The column was designed to simulate the chemical and physical environments available at different depths along an oxygenated Precambrian continental margin subjected to upwelling of Fe(II)-rich water, from which deepwater IF were depositing (Fig. 1). While such a margin has spatial variation in the horizontal plane that our column cannot account for, the primary physical and chemical forcing we are applying to our system is upwelling/advection, which was thought to have happened along such margins26,27,28. This has been simulated through reaction modeling in one (vertical) dimension29,30, and our column seeks to provide a laboratory-scale analog of this16.

Within this context, the column simulated a habitat for planktonic marine cyanobacteria (Fig. 1). Secondary electron microscopy (SEM) of liquid extracted from the column documents the presence of cells in the liquid phase (Supplementary Information). The inclusion of glass beads was necessary to stabilize the chemical gradients developed through advection and molecular diffusion from disturbance by external perturbations (e.g. vibrations). However, the beads provided an additional benthic habitat, such is analogous to that inhabited by cyanobacteria that may have been responsible for formation of stromatolites, which are common in Archean-aged carbonate platforms31,32,33. Fluorescence microscopy documents that cells also adhered to glass beads (Supplementary Information), such as would be expected of benthic cyanobacteria34.

Fe(III) reduction likely occurred using Fe(III) from accumulated Fe(III) (oxyhydr)oxide minerals produced from Fe(II) oxidation. While maximally a few tens of μM Fe(III) was detectable in the aqueous samples from the column at most times, this was likely present as Fe(III) (oxyhydr)oxide minerals and Fe(III) adsorbed to the surface of Synechococcus PCC 7002 cells (ref.17 and Supplementary Information). Fe(III) (oxyhydr)oxide minerals were abundantly associated with glass beads, as determined by iron extraction following cessation of a replicated column experiment (Supplementary Information).

While Fe(II) concentrations were elevated within the photic zone from the concentrations in the reservoir during light incubation, the Fe(II) and Fe(III) concentrations in aqueous samples were observed to spike to their highest values during the dark incubation (day 21.2; Fig. 2). As this spike occurred within the colonized portion of the column in the dark, the result suggests a biological Fe(III) reduction process was occurring independent of light. For this reason, additional experiments were conducted to explore the biological and physical controls on Fe(III) reduction in the presence of Synechococcus PCC 7002.

The batch experiments with synthetic ferrihydrite document that Synechococcus PCC 7002 is capable of Fe(III) reduction. Fe(II) was produced from ferrihydrite in the presence of live cells growing in light (Fig. 3). However, heat-killed cells no longer produced additional Fe(II), regardless of whether or not they were incubated in the light or dark. This result indicates that Fe(III) reduction was a process attributable to only live cells. Furthermore, Fe(II) present when cells were heat-killed was subsequently oxidized by residual oxygen produced during initial growth. This is in contrast to light grown cells that were moved to a dark incubation, where Fe(II) produced in the light did not experience as much subsequent oxidation as in heat-killed incubations, despite similar amounts of oxygen. Production of Fe(II) from cells always incubated in light occurred until the end of the experiment, when cells were likely in stationary phase17,30. Our results indicate that Fe(III) reduction is attributable to live cells, and can occur under light or dark conditions.

Extracellular Fe(III) reduction has been proposed as a pathway necessary for uptake of iron as Fe(II), and such an iron acquisition pathway may be widespread among phytoplankton35,36. Fe(III) reduction has been observed in several cyanobacteria13,14, yet the reported mechanisms of Fe(III) reduction vary36. Therefore, several Fe(III) reduction pathways may be relevant to interpret the results of our experiment. Siderophores complex and dissolve Fe(III), and are synthesized and secreted by microorganisms under iron limitation37. Siderophores are widely used by marine photosynthetic bacteria as a substrate for Fe(III) reduction before uptake of Fe(II)38. Synechococcus PCC 7002 is known to secrete siderophores39, and other cyanobacteria do as well37. Siderophore production was characterized in Synechococcus PCC 7002 under iron-limiting growth conditions17,37,40. No siderophores were detected in either our column or batch experiments. This is consistent with the lack of siderophore-related proteins produced by this strain under non-limiting iron concentrations similar to the current experiments17. Thus, reduction of siderophore-bound Fe(III) seems unlikely to be significant in the column.

Another possible Fe(III) reduction pathway is via the extracellular production of the reactive oxygen species (ROS) superoxide. Superoxide can oxidize Fe(II), but can also reduce Fe(III), especially if Fe(III) is in aqueous or ligand-bound form11. Many marine algae41 and a wide range of marine bacteria42 are capable of producing superoxide, suggesting marine superoxide cycling can occur independent of light. The coastal marine cyanobacterium Lyngbya majuscule generates superoxide for Fe(III) reduction and Fe(II) uptake14. NAD(P)H oxidoreductases are known or thought to be responsible for superoxide production in many biological systems14,42,43, as superoxide production can be inhibited by the addition of diphenyleneiodonium (DPI), which is known to act on these enzymes23,40. Addition of DPI to incubations of Synechococcus PCC 7002 completely inhibited growth (Supplementary Information), indicating that NAD(P)H oxidoreductases are also present and essential to the growth of this strain. Furthermore, Fe(III) is known to bind to the surface of Synechococcus PCC 7002 cells under similar conditions as within the column, perhaps to a capsular exopolysaccharide17, which could make it more susceptible to superoxide-mediated reduction11. Therefore, superoxide production by Synechococcus PCC 7002 seems to be a plausible light-independent pathway for Fe(III) reduction within the column.

Fe(III) reductases have also been suggested as an alternative mechanism for diverse planktonic cyanobacteria to reduce Fe(III) before uptake13,44. In one molecular model for Synechocystis 6803, Fe(III) is transported through the outer membrane, reduced to Fe(II) in the periplasmic space, and then transported into the cell45. When Synechococcus PCC 7002 is grown in Fe(II)-rich conditions, similar to the conditions within the column, Fe(III) is observed at the surface of the cell, and a number of iron uptake and receptor proteins were abundant17. These observations suggest that this strain may actively bind iron at its surface, which could make it subject to active reduction as well.

Finally, photochemical reduction may also enhance the biologically mediated Fe(III)-reducing activity we observed. Ligand-bound Fe(III) is reduced to Fe(II) and released in LMCT, which has been shown to promote Fe(II) uptake in the freshwater cyanobacterium Microcystis aeruginosa46. Furthermore, photochemical reduction of Fe(III) bound to ligands, including siderophores, is common in the open ocean47. Generally, these processes are thought to occur with UV light47, but up to 30% of photochemical Fe(III) reduction may be attributed to visible wavelengths48. Our light spectrum profile of the column indicates that visible wavelengths are still abundant at 2 cm below the surface (Supplementary Information), within the zone of Fe(II) production (Fig. 2). Furthermore, Fe(II) concentrations in the dark incubation of live cells stayed relatively constant until the end of the experiment, when cells may have died, consistent with continued biological Fe(II) production even in the dark. Therefore, we cannot rule out that photochemical processes might have also contributed to the Fe(III) reduction occurring in the column.

Our laboratory simulation shows that cyanobacterial enzymatic reactions can reduce Fe(III) in the presence of free oxygen. In our experiments, oxygen values ranged from well above air saturation (224 μM in this experiment) down to a few μM when and where Fe(III) reduction occurred (Figs 2 and 3), indicating that oxic Fe(III) reduction is possible under a wide range of oxygen concentrations. Oxygen concentrations in the late Archean ocean may have reached 1–10 μM49, or even up to 35 μM locally29. Surface values below 5 μM are suggested into the Proterozoic50, while in the Neoproterozoic, poorly oxygenated zones (ca. 10 μM) may have persisted at interfaces with upwelling Fe(II)-rich water below more oxygenated conditions51. Furthermore, oxygen minimum zones with tens of μM oxygen were present in the Proterozoic52, which is characterized by ferruginous conditions throughout much of the oceans1,2,3,4. The expansion of oxygen minimum zones in the modern ocean53 raises the possibility that cyanobacterial-driven Fe(III) reduction may be relevant in deeper waters, as deep chlorophyll maxima are sometime observed at the boundary to more nutrient-rich and often oxygen-depleted deep waters54. Thus, our results are relevant to mildly to fully oxygenated interfaces with ferruginous waters throughout Earth’s history, following the possible appearance of oxygen in the surface oceans as early as 3 Gy ago55.

While a convergence of evidence documents the importance of light dependent Fe(III) reduction in modern oxic seawater conditions8,38,48,56, our work indicates that this pathway, in combination with Fe(III) reduction at cyanobacterial surfaces13, is also significant when and where iron concentrations are much higher (e.g. hundreds of micromolar) than those in the modern surface ocean (pico to nanomolar range). As our experiments involved growth of cyanobacteria, and due to varying numbers of cells, the oxic Fe(III) reduction we observed was unlikely to be in steady-state with abiotic Fe(II) oxidation. Therefore, it was impossible to parse the quantitative impact of oxic Fe(III) reduction on iron turnover. However, prior estimates for Fe(III) reduction in the presence of cyanobacteria allow for us to make an estimate. A rate of 115 × 10−21 mol cell−1 hr−1 of Fe(III) reduced in light, which integrates abiotic photochemical Fe(III) reduction, was measured for Synechocystis 680313. Given a concentration of cyanobacteria in the surface ocean of ca. 105 cells mL−1 (ref.57), the combination of these processes could reduce Fe(III) at a rate of 1.15 × 10−11 mol hr−1. Oxidation rates for Fe(II) are extremely sensitive to oxygen and iron concentrations, as well as pH and temperature. Using a half-life for similar pH and Fe(II) concentration (ca. 200 nM) of 1413 min−1, a comparable Fe(II) oxidation rate is 1.86 × 10−9 mol hr−1 (ref.58) . These calculations predict that for oxic Fe(III) reduction to be effective in maintaining Fe(II) in oxic waters, cells would need to be quite dense, such as might occur within a mat, a bloom, or a deep chlorophyll maximum. However, we note that comparable rates for the higher Fe(II) concentrations we utilized are lacking. Our empirical observations of the surface maximum of Fe(II) (Fig. 2) suggest that oxic Fe(III) reduction can outpace Fe(II) oxidation in the conditions we tested.

While Fe(III) reduction is thought to be a mechanism used by cyanobacteria for iron uptake in iron-limiting conditions13,14, which characterize many modern aquatic habitats, it also appears to be a widespread phenomenon in algae36,41. This likely stems from the large demand for iron in the photosynthetic machinery59, which may harken back to the iron-replete Precambrian oceans from which many oxygenic photosynthetic organisms evolved. Deep hydrothermally-sourced Fe(II), supplied via upwelling to some shallow-water settings27,28,31, would have been oxidized with photosynthetic oxygen, thus titrating iron out of solution60. Therefore, an Fe(III) reduction strategy might have been necessary for even the earliest cyanobacteria to acquire iron as they modified the redox potential, and thus availability of iron in their environment.

Clearly, oxic Fe(III) reduction could have played a distinct role in Precambrian iron cycling. Yet rapid iron redox cycling has been invoked primarily within the interface of oxic to anoxic sediments or waters (e.g. refs31,61,62), mediated either by dissimilatory Fe(III)-reducing microbes or reductive dissolution of Fe(III) (oxyhydr)oxide minerals by hydrogen sulfide. There are several examples where oxic Fe(III) reduction may help to explain conflicting interpretations of oxygen levels based on different redox proxies. The 2.5 Gy old Campbellrand-Malmani Platform in South Africa preserves subtidal to supratidal depositional settings63, and contains encrustations of decimeter to meter-thick bedding of aragonite and calcite crystals, while lacking significant micrite9,12. This has been attributed to Fe(II) inhibiting the crystal growth of calcite and aragonite, as Fe(II) suppresses new crystal nucleation rates64,65,66,67. Yet the geochemistry of the succession suggests it was an oxygen oasis29,33,68,69,70. Iron and molybdenum isotope systematics indicate the presence of Fe(II) in shallow seawater29,60, and detailed mineralogical investigation indicates that iron was incorporated into shallow-water carbonate minerals as Fe(II) from coeval seawater60. The conflicting evidence for Fe(II) in seawater in the presence of oxygen could be reconciled by invoking oxic Fe(III) reduction by organisms similar to modern cyanobacteria. Importantly, there were extensive microbial mats, recorded as stromatolites from the section, and photosynthetic lifestyles cannot be ruled out33. Even earlier in the Archean, deposition of iron-bearing stromatolites from the ca. 2.8 Gy old Steep Rock carbonate platform also indicate periodic incursion of Fe(II)-rich seawater into an oxygen oasis, signified by the deposition of iron-poor limestone and rare earth element patterns consistent with oxygen31,71. These stromatolites also have features parsimonious with the presence of cyanobacteria-like organisms71, highlighting a role for oxic Fe(III) reduction at such settings.

Our work extends the implications that Fe(III) reduction can occur under oxic conditions of modern marine systems to Precambrian marine settings that are typified by ferruginous deep seawater overlain by oxygenated seawater. Oxic Fe(III) reduction, mediated enzymatically and perhaps augmented by photochemistry, can persistently generate Fe(II) from Fe(III) (oxyhydr)oxides and/or ligand-bound Fe(III), even from Fe(II)-rich water masses. As redox interfaces between deep, Fe(II)-rich seawater and oxygenated seawater persisted for billions of years, oxic Fe(III) reduction may have played an important role in marine iron cycling for as long.