L. plantarum consumes current when supplied with nitrate as an electron acceptor
Some microorganisms that endogenously uptake electrons from extracellular electron donors can also uptake electrons from cathode poised at an equivalent redox potential, coupling this electron uptake to reduction of an electron acceptor9. To determine if L. plantarum can uptake electrons from a cathode, we first tested whether it could consume current under different cathode potentials with an external electron acceptor present. Since L. plantarum can reduce nitrate under anaerobic conditions24, we chose to use nitrate as an electron acceptor. To promote growth without usurping the role of the cathode as the electron donor, we supplied 5 mM glucose as a carbon source, since this concentration of glucose can only marginally be used as electron donor for L. plantarum24. After culturing L. plantarum cells in a minimal medium25 (Supplementary Table 1) in bioelectrochemical reactors, we poised the cathode polarized at different potentials (-0.15, -0.25, -0.40 and − 0.60 V vs Ag/AgClKCl sat.), added nitrate, and monitored current at the cathode (Supplementary Fig. 1A). We observed current consumption by L. plantarum under all the conditions tested, with the highest current at -0.60 V (all potentials are reported vs Ag/AgClKCl sat). No current consumption was observed with heat killed cells under identical conditions (Supplementary Fig. 1B), indicating that electron uptake required metabolically active cells. We also analyzed the impact of removing the carbon source from the medium on this EET (Supplementary Fig. 1C). We observed higher current consumption when glucose was provided, suggesting that cells could maintain a higher metabolic activity that resulted in a greater EET from the cathode. Because of all these observations, we chose to use a potential of -0.6 V and to provide 5 mM glucose to the medium for further experiments to maximize EET from the cathode.
To probe whether this electron uptake was coupled to reduction of an electron acceptor, we next monitored current in the presence and absence of nitrate (Fig. 1A). Approximately 18 h after inoculation of cells with nitrate, a maximum current consumption of ~-20 µA/cm2 at -0.60 V (Fig. 1A) was observed. However, no significant current consumption occurred in the absence of nitrate (Fig. 1A), indicating that the extracellular electron uptake required an external electron acceptor. The electron uptake also coincided with the reduction of ~ 7 mM nitrate to nitrite (Fig. 1B). We compared these data to nitrate reduction by L. plantarum under open circuit (OC) conditions, in which the cathode is not electrically connected to the anode and thus electrons from electrical current cannot be supply. Under OC conditions, L. plantarum reduced significantly less nitrate to nitrite (8.98 ± 2.29 mM vs 1.19 ± 1.16 mM in one day, p < 0.050) (Fig. 1B). Using differential pulse voltammetry (Supplementary Fig. 1D), we identified a redox active center at -0.52 V (pH = 6.3, vs Ag/AgCl sat. KCl) which was not observed when nitrate or cells were not present in the medium. Thus, this result confirms the existence of a redox process associated with the bioelectrochemical reduction of nitrate or nitrite, similar to other observations26. All together, these results indicate that L. plantarum can uptake electrons from a cathode and couple this uptake to reduction of nitrate.
Since L. plantarum can reduce nitrite to ammonia under respiratory conditions24, we hypothesized this reaction might also occur under electron uptake conditions. Initial experiments of heat-killed cells in bioreactors indicated that nitrite reduction occurred under both biotic and abiotic conditions at -0.60 V (Supplementary Fig. 2A and 2B). To minimize the abiotic reduction of nitrite, we polarized the cathode at more positive potentials (-0.25 V and − 0.40 V) and again probed nitrite reduction under both biotic and abiotic conditions (Supplementary Fig. 2C and Fig. 2D). From this data, we estimated that nitrite reduction coupled to electron uptake by L. plantarum was minimal (< 2.5%), and that instead, most of this reduction (> 91%) was catalyzed by L. plantarum, but without use of the cathode as electron donor (Supplementary Table 5). Intriguingly, these data suggest that L. plantarum predominately uses electron uptake from a cathode to drive the reduction of nitrate to nitrite, but that a separate pool of electron donors are used by L. plantarum to reduce nitrite to ammonia.
Through its fermentative pathways, L. plantarum produces organic molecules, e.g. acetate, that can also serve as electron donors. To establish to what degree the cathode vs. endogenous molecules served as the electron donor for L. plantarum to reduce nitrate, we quantified the electron uptake needed for nitrate reduction. We first calculated the electrons consumed from the cathode from the current consumption and compared this value to the number of electrons theoretically needed to reduce nitrate to nitrite in the bioreactor medium (Fig. 1B). More electrons were used to reduce nitrate than electrons supplied by the cathode: only 6.9 ± 1.6% of the electrons needed for nitrate reduction were supplied by the cathode (i.e. coulombic efficiency). This difference indicates additional electron donors, such as an organic fermentation product, are utilized by source for L. plantarum to reduce nitrate.
EET from a cathode in L. plantarum induces a metabolic shift that enhances cell viability
When L. plantarum donates electrons to an extracellular anode, it conserves energy more rapidly and increases its fermentative flux22. To probe the impact of the electron uptake from a cathode on energy conservation and metabolism, we measured metabolites of L. plantarum under OC and cathodic EET conditions. Under both conditions, L. plantarum predominantly produced the end products of fermentation lactate, ethanol, acetate and succinate (Fig. 2A and 2B). Formate (< 0.02 mM) and pyruvate (< 0.06 mM) were detected in all bioreactors at trace levels, and no acetoin or 2,3-butanediol were detected at any time. By day 5, these fermentation products contained > 98% of the initial carbon supplied as glucose, indicating these measurements provide a complete view of L. plantarum carbon metabolism under these conditions.
Interestingly, the fermentation process was affected by electron uptake in L. plantarum. Over the first ~ 1.7 days, fermentation products were similar under EET and OC conditions (Fig. 2A and Supplementary Fig. 3A). Glucose was fully consumed, lactate was the main fermentation product (~ 22 mM), and the nitrate concentration was unchanged. After day ⁓1.7, however, L. plantarum metabolism in bioreactors under EET conditions began to change. Between day 1.7 and day 4, there was a gradual increase in current consumption and significant concentrations of nitrate were consumed (238 µmol day − 1 vs 5.5 µmol day − 1 under OC conditions) (Supplementary Fig. 3B). This was accompanied by a significantly increased production of acetate and succinate and increased consumption of lactate. For instance, lactate consumption rates were 35.8 µmol day − 1 under EET conditions and of 9.8 µmol day − 1 under OC conditions. Thus, electron uptake and nitrate reduction coincide with a shift of L. plantarum metabolism towards a more heterofermentative-like pattern (Fig. 2B, 46% lactate, 38% acetate, 15% ethanol and 2.3% succinate). In contrast, under OC conditions, there was no further evolution of fermentation products from L. plantarum after day 1.7 and lactate remained the dominant product. Thus, the fermentation pattern of L. plantarum under OC remained homofermentative (Fig. 2B). This strongly suggests that, after exhausting glucose, L. plantarum utilizes the cathode as an electron donor and nitrate as an electron acceptor, allowing lactate to be sequentially oxidized to pyruvate then acetate (Fig. 2E). A similar metabolic shift towards acetate production was previously observed for L. plantarum when oxygen27 or citrate27 were present28 as electron acceptors.
During fermentation, the production or consumption of organic acids and other media components such as ammonia usually changes the pH of the media. To probe whether the metabolic shift observed impacted pH of the medium, we measured pH at day ⁓5. The medium of cultures under cathodic EET condition had a significantly higher pH than L. plantarum cultures lacking nitrate or under OC conditions (Supplementary Fig. 3C). We suggest this pH increase could arise from nitrate reduction as protons are consumed during this reaction. All together, these results suggest that electron uptake and nitrate reduction trigger the conversion of lactate to acetate (Fig. 2E).
In other microorganisms20 a polarized cathode can impact fermentative metabolism solely by altering the intracellular redox potential. These observations suggest an alternative hypothesis for the observed metabolic shift. To test this alternative hypothesis, we also examined end-fermentation products of L. plantarum in the presence of a cathode at -0.6 V without nitrate (Fig. 2B and Supplementary Fig. 3A). Under these conditions, L. plantarum did not consume current, as previously observed (Fig. 1A), nor did the concentrations of acetate or succinate increase (Fig. 2B and Supplementary Fig. 3A). Since increased acetate and succinate production was only triggered when both nitrate and a polarized cathode were present (Fig. 2B), we attribute this metabolic shift to electron uptake coupled with nitrate reduction. Overall, these results indicate that uptake of electrons from a cathode to reduce nitrate reroutes the fermentation fluxes in L. plantarum from lactate towards acetate generation and towards the reductive branch of the tricarboxylic acid cycle (Fig. 2E).
Overall, our results indicate a complex electron flow during extracellular electron uptake: the cathode and an endogenous fermentation product (lactate) serve as electron donors, while exogenous nitrate acts as an electron acceptor. To understand the impact of this electron flow on the intracellular redox state of L. plantarum, we analyzed how the reducing equivalents generated from glycolysis, i.e. NADH, and the electrons from the cathode were used. For this analysis, we estimated the NADH regenerated via fermentation and via the reduction of nitrate using the measured concentrations of end-fermentation products and nitrate (see Methods section for methodology). L. plantarum can consume NADH in three ways: by reducing pyruvate to lactate (1 mol NADH/mol lactate produced) or to ethanol (2 mol NADH/mol ethanol produced), by reducing pyruvate or phosphoenolpyruvate to succinate via the reduced branch of the tricarboxylic acid cycle (2 mol NADH/mol lactate produced), and by reducing nitrate to nitrite (2 mol of NADH/mol nitrate consumed). Under OC conditions, we estimated that ⁓80% and ⁓20% of all the NADH was regenerated via lactate and ethanol production, respectively (Fig. 2C), in agreement with prior observations of homofermentation29. However, when cells consumed electrons from the cathode (EET + nitrate), we estimated that NADH regeneration via lactate was reduced to ⁓50%, and ⁓20% of the NADH was regenerated through the reduction of nitrate to nitrite (Fig. 2C). Thus, electron uptake by L. plantarum changes the strategy that it uses to balance its intracellular redox state.
Under cathodic EET conditions, acetate production is stimulated in L. plantarum, a pathway that generates extra ATP via substrate-level phosphorylation in glycolysis. To quantify how the cathodic EET impacts energy conservation, we estimated the total ATP produced from glucose fermentation end-products by day ∼5. Our calculations showed a mild increase in total ATP production when cells performed EET in the presence of nitrate (Fig. 2D) but these differences were not significant compared to OC conditions (p > 0.05). Further studies are needed to elucidate whether cells can generate ATP addition from nitrate reduction via oxidative phosphorylation. Overall, our observations show that current consumption coupled to nitrate reduction induces a metabolic rerouting of fermentation towards ATP producing pathways rather than towards NADH consuming routes in L. plantarum.
Extracellular electron transfer can affect catabolic processes and how they are coupled to anabolic processes, including cell growth22. To examine how this electron uptake impacted L. plantarum anabolism, we measured its cell viability, OD600nm, and biomass under EET and OC conditions. When L. plantarum cultures produced the maximal current density (~ day 2), they contained a higher number of viable cells compared to OC conditions (Fig. 3A). However, the maximal current consumption (top panel, Fig. 3C) coincided with the entry of L. plantarum into the stationary phase (top panel, Fig. 3C). Running counter to the prevailing idea that increasing EET drives an increase in anabolism30,31, this observation suggests that when L. plantarum anabolism slows, electron uptake increases. Further supporting this observation, there were no significant differences in the cell density over time (OD600nm, Fig. 3C) or final biomass (Fig. 3B) between OC and EET conditions. Moreover, the final biomass under EET conditions was also unchanged whether nitrate is present or absent (Supplementary Fig. 4). These results show that electron uptake coupled to nitrate reduction increases cell viability, but does not increase final biomass. Taken together, our data suggest that substrate depletion and slowed anabolism triggers electron uptake in L. plantarum, which re-routes metabolic flux towards ATP generating pathways and thus improves cell viability.
A) Cell viability at day 2 and B) cell density (OD600nm) in the presence of a cathode (EET) and in the presence of an electrode maintained at open circuit (OC). Nitrate was supplied as an electron acceptor in the medium. In panel B the evolution of current density of one of the bioreactors is shown (gray dots). C) Biomass (dry weight) collected at day ∼5 for both inward and OC conditions. For all experiments, nitrate was already present in the medium when the electrodes were polarized to ΔE = -600 mVAg/AgCl, sat. KCl. The error bars indicate the standard deviation from three biological replicates. Significant differences in iron reduction were determined by one-way ANOVA with Dunn-Sidak post-hoc test (n = 3), * p ≤ 0.05.
Inward EET does not require the cofactors employed by respiratory and EET chains
With this understanding of the effects of electron uptake on L. plantarum, we next explored the molecular mechanisms supporting electron uptake. We studied whether cathodic EET requires molecules that activate aerobic and anaerobic respiration (Fig. 4A)32, heme and 1,4-dihydroxy-2-naphthoic acid (DHNA), respectively, by adding these molecules exogenously and examining current consumption and nitrate reduction. Heme addition did not affect the magnitude of the current consumed, but delayed the start of electron uptake by cells (Fig. 4B). Heme addition did not affect nitrate reduction either (Fig. 4C). Similarly, the addition of 1,4-dihydroxy-2-naphthoic acid (DHNA) did not impact current consumption (Fig. 4D) or nitrate reduction (Fig. 4E). Thus, menaquinone and heme are not components of the EET chain for electron uptake, indicating that the required molecules do not overlap with other known electron transport chains in L. plantarum.
We next studied the role of riboflavin, a required cofactor for L. plantarum to reduce insoluble electron acceptors22. Since riboflavin is essential for growth of L. plantarum, we relied on electrochemical analyses to determine its role in EET. To probe whether riboflavin acts as a freely-diffusing electron shuttle, we performed differential pulse voltammetry on the cathode in bioreactors containing L. plantarum cultured with nitrate under EET conditions. The characteristic redox peak of flavins at -0.4 V was absent from the cathodic chamber (inset of Fig. 4E), indicating riboflavin was not present as a free mediator. Omitting riboflavin from the bioreactor medium did not affect the current density consumption (Fig. 4F) or nitrate reduction (Fig. 4G). Since L. plantarum cannot synthesize riboflavin24, these results indicate riboflavin is not a mediator that cells use for electron uptake from a cathode, and they suggest riboflavin may not be a cofactor of proteins within this electron transport chain. Since EET to an anode significantly benefits from riboflavin supplementation22, these results also strongly suggest that electron uptake from a cathode and electron donation to an anode require different molecules in L. plantarum.
An alternative hypothesis is that as-yet unidentified molecules are used by L. plantarum to support both cathodic and anodic EET. To test this hypothesis, we probed whether L. plantarum cells uptaking electrons from a cathode could donate electrons to an anode (+ 0.2 V) using the spent media employed for the cathodic EET tests. We supplemented the medium with mannitol, as it is a suitable electron donor for L. plantarum while performing EET with anodes22. The current production observed was very low (~ 3 µA/cm2) compared to that one reported for this species when the conditions for anodic EET are provided (~ 129 µA/cm2), indicating that the soluble and insoluble molecules involved in cathodic EET could not support EET to an anode (Supplementary Fig. 5). Thus, the elements that L. plantarum uses for uptaking electrons from a cathode are distinct from those required when donating electrons to an anode.
So far, our results showed that L. plantarum does not use quinones and flavins for extracellular electron uptake, compounds that support mediated EET in other species33,34. If L. plantarum uptakes electrons via mediated EET, either other molecules present in the culturing medium act as electron shuttles, or cells synthesize a redox mediator. To further explore whether other exogenous mediators were used by cells while uptaking electrons, we closely examined the composition of the CDM medium to identify other compounds previously reported to act as electron shuttles for bacteria. We found that cysteine, a redox active compound that can behave as an electron shuttle between other electroactive species and minerals35,36 was present at 0.8 mM in the medium used. We tested the role as mediator in the electron uptake by removing cysteine from the medium and measuring the current consumption response. L. plantarum consumed similar levels of current in the presence and absence of cysteine (Supplementary Fig. 6A-B). Thus, cysteine is not a mediator for cathodic electron uptake. Since no other compounds present in the CDM are known to act as electron shuttles, we posit that cells were performing direct EET with the cathode or were using a yet unidentified self-synthesized shuttle molecule or detached enzyme.
Overall, these results demonstrate that the extracellular electron uptake machinery does not require any of the external cofactors, i.e. heme, quinone-precursors, or free riboflavin, necessary to activate the other known electron transfer chains in L. plantarum and other LAB. Therefore, this EET pathway constitutes a distinct electron transport chain not previously described for these species.
Inward EET requires different genetic elements than outwards EET
Lactic acid bacteria, including L. plantarum, and many other species from the Firmicutes phylum possess a flavin-based extracellular electron transfer (FLEET) locus (Fig. 5A) that is associated with an ability to perform EET with insoluble electron acceptors22,23,37. Because certain proteins in other bacterial species can be used for both interacting with insoluble donors and acceptors38, we next interrogated the involvement of certain genetic elements within the FLEET locus in the cathodic EET chain.
Since DHNA is not required for electron uptake, we reasoned that DmkB (heptaprenyl diphosphate synthase complex II), an enzyme which catalyzes terminal steps in the production of menaquinone-7 from DHNA, is unlikely to be required for it. Instead, we first targeted the type II NADH-dehydrogenase (Ndh2) and the PplA proteins, since they have a role in reducing insoluble electron acceptors in this species22. We observed that electron uptake, quantified as total charge consumed from the cathode (Fig. 5B), and nitrate reduction by Δndh2 strain (Supplementary Fig. 7A) were similar to wild-type. Likewise, the ΔpplA strain also showed similar levels of current density consumption (Fig. 5B) and nitrate reduction compared to the wild-type strain (Supplementary Fig. 7A). These results indicate that neither PplA or Ndh2 are components of the cathodic EET chain. Because of this finding and our previous result showing that riboflavin is not required for electron uptake, we posit that FmnA and FmnB, proteins involved in the flavinilyzation of PplA23 and with their respective coding-genes within the FLEET locus, are unlikely to be essential elements in the cathodic EET chain.
The FLEET locus also contains genes encoding the proteins EetA and EetB, with a yet unknown role in the EET to anodes in L. plantarum but known to have a role in reducing iron in Listeria monocytogenes23. We next studied their role in the cathodic EET chain by constructing a mutant strain with both coding regions deleted (ΔeetA/B). The ΔeetA/B mutant showed similar total electron uptake levels (Fig. 5B) and nitrate reduction as wild-type (Supplementary Fig. 7B), indicating that these proteins do not have a role in electron uptake. Overall, our analysis on the requirement of the FLEET components for the electron uptake capacity in L. plantarum shows that the cathodic and anodic EET mechanisms are genetically distinct. Thus, the presence or lack of the FLEET gene locus, which is present in many Gram-positive fermentative bacteria23, may not be indicative of the possession of an extracellular electron uptake capacity of a species.
To understand the terminal oxidoreductase involved in nitrate-dependent EET from a cathode, we next explored the role of the nitrate reductase A, the only enzyme known to catalyze nitrate reduction in L. plantarum. To this end, we tested the electron uptake capacity of a strain that lacks the operon ΔnarGHJI encoding the sole nitrate reductase. We observed that current consumption and nitrate reduction were completely inhibited in the ΔnarGHJI strain (Fig. 6A and Supplementary Fig. 8A). Furthermore, the catalytic redox center with a midpoint potential of -0.52V that we previously observed in wild type strain (Supplementary Fig. 1D) was absent from the voltammograms obtained using ΔnarGHJI strain (Supplementary Fig. 8B). Complementation of narGHJI via plasmid expression in the deletion background (narGHJI+ strain) restored the current consumption and nitrate reduction levels capacity to wild type levels (Fig. 6A and Supplementary Fig. 8C). These results indicate that nitrate reductase is essential for both the electrons to flow through the cathodic electron transport chain and for the reduction of nitrate.
Nitrate reductase A is a membrane bound enzyme that contains 3 subunits: NarI (subunit γ), NarH (subunit β), and NarG (subunit α)39,40 (Fig. 6C). When L. plantarum employs a soluble electron donor to perform anaerobic respiration on nitrate, both an external quinone and heme source are needed to restore NarI activity to receive electrons from a NADH-dehydrogenese24. Electrons are then transferred to NarH, which then transfers them via Fe-S clusters to the NarG subunit, the catalytic site of the enzyme (Fig. 6C). To understand how electrons were transported across this enzyme during EET from a cathode, we analyzed the requirement of each subunit in the process. Since L. plantarum is heme auxotroph and NarI is a bd-cytochrome, the transport of electrons cannot occur in the absence of exogenous heme. Thus, electrons from the cathode cannot be transported through this subunit. Moreover, NarI is described to accept electrons from membrane-embedded menaquinones41, which are not present under our experimental conditions. We conclude that NarI is not involved in the cathodic EET chain under our conditions.
We then studied the electron transport across NarH and NarG during electron uptake from a cathode. These subunits harbor Fe-S clusters, described to be electron transport centers in nitrate reductase A in L. plantarum and other species39,41. To study the requirement of these subunits, we mutated specific cysteine residues within NarH that bind three 4Fe-4S to alanine, a mutation that results in the loss of several Fe-S clusters and full enzymatic activity41. To build this mutant we constructed a plasmid to overexpress the narGH*JI genes in a ∆narGHJI background. Eliminating electron transport (Fig. 6B) of NarH subunit did not affect current consumption capacity in L. plantarum nor nitrate reduction (Supplementary Fig. 8C). This suggests that electrons do not flow through the Fe-S clusters of the NarH subunit when provided by a cathode. Thus, these results imply that electrons flow only via NarG subunit within the nitrate reductase when L. plantarum uptakes electrons from a cathode. Therefore, the activity of the nitrate reductase in L. plantarum is mechanistically distinct when the cathode is the electron donor compared to when a soluble molecule is. All together, these observations indicate that EET from a cathode involves the nitrate reductase A, and NarG is the key enzyme for electron uptake. We therefore propose a model for this EET chain in L. plantarum in which a fraction of the electrons used to reduce nitrate flow from the cathode to the nitrate reductase A and, specifically, to NarG subunit through a yet unknown intermediate step(s) (Fig. 6A).