sign}'.c) Lack of DHNA is always used as a reliable negative control in the other experiments, provide any data that shows if the addition of DHNA is essential for the mV drop in this assay, so we know it is also an assay of EET.Lacking better evidence/controls, this is one of the weaker bits of data in the main body, especially next to the more robust Fe/current data. Appropriate for supplements, but not conclusive as is.4) An idea regarding the ∆pplA phenotype-the two conditions require EET, but iron oxides should be lower potential acceptors than the anode which is poised at a high driving force of >+400 mV. The anode is plenty high to oxidize something like DHNA (E{degree sign}' ~70 mV) or chelated metals. PplA could be more necessary near the low potential of flavins. The supplied data can't be used to compare the effect of specific riboflavin concentrations on iron reduction rates (only extents) vs current production rates, but it might be available.5) Line 311, More data would be needed to use succinate production as evidence for or against respiration. A mol of pyruvate could be excreted as succinate due to TCA cycle oxidation (indicating respiration), manifesting as excess electrons relative to the amount of succinate produced . Conversely, succinate could be due to electron disposal via a reductive pathway (indicating fermentation). Lacking good carbon or electron recovery, neither can be ruled out, though the non-respiratory reductive pathway is most logical.6) Table 1/line 403-: The low YATP in cells using EET is a possible 'different energy conversion strategy'. . I'd suggest uncoupling of acid production from growth by low pH seems likely. In most strains, simple glycolytic pathway functions at low external pH, but complex protein synthesis and replication processes become inhibited, ATP from substrate turnover is needed to maintain pmf and internal pH, and high internal organic acids become toxic. Citable yield/pH data can be hard to locate as it comes from the 80's in the age of calling every lactic acid bacterium a Streptococcus, but since the non-EET cultures at neutral pH got close to the Bauchop and Elsden YATP, and EET cultures at pH 4 were a fraction of this, these data look like common behaviour of many lactic acid bacteria. In future comparisons, pH control could help estimate energy yields from EET. The first paper I found in a quick search to illustrate pH effects was: Russell and Dombrowski, DOI: 10.1128/aem.39.3.604-610.1980.7) The value for Geobacter ATP/substrate yield in Table S2 seemed very high. Mahadevan says Geobacter produces 0.5 mol ATP/mol acetate when using extracellular acceptors . If I understand the adjustment for 6-carbon compounds, would this make the value for Geobacter using EET to be 1.5 mol ATP/mol 6-C substrate?Reviewer #2 (Recommendations for the authors):Line 21 – acronyms like this make our field more challenging to understand and are unnecessary. Does the work presented actually support 'energy conservation'? Growth seems to occur after EET activity takes place rather than with it as discussed below.Line 27 – 'fermentation yield' referring to biomass yield or fermentation product yield?Line 29, Line 45, Line 75, Line 86, Line 479 – blending fermentation and respiration has been known for over a decade in Shewanella . The description on Line 517 regarding the major finding from this reference is incorrect. PMF is not used by S. oneidensis anaerobically to generate ATP as demonstrated by the robust anaerobic growth of an ATP synthase mutant.Line 61 – implied here is that LAB are auxotrophic for heme and menaquinone, but should be explicitly stated.Page 8 (top) – the comparison to other organisms is not particularly helpful unless current per unit cell or unit biomass is quantified, nor does it seem particularly relevant to the overall theme of this manuscript.Line 152 – predicted to encodeLine 182 – predicted to encodeLine 183 – under the condition tested.Listeria work?Line 200 – isn't this already known from the Line 206 – unclear what 'extracellular redox potential' is, unclear to me if any differences are significant in Figure 3B. I guess this is a measurement of Fe(II) / Fe(III) in the medium?Figure 3D – did the authors include a strain that lacked the cluster?Line 252 – unclear to me how ICP-MS can distinguish between iron inside a cell and associated with the cell surface.Line 256 – have other metals been tested? Frequently manganese substitutes for iron in enzymes from non-iron dependent bacteria. Manganese oxide is also a known substrate for EET in Geobacter and Shewanella and could be nutritionally significant to LAB.Line 262 – does this mean a large amount of the cells that grew are now dead?Figure 4  – I don't understand why maximum current output is completely decoupled from when biomass spikes. At 24h (maximum current), basically no growth has occurred. At 2 days and 5 days, when biomass is the greatest (in the WT culture), very little current is produced. This seems like direct evidence that EET is not coupled with metabolism in these bacteria. The ATP measurements were made at 24h, where OD of the mutant is decreasing (thus less ATP would be expected). Same issue with NAD/NADH measurement.Figure 4C – Lines are undefined in the figure legend. Also, growth should be plotted on a semi-log scale, see: https://schaechter.asmblog.org/schaechter/2018/07/why-you-must-plot-your-growth-data-on-semi-log-graph-paper.htmlFigure 5 – based on the growth curve shown in Figure 4, the OC culture lags behind and does not reach maximum OD until day 5. What do these measurements look like at day 5 (or 6)? It seems likely that the authors are drawing conclusions from cells in two different states  rather than focusing on the finding that having an external electron acceptor appears to accelerate the metabolism of cells.Line 465 – Description of the experiment setup in 2B does not include addition of flavins for the iron reduction assay, so it is unclear why the authors claim that both flavins and quinones are necessary for EET in diverse LAB species.Line 473 – Iron and electrode reduction is dependent on addition of DHNA. Are these cells likely to ever encounter 20 ug/mL DHNA in the environment? Is the EET activity observed relevant to normal physiology for these organisms, or a lab artifact caused by providing a non-native electron carrier? The argument beginning on line 491 underscores the artificial nature to me.Line 559 – none of the references here provide any quantitation quinone concentration in the digestive tract. (Relates to the comment above from line 473).Line 637 – is ferrozine toxic to these cells?Line 646 – Were these mutants complemented?Line 653 – the suicide vector should be identified and referenced here.General comments:Unclear why Figure 1C shows twice as much current twice as fast as Figure 3C in what appears to be identical conditions?Reviewer #3 (Recommendations for the authors):Figure 1A – though the tubes are aligned with the plot below, it may be helpful for some readers to have labels on this image to clarify the conditions of each.Figure 1C – for the top line, the "+DHNA+mannitol" label should be on one lineFigure 4A – labeling should be more consistent ("anodic" vs. "EET")Figure 4A – "log" should be removed from the y-axis labelFigure 4C – points on this plot should be connected with straight linesFigure 5D – axis label is cut offFigure 5G – FLEET should not be mentioned in the model at all. Instead, Ndh2 should be labeledFigure 6A – Label differences between the two kale juice bottlesAuthors should also provide more information for the definitions of YATP and the values in Table 1L. plantarum to that of S. oneidensis--are the quoted values for S. oneidensis using (an) endogenously produced shuttle(s), or are they for S. oneidensis + (an) added shuttle(s)? This should be clarified, because L. plantarum has no current density capacity without added DHNA.Line 137: Where the authors compare current density capacity for Authors should offer a mechanism for the conditional pplA phenotypeSwitching between the terms "electron donor" and "carbon source" may be confusing for some readers; one term should be used consistently.Figure 3-—figure supplement 1F is not discussed in the text.Line 204: typo in "pplA"The ndh2 mutant should be included in the kale juice fermentation experiments to test the contribution of Ndh2 in this context.Readers may not be familiar with the term "Crabtree-positive""SLP" should be defined on page 3L. plantarum metabolism  was moved from the results/discussion to the introduction. If L. plantarum is able to grow via respiration (as suggested in lines 304-308), this should also be mentioned in the introduction. The introduction should also be revised to ensure that terms are used unambiguously and to remove inaccurate generalizations. Some examples are given in the comments below.The introduction does not provide enough detail for a broad audience to understand the metabolic pathways being discussed. It would help if some of the detail about It is not totally accurate to say that energy per se is generated or created during metabolism. The authors should revise these statements to say that energy is obtained or conserved or that ATP is generated.This paper discusses detailed metabolic pathways and the terms used to describe these pathways should be used carefully and precisely to avoid confusion for the reader. For instance, the authors should precisely define "substrate-level phosphorylation" and "fermentation" and should not use these terms interchangeably. "Substrate-level phosphorylation" refers to a reaction that generates ATP directly, without the use of the proton motive force and the membrane-associated ATP synthase enzyme. "Fermentation" refers to the pathways that restore the NAD+/NADH ratio and allow growth with substrate-level phosphorylation as the sole mechanism of ATP generation.Lines 50-51 and 373-374: Some organic compounds can also be respired (for example fumarate).Lines 51-52 should say "Fermentative bacteria produce ATP solely via substrate-level phosphorylation" because substrate-level phosphorylation also occurs when bacteria are growing by respiration .Lines 71-73: It is unclear how the property of being "exergonic" or "endergonic" is relevant in the context here.Lines 85-86: "…EET is associated with non-fermentative respiratory organisms…" Does this refer specifically to endogenous EET . This should be clarified because there is a long history of research on fermentation+EET in the presence of added mediators (see next comment).Related to the above point: In the introduction or discussion, the authors should mention or acknowledge other work examining how growth in the presence of an electrode affects fermentative metabolisms. Some examples are:Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force. Mol Microbiol. 2014 Apr;92(2):399-412. doi: 10.1111/mmi.12566.Glasser NR, Kern SE, Newman DK. Phenazine redox cycling enhances anaerobic survival in Emde R, Schink B. Enhanced Propionate Formation by Propionibacterium freudenreichii subsp. freudenreichii in a Three-Electrode Amperometric Culture System. Appl Environ Microbiol. 1990 Sep;56(9):2771-6. doi: 10.1128/aem.56.9.2771-2776.1990.And other references discussed in reviews such as:Moscoviz R, Toledo-Alarcón J, Trably E, Bernet N. Electro-Fermentation: How To Drive Fermentation Using Electrochemical Systems. Trends Biotechnol. 2016 Nov;34(11):856-865. doi: 10.1016/j.tibtech.2016.04.009.Vassilev I, Averesch NJH, Ledezma P, Kokko M. Anodic electro-fermentation: Empowering anaerobic production processes via anodic respiration. Biotechnol Adv. 2021 May-Jun;48:107728. doi: 10.1016/j.biotechadv.2021.107728.Comparing Figure 1A to Figure 3A, the extent of Fe3+ reduction seems quite different. Were the conditions the same for these two experiments? If so, an explanation should be added to the text.Lines 104-106: Can DHNA itself be used as an electron shuttle? This should be addressed.L. plantarum to that of S. oneidensis--are the quoted values for S. oneidensis using (an) endogenously produced shuttle(s), or are they for S. oneidensis + (an) added shuttle(s)? This should be clarified, because L. plantarum has no current density capacity without added DHNA.Line 137: Where the authors compare current density capacity for Line 139: "current production was not dependent on the carbon source or growth medium, and current increased after supplementation of riboflavin in the growth medium". Both of these claims are hard to evaluate because the plots in figure 1 and figure1-—figure supplement 2 are shown for different lengths of time, different carbon sources (electron donors), and different media. To make it easier for the reader to compare, it would help to have the name of the medium as a title for each current density plot. To judge whether riboflavin truly increases current, we should compare figure 1c to figure 1-—figure supplement 2D. In figure 1c, with no riboflavin added, current increases to ~120 uA/cm2 at around 1 day. In the supplemental figure, this increase is not seen until after riboflavin is added. Without a control, it is hard to know whether this increase was dependent on the riboflavin addition.Line 154: Please define homofermentative.Line 160: "the percentage of" should be removedfigure 2-—figure supplement 2C: I am confused about how this experiment controls for the possibility that reduction of soluble iron , which wouldn't necessarily require EET, contributes to the higher levels of ferrous iron observed when DHNA and ferric ammonium citrate are both added. Maybe a control with no ferrihydrite added would tell us whether ferric ammonium citrate is being reduced.Line 194: more background should be provided for the FLEET-inducing conditionsLines 304-309: More background information is needed to understand the motivation behind these experiments. Were oxygen and nitrate available as electron acceptors?Line 305: "…needed for PMF generation in aerobic…"Line 414: "previous results"; does this refer to results in this paper or a prior publication? If a prior publication, it should be cited here. Essential revisions:1) The methods and results of metabolite analyses are not sufficiently described. Further explanation is required if conclusions are to be made about yield, fermentation, and physiology.We thank the Editor and Reviewers for raising these important concerns. We revised the Materials and methods and Results sections to more comprehensively describe the methods and results of the metabolite analyses. Within the Results, we included new data  showing the levels of formate and pyruvate under extracellular electron transfer and open circuit conditions. We also clarify that we included standards for a number of metabolites, but that our measurements show that these compounds were absent or sufficiently low to not be within the range of detection.L. plantarum. Our new data on formate and pyruvate levels allowed us to track carbon and electrons at ~80% and ~85%, respectively. With this information, we show that EET increases the yield of ATP and fermentation products per mole of mannitol  by ~1.75x. This result indicates that EET enables more efficient energy conservation and greater mannitol fermentation in L. plantarum. The better accounting of electrons reveals that L. plantarum uses an ~2:1 ratio of endogenous to exogeneous electron acceptors during EET. Interestingly, the yield of biomass per ATP and per mannitol  decreases by ~1.5x, showing that while EET triggers faster, more efficient energy conservation, these catabolic processes are also less coupled to anabolic processes. Thus, the revised manuscript provides an improved, multifaceted view of this hybrid metabolism.Second, we also now provide a more complete picture which allows us to make quantitative conclusions about yield, fermentation, and physiology during EET in 2) Some claims are overstated and need to be revised or, better yet, backed up with more experimental data.We revised claims to more precisely reflect our findings and, for other claims, provided additional experimental data to further support them as follows:(1) We provide new data on formate and pyruvate and the implications of those data on yield of ATP and fermentation products (discussed in our response to point 1 above).L. plantarum performs EET with physiologically-relevant (low) concentrations of DHNA (concern raised by Reviewers #2 and #3). DHNA is present at a range of concentrations (0.09-0.5 ug/mL) in foods and other environments inhabited by L. plantarum. Our new data  shows that L. plantarum supplied with 0.010 ug/mL DHNA, which is on the lower end of this range, still produces significant current and iron reduction. Additionally, we now show that this DHNA-dependent EET causes a significant decrease in pH , indicating EET affects the physiology of L. plantarum.(2) We provide new evidence that L. plantarum in laboratory culture media, we also qualify our conclusions to explain that the extent and physiological impact of EET will depend on the environmental and nutritional conditions of the niche L. plantarum is inhabiting (lines 153-162).Lastly, while these data indicate that physiologically relevant levels of DHNA have physiological effects on L. plantarum, further linking EET activity to its ability to modulate extracellular ORP during fermentation.(3) More background information is provided on the rationale and need for measuring oxidation-reduction potential (ORP) (concern raised by Reviewers #1 and 2) (lines 213-217). Appropriate statistical tests are also now applied to the ORP data. Lastly, new data are included  showing that DHNA supplementation is required for robust ORP reduction by wild-type (4) Reviewer #3 suggested that the claim that EET proceeds independently of respiration may be overstated. We now explain our current understanding and the limits on that understanding. Our results show EET proceeds via a respiratory protein, but ATP generation during EET is mainly produced by substrate-level phosphorylation, not oxidative phosphorylation. We also acknowledge that EET could involve other as-yet unidentified respiratory components (lines 478-485 and 504-5) and that we cannot exclude ATP generation by oxidative phosphorylation (lines 482-485).Reviewer #1 (Recommendations for the authors):1) The only weakness: The methods and results of metabolite analyses are not sufficiently described. This requires explanation if conclusions are to be made about yield, fermentation, and physiology.The paper provides strong evidence that electron diversion to external acceptors is an electron sink. However, while quantitative conclusions are made about fermentation, only ~30% of the mannitol can be accounted for in the EET condition that is the focus of this paper. Put another way, the paper is about where electrons go, but the majority of the electrons are missing.Clarify if this strain can excrete products such as sugar polymers, sugars, pyruvate, formate, glycerol, acetoin, or butanediol. State if standards for these were included in the product analysis . Explain why formate isn't present (was it not measured?), as this would allow determination of flux through PFL vs. a pyruvate excretion, a carbon sink of acetoin, or butanediol. Could missing carbon be due to excretion of a partially oxidized sugar like fructose , or accumulation of storage polysaccharide?We thank the Reviewer for the Reviewer’s strong endorsement of our manuscript and clear recommendations for improvements.In the revised manuscript, we provide additional data showing where ~80% of the carbon goes under EET conditions (Supplement File 1) and where the majority of the carbon goes under open circuit conditions. With this more complete information, our balances account for between 77-96% of the electrons (see Supplement file 1). These new data more completely support our conclusion that EET increases fermentation product yield, metabolic flux, and environmental acidification.A) Address these questions in the section around line 299; where fermentation products are mentioned as being present or absent. What was looked for, but not found , vs which were not looked for, . This makes it clear that carbon is not missing, just that the methods were unable to detect it.We agree with the Reviewer on this recommendation to provide more clarity on the methods and results of the metabolite analyses. As a result, we have included additional information in the Materials and Methods (lines 712-720) and in the Results (lines 323-325 and 328-335). As described in detail below, we can account for ~80% of the carbon and electrons under EET conditions, which captures a majority of both the carbon and electron flow.L. plantarum have not been reported to make storage polysaccharides, and thus it is very unlikely that storage polysaccharides could account for the remaining missing carbon.With our HPLC methods, we were able to detect the following compounds using standards: acetate, formate, pyruvate, malate, lactate, succinate, oxalacetate, fumarate, ethanol, acetoin, butanediol, mannitol, glucose and fructose . Despite being extensively studied, We detected large and significant changes in the concentration of main fermentation end-products, acetate, lactate and ethanol . We detected significant, but small changes, in pyruvate and formate production . We did not detect fructose, malate, glucose, oxaloacetate, fumarate, acetoin or 2,3-butanediol in the samples. We have included this new information in the revised Results section (lines 333-334). We also now include carbon in biomass as part of the carbon recovery in an updated Table 1 and updated Table S1. 2 production because our bioelectrochemical reactors are not gas tight. Thus, we suggest the missing carbon is likely due to CO2 production, but we cannot rule out production of other unidentified metabolites.Taken together, these updated measurements increase the carbon recovery observed under both EET and OC conditions. Under EET conditions, ~80% of the carbon is recovered in fermentation products, while under OC conditions, we can account for ~55% of the carbon. We did not measure COB) Table 1 and all other tables making calculations need significant qualifiers-- since NADH flux is guessed from mannitol disappearance , and NADH fates + ATP yields are based on a small subset of fermentation products detected. Statements like line 347 "EET allowed cells to produce more fermentation products per mol mannitol" can't be made. Nor can one conclusively say what % of electrons went to electrodes, nor what the YATP is. If anything, the fact that the mix of lactate, acetate and ethanol do not shift dramatically to acetate production , is surprising and indicates something unexplained is going on.L. plantarum fermentation :, .We agree with the Reviewer that the qualitative observations are quite surprising and that our quantitative calculations need to carefully state what uncertainties remain. We also believe that our calculations need to be considered in the context of the prior, extensive literature on L. plantarum, allowing us to make quantitative assessments of changes in ATP and NADH. Specifically, our findings now have carbon recoveries (~80%) within 10% of previous studies of L. plantarum metabolism . Additionally, our studies probe the same spectrum of metabolites found in these prior studies. with the sole exception of CO2 . Because CO2 production does not regenerate NADH or produce ATP, the lack of CO2 accounting will not affect ATP or NADH balances. Thus, the conclusions we draw about quantifying ATP generated by substrate-level phosphorylation aligns with other quantitative conclusions from prior work on L. plantarum metabolism .Additional context to the changes made in the manuscript: our improved data (Table 1) now aligns with existing literature on In response to the Reviewer’s comment, we now use two different sets of assumptions to NADH generation and present the resulting values as a range of NADH regeneration. As described in Supplementary file 1, Method 1 uses the concentration of metabolites to calculate NADH production, essentially assuming that all NADH production stems from production of metabolites. Because it does not include NADH produced from mannitol consumption directed towards biosynthesis or other unidentified fermentation products, Method 1 likely underestimates total NADH produced, leading to higher values of NADH regeneration. Conversely, Method 2 estimates the NADH produced from mannitol consumption, essentially assuming that NADH is produced whenever mannitol is consumed. Since mannitol consumption may lead to biosynthesis or unidentified fermentation products without NADH generation, Method 2 likely overestimates the total NADH produced, leading to lower NADH regeneration values. In light of this uncertainty, we now use both Methods and present a range for NADH regeneration.Again, the lack of a carbon balance takes nothing away from qualitative conclusion that mannitol electrons are diverted to EET, or the strength of the EET evidence. The electrons at the electrode or in the iron are real and countable, this just limits the quantitative statements that can be made.We recognize that our measurements do not account for all the carbon, however, in the revised manuscript, these quantitative uncertainties are now limited to ~20% because of the much improved carbon and electron balance. To address the Reviewer’s concern, we have now indicated the limited extent of these uncertainties in the manuscript (lines 363-365).2) L136; Reported Geobacter current densities easily reach 1000 µA/cm2 for flat surfaces, not 100-200.Following Reviewer #2’s suggestion, we now compare the current production of different species normalized per g of protein biomass (line 140-145). This normalized value is a better metric to compare EET at a cellular level among different species.3) Figure 3B: Many questions about this experiment. It is the only time it appears, yet is used as a quantitative way to compare mutants.a) State if the iron(III) in the medium was reduced by mutants during this assay-for redox potential to drop equally suggests that it was (which contradicts 3A).b) Provide the final pH during these experiments-- pH can affect E{degree sign}'.c) Lack of DHNA is always used as a reliable negative control in the other experiments, provide any data that shows if the addition of DHNA is essential for the mV drop in this assay, so we know it is also an assay of EET.Lacking better evidence/controls, this is one of the weaker bits of data in the main body, especially next to the more robust Fe/current data. Appropriate for supplements, but not conclusive as is.et al., 2018 – Am. J. Enol. Vitic.). Redox reactions are prevalent in food fermentations  and ORP directly influences the outcome of fermentations with lactic acid bacteria .We now provide background and justification for our use of ORP experiments (lines 213-217). Extracellular redox potential is also referred to as oxidation-reduction potential (ORP), which is the ratio of all oxidative to reductive components in the environment  possess NADH oxidase and therefore are able to reduce O2, thereby also contributing to ORP reduction . Hence, although we expect that iron was reduced by the wild-type L. plantarum strain, it was not the only redox active compound. We revised the text (lines 219-228) and Figure 3B to clarify these distinctions.A) Already after one hour incubation in mMRS there was a significant difference in ORP between the wild-type and B) pH values along with the experimental data collected in response to comment C (below), have been added to a new Figure 3 – supplement figure 1. The culture medium pH was identical between the wild-type and mutant strains. Therefore, it is very unlikely that pH alone accounts for the differences in the observed ORP values.L. plantarum growth led to significantly higher ORP values.C) We appreciate the Reviewer’s suggestion of including a control group lacking DHNA. We performed that experiment and the results are included as Figure 3 – supplement figure 1. As expected, the absence of DHNA during 4) An idea regarding the ∆pplA phenotype-the two conditions require EET, but iron oxides should be lower potential acceptors than the anode which is poised at a high driving force of >+400 mV. The anode is plenty high to oxidize something like DHNA (E{degree sign}' ~70 mV) or chelated metals. PplA could be more necessary near the low potential of flavins. The supplied data can't be used to compare the effect of specific riboflavin concentrations on iron reduction rates (only extents) vs current production rates, but it might be available.L. plantarum likely uses different mechanisms and proteins for EET depending on the redox potential of the extracellular electron acceptor. Indeed, PplA uses FMN as its redox co-factor (E0’ ~-150 mV and -350 mV), and so it may only reduce electron acceptors with lower potentials (for ferrihydrite E0’ ~ -100 mV -+100 mV). We also believe that the mechanisms and proteins for EET depend on the prior growth conditions for L. plantarum before addition of the extracellular electron acceptor. In the revised manuscript, we provide these hypotheses and indicate that testing them is the subject of future work in our laboratories (lines 551-553).We agree with the Reviewer that 5) Line 311, More data would be needed to use succinate production as evidence for or against respiration. A mol of pyruvate could be excreted as succinate due to TCA cycle oxidation (indicating respiration), manifesting as excess electrons relative to the amount of succinate produced . Conversely, succinate could be due to electron disposal via a reductive pathway (indicating fermentation). Lacking good carbon or electron recovery, neither can be ruled out, though the non-respiratory reductive pathway is most logical.L. plantarum, like other lactic acid bacteria, only possesses the reductive branch of the TCA cycle; it does not possess an oxidative branch . Thus, we can rule out the possibility of TCA cycle oxidation, leaving the reductive branch as the only way to produce succinate. In agreement with the prior literature, we detected only trace amounts of succinate amongst the many TCA cycle metabolites we looked for: succinate, fumarate, oxaloacetate, citrate, malate. Thus, we are confident that the metabolic flux through the reductive branch of the TCA cycle was marginal, if present. In the revised manuscript, we now explain that L. plantarum does not possess an oxidative branch of the TCA cycle and that our measurements were sensitive to, but did not detect, these other TCA cycle intermediates (lines 308-310 and 312-314).While the Reviewer’s reasoning is sound, 6) Table 1/line 403-: The low YATP in cells using EET is a possible 'different energy conversion strategy'. . I'd suggest uncoupling of acid production from growth by low pH seems likely. In most strains, simple glycolytic pathway functions at low external pH, but complex protein synthesis and replication processes become inhibited, ATP from substrate turnover is needed to maintain pmf and internal pH, and high internal organic acids become toxic. Citable yield/pH data can be hard to locate as it comes from the 80's in the age of calling every lactic acid bacterium a Streptococcus, but since the non-EET cultures at neutral pH got close to the Bauchop and Elsden YATP, and EET cultures at pH 4 were a fraction of this, these data look like common behaviour of many lactic acid bacteria. In future comparisons, pH control could help estimate energy yields from EET. The first paper I found in a quick search to illustrate pH effects was: Russell and Dombrowski, DOI: 10.1128/aem.39.3.604-610.1980.We thank the Reviewer for these insights. We removed the energy-spilling reference from the manuscript. Since our data shows that final biomass is not affected by EET whereas energy conservation is, it seems likely that growth is not strongly coupled to energy conservation under EET conditions. We strongly agree with the Reviewer’s suggestion that the low pH under EET conditions may uncouple ATP generation and growth. We also hypothesize that this ATP produced from substrate-level phosphorylation might be used to create proton motive force to maintain intracellular pH and biosynthetic functions. We have included this hypothesis in the main manuscript (lines 406-409).7) The value for Geobacter ATP/substrate yield in Table S2 seemed very high. Mahadevan says Geobacter produces 0.5 mol ATP/mol acetate when using extracellular acceptors . If I understand the adjustment for 6-carbon compounds, would this make the value for Geobacter using EET to be 1.5 mol ATP/mol 6-C substrate?Appl Environ Microbiol) in Supplement File 2.We thank the Reviewer for this correction. We incorrectly provided the thermodynamic boundary for the ATP/substrate yield from the referenced study. We now provide the value of 1.5 mol ATP/mol 6 carbon substrate (0.5 mol ATP/mol acetate) :Line 21 – acronyms like this make our field more challenging to understand and are unnecessary. Does the work presented actually support 'energy conservation'? Growth seems to occur after EET activity takes place rather than with it as discussed below.We agree with the Reviewer that a broad scientific audience will find the FLEET acronym confusing and have eliminated it from the manuscript.L. plantarum. In biochemistry, energy conservation is defined as the conversion of light or chemical energy into cellular energy in the form of ATP by an organism (Russell and Cook 1995 – Microbiol Rev). In rapidly dividing cells, energy conservation, a catabolic process, is associated with growth, an anabolic process. However, catabolism need not be coupled with anabolism, and energy conservation can still occur in cells that are not rapidly dividing, e.g. in resting cell suspensions (Russell and Cook 1995 – Microbiol Rev).The Reviewer also questions whether our findings indicate that EET supports energy conservation in L. plantarum, EET increases the rate at which chemical energy (in mannitol) is converted to cellular energy (in ATP) per cell by ~1.75-fold , indicating that EET increases the rate of energy conversation per cell. EET increases the amount of cellular energy obtained per chemical energy (Ymannitol) (Table 1) by ~1.75-fold, indicating that EET is also a more efficient energy conservation strategy in L. plantarum.In To understand how the anabolic process of energy conservation is coupled to the catabolic process of growth, we can compare changes in ATP to changes in biomass. At the start of EET, increased ATP levels are accompanied by a significant shortening of lag phase . Since lag phase is the period microorganisms use to accumulate ATP for exponential growth, this shortened period suggests catabolic and anabolic processes are well coupled at this stage. However, by stationary phase, the biomass yield (Table 1) decreased under EET conditions, suggesting a much poorer coupling of catabolic and anabolic processes.Line 27 – 'fermentation yield' referring to biomass yield or fermentation product yield?The text was revised to clarity that ‘fermentation product yield’ is being discussed here.Line 29, Line 45, Line 75, Line 86, Line 479 – blending fermentation and respiration has been known for over a decade in Shewanella . The description on Line 517 regarding the major finding from this reference is incorrect. PMF is not used by S. oneidensis anaerobically to generate ATP as demonstrated by the robust anaerobic growth of an ATP synthase mutant.S. oneidensis and other work, so we apologize for the inadvertent omission of the Hunt et al. J. Bacteriology 2010 reference in the introduction and mis-stating of the ATP synthesis result. In the revised manuscript, we removed the reference to S. oneidensis as a well understood respiratory microorganism (line 45), have replaced reference 14 with the Hunt et al. J. Bacteriology 2010  (line 80), removed the word recent when describing these findings (line 85), and revised the discussion to correctly describe that fumarate reduction supports the creation of ATP via substrate level phosphorylation, not PMF in S. oneidensis (line 523).First, our work was very much inspired by the observations in S. oneidensis), our work demonstrates that a blending of fermentation and respiration occurs during reduction of solid electron acceptors in a primarily fermentative organism (L. plantarum) . Going beyond prior qualitative observations, we show that this hybrid metabolism leads to ~1.75x-more efficient and ~1.75x-faster energy conservation , but an overall ~1.5-fold weaker coupling between anabolism and catabolism (lower biomass yield). Additionally, we discover that this hybrid metabolism substantially affects the intracellular redox state and show redox change arises from a ~2:1 use of endogenous to extracellular electron acceptors. This fuller discussion is included in the manuscript (lines 525-535).Second, we do not claim that our work is the first observation of a hybrid metabolism that blends fermentation and respiration. Rather, this work quantitatively elucidates a different blending of fermentation and respiration that both contrasts with and expands upon previous work. As a counterpoint to the prior observation that substrate-level phosphorylation is the major energy conservation strategy during anaerobic fumarate reduction in a non-fermentative organism .Page 8 (top) – the comparison to other organisms is not particularly helpful unless current per unit cell or unit biomass is quantified, nor does it seem particularly relevant to the overall theme of this manuscript.L. plantarum per unit protein and compare this value to reported values for Geobacter sulfurreducens and Shewanella oneidensis MR-1 (lines 140-147).We agree with the Reviewer that current should be normalized per unit biomass, and we believe that this normalized value provides context for how significant EET is as a metabolic activity. In the revised manuscript, we normalize the current from Line 152 – predicted to encodepplA.The text was revised to state the predicted protein encoded by Line 182 – predicted to encodendh2.The text was revised to state the predicted protein encoded by Line 183 – under the condition tested.The text was revised to incorporate the Reviewer’s suggestion.Line 200 – isn't this already known from the Listeria work?ListeriaListeria monocytogenes  showed that iron reductase activity was associated with the presence of PplA and Ndh2. However, as a respiratory species, L. monocytogenes has a metabolic strategy that is distinct from lactic acid bacteria. Thus the finding that iron reduction is associated with PplA and Ndh2 in lactic acid bacteria is new. Additionally, our work shows for the first time in any bacterium that ndh2 and pplA are upregulated during growth in the presence of DHNA and iron.Prior work in Line 206 – unclear what 'extracellular redox potential' is, unclear to me if any differences are significant in Figure 3B. I guess this is a measurement of Fe(II) / Fe(III) in the medium?et al., 2018 – Am. J. Enol. Vitic.). The text has been revised to provide a definition of ORP and this additional background to highlight its importance (lines 213-217).Extracellular redox potential is also referred to as oxidation-reduction potential (ORP), which is the ratio of all oxidative to reductive components in the environment  . This important facet of the experiment is now described in the manuscript (lines 219-223).The Reviewer is correct in that iron (II) / iron (III) complexes influence ORP, but there are many other drivers of ORP reduction in the MRS medium, such as LAB consumption of oxygen with NADH oxidase . An ICP-MS section was added to the Materials and methods (lines 811-821) to clarify these points.We appreciate the Reviewer’s question here. While ICP-MS cannot spatially resolve iron that is intracellular vs. extracellular, we removed surface-associated metals by washing the cells twice in PBS before performing ICP-MS. This washing procedure has been shown in other bacteria to remove loosely bound metals  compared to the mMRS control medium. In addition, these two metals are not expected to be redox-active metals for EET.For simplicity, we focused on intracellular iron concentrations within the paper, but indeed ICP-MS was also able to detect other metals in Line 262 – does this mean a large amount of the cells that grew are now dead?600 suggest that cells reach stationary phase shortly after reaching maximum current density. Additionally, shortly after maximum current density , the rates of mannitol consumption, lactate production, and ethanol production decrease . Thus, we hypothesize that at the point of maximum current density, when dry weight and CFU measurements were taken, the cells are entering stationary phase and a fraction of them are metabolically inactive or dead. In the revised manuscript, we point out that this discrepancy could be due to the increased acid stress on L. plantarum resulting from EET condition (lines 337-340).The Reviewer makes an interesting observation. Indeed, the combination of a 4-fold increase in dry weight and only 2-fold increase in viable cells may indicate that a fraction of the biomass was dead or not metabolically active enough to form a colony at the time point sampled. In support of this idea, our measurements of cell density by ODFigure 4  – I don't understand why maximum current output is completely decoupled from when biomass spikes. At 24h (maximum current), basically no growth has occurred. At 2 days and 5 days, when biomass is the greatest (in the WT culture), very little current is produced. This seems like direct evidence that EET is not coupled with metabolism in these bacteria. The ATP measurements were made at 24h, where OD of the mutant is decreasing (thus less ATP would be expected). Same issue with NAD/NADH measurement.We apologize for introducing confusion here, which stemmed from a lack of clarity about which current density and biochemical measurements were from different experiments. We have clarified in the Figure Legends that the same set of experiments yielded the current density plots in Figure 3 and the metabolite and pH measurements shown in Figure 5. We have also clarified that another set of experiments were used to generate the current density shown in Figure 1C and the ATP and NAD/NADH ratios shown in Figure 4.L. plantarum cell density under EET conditions.Comparison of the data shown in Figure 1C and 4C show maximum current is closely followed by maximum OD. After one day, the current density has reached its maximum, but no significant increase in planktonic biomass was observed. Between day one and day two, lower but significant current is produced, the cell density reaches ~90% of its maximum, and the consumption of mannitol and production of lactate also increases dramatically. Overall, these observations show that current production is followed by increases in growth and metabolic flux. We hypothesize that the delay observed between current production and the boost in growth and metabolites could be the time cells need to accumulate ATP and compounds needed to enter exponential growth (now included on lines 274-278). In future experiments, we seek to more precisely relate current produced and growth by continuously monitoring of Figure 4C – Lines are undefined in the figure legend. Also, growth should be plotted on a semi-log scale, see: https://schaechter.asmblog.org/schaechter/2018/07/why-you-must-plot-your-growth-data-on-semi-log-graph-paper.htmlWe have addressed the reviewer recommendation and edited Figure 4C accordingly.Figure 5 – based on the growth curve shown in Figure 4, the OC culture lags behind and does not reach maximum OD until day 5. What do these measurements look like at day 5 (or 6)? It seems likely that the authors are drawing conclusions from cells in two different states  rather than focusing on the finding that having an external electron acceptor appears to accelerate the metabolism of cells.L. plantarum affected growth, ATP and intracellular redox state. To show this connection, we chose to measure ATP and NAD+/NADH when EET rate is maximum (maximum current density). These measurements show that energy metabolism is greatly accelerated and changed by EET: cells exhibit higher viability, biomass and ATP accumulation and maintain a higher NAD+/NADH ratio. We agree that performing those measurements at other time points would provide a more dynamic picture of the relationship between EET, energy conservation and redox balancing. Our laboratories will address these questions in future studies.The Reviewer is correct that at the time of ATP and NAD/NADH measurements, the cells performing EET and cells under OC were at different growth stages. In Figure 4, we probed how EET in Line 465 – Description of the experiment setup in 2B does not include addition of flavins for the iron reduction assay, so it is unclear why the authors claim that both flavins and quinones are necessary for EET in diverse LAB species.L. plantarum and several other LAB which we found to be EET-active are auxotrophic for riboflavin  so riboflavin was already supplemented in the growth medium as the vitamin itself (in CDM) or within yeast extract (in MRS). In the iron reduction assay, cells are metabolically active, but not growing, and would have sufficient holo-Ndh2 and holo-PplA carried over from their growth medium to perform EET. This is supported by new data in Figure 1figure supplement 1E where additional riboflavin supplementation into mMRS did not increase subsequent iron reduction. As such, we decided to not include riboflavin in the iron reduction assay used for Figure 2B.We included both flavins and quinones in this statement because we show that flavins are required for EET in Figure 1—figure supplement 1D and Figure 1—figure supplement 2D. Flavins are required as cofactors for Ndh2  and PplA . Line 473 – Iron and electrode reduction is dependent on addition of DHNA. Are these cells likely to ever encounter 20 ug/mL DHNA in the environment? Is the EET activity observed relevant to normal physiology for these organisms, or a lab artifact caused by providing a non-native electron carrier? The argument beginning on line 491 underscores the artificial nature to me.L. plantarum to encounter 20 µg/mL DHNA in its native environment. DHNA is found in concentrations of 0.089-0.44 μg/mL in commercial fermented beverages, and under laboratory conditions, microbes can synthesize and secrete DHNA leading to concentrations of 0.37-48 μg/mL (lines 153-156). To test if EET in L. plantarum is relevant under these physiological concentrations, we probed whether L. plantarum can perform EET with a sub-physiological DHNA concentration of 0.01 μg/mL. Indeed, L. plantarum produced significant current density and a significant decrease in pH . Far from being artificial, these results show that the concentrations of DHNA in environments known to support L. plantarum growth have significant effects on its physiology and mechanisms to outcompete neighboring organisms.The Reviewer is correct in stating that it is unlikely for Line 559 – none of the references here provide any quantitation quinone concentration in the digestive tract. (Relates to the comment above from line 473).L. plantarum is known to reside. To our knowledge, no paper has quantified DHNA concentrations in the gastrointestinal tract. Other forms of quinones have been quantified in the GI tract. For instance, menaquinone concentrations of 5.5±2.7-14.54±0.29 μg/g were quantified in distal colonic contents , and of 25.3-34.4 nmol/g were quantified from fecal samples . Although we mainly focused on DHNA in this manuscript, it is reasonable to anticipate that L. plantarum can perform EET in the GI tract by responding to different forms of quinones due to their shared molecular structures. We will consider testing this hypothesis in our future studies.As mentioned above, we have included in the manuscript the physiological levels of DHNA found in environments in which Line 637 – is ferrozine toxic to these cells?L. plantarum growth. Therefore, there is no evidence that ferrozine is toxic to L. plantarum, nor do we expect that compound to impact the ferrihydrite reduction assay, which takes place over three hours and requires metabolically active, but not actively growing cells.While not included in the data for this manuscript, we found that ferrozine does not inhibit Line 646 – Were these mutants complemented?L. plantarum EET pathway and are including complementation analysis in order to identify the effects of gene proximity and synteny on EET activity.Efforts for mutant complementation were made, but restoration of EET activity was variable, potentially because critical epigenetic regulatory conditions were not provided or because of the challenges of over-expression of membrane-bound proteins . We are currently in the process of mutating all genes in the Line 653 – the suicide vector should be identified and referenced here.The text has been revised to specify the suicide vector along with a citation.General comments:Unclear why Figure 1C shows twice as much current twice as fast as Figure 3C in what appears to be identical conditions?L. plantarum before introducing the cells into bioelectrochemical reactors, the inoculum did show mild growth differences. This can cause significant differences in the electrochemical output (current density). Thus, we suggest the differences in the current density production by the wild-type strain between Figure 1C and Figure 3C result from possible differences on the initial number and activity of cells. Future work will seek to uncover how initial conditions affect EET in L. plantarum.We observe somewhat variable current production depending on the exact conditions used for inoculation of bioreactors. Although we maintained identical culturing conditions for Reviewer #3 (Recommendations for the authors):Figure 1A – though the tubes are aligned with the plot below, it may be helpful for some readers to have labels on this image to clarify the conditions of each.Numbered labels are now provided in Figure 1A to connect the image and the graph.Figure 1C – for the top line, the "+DHNA+mannitol" label should be on one lineFigure 1C has been revised to include this suggestion.Figure 4A – labeling should be more consistent ("anodic" vs. "EET")We have revised the Figure accordingly and replaced anodic with EET.Figure 4A – "log" should be removed from the y-axis labelWe thank the reviewer for noticing this. We have corrected this typo.Figure 4C – points on this plot should be connected with straight linesWe have addressed this suggestion accordingly, and we have also plotted Figure 4C in a semi-logarithmic scale following another reviewer’s suggestion.Figure 5D – axis label is cut offWe thank the reviewer for noticing this and we have corrected this typo.Figure 5G – FLEET should not be mentioned in the model at all. Instead, Ndh2 should be labeledWe have edited the Figure following the recommendations of the reviewer.Figure 6A – Label differences between the two kale juice bottlesWe apologize for omitting some of the details of the juice preparation in that plot. We have indicated the differences between the two juices in Figure 6D by adding details on the preparation steps.Authors should also provide more information for the definitions of YATP and the values in Table 1We have provided the definition of each yield at the bottom of Table 1.Line 137: Where the authors compare current density capacity for L. plantarum to that of S. oneidensis—are the quoted values for S. oneidensis using (an) endogenously produced shuttle(s), or are they for S. oneidensis + (an) added shuttle(s)? This should be clarified, because L. plantarum has no current density capacity without added DHNA.L. plantarum vs S. oneidensis (mA/mg-protein) corresponds to a study in which flavins were self-secreted. We have also clarified that S. oneidensis and G. sulfurreducens do not require addition of riboflavin and quinones, unlike L. plantarum (lines 141-147). However, because L. plantarum possesses genetic elements involved in EET (i.e. ndh2) and performs EET with levels of DHNA similar to those found in its environment  L. plantarum does perform endogenous EET.We agree with the reviewer that this point should be clarified. The new data we provide from the literature to compare Authors should offer a mechanism for the conditional pplA phenotypeL. plantarum (lines 551-535). We have preliminary data that suggest there is a conditional need for PplA in L. plantarum to perform EET depending on the culturing conditions and will address this fully in future work.We briefly discussed the differences in the conditional need of PplA for EET across lactic acid bacteria and now have included an additional hypothesis for the conditional need of PplA in Switching between the terms “electron donor” and “carbon source” may be confusing for some readers; one term should be used consistently.We agree with the Reviewer, and we now appropriately use the term “electron donor” throughout the manuscript.Figure 3—figure supplement 1F is not discussed in the text.We apologize for this omission. We have included a brief discussion of this supplementary Figure (lines 355-359). This figure now is Figure 3 — figure supplement 2C.Line 204: typo in “pplA”The spelling error has been corrected.The ndh2 mutant should be included in the kale juice fermentation experiments to test the contribution of Ndh2 in this context.While this is a helpful suggestion, we did not use the Ndh2 mutant in our kale juice fermentation experiment since this experiment focused on the metabolic impact of EET during fermentation. However, we envision future experiments addressing the EET mechanism under ecological conditions will utilize this mutant.ndh2 is necessary for EET in kale juice (line 27).Since we do not have this data, we have revised the Abstract to avoid concluding that Readers may not be familiar with the term “Crabtree-positive”We agree with the Reviewer and have removed this jargon from the Discussion.“SLP” should be defined on page 3We have replaced the acronym ‘SLP’ with the term ‘substrate-level phosphorylation’.The introduction does not provide enough detail for a broad audience to understand the metabolic pathways being discussed. It would help if some of the detail about L. plantarum metabolism  was moved from the results/discussion to the introduction. If L. plantarum is able to grow via respiration (as suggested in lines 304-308), this should also be mentioned in the introduction. The introduction should also be revised to ensure that terms are used unambiguously and to remove inaccurate generalizations. Some examples are given in the comments below.L. plantarum and LAB metabolism, the Introduction text (lines 55-71 and 97-104) has been revised to incorporate definitions of homofermentation and heterofermentation as well as stating that L. plantarum is primarily homofermentative but also a respiratory-capable LAB.We appreciate the Reviewer’s suggestions regarding the introduction. Regarding the question of providing additional background on It is not totally accurate to say that energy per se is generated or created during metabolism. The authors should revise these statements to say that energy is obtained or conserved or that ATP is generated.We agree with the Reviewer. We have carefully revised the manuscript to consistently use the term ‘energy conservation.’This paper discusses detailed metabolic pathways and the terms used to describe these pathways should be used carefully and precisely to avoid confusion for the reader. For instance, the authors should precisely define “substrate-level phosphorylation” and “fermentation” and should not use these terms interchangeably. “Substrate-level phosphorylation” refers to a reaction that generates ATP directly, without the use of the proton motive force and the membrane-associated ATP synthase enzyme. “Fermentation” refers to the pathways that restore the NAD+/NADH ratio and allow growth with substrate-level phosphorylation as the sole mechanism of ATP generation.Per the Reviewer’s helpful suggestion, the Introduction text has been revised to define substrate-level phosphorylation and fermentation (lines 50-54).Lines 50-51 and 373-374: Some organic compounds can also be respired (for example fumarate).The Reviewer is correct here. The introduction (lines 41-49) includes a broad definition of anaerobic respiration, which includes using both organic and inorganic compounds. We acknowledge that the original text (lines 373-374) may lead the reader to think that respiration is only associated with the use of inorganic electron acceptors. To avoid this confusion, we now state that another major difference in fermentation and anaerobic respiration is the use of the endogenous versus external organic electron acceptors (lines 377-378).Lines 51-52 should say “Fermentative bacteria produce ATP solely via substrate-level phosphorylation” because substrate-level phosphorylation also occurs when bacteria are growing by respiration .We have revised this sentence (lines 50-52). Instead of using the word ‘solely’, we prefer to employ the word ‘mainly’ since ATP can also be generated via respiration in some primarily fermentative bacteria, like lactic acid bacteria, under specific conditions.Lines 71-73: It is unclear how the property of being “exergonic” or “endergonic” is relevant in the context here.We agree with the Reviewer that providing these details is not relevant and have removed them.Lines 85-86: “…EET is associated with non-fermentative respiratory organisms…” Does this refer specifically to endogenous EET . This should be clarified because there is a long history of research on fermentation+EET in the presence of added mediators (see next comment).The Reviewer makes a good point, and we revised the sentence to clarify that we are describing that endogenous EET is mainly associated with respiratory species.Related to the above point: In the introduction or discussion, the authors should mention or acknowledge other work examining how growth in the presence of an electrode affects fermentative metabolisms. Some examples are:Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force. Mol Microbiol. 2014 Apr;92(2):399-412. Doi: 10.1111/mmi.12566.Glasser NR, Kern SE, Newman DK. Phenazine redox cycling enhances anaerobic survival in Emde R, Schink B. Enhanced Propionate Formation by Propionibacterium freudenreichii subsp. Freudenreichii in a Three-Electrode Amperometric Culture System. Appl Environ Microbiol. 1990 Sep;56(9):2771-6. Doi: 10.1128/aem.56.9.2771-2776.1990.And other references discussed in reviews such as:Moscoviz R, Toledo-Alarcón J, Trably E, Bernet N. Electro-Fermentation: How To Drive Fermentation Using Electrochemical Systems. Trends Biotechnol. 2016 Nov;34(11):856-865. Doi: 10.1016/j.tibtech.2016.04.009.Vassilev I, Averesch NJH, Ledezma P, Kokko M. Anodic electro-fermentation: Empowering anaerobic production processes via anodic respiration. Biotechnol Adv. 2021 May-Jun;48:107728. Doi: 10.1016/j.biotechadv.2021.107728.In the original Discussion, we state that fermentation can be modulated using electrodes and cite the Moscovitz reference. However, we acknowledge this statement was brief and have expanded this discussion and included the Vassilev and Emde and Schink references in the revised manuscript (line 594 and lines 495-496). We also included the Glasser et al. reference (lines 93-96) as an example of a primarily respiratory organisms that also ferments performing EET.Comparing Figure 1A to Figure 3A, the extent of Fe3+ reduction seems quite different. Were the conditions the same for these two experiments? If so, an explanation should be added to the text.L. plantarum after growth in mMRS. The results shown in Figure 3A were obtained after L. plantarum growth in mMRS supplemented with DHNA and ferric ammonium citrate. The inclusion of DHNA and ferric ammonium citrate in the mMRS growth medium was found to result in increased L. plantarum EET activity . To clarify the rationale for the change in mMRS growth medium, we moved that supporting data to Figure 1—figure supplement 1 and revised the Results section to better describe these results and how they influenced our experimental methods (lines 123-133).The conditions differed between Figures 1A and 3A. The iron reduction assay results shown in Figure 1A were obtained for Lines 104-106: Can DHNA itself be used as an electron shuttle? This should be addressed.eLife; Glasser NR et al., 2017 – Annu Rev Microbiol). DHNA can also be synthesized into menaquinone or demethylmenaquinone, which can transfer electrons in an electron transfer chain. However, our current data does not allow us to assess which of these roles DHNA is playing. Future work will elucidate the role of DHNA and flavins in EET in L. plantarum.Yes, DHNA can be used as an electron shuttle  endogenously produced shuttle(s), or are they for S. oneidensis + (an) added shuttle(s)? This should be clarified, because L. plantarum has no current density capacity without added DHNA.L. plantarum, Shewanella oneidensis and Geobacter sulfurreducens can synthesize riboflavin and quinones and do not require the addition of either for EET activity (lines 146-147).We revised the comparison of EET among species to indicate that, unlike Line 139: “current production was not dependent on the carbon source or growth medium, and current increased after supplementation of riboflavin in the growth medium”. Both of these claims are hard to evaluate because the plots in figure 1 and figure1—figure supplement 2 are shown for different lengths of time, different carbon sources (electron donors), and different media. To make it easier for the reader to compare, it would help to have the name of the medium as a title for each current density plot.We acknowledge the lack of clarity here and have revised the text to more accurately convey our point (lines 148-152). Our intention is to qualitatively, rather than quantitatively, evaluate current production with different media. We have followed the Reviewer’s suggestion and added a title for each plot of Figure 1—figure supplement 2 to make it easier for the reader to understand the medium used.To judge whether riboflavin truly increases current, we should compare figure 1c to figure 1—figure supplement 2D. In figure 1c, with no riboflavin added, current increases to ~120 uA/cm2 at around 1 day. In the supplemental figure, this increase is not seen until after riboflavin is added. Without a control, it is hard to know whether this increase was dependent on the riboflavin addition.We agree with the Reviewer on this point. We provide additional data in Figure 1—figure supplement 2D that includes a control in which riboflavin was not added. This control shows that the increase in current is due to the addition of riboflavin.Except for Figure 1—figure supplement 2D, all the experiments performed in bioelectrochemical reactors contain riboflavin in the media (as part of the vitamin stock solution in the CDM). This is now explained in the manuscript text (lines 148-152).Line 154: Please define homofermentative.The Introduction text has been revised to include definitions of both homofermentation and heterofermentation (lines 60-64).Line 160: “the percentage of” should be removedThe legend text of Figure 2 has been revised to incorporate this suggestion.Figure 2—figure supplement 2C: I am confused about how this experiment controls for the possibility that reduction of soluble iron , which wouldn’t necessarily require EET, contributes to the higher levels of ferrous iron observed when DHNA and ferric ammonium citrate are both added. Maybe a control with no ferrihydrite added would tell us whether ferric ammonium citrate is being reduced.L. plantarum was grown in mMRS containing ferric ammonium citrate but not DHNA (noting that DHNA was still included in the ferrihydrite assay medium) . Therefore, our data show that ferric ammonium citrate is not contributing to the color formation in the ferrihydrite reduction assay. Additionally, we agree that it is notable how L. plantarum exposure to DNHA and ferric ammonium citrate during growth results in an improved capacity for that organism to reduce extracellular ferrihydrite and that the expression of ndh2 and pplA is induced under those conditions (lines 199-206). We are pursuing these questions in studies investigating the transcriptional control and downstream regulation of EET in L. plantarum.Prior to performing the ferrihydrite reduction assay, the cells were washed twice (in PBS), thereby removing any ferric ammonium citrate that was present in the mMRS growth medium (line 744). Moreover, there was very little color change in the ferrihydrite reduction assay when wild-type Line 194: more background should be provided for the FLEET-inducing conditionsThe Results section was revised to clarify and provide background on what was intended by the term “FLEET-inducing conditions” (lines 126-133). For clarity, we have also removed that term from the text and explained directly which culture conditions were used.Lines 304-309: More background information is needed to understand the motivation behind these experiments. Were oxygen and nitrate available as electron acceptors?To clarify further the motivation behind this experiment for the reader we have included a more detailed explanation (lines 295-301). Neither oxygen nor nitrate were used as electron acceptors, as their presence would be expected to compete with the anode as an electron acceptor.Line 305: “…needed for PMF generation in aerobic…”We have corrected this typo.Line 414: “previous results”; does this refer to results in this paper or a prior publication? If a prior publication, it should be cited here.The text has been revised to incorporate this suggestion.