EEU is linked to photosynthetic electron transfer

EEU from metal oxides or poised electrodes into bacterial cells has been observed in pure cultures3,4,5,6,12,14,15,16,17,18,19,20, and mixed microbial communities4,5,21,22,23. However, the electron transfer pathways that underlie EEU have only been probed in chemotrophic microbes14,15,18,24. In phototrophic microbes, it is unknown if electrons from a cathode enter the pETC and if this activity is important for the establishment of a proton motive force (PMF), ATP synthesis, or the generation of reducing equivalents. Bioelectrochemical studies traditionally rely upon macroscale (>500 mL) or mesoscale (0.2–500 mL) BESs that are scaled for biomass production25. In such BESs it is difficult to isolate the response of surface-attached cells. This is because other factors like the influence of planktonic cells3,10, extracellular enzymes26, and abiotic reactions confound the interpretation of electrochemical data3,10. Being able to collect electrochemical data from surface-attached cells exclusively would shed light on whether EEU leads to electron transfer into the pETC.

To achieve this, we designed and constructed a microfluidic bioelectrochemical cell (µ-BEC) (Fig. 1a, Supplementary Figure 1). The µ-BEC is a four-chamber, three-electrode, small-volume (1.6 µL per well) BES that is compatible with confocal microscopy (Fig. 1a) (see Methods for a complete description of the µ-BEC design and assembly). Its major advantage is that it allows us to study surface-attached cells exclusively as planktonic cells can be washed out with microfluidic control (Fig. 1b). Appropriately grown microbial cells were incubated in µ-BECs for ~120 h at +100 mV vs. standard hydrogen electrode (SHE) under continuous illumination. Once we obtained stable current densities under illuminated conditions (~ −100 nA cm−2), planktonic cells were washed out of the system with microfluidic control. Medium flow was turned off following this wash because constant flow led to excessive noise in the electrochemical data. To determine that we only had surface-attached cells and no plankton, we performed confocal fluorescence microscopy with LIVE/DEAD® staining in the intact µ-BEC. We observed surface-attached cells in single-layer biofilms (Fig. 1c and Supplementary Figure 2a). Previous studies have shown that EEU-capable microbes, including TIE-1, make single-layer biofilms on electrodes3,9,27,28,29. Furthermore, we were unable to detect the presence of any motile planktonic cells in the µ-BEC.

Fig. 1 Extracellular electron uptake in the micro-bioelectrochemical cell. a Schematic drawing of a single, four-chamber micro-bioelectrochemical (µ-BEC) with b microbial cells attached to the indium tin oxide (ITO) working electrode (WE). The reference (RE) and counter (CE) electrodes are silver and platinum wires, respectively (not drawn to scale). c Confocal micrograph of Rhodopseudomonas palustris TIE-1 biofilms attached to the WE under poised conditions using LIVE/DEAD® staining. Green cells are viable. Scale bars are 10 µm. d Current densities for TIE-1 wild-type (WT) (black) in the µ-BEC under illuminated and dark conditions (shaded regions) compared to a ‘No cell control’ reactor (red). Data shown are representative of three experiments. Source data are provided as a Source Data File Full size image

We used the above approach to obtain surface-attached cells in the µ-BEC and used these biofilms to study the influence of light and chemical inhibitors on EEU. Confocal imaging using LIVE/DEAD® staining was performed in the intact µ-BEC after these tests that typically lasted for a few minutes (see Methods for details). We observed light-stimulated EEU by pre-established wild-type (WT) TIE-1 biofilms (Fig. 1d). Upon illumination, biofilms reached stable current densities within ~1–2 s and typically reached a maximum of ~ −100 nA cm−2 (Supplementary Table 1,2,3). Overall, the µ-BEC replicates the biofilm architecture reported in bulkier systems and permits reproducible measurements of EEU by surface-attached cells.

To better understand electron flow during EEU we pursued a chemical probe-based approach to selectively inhibit key proteins involved in cyclic pETC. TIE-1 and related anoxygenic phototrophs use cyclic photosynthesis30 to generate energy (Fig. 2). The photosystem (P 870 ) is reported to be at the potential of +450 mV30. Quinones reduced by the photosynthetic reaction center (P 870 *) donate electrons to the proton-translocating cytochrome bc 1 31. Electrons are then transferred to cytochrome c 2 , and cycled back to the reaction center30. To test whether cytochrome bc 1 is involved in EEU, we used antimycin A, a specific inhibitor of cytochrome bc 1 32 to block cyclic pETC (Fig. 2a). Antimycin A is a quinone analog that blocks the Q i site of cytochrome bc 1 , inhibiting electron transfer from ubiquinol to cytochrome b, thus disrupting the proton motive Q cycle31,32. We observed a decrease in current uptake with antimycin A treatment (Fig. 2a, Supplementary Table 1). Current density became anodic (positive current) under phototrophic conditions (12.46 ± 1.34 nA cm−2; P < 0.0001, one-way ANOVA) relative to untreated controls (−85.5 ± 5.42 nA cm−2) but reverted to cathodic (negative current) densities under dark conditions (−3.46 ± 1.80 nA cm−2; P = 0.0006, one-way ANOVA) (Fig. 2a). Importantly, we did not observe a difference in the number of live/dead cells attached to electrodes in inhibitor treated vs. untreated control reactors (Supplementary Figure 2). These data suggest that electrons enter the pETC and that cytochrome bc 1 is involved in electron flow during EEU.

Fig. 2 Photosynthetic electron transfer is required for extracellular electron uptake. Current densities of TIE-1 wild-type (WT) in response to inhibition of the photosynthetic ETC under illuminated and dark (shaded regions) conditions with (a) antimycin A, (b) carbonyl cyanide m-chlorophenyl hydrazine (CCCP), and (c) rotenone. Data shown are representative of three experiments. Each current density diagram (left) is followed by the proposed path of electron flow (right). The site of chemical inhibition is indicated by a red halo on the electron path diagrams. P 870 (photosystem), P 870 * (excited photosystem), UQ (ubiquinone), bc 1 (cytochrome bc 1 ), c 2 (cytochrome c 2 ), NADH-DH (NADH dehydrogenase), Δp (proton gradient), H+ (protons), hv (light), ? (currently unknown), PMF (proton motive force) and ATP (adenosine triphosphate). Source data are provided as a Source Data File Full size image

Cyclic electron flow by the pETC is important for the establishment of a PMF that drives ATP production30. To investigate whether a proton gradient is important for EEU, we exposed TIE-1 biofilms to the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (Fig. 2b). CCCP is a lipid-soluble molecule that dissipates the PMF such that electron transfer is uncoupled from ATP synthesis30,33. We observed a decrease in current uptake heading toward anodic current under illuminated conditions upon CCCP treatment (21.2 ± 9.13 nA cm−2; P < 0.0001, one-way ANOVA) compared to untreated controls (−113.5 ± 21.7 nA cm−2) (Fig. 2b, Supplementary Table 2). Current uptake was not different between CCCP (−18.4 ± 14.0 nA cm−2; P = 0.8666, one-way ANOVA) and untreated controls (−17.52 ± 3.41 nA cm−2) under dark conditions (Fig. 2b). These results demonstrate that a PMF is required for EEU. Furthermore, dark EEU is not PMF-dependent as EEU can occur in the presence of CCCP.

The proton-translocating NADH dehydrogenase oxidizes NADH to generate a PMF for ATP production30. NADH dehydrogenase can also function in reverse to catalyze uphill electron transport from the ubiquinone pool to reduce NAD+ in the anoxygenic phototrophs Rhodobacter capsulatus34 and R. sphaeroides35. Its activity is linked to redox homeostasis and carbon metabolism in these organisms36. To investigate whether NADH dehydrogenase has a role in EEU in TIE-1, we treated cells with the NADH dehydrogenase inhibitor rotenone37. Rotenone blocks electron transfer from the iron-sulfur clusters in NADH dehydrogenase to ubiquinone38 (Fig. 2c). In illuminated biofilms, we observed a ~20% decrease in current uptake with low rotenone concentrations (25 µM; −71.8 ± 2.02 nA cm−2; P < 0.0001, one-way ANOVA) compared to untreated controls (−94.7 ± 3.61 nA cm−2), and up to a ~50% decrease with exposure to high rotenone concentrations (100 µM; −41.6 ± 4.55 nA cm−2; P < 0.0001, one-way ANOVA) (Fig. 2c, Supplementary Table 3). The current uptake maxima were markedly lower under these conditions (Supplementary Table 3). After initial current uptake, we observed that rotenone-treated cells showed lowered current uptake post light exposure (Fig. 2c). It is unclear if this reduction is solely due to lowered current uptake or a combination of both lowered current uptake and increased electron donation to the electrode. The reduction in current uptake could also be a consequence of overreduction of the ubiquinone pool as has been observed in R. sphaeroides NADH dehydrogenase mutants38,39. Because we observe only a partial lowering of current uptake with NADH dehydrogenase inhibition (Fig. 2c), the cell likely has additional sinks for using reduced ubiquinone.

CCCP and antimycin A treatment both resulted in anodic current generation under illuminated conditions. Although the magnitude of the electrochemical response was different in the two cases, these data suggest that when the pETC is inhibited, TIE-1 cells likely transfer electrons to the poised electrodes by using them as an electron sink. Overall, our inhibitor studies show that (1) electrons enter the pETC of TIE-1 following EEU; (2) PMF is required for light-dependent EEU; (3) cytochrome bc 1 is involved in electron flow; and that (4) NADH dehydrogenase plays an important role in EEU.

EEU leads to an imbalance in intracellular redox

NAD and its reduced state NADH are essential cofactors for microbes30. NADH can be converted to NAD(P)H via NAD(P)+ transhydrogenase40 (Rpal_4660-4662). NADH and NAD(P)H are key electron donors for biosynthetic reactions, including CO 2 fixation. To better understand how the intracellular redox pool is affected by EEU, we examined the NADH/NAD+ and NAD(P)H/NAD(P)+ ratios in planktonic cells41. We compared these ratios to aerobic chemoheterotrophy (i.e., the inoculum) and phototrophic conditions where other electron donors were provided. We observed that the NADH/NAD+ ratio in the WT during EEU was higher than aerobic chemoheterotrophic growth (Fig. 3a). The NADH/NAD+ ratio was also higher than phototrophic growth on hydrogen (H 2 ) or photoheterotrophic growth on acetate or butyrate (P < 0.0001; Fig. 3a, one-way ANOVA). The NAD(P)H/NAD(P)+ ratio was also highest during EEU compared to other conditions (P < 0.01, one-way ANOVA; Fig. 3b).

Fig. 3 Extracellular electron uptake leads to a reducing intracellular redox environment. a TIE-1 WT NADH/NAD+ and b NAD(P)H/NAD(P)+ ratios under various growth conditions. Conditions tested: yeast-extract peptone (blue); photoheterotrophy with acetate (red) and butyrate (green); and photoautotrophy with H 2 (yellow) or a poised electrode (black). Data are means ± s.e.m. of three biological replicates assayed in triplicate. The P values were determined by one-way ANOVA followed by a pairwise test with Bonferroni adjustment (*P < 0.05, **P < 0.01, ***P < 0.0001; ns, not significant). c Transcriptomic analysis of the de novo NAD biosynthesis pathway under various photoautotrophic and photoheterotrophic growth conditions. d Genome-wide transcriptomic analysis of NAD(P)+/H-requiring reactions. Source data (and reactions not mentioned in text) are provided as a Source Data File Full size image

Analysis of intracellular redox suggests that EEU may lead to a highly-reduced environment in the cell. The lack of NAD+ or NAD(P)+ might require de novo NAD synthesis for cellular survival. Therefore, NAD biosynthesis might increase during EEU. We analyzed the expression of the de novo (aspartate-dependent) NAD biosynthesis pathway42 in the WT transcriptome encoded by nadABCDE. This pathway was not differentially expressed under any phototrophic condition, including EEU (Fig. 3c). NAD kinase which converts NAD+ to NAD(P)+ was also not differentially expressed under the conditions tested (Fig. 3c). These data suggest NAD biosynthesis does not increase at the level of gene expression during EEU despite a highly-reduced redox pool.

We reasoned that NAD(P)+ consuming and/or producing reactions might be upregulated during EEU to maintain redox balance. Therefore, we assessed the expression of NAD(P)+/H-requiring reactions across the TIE-1 genome. We observed that the majority of NAD(P)+/H-requiring reactions were downregulated under phototrophic conditions (Fig. 3d). Interestingly, an NADP-dependent FMN-binding flavin reductase-like protein (fre) was upregulated during photoautotrophic growth, increasing ~4-fold during EEU (Fig. 3d). A pair of NAD(P)+/H-dependent oxidoreductases (akr3 and akr4) were also differentially expressed (Fig. 3d). Akr3 was upregulated under all phototrophic conditions whereas akr4 was specifically upregulated during phototrophic H 2 oxidation and EEU. These data suggest that under EEU the cells are highly reduced and that the lack of oxidized NAD+ and/or NAD(P)+ is not relieved by de novo NAD biosynthesis. However, several NAD(P)+/H-dependent reactions are upregulated.

EEU is linked to CO 2 fixation via the CBB cycle

Our data shows that EEU results in electron transfer to the pETC (Fig. 2), eventually producing NADH and NAD(P)H (Fig. 3). In anoxygenic phototrophs CO 2 fixation is a major sink for NAD(P)H30. In our initial study on EEU by TIE-1, we observed that mRNA transcripts for genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) increased during EEU3. RuBisCO catalyzes CO 2 fixation in many autotrophic organisms as part of the CBB cycle30. Therefore, we asked whether CO 2 fixation occurs during EEU via RuBisCO. TIE-1 encodes two forms of RuBisCO: forms I (cbbLS) and II (cbbM)43. Using transcriptomic analysis, we analyzed the expression of the CBB cycle in TIE-1 and observed that form I ruBisCO was upregulated under all phototrophic conditions, but its expression was highest during EEU (~6-fold, P < 0.0001, one-way ANOVA) and phototrophic iron oxidation (~7-fold, P < 0.0001, one-way ANOVA) (Fig. 4a). Form II ruBisCO was expressed at similar levels across all phototrophic conditions (Fig. 4a). The other enzyme unique to the CBB cycle, phosphoribulokinase (Prk), was also upregulated during EEU (P < 0.0001, one-way ANOVA; Fig. 4a). Prk catalyzes the synthesis of the CO 2 acceptor molecule, ribulose 1,5-bisphosphate (RuBP)30.

Fig. 4 Extracellular electron uptake leads to carbon dioxide fixation. a Differential expression analysis of genes encoding Calvin-Benson-Bassham (CBB) cycle enzymes in TIE-1 wild-type (WT) under various photoautotrophic (poised electrodes, iron oxidation, and H 2 oxidation) and photoheterotrophic growth conditions (acetate and butyrate). b 13CO 2 incorporation under cathodic conditions in TIE-1 WT and the ruBisCO double mutant (∆form I ∆form II) biofilms and planktonic cells determined by secondary ion mass spectrometry (SIMS). Data are means ± s.e.m. of at least 25 cells. The P values were determined by one-way ANOVA followed by a pairwise test with Bonferroni adjustment (*P < 0.05, **P < 0.01, ***P < 0.0001; ns, not significant). c Differential expression analysis of CO 2 and HCO 3 − consuming reactions in TIE-1 WT. RuBP (Ribulose 1,5-bisphosphate), 1,3 BPG (1,3-bisphosphoglycerate), G3P (Glyceraldehyde 3-phosphate), FBP (Fructose 1,6-bisphosphate), F6P (Fructose 6-phosphate), X5P (Xylulose 5-phosphate), Ru5P (Ribulose 5-phosphate) and R5P (Ribose 5-phosphate). Source data (and reactions not mentioned in text) are provided as a Source Data File Full size image

The expression of genes encoding CBB cycle-specific enzymes, including form I ruBisCO, suggests that CO 2 fixation occurs during EEU. There are established methods for answering whether CO 2 fixation is occurring in planktonic cells that can be grown in bulk44,45. However, in the case of EEU the cells attach to electrodes, which precludes us from using standard methodology. To overcome this, we employed secondary ion mass spectrometry (SIMS), and traced 13CO 2 assimilation in TIE-1. The WT and a ruBisCO double mutant (∆cbbLS ∆cbbM) (Supplementary Table 4) were subjected to four treatments in BESs as follows: (1) poised electrodes with 12CO 2 ; (2) poised electrodes with 12CO 2 supplemented with 10% 13CO 2 (poised + 13CO 2 ); (3) electrodes at open circuit with 12CO 2 (passing no current; control); and (4) electrodes at open circuit with 12CO 2 supplemented with 10% 13CO 2 (control + 13CO 2 ) (Supplementary Figure 3). We chose to pre-grow cells under aerobic chemoheterotrophic conditions because the ruBisCO double mutant did not have a growth defect here compared to the WT (Supplementary Table 5). We used bulk BESs (~70 mL) here because they are closed systems, and do not lose CO 2 , unlike the μ-BEC, which is an anoxic microfluidic system under intermittent microfluidic flow.

Cells were cultivated for ~60 h, and planktonic and surface-attached cells (biofilms) were harvested for SIMS analysis. WT cells under poised conditions were enriched in 13C relative to the nonamended cells, indicating the assimilation of 13CO 2 by both surface-attached and planktonic cells (Fig. 4b, Supplementary Table 6). The WT also increased in biomass above open circuit (Supplementary Figure 4). In contrast, the ruBisCO double mutant had a 96% reduction in 13CO 2 assimilation compared to WT (Fig. 4b, Supplementary Table 7), a reduced capacity to take up electrons (Supplementary Figure 3) and no biomass increase (Supplementary Figure 4). These data demonstrate that EEU and CO 2 assimilation are connected, and that RuBisCO catalyzes the major CO 2 assimilation reaction in this system.

The planktonic and the surface-attached cells show the same level of 13C assimilation. This might be due to surface-attached cells and the plankton interacting dynamically with the electrode. To address this, we devised an experiment where pre-established biofilms (from 48 h bioreactor runs) on poised electrodes (biocathodes) were transferred into “plankton-free” bioreactors with fresh medium (Supplementary Figures 5). We observed that after 48 h current densities in “plankton-free” bioreactors were ~70% lower than the plankton-containing bioreactors (P < 0.05, one-way ANOVA; Supplementary Figure 5a–e). Plankton increased to nearly 0.06 OD 660 , while the biocathode remained fully colonized (Supplementary Figure 5a–c, f). In a reciprocal experiment, when new cell-free cathodes were installed in the plankton-containing bioreactors (used to obtain the biocathodes), current densities resembled the original levels (Supplementary Figure 5a–e). This suggests that the plankton retains the ability to attach to the electrodes after 48 h. These data, along with 13CO 2 assimilation, suggests that planktonic cells in the bioreactors are interacting dynamically with the poised electrodes.

The uptake of 13CO 2 in the ruBisCO double mutant (Fig. 4b) likely represents CO 2 consuming reactions such as non-autotrophic carboxylases shown in Fig. 4c. Multiple carboxylases in the TIE-1 genome are expressed during EEU, however, many of these reactions are downregulated relative to chemoheterotrophic growth (Fig. 4c). cynS, which encodes cyanase is upregulated during EEU (P < 0.05, one-way ANOVA; Fig. 4c). Cyanase catalyzes the bicarbonate-dependent metabolism of cyanate, that accumulates as a byproduct of urea dissociation and/or carbamoyl phosphate decomposition46. Overall, our data suggest that RuBisCO is the primary reaction that is catalyzing CO 2 fixation during EEU.

The CBB cycle is a primary electron sink for EEU

RuBisCO catalyzes a reaction between RuBP and CO 2 that results in the formation of two molecules of 3-phosphoglycerate (3-PGA), with no requirement for reducing equivalents30. The reactions that follow, however, require ATP and NAD(P)H. Phosphoglycerate kinase (PGK) catalyzes the phosphorylation of 3-PGA by ATP, which is converted in the reductive phase of the cycle by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) into glyceraldehyde 3-phosphate (G3P). Thus, the CBB cycle, and not RuBisCO directly, is likely the electron sink for EEU. Because ruBisCO is the primary autotrophic carboxylase (Fig. 4b) and because form I ruBisCO was upregulated during EEU (Fig. 4a), we tested the effect of the lack of ruBisCO on this process.

We grew WT and the ruBisCO double mutant in bulk BESs. We chose this bioelectrochemical format because of the need for more biomass for downstream studies. After ~60 h of incubation in bulk BESs, the peak current density in the WT remained stable at ~ −1.5 µA cm−2 (Fig. 5a). The ruBisCO double mutant had a 90% reduction in current uptake vs. WT (P < 0.0001, one-way ANOVA; Fig. 5a). To assess ruBisCO gene expression, we performed reverse transcription quantitative PCR (RT-qPCR) on the planktonic cells. In the WT, form I ruBisCO was upregulated ~8-fold with an associated downregulation of form II ruBisCO (P < 0.0001, one-way ANOVA; Fig. 5b). These expression data in the WT coincide with previous studies on EEU by TIE-13.

Fig. 5 RuBisCO is required for extracellular electron uptake. a Endpoint current densities for ruBisCO deletion mutants compared to TIE-1 wild-type (WT). Data are means ± s.e.m. of three biological replicates. b ruBisCO mRNA log 2 fold change under poised current (cathodic) and no current (open-circuit) conditions for TIE-1 WT and ruBisCO deletion mutants. c LIVE/DEAD® staining of electrode-attached cells under cathodic conditions. Data are means ± s.e.m. of three biological replicates assayed in triplicate. % represents the percent cells in relation to the total number of cells counted. d Endpoint current densities for ruBisCO complementation mutants. Data are means ± s.e.m. of three biological replicates. e ruBisCO mRNA log 2 fold change under cathodic conditions for TIE-1 WT and ruBisCO complementation mutants. f LIVE/DEAD® staining of electrode-attached cells under cathodic conditions. Data are means ± s.e.m. of three biological replicates assayed in triplicate. g Endpoint current densities under standard conditions (WT) and when treated with gentamicin (WT + gentamicin). Data are means ± s.e.m. of three biological replicates. h Log 10 colony forming units (CFU) and generation time (h) of planktonic cells incubated under standard conditions (WT) and when treated with gentamicin (WT + gentamicin). Data are means ± s.e.m. of at least two biological replicates assayed in triplicate. i mRNA log 2 fold change of photosynthetic reaction center (pufL), pio operon (pioA), and ATP synthase homologs (atp1, atp2) in TIE-1 WT and the ruBisCO double mutant. RT-qPCR data are means ± s.e.m. of two biological replicates assayed in triplicate. The P values were determined by one-way ANOVA followed by a pairwise test with Bonferroni adjustment (*P < 0.05, **P < 0.01, ***P < 0.0001; ns, not significant). Source data are provided as a Source Data File Full size image

The ruBisCO mutants did not have a cell viability defect across incubations compared to the WT (P = 0.3691, one-way ANOVA; Fig. 5c, Supplementary Figure 6). We also assessed NADH/NAD+ and NAD(P)H/NAD(P)+ ratios in the ruBisCO double mutant and observed that these cells were more reduced under EEU compared to aerobic chemoheterotrophic conditions (Supplementary Figure 7). However, because these cells show very low current uptake (Fig. 5a), these data are difficult to interpret. Additionally, we did not observe a difference in ATP levels in the WT and the ruBisCO double mutant planktonic cells during EEU (P = 0.2612, one-way ANOVA; Supplementary Figure 8).

Upon complementation of the ruBisCO double mutant with form I and/or form II ruBisCO (Supplementary Table 4), current uptake reached ~ −0.75 µA cm−2, similar to EEU by the WT (Fig. 5d). This was above current uptake levels by the ruBisCO double mutant (P < 0.01, one-way ANOVA; Fig. 5d). We observed that form I and form II ruBisCO were expressed at levels similar to the WT (Fig. 5e). Similar to the ruBisCO deletion mutants, the ruBisCO complementation mutants did not have a cell viability defect compared to the WT (P = 0.0572, one-way ANOVA; Fig. 5f, Supplementary Figure 6).

RuBisCO deletion does not affect EEU due to a growth defect

To determine whether the EEU defect in the ruBisCO double mutant was growth-dependent, we inoculated WT cells into bioreactors containing a sub-lethal concentration of gentamicin to inhibit protein synthesis (Supplementary Figure 9). We observed that gentamicin-treated WT cells accepted 80% more electrons during EEU compared to the ruBisCO double mutant (P < 0.0001, one-way ANOVA; Fig. 5g). To assess a potential growth defect in the ruBisCO double mutant, we harvested the electrodes at the end of the incubations and used 5 mm sections as inoculum for chemoheterotrophic growth. We did not observe a growth defect in the ruBisCO double mutant upon re-growth compared to the WT (P = 0.8232, one-way ANOVA; Fig. 5h). Planktonic colony forming units (CFUs) for the ruBisCO double mutant harvested at the end of incubations in the bulk bioreactors were not different from the WT (P = 0.0804, one-way ANOVA; Fig. 5h). These data suggest that the lower EEU activity of the ruBisCO double mutant is not due to a growth defect.

We performed gene expression analysis using a set of genes that have been reported to be involved in EEU from electrodes3. We first assessed the expression level of the photosynthetic reaction center large subunit (pufL). Gene expression analysis showed a ~5-fold upregulation of pufL in the ruBisCO double mutant, very similar to the WT expression (P = 0.0559, one-way ANOVA; Fig. 5i). Because previous mutant studies have shown that the pioABC system, a gene operon essential for phototrophic iron oxidation47, also has a role in electron uptake3, we performed expression analysis of pioA in the ruBisCO double mutant and the WT. We observed that the expression level of pioA in the ruBisCO double mutant was not different from the WT (P = 0.0759, one-way ANOVA; Fig. 5i).

We also assessed the expression of the systems responsible for energy transduction. The TIE-1 genome contains two F-type ATPases: Atp1 and an “alternate” Atp2. atp1 showed lower upregulation (~4-fold) than atp2 (~7-fold) in both the WT and the ruBisCO double mutant (Fig. 5i). The WT transcriptomic data corroborate the RT-qPCR data where atp1 is downregulated during phototrophic growth conditions, including EEU, whereas atp2 is specifically upregulated during EEU (Supplementary Tables 8, 9). These results suggest that the atp2 operon plays an important role in ATP synthesis during EEU. Overall, our data suggest that the WT and the ruBisCO double mutant do not show any differences in the level of gene expression for critical genes required for EEU, pETC, and energy generation. These data, in conjunction with the lack of 13CO 2 assimilation (Fig. 4b), suggests the ruBisCO double mutant cells may be using cellular reserves to stay viable under the conditions tested.

The CBB cycle is important for phototrophic H 2 oxidation

The inability of the ruBisCO double mutant to take up electrons from solid electrodes suggests that the CBB cycle is the primary electron sink during EEU. This finding underscores that CO 2 fixation is tightly linked to EEU in these bacteria. In order to probe whether this coupling extends to other growth conditions, we examined the ability of the ruBisCO double mutant to oxidize H 2 under phototrophic conditions. We observed ~80% lower H 2 consumption in the ruBisCO double mutant compared to the WT (P < 0.05, one-way ANOVA; Fig. 6a, Supplementary Table 10) with a concomitant reduction in CO 2 consumption (P < 0.05, one-way ANOVA; Fig. 6b, Supplementary Table 10). We also observed an increase in biomass in the WT compared to the ruBisCO double mutant during phototrophic H 2 oxidation (P < 0.0001, one-way ANOVA; Supplementary Figures 10, 11). These data suggest that CO 2 fixation is an important electron sink under photoautotrophic conditions, where electron donors, such as H 2 , are oxidized to provide cellular reducing power.

Fig. 6 RuBisCO is important for phototrophic hydrogen (H 2 ) oxidation. a Hydrogen (H 2 ) oxidation and b carbon dioxide (CO 2 ) consumption by the ruBisCO double mutant (∆form I ∆form II) as a percent of consumption by TIE-1 wild-type (WT). Data are means ± s.e.m. of two biological replicates assayed in triplicate. c mRNA log 2 fold change of photosynthetic reaction center (pufL), NiFe hydrogenase (hupL), and ATP synthase homologs (atp1, atp2) in WT and the ruBisCO double mutant. RT-qPCR data are means ± s.e.m. of two biological replicates assayed in triplicate. The P values were determined by one-way ANOVA followed by a pairwise test with Bonferroni adjustment (*P < 0.05, **P < 0.01, ***P < 0.0001; ns, not significant). Source data are provided as a Source Data File Full size image