Survival of cyanobacteria and mitigation of Fe(II) toxicity effects in a silica-rich Archean ocean

Domain: chemistry
Published: 2026-02-21
Editorial outcome: major_revision

Abstract
Banded iron formations (BIF) were deposited abundantly between 2.7-2.4 Ga from iron- and silica-rich oceans, with cyanobacterial oxygen (O2) as a possible oxidant for Fe(II) oxidation and Fe(III) oxyhydroxide precipitation. However, toxic reactive oxygen species (ROS) from Fe(II)/O2 interactions might have inhibited cyanobacterial growth, contributing to the delay between cyanobacterial evolution (>3.0 Ga) and the Great Oxidation Event (2.5 Ga). Here, we explored the impact of Fe(II)(aq) and SiO2(aq) on Synechococcus sp. PCC 7002. High Fe(II)(aq) (>500 µM) increased ROS formation, but elevated SiO2(aq) (2200 µM) suppressed ROS formation, promoting growth and O2 production. Diel light cycles further reduced ROS formation compared to continuous illumination. Modelling O2 distribution based on experimental rates revealed oxygenated surface waters at relevant upwelling rates. Together, our results indicate that high SiO2(aq) and day-night-light cycles in Archean oceans mitigated ROS stress, enabling cyanobacterial proliferation and enhancing their role in Earth’s oxygenation and BIF deposition.

Introduction
Sometime between the evolution of oxygen (O2)-producing cyanobacteria, possibly as early as 3.4 billion years ago1,2 and the permanent rise of atmospheric oxygen during the Great Oxidation Event (GOE) at 2.5–2.3 Ga3, free O2 began to accumulate in seawater. This accumulation likely began in the form of localized and/or transient oxygen oases, with estimated O2 concentrations ranging from 5 to 100 µM4–7, before episodically expanding across broader regions of the continental shelf8–10. The reasons for the protracted delay between the emergence of cyanobacteria and the GOE remain unresolved but may, at least in part, reflect environmental constraints that inhibited cyanobacterial growth and dispersal11,12.

One of the most distinctive sedimentary archives of the Archean, BIFs, may hold clues to these limiting environmental conditions (see 13 for review). These chemical sediments consist predominantly of iron (15–40 wt.% Fe) and silica-rich (40–60 wt.% SiO2) layers that precipitated from seawater following the oxidation of Fe(II)(aq), with estimated concentrations ranging from 0.03 to 0.5 mM14,15, and possibly exceeding 1.0 mM in some settings16,17. Prior to the emergence of silica-precipitating microorganisms (e.g., diatoms), silica inputs from chemical weathering of early continental crust and hydrothermal vents led to elevated dissolved silica concentrations in the seawater. These concentrations were constrained chiefly by saturation with respect to cristobalite or amorphous silica, on the order of 0.67-2.2 mM18,19. Recent studies further suggest that under these ‘high silica’ concentrations, the primary precipitate may have been a gel-like composite of ferric oxyhydroxide and silica20–22.

While the deposition of the oldest BIF was probably linked to anoxygenic phototrophic Fe(II)-oxidizers23,24, photochemical Fe(II) oxidation25,26 or abiotic deposition processes such as chemical greenalite deposition27,28, the proliferation of cyanobacteria would have accelerated Fe(II) oxidation and thus BIF deposition13,29. Cyanobacteria fix and reduce CO2 using water as electron donor to form biomass, also yielding O2 as by-product (reaction 1). As such, they were key players in the oxygenation of Earth30,31.

6CO2 + 12H2O ! hν C6H12O6 + 6O2 + 6H2O (1)

In early Precambrian oceans, the O2 may have reacted with Fe(II)(aq) to form Fe(III)(aq), which hydrolyzed into Fe(III) oxyhydroxide minerals. The reaction between dissolved Fe(II) and O2(aq) may also yield reactive oxygen species (ROS), that is, highly reactive oxygen-containing molecules (i.e., the Fenton reaction32):

O2 + Fe2+ ! Fe3+ + O2(•-) (2)

O2(•-) + Fe2+ + 2H+ ! Fe3+ + H2O2 (3)

Fe2+ + H2O2 ! Fe3+ + OH- + OH• (4)

Briefly, superoxide (O2•-), generated by the reaction of O2 and Fe(II)(aq) (reaction 2; note we provide actual chemical species here versus the generic Fe(II)), further reacts with Fe(II)(aq) to produce Fe(III) and hydrogen peroxide (H2O2) (reaction 3). The H2O2 then oxidizes the remaining Fe(II)(aq) to Fe(III), along with the formation of hydroxide anions (OH-) and hydroxyl radicals (OH•) (reaction 4). While the Fe3+ drives the formation of Fe(III) minerals, the generated radicals can undergo further reactions, forming additional ROS like ozone (O3) and hydroperoxyl radicals (OOH•) (for a review see ref. 33).

ROS are harmful to cells; extracellular ROS can damage cell membranes through oxidative stress34, while intracellular ROS can oxidize critical biomolecules such as RNA, DNA, proteins, and lipids35. Therefore, ROS formation in Archean oceans may have profoundly influenced microbial activity and evolution. Previously, Swanner and colleagues investigated ROS production under early ocean analog conditions using phosphate-buffered saline (PBS) media and varying concentrations of Fe(II)(aq) (0.1 and 1 mM) in the presence of Synechococcus sp. PCC 7002 at a density of 2 × 107 cells/mL12. Under continuous light, they found a 4-fold increase in intracellular ROS levels in the presence of 1 mM Fe(II)(aq) compared to 0.1 mM Fe(II)(aq), suggesting potential toxicity to cyanobacteria at elevated Fe(II)(aq) concentrations. Additionally, in incubation experiments of cyanobacteria under initially anoxic conditions (in the presence of up to 300 µM Fe(II)(aq)) reduced autofluorescence of the cyanobacteria was reported, accompanied by significantly lower actinic yield, and decreased growth rates at Fe(II)(aq) concentrations >180 µM.

In a follow-up study, Swanner and colleagues conducted Fe(II)(aq) oxidation experiments with Synechococcus sp. PCC 7002 under continuous illumination and a range of Fe(II)(aq) concentrations (7.5, 29, 577, and 4805 µM)11. At lower Fe(II)(aq) concentrations, cultures changed from colorless to green, indicating a high density of cyanobacterial cells. By contrast, higher Fe(II)(aq) concentrations produced an orange coloration, consistent with the formation of Fe(III) oxyhydroxide minerals. These observations were supported by direct cell counts, which confirmed reduced growth at higher Fe(II)(aq) concentrations, again suggesting toxic effects. Microscopic analyses further revealed that cells grown under high Fe(II)(aq) concentrations were smaller and exhibited reduced pigment content, as indicated by lower carotenoid and chlorophyll concentrations inferred from optical density measurements. Although Fe(II)(aq) oxidation rates increased with rising Fe(II)(aq) concentrations (from 13 µM/day at 29 µM to 475 µM/day at 4805 µM) the rate of O2 production in the liquid phase decreased from 120 µM/day to 47 µM/day, indicating diminished cellular activity. Notably, O2 was already detectable during Fe(II)(aq) oxidation at 29 and 475 µM Fe(II)(aq) but absent at 4805 µM Fe(II)(aq). Taken together, these findings suggest that elevated Fe(II)(aq) concentrations in Archean seawater could have imposed oxidative stress, limiting cyanobacterial productivity and expansion. This could potentially explain the lag between the evolution of cyanobacteria and the GOE11,12.

However, the potential mitigating impact of high SiO2(aq) concentrations on Fe(II)(aq) toxicity has not been previously considered. Our working hypothesis is that silica binds Fe(II)(aq), forming Fe-Si-aggregates that slow Fe(II) oxidation and consequently inhibit ROS formation. Previous studies have shown that reduced Fe(II) oxidation rates lead to lower ROS formation36. Consistent with this, Dreher and colleagues37 showed, using SEM/EDS, that Fe-Si aggregates precipitate under similar experimental conditions, further supporting our hypothesis. To test this hypothesis, we experimentally examined the combined effects of Fe(II)(aq) and SiO2(aq) on cyanobacterial O2 production, Fe(II) oxidation, and potential ROS-related toxicity. Specifically, we incubated Synechococcus sp. PCC 7002 under alternating day-night-cycles (16 h light; 8 h dark) in artificial seawater medium, in the absence of SiO2(aq) (0 µM, which we refer to as ‘no-silica’) and in the presence of SiO2(aq) (2200 µM, which we refer to as ‘high-silica’) and varying initial Fe(II)(aq) concentrations (0, 500, 2500, 5000 µM). Over the course of the experiments, we monitored O2 production, Fe(II) and SiO2(aq) concentrations, total iron (Fe(tot)), cell numbers, and ROS production to assess the interdependent effects of Fe(II)(aq) and SiO2(aq) on cyanobacterial physiology and oxidative stress.

Results
Effects of different silica concentrations on Fe(II) oxidation by cyanobacterial O2 under alternating day-night-cycles
We conducted Fe(II) oxidation experiments, under initially anoxic conditions, using Fe(II)(aq) concentrations ranging from 0.5 to 5 mM. Following inoculation with the cyanobacterial strain Synechococcus sp. PCC 7002, we monitored Fe(II), Fe(tot), cyanobacterially produced O2, dissolved Si, and cell counts over time (for sterile controls see Fig. S3). The results are plotted in Fig. 1 on the respective timescale of the active Fe(II) oxidation, as this sets the focus on the period when there was still Fe(II) in the system, which is the most relevant for determining toxicity effects and the latter modeling. In setups without amendment of additional Fe(II)(aq), all cultures exhibited a color change from clear to green within two days, regardless of SiO2(aq) concentration (Fig. 1C), indicating cyanobacterial growth. In the ‘high silica’ setups compared to the ‘no silica’ setups, cell growth was initially more rapid but reached a slightly lower maximum cell density (Fig. 1C, Table 1). Under both experimental conditions, O2 concentrations peaked at 500 µM after 60 days (Fig. 1B). In the setups without additional Fe(II)(aq) amendment, the Fe(II) concentration remained relatively stable throughout the experiments, averaging approximately 50 µM in with and without both silica setups (Fig. 1A), a level attributable to the Fe(II) added as nutrient in the medium. In the ‘high silica’ setups, the initial SiO2(aq) was ca. 1600 µM (Fig. S1A). However, after 30 days, solid silica precipitation started, ultimately reducing the SiO2(aq) concentration to several hundred µM by the end of the experiment.

In experiments with amendment of 500 µM of Fe(II)(aq), all bottles turned green within two days, regardless of the SiO2(aq) concentration (Fig. 1F). Growth curves were similar across setups, with no discernible effect of SiO2(aq) (Fig. 1F, Fig. 2, Table 1), and maximum cell densities were reached after 87 days (Fig. 2). In both ‘no silica’ and ‘high silica’ setups, O2 accumulated in the headspace following complete Fe(II) oxidation, peaking at 450 µM after 60 days (Fig. 1E).

Within the first five days of the experiment, 80–90% of the added 500 µM Fe(II)(aq) was oxidized in both ‘no silica’ and ‘high silica’ setups (Fig. 1D). With and without silica, the Fe(II) oxidation rates were similar within the calculated error (Table 1). In the ‘no silica’ and ‘high silica’ setups, Fe(II) oxidation rates were similar at about 100 µM/day (Fig. 1D; Table 1). In the ‘high silica’ setups (Fig. 1B), silica precipitation began after 60 days, reducing SiO2(aq) to approximately 100 µM.

In experiments amended with 2500 µM of Fe(II)(aq), the produced O2, bottles turned orange in the absence of added silica (Fig. 1I), contrasting with experiments with 0 and 500 µM added Fe(II)(aq). However, in the presence of silica, the 2500 µM Fe(II) bottles turned yellow by day 21 and green by day 31. The orange coloration in the ‘no silica’ setup suggests abiotic Fe(II) oxidation and the formation of Fe(III) oxyhydroxide minerals, likely caused by cyanobacterially but with limited cell growth and metabolic activity. By contrast, the green coloration in the ‘high silica’ setups indicates extensive cyanobacterial proliferation. It is important to interpret these color changes cautiously, as higher Fe(II) concentrations can intensify the orange hue regardless of biological activity, potentially confounding visual comparisons between setups.

Cell counts revealed that in the ‘no silica’ setups, cyanobacterial growth was delayed, with cell numbers increasing only after 25 days (Fig. 1I). Conversely, cell proliferation in the ‘high silica’ setups commenced immediately after inoculation and ultimately reached similar densities by day 150 (Fig. 1I). Silica availability had a marked influence on O2 accumulation. In the ‘no silica’ setups (Fig. 1H), O2 accumulated only after day 20 (ca. 50 µM by day 31). In comparison, in the ‘high silica’ setups (Fig. 1H) O2 accumulated as early as day 10, reaching 150 µM by day 31.

In the ‘no silica’ setups, the Fe(II) oxidation rate was lower compared to the ‘high silica’ setups (Fig. 1G; Table 1). Initial SiO2(aq) concentration in the ‘high silica’ setups were 1500 µM (Fig. S1C, Fig. 1C) and decreased to 700 µM within the first 20 days. By day 90, variability emerged among the triplicate bottles: one replicate maintained a stable SiO2(aq) concentration of 700 µM, whereas the other two showed continued declines to 300 µM and 100 µM, respectively.

In experiments amended with 5000 µM of Fe(II)(aq), the ‘no silica’ setups remained orange until day 87 (Fig. 1L), indicating Fe(III) oxyhydroxide formation and limited cyanobacterial growth. By contrast, the ‘high silica’ setups turned yellow by day 21 and green by day 87, reflecting more substantial cyanobacterial growth. Consistent with these color changes, total cell numbers were higher in the ‘high silica’ setups. In the ‘no silica’ setups, cell growth was delayed until Fe(II) oxidation was complete, after which cell densities increased to 5 × 10⁷ cells/mL (Fig. 1L). In one replicate, cyanobacteria eventually recovered, reaching 1.5 × 10⁸ cells/mL by day 140 Fig. 2. In the ‘high silica’ setups, final cell densities varied among the three replicates, reaching 2.5 × 10⁸, 1.5 × 10⁸, and 5 × 10⁷ cells/mL, respectively (Fig. 1L).

O2 accumulated in the ‘no silica’ setups after 60 days, reaching ca. 80 µM by day 87 (Fig. 1K), although replicate variability was high: one bottle showed no detectable O2, while another reached 300 µM O2 by day 140. In contrast, O2 concentrations in the ‘high silica’ setup increased after 57 days (Fig. 1K) and reached 200 µM by day 87. Two out of three replicates further reached 400 µM O2 by day 100, whereas in the third, in which only 60% of the Fe(II) was oxidized, showed no detectable O2.

The Fe(II) oxidation rates were similar between the ‘no silica’ and ‘high silica’ setups (Fig. 1J; Table 1), but were notably lower than the rates observed in experiments with 2500 µM Fe(II)(aq). In the ‘high silica’ setups, the initial SiO2(aq) concentration of 1500 µM declined to 500 µM by day 31 (Fig. S1D), after which it either stabilized or continued to precipitate, reaching final concentrations between 100 and 200 µM.

Improvement of long-term cyanobacterial cell viability by silica
In addition to experiments focusing on cyanobacterial growth and activity i.e., cell numbers, O2 production, resulting Fe(II) oxidation and dissolved silica (see Fig. S1), we explored the role of silica in promoting long-term cell viability experiments by observing potential mitigation effects of the harmful effects of ROS generated in the presence of both Fe(II)(aq) and O2. In all setups, the presence of Fe(II)(aq) delayed both cell growth and O2 production (Fig. 2). In the 500 µM Fe(II)(aq) (Fig. 2E), ‘high silica’ setups initially showed higher O2 accumulation during the first 70 days, with both setups converging at 350 µM by day 60. In the 2500 µM Fe(II) experiments (Fig. 2G–I), the presence of SiO2(aq) led to significantly enhanced cyanobacterial activity: by day 150, O2 concentrations reached 500 µM and cell densities ca. 2.4 × 108 cells/mL, compared to 350 µM O2 and 1.8 × 108 cells/mL in the absence of SiO2(aq). The beneficial effect of silica was even more pronounced in the 5000 µM Fe(II) setups. After 250 days, O2 accumulation in the ‘high silica’ setups reached 250 µM, with cell densities of 2 × 108 cells/mL, while the ‘no silica’ setups showed limited cell growth, reaching only 5 × 107 cells/mL and 100 µM O2.

Formation of ROS in incubations of strain PCC 7002 with Fe(II)(aq)
Based on the lower cell growth and Fe(II) oxidation rates at higher Fe(II)(aq) concentrations (500, 2500–5000 µM), we hypothesized that cyanobacterially produced O2 reacted with Fe(II)(aq) to generate ROS, potentially leading to cellular stress or toxicity. However, our results also indicated that SiO2(aq) mitigates these negative effects, as evidenced by higher cell densities, earlier O2 accumulation, and increased Fe(II) oxidation rates in silica-amended setups. To further evaluate the impact of varying Fe(II)(aq) and SiO2(aq) concentrations on ROS production, we subsequently constructed a series of experiments using Synechococcus sp. PCC 7002 cell suspensions (5 × 109 cells/mL) exposed to different Fe(II)(aq) concentrations (0, 500, 2500 or 5000 µM), both in the absence and presence of 2200 µM SiO2(aq). Our results (Fig. 3, Fig. S2, Table S1) showed that in the absence of Fe(II)(aq), ROS fluorescence signals were generally very low, yet slightly elevated in the presence of SiO2(aq) compared to setups without SiO2(aq) (ca. 500 a.u. and 400 a.u., respectively) (Fig. 3). After the amendment of 500 µM Fe(II)(aq), the ROS fluorescence signal, with and without silica, was similar to the setups without Fe(II)(aq), suggesting no significant ROS formation at this Fe(II)(aq) concentration. However, in the presence of 2500 µM of Fe(II)(aq), we observed a pronounced increase in ROS fluorescence in the absence of silica (2000 a.u.), while the signal remained near baseline in silica-amended setups. A similar trend was observed at 5000 µM Fe(II)(aq), where ROS levels reached 1300 a.u. without silica, but remained negligible in the presence of silica (Fig. 3). These results suggest that high Fe(II)(aq) concentrations promote ROS generation, and that dissolved silica effectively suppresses ROS formation under these conditions.

Numerical modeling of the experimental O2 data
In order to simulate the total O2 production, we incorporated our raw laboratory data into a numerical model to simulate the total O2 production. To account for the observed plateau of O2 concentration at approximately 450 µM (Fig. 4A–E), we differentiated between ‘active’ cells (O2-producing; green solid line) and ‘inactive’ cells (non–O2-producing; calculated as the difference between total cells and measured active cells). The ‘inactive’ group included both dead cells and those estimated to have received insufficient light due to increased cell density during the experiment. While cultures were initially colorless, they gradually developed an intense dark green coloration, increasing turbidity, and thereby reducing the average light intensity available to each bacterial cell.

We also calculated the total O2 production in our experiments, accounting for both the dissolved O2 in the liquid and free O2 present in the headspace. Based on the equilibrium relationship between the aqueous solution and headspace (see Supplementary Information, formula 9), we estimate that O2 production (including the headspace) is approximately 42 times greater than measurements based solely on dissolved O2. This correction is crucial for accurately quantifying true O2 production in our experimental setup.

Oxygen and Fe(II) distribution modeled for the ancient water column
To scale our laboratory-derived O2 production to early ocean conditions, we calculated the oxygen production per cell (rphoto) from the first two days of the experiment, a period when no detectable O2 accumulated in the liquid phase and no measurable cell growth occurred (see “Methods” for details). We calculated the oxygen and Fe(II)(aq) distribution for three upwelling rates: 4, 95, and 473 m/yr. 4 m/yr represents the average upwelling velocity of modern oceans, while 95 m/yr and 473 m/yr are modeled average and enhanced oceanic upwelling velocities in the mid-Cretaceous North Atlantic Ocean, respectively38. Using an upwelling rate of 4 myr⁻¹ (Fig. 5A–D), our model predicted O2 concentrations of at least 400 µM between 50 and 100 m depth in the water column, with the potential for substantially higher values. Given that the maximum O2 solubility in an open systems at room temperature and atmospheric pressure is ~300 µM39,40 (higher in closed systems), these results indicate that such upwelling rates would saturate and fully oxidize the photic zone, leading to complete oxidation of Fe(II)(aq) and excess O2 outgassing to the atmosphere.

By increasing the upwelling rate to 95 myr⁻¹, our model predicts that, at a cyanobacterial abundance of 10⁵ cells mL⁻¹ (Fig. 5E, G), Fe(II)(aq) would have reached the ocean surface. Oxygen concentrations at the surface would have reached between 0.04 and 0.4 µM maximum concentration. At a higher theoretical abundance of 10⁶ cells mL⁻¹, however, the photic zone remained Fe(II)(aq)-free down to depths of ~200 m or ~125 m, respectively. In these high-cell-density scenarios, O2 accumulated to much higher concentrations within the upper 0–50 m of the water column, with modeled values reaching (200 µM) or exceeding (800 µM) maximum O2 saturation.

At an upwelling rate of 473 m/yr, Fe(II)(aq) reached the water surface in most scenarios (Fig. 5I, K, L). Under these conditions, O2 accumulation at the top of the water column was limited, ranging from 0.03 µM (Fig. 5K) to 0.8 µM (Fig. 5L). In scenario J, however, Fe(II)(aq) was absent only from the upper 50 m, allowing O2 to accumulate to 80 µM near the water surface. Overall, the results highlight the strong control of upwelling rate on O2 and Fe(II)(aq) dynamics. At low upwelling rates (4 m/yr), the photic zone was fully oxidized, whereas at 96 m/yr, O2 accumulated only near the surface. In contrast, most scenarios with rapid upwelling (473 m/yr) resulted in Fe(II)(aq) reaching the surface, suppressing O2 buildup and preventing full saturation.

Methods
Cultivation of microorganisms and growth conditions
The cyanobacterium Synechococcus sp. PCC 7002, provided by Gen Enomoto from the University of Freiburg, was cultivated in liquid oxic A+ medium. This medium was prepared using the following stock solutions: Stock 1 (2 mL) (1:2 dilution, containing 3.89 g FeCl3·6H2O, 34.3 g H3BO3, 4.3 g MgCl2·4H2O, 0.315 g ZnCl2, 0.03 g MoO3, 0.003 g CuSO4·5H2O, and 0.0122 g CoCl2·6H2O in Milli-Q water to 1 L), stock 2 (10 mL) (100 g Trizma base adjusted to pH 8.2 with HCl in Milli-Q water to 1 L), stock 3 (100 mL) (0.5 g KH2PO4, 10 g NaNO3, 24.4 g MgSO4, 180 g NaCl, and 6 g KCl in Milli-Q water to 1 L), stock 4 (10 mL) (0.3 g Na2EDTA in Milli-Q water to 100 mL), stock 5 (10 mL) (2.8 g CaCl2 in Milli-Q water to 100 mL). The PCC 7002 culture was grown on a shaker at 60 rpm to a cell density of approximately 10⁸ cells/mL at 25 °C in an Erlenmeyer flask, under a 40-watt halogen light bulb providing an intensity of 300–500 lux.

Setup of Fe(II) oxidation experiments
The Fe(II) oxidation experiments aimed to culture the planktonic cyanobacterium Synechococcus sp. PCC 7002 in the presence of 0, 500, 2500, and 5000 µM Fe(II), along with 0 and 2200 µM dissolved monomeric silica. These experiments were setup in triplicate bottles each to analyze cell growth and accumulated oxygen concentration after complete Fe(II) oxidation by the O2 produced by the cyanobacteria. The bottles were kept at room temperature on a shaker at 60 rpm with day-night-cycles of 8/16 h at intensities of 300–500 lux. Each experiment was conducted in 250 mL Schott bottles containing 100 mL of liquid medium. Anoxic artificial seawater medium (ASW) was prepared with the following composition: 17.3 g NaCl, 8.6 g MgCl2·6H2O, 0.025 g MgSO4·7H2O, 0.99 g CaCl2·2H2O, 0.39 g KCl, 0.059 g KBr, 0.05 g KH2PO4, 0.25 g NH4Cl, and 2.5 g NaHCO3 per 1 L of Milli-Q (MQ) water. Salts, the bicarbonate buffer (30 mM), and the monomeric silica stock solution were separately purged with N2 (or N2/CO2 at 90:10% for the buffer) and autoclaved. After autoclaving, the salt solution in a Widdel flask was flushed with N2/CO2 while still hot (~70–80 °C), and the sterile silica solution was immediately added, resulting in the formation of white flocs. The medium was cooled to 20 °C, the bicarbonate buffer was added, and the pH was adjusted to 7.0 using 1 M anoxic HCl. Due to precipitate formation, the medium was transferred to 2 L Schott bottles and kept at room temperature for two days. It was then filtered through a 0.22 µm PES bottle-top filter inside a glove box. Vitamins and trace element solutions were added under sterile conditions, and the pH was re-adjusted to 7.0. The prepared medium was aliquoted into sterile 250 mL Schott bottles, with each bottle containing a total volume of 100 mL, which included 5 mL of washed cyanobacteria and the respective FeCl2 solution. During this process, a constant stream of N2/CO2 was maintained. Oxygen concentrations were measured, and any bottles with more than 3 µM oxygen were flushed with N2/CO2 again. Subsequently, FeCl2 was added from an anoxic and sterile 100 µM or 1 M stock solution. The pH values were rechecked and remained stable at 7.0. Cyanobacteria were washed three times with ASW before being added to the medium as a 5% inoculum to achieve a final cell density of 10⁶ cells/mL. The experiments were initially fully anoxic, so all oxygen detected during the course of the experiments was produced solely by the inoculated cyanobacteria.

Quantification of Fe(II) and Fe(III) by the spectrophotometric Ferrozine assay
The ferrozine assay is a spectrophotometric method used to quantify the concentration of Fe(II). Samples were first dissolved and diluted in 1 M HCl to achieve a concentration of up to 1 mM Fe. For Fe(II) analysis, 80 µL of 1 M HCl was pipetted into a 96-well plate, followed by the addition of 20 µL of the sample. After a 15-min incubation, 100 µL of ferrozine solution (0.1 w/v) was added. The mixture was allowed to complex for 5 min before being analyzed using a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, USA). Fe(II) is forming a purple complex with ferrozine molecules, which absorbs light at 562 nm. For total iron (Fe(tot)) determination, the procedure was modified by replacing the 1 M HCl with 80 µL of 10% (v/v) hydroxylammonium chloride (HAHCl) before adding the sample. This step was followed by a longer incubation period of 30 min to ensure the complete reduction of Fe(III) to Fe(II). To prevent degradation, both the ferrozine and HAHCl solutions were stored and incubated in the dark, as they are highly light-sensitive. Different standards between 0 and 1000 µM were used to calculate the respective iron concentration.

Quantification of dissolved silica by the molybdenum blue method
Dissolved silica, including monomeric silica and colloidal silicic acid (Si-Si-colloids), was quantified by the Molybdenum Blue Method70. The samples were filtered (0.22 µm, PES) and diluted in MQ to 1 mL. Subsequently, 40 µL of an ammonium heptamolybdate tetrahydrate (6.33 g) together with 50 mL of 4.5 M sulfuric acid solution were added to form a blue complex with the silica. To avoid complexing with phosphate, 40 µL of a 10 g/100 mL MQ oxalic acid solution was added. Lastly, 20 µL of 0.16 M ascorbic acid was added as reductant. After 30–60 min of incubation, the blue silica-molybdenum complexes were quantified at 810 nm. Different standards between 1 and 100 µM were used to calculate the silica concentration. Due to the filtration of the samples (0.22 µm) and removal of Si precipitates, the measured remaining silica fraction is named ‘dissolved silica’.

Oxygen quantification by optode sensors
Free dissolved oxygen was quantified using a luminophore optode foil from PreSens. Small 3 by 3 mm pieces of foil were affixed with silicone glue within the lower 2 cm of the bottles to ensure direct contact with the liquid phase. The interaction between the optode foil (luminophore) and oxygen molecules (quencher) results in an energy transfer that reduces the luminescence signal of the foil. The oxygen concentration in the liquid phase was then calculated using the Stern-Volmer equation. A temperature sensor was placed in a co-incubated Schott bottle under identical experimental conditions to account for temperature variations. Each container and optode foil setup underwent a fresh two-point calibration before measurements. The calibration included one data point at full oxygen saturation and another one under oxygen-free conditions. For the first calibration point, 100 mL of pure ASW medium was added to a 250 mL Schott bottle containing the optode foil and stirred vigorously until complete O2 saturation was achieved. For the second calibration point, the reductant sodium dithionite (Na2S2O3) was added to the same bottle to eliminate all remaining dissolved oxygen.

Intracellular ROS measurements
The membrane-permeable dye CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; Life Technologies GmbH) was used to quantify intracellular reactive oxygen species. Log-phase cultures were harvested via centrifugation and washed twice with TRIS buffer (pH 7.0). Cells were adjusted to a concentration of 5 × 108 cells/mL in TRIS buffer and degassed in the dark using a 90:10 N2/CO2 gas mixture. FeCl2 was added from an anoxic stock solutions to achieve final concentrations of 0, 500, 2500, and 5000 µM We then added pH-adjusted silica from a metasilicate stock solution, pre-heated to 90 °C in a water bath to monomerize the silica, leading to a final concentrations of 0 and 2200 µM in duplicates in two different experimental runs. The suspensions were shaken for one hour. After incubation, cells were resuspended in fresh buffer containing 5 µM CM-H2DCFDA and incubated in the dark for 30 min. Following a second resuspension in fresh buffer, fluorescence emission at 519 nm (excitation at 490 ± 5 nm) was recorded using the infinite 200 PRO Fluorometer from Tecan Life Sciences. Background fluorescence from untreated cells and Fe minerals was subtracted to ensure accuracy.

Cell numbers by hemocytometry
The cell density was visually counted under a Leica CTR5500 microscope using a Neubauer-improved hemocytometer from Hirschmann. Particularly, 10 µL of sample was pipetted between sample holder and coverslip. At a magnification of 400x, the cells were counted in the smallest squares until a final count of 100 cells. 180 µL cells were fixed by the addition of 20 µL 21% PFA solution. The minerals were dissolved by the addition 200 µL of 100 mM Fe(II)-EDAS solution and 600 µL of ca. 0.8 M oxalic acid The cell count per small square was then multiplied by the dilution factor and by 2.5 × 107 as volume factor to get the real cell count in mL/L.

MATLAB model from lab experimental data
We modeled our lab experimental data to quantify total oxygen production in the system in MATLAB (version R2023b), considering both dissolved oxygen and the contribution from the headspace. Based on the equilibrium partitioning between the aqueous solution in the head space based on the Henry-Law (see SI formula 9), oxygen production including the headspace is approximately 42 times greater than what is measurable in the dissolved liquid phase alone. Additionally, increasing cell density caused greater turbidity in the liquid phase, which led to shading effects reducing the overall photosynthetic rate per cell. We accounted for this by modeling the proportion of active versus inactive cells in the system. Detailed methods and the corresponding MATLAB code are provided in the Supplementary Information (SI).

MATLAB model to mimic the oxygen and Fe(II) distribution in the ancient ocean
To simulate oxygen distribution in ancient ocean water columns, we used the photosynthetic rate (rphoto) derived from our lab experiments under the most realistic conditions—500 µM Fe(II) and 2200 µM silica. We divided the experiment into two phases: Phase 1, when dissolved Fe²⁺ was still present, mimicking early ocean conditions, and Phase 2, after Fe²⁺ oxidation, to analyze the long-term bacterial response following Fe²⁺ exposure. From Phase 1, we calculated the daily oxygen production per cell (rphoto). According to reaction (5), one molecule of O2 can oxidize four Fe²⁺ ions.

O2 + 4Fe2+ + 8OH- $ 4FeOOH + 2H2O (5)

We determined rphoto from the first two days of the experiment, during which no free oxygen was detected, and cyanobacteria were still in the lag phase without measurable growth, allowing the simplification that all produced oxygen was used for Fe(II) oxidation and the cell count stayed the same. Starting with 529 µM Fe(II) at day 0, 150 µM was oxidized over two days, corresponding to a rate of 75 µM/day Fe(II) oxidation, which equates to 18.75 µmol O2/day based on reaction (5). Due to uncertainty in manual cell counts, we calculated rphoto for two different cell densities observed in our experiments: 6.48 × 10⁶ cells/mL (experimental cells 1) and 1.6 × 10⁷ cells/mL (experimental cells 2). Reported early ocean cyanobacterial concentrations range between 10⁵ cells/mL (target cells 1) and 10⁶ cells/mL (target cells 2), so we modeled four scenarios combining these experimental and target cell densities to calculate rphoto values (see Table 3) using formula 6. Based on the study of Swanner and colleagues12, we further estimated oxygen distribution in the early ocean water column for upwelling rates of 4, 94, and 473 m/year.

18:75 µM * target cells / experimental cells = rphoto (6)

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors investigate the hypothesis that elevated Fe(II) concentrations in the Archaean oceans may have limited cyanobacterial growth, potentially explaining the ~500 million-year gap between the emergence of oxygenic photosynthesis and the Great Oxidation Event. Through a series of experiments in which a cyanobacterial strain was inoculated in media with varying Fe(II) and SiO₂ concentrations, the authors conclude that SiO₂ could have mitigated Fe toxicity in the early oceans.\nThis study follows up on Swanner et al. (2015a, 2015b) and adopts much of their experimental design and modeling framework. Consequently, its main novelty lies in introducing SiO₂ as an additional variable in the system. Overall, I find the study insufficiently novel. Moreover, the presentation of the results and discussion could be streamlined to improve clarity and flow, as the current structure is difficult to follow. In its present form, I cannot recommend publication in Nature Communications.\n1.- While I understand the authors support of their model on oxygenic photosynthesis driving the origin of banded iron formations. The abstract and introduction include strong claims such as “Banded iron formations (BIF) were deposited… with cyanobacterial oxygen (O2) as the main oxidant for Fe(II)(aq) oxidation” which could be nuanced to include the possibility of abiotic processes contributing to the formation of BIF deposits (e.g. photooxidation of Fe(II) or greenalite precursor models)\n2.- The hypothesis regarding elevated SiO₂ conditions and their potential mitigating effects is not well motivated in the introduction, leaving the reader uncertain about why this variable is important to investigate in the first place. Much of the content in lines 456–475 does not directly discuss the results but instead introduces a relevant body of background work that should be presented earlier in the manuscript to establish a stronger rationale for the study.\n3.- The authors tested multiple conditions, which increases the robustness of the study. However, the results are presented in a manner that makes it difficult for the reader to discern the major conclusions and differences between the various conditions. Since the main focus of the paper is on the effects of SiO₂ on Fe(II) toxicity, it may be more effective to first present the overall trends associated with SiO₂ presence or absence, followed by a more detailed analysis of the specific Fe(II) conditions. This approach would help the reader better appreciate the key patterns in the data.\n3.- Following the same reasoning, the presentation of the figures could be improved to better facilitate comparisons between the different experimental conditions. For example, lines 145–146 state that “...This O₂ accumulation peaked earlier in the ‘no silica’ setup (after 50 days) compared to the ‘high silica’ treatment (after 60–70 days) (Fig. 1E),” yet Fig. 1E only shows the first ~10 days of data, while the complete dataset is actually presented in Fig. 2. Similar issues occur throughout the remainder of the section, making it difficult to link the authors’ claims to the data shown in the figures, as each Fe condition is plotted on a different time scale.\nSimilarly, the water-column modeling section presents three figures (Figures 5–7) comprising a total of 24 panels, which makes data comparison and identification of the main conclusions challenging for the reader.\nHere are some suggestions that I believe would improve the figure presentation:\n- Figures 1 and 2 could be combined into a single figure. It seems the authors intended to highlight the timescales at which Fe(II) concentrations approached zero in solution. I suggest using a consistent time scale for each Fe condition (e.g., displaying the full 150-day dataset) and including insets of the Fe(II) concentration curves to show the early stages in more detail (e.g., the first 10, 30, or 90 days).\n- Figures 5, 6, and 7 could also be combined into a single figure, as most panels share similar Fe(II) and O₂ scales for the 10⁶–10⁷ cells/ml. Incorporating colored curves and clear legends would greatly improve the visual comparability of the data.\n3.- The discussion section presents mixed results. In particular, lines 382–394 introduce a new dataset on day-night cycles well after the main results and several paragraphs of discussion, making it difficult to connect these findings to the broader implications for the system.\nMinor comments/suggestions\n1. Line 129.- Should be Fig S1 A, not Fig 1A\n2. In Fig 3. The presented intensities relative to the abiotic and anoxic 5000 uM Fe condition is creating negative intensities/counts for the 2500 and 5000 uM Fe conditions, which can be confusing to the reader and at first glance might be interpreted as if there is something interfering with the spectrophotometric measurements. I would suggest normalizing the results instead to remove this artifact.\n3. For the ROS measurements, why was a metasilicate solution used instead of the monomeric silica applied in the long-term experiments?", "reviewer_2": "This study seeks to understand the chemical factors that may have influenced the proliferation of cyanobacteria and the Great Oxidation Event, topics of broad interest to the geoscience community. The study specifically focuses on the potential connections among iron toxicity, oxidative stress, and elevated Fe(II) concentrations in Archean oceans as factors that may have limited cyanobacterial proliferation and the impact of high SiO2 concentrations in Archean seawater as a potential mitigating factor in iron toxicity and ROS generation.\nThe study uses an experimental approach to test the impact of Fe(II) concentrations, oxidative stress, and SiO2 concentrations on cyanobacterial growth and O2 production. The authors measure O2 concentrations, Fe(II) and SiO2 concentrations, Fetotal, cell numbers, and ROS production to characterize the interactive effects of Fe(II) and SiO2 on cyanobacterial physiology, oxygen production, and growth. These experimental findings are paired with geochemical modeling to characterize total O2 production under Archean-type conditions and scale the laboratory results to early oceans. Together, the experimental and modeling results demonstrate how day/night cycles and elevated SiO2 concentrations in Archean oceans could have mitigated ROS stress and Fe(II) toxicity, playing instrumental roles in the proliferation of cyanobacteria and the GOE.\nOverall, the manuscript is well written, the experiments are well designed, and the results are compelling and novel. This study provides important new experimentally derived insights into the ways in which Fe(II) toxicity and mitigation of toxicity and ROS generation by SiO2 may have impacted cyanobacterial proliferation, oxygen accumulation, and the GOE. The experimental results convincingly demonstrate that Fe(II) toxicity negatively impacts cyanobacterial growth and O2 production. The experiments also clearly demonstrate that under elevated concentrations of SiO2 (similar to estimates of Archean oceans), the impact of Fe(II) toxicity and ROS stress is diminished, evidenced by higher growth and O2 production and diminished ROS in high SiO2 cultures. Using these experimental findings, the study refines models that predict O2 accumulation and ocean Fe(II) concentrations during the Archean and demonstrates that elevated SiO2 concentrations in Archean oceans may have played a key role in cyanobacterial proliferation and the GOE.\nThe findings are novel, and the results will be of broad interest. I support publication of the manuscript, but I have several comments and suggestions outlined below and in the attached document regarding details of experimental design, presentation of the data, and potential discussion points that would strengthen the manuscript. Broadly, 1) the introduction and discussion could be strengthened by posing a possible mechanism to explain *how* silica is mitigating the impacts of Fe(II) toxicity and ROS, 2) some details of the experimental design should be mentioned in the main text (see below), 3) the data in figure 1 should be plotted on the same timescales to more robustly show trends highlighted in the text, 4) there are some mentions of the precipitates but no data characterizing what these precipitates are – if these data were collected, it would be useful to show them. These comments and other minor suggestions are outlined in the attached document with line references.", "reviewer_3": "Dreher et al. report new experimental data testing the effects of dissolved Fe(II) and SiO2 on Synechococcus O2 production rates. The study is a more detailed and improved version of earlier ones (e.g., Swanner et al. 2015). The study results are neat and novel and could have important implications for Archean biogeochemical cycles. The results could also shed some light on enigmatic Archean iron formation depositional conditions.\nI am not an experimentalist by training; I hope at least one other reviewer is. But I do study Archean Earth. My personal opinion is that the findings are probably novel enough to merit publication in Nature Communications. Many knowledge gaps remain unfilled in our understanding of Archean Fe cycling—and many scientists are active in this area. This paper could provide new perspective.\nMy most impactful comments are:\n1. The authors need to dedicate at least some text in the introduction to explaining why high Archean dissolved Si concentrations are important. Why do we think this was the case, and why is that information important? This is incredibly important because it is the true novelty of these new experiments.\n2. Salinity does not seem to have been considered in the O2 solubility calculations. I think this should change.\n3. How might strictly benthic Archean cyanobacteria impact their findings? Some researchers would argue that all Archean cyanos were benthic and stuck to the seafloor (i.e., not planktonic and free-floating; Sanchez-Baracaldo, 2015). How might this assumption change their hypotheses, if at all?\nSome line-by-line comments:\nLine 106: Why are high silica contents important for the problem? I know why, but the average reader may not. A few sentences explaining the motivation for exploring high silica seem necessary.\nLine 109: Was a synthetic seawater-like solution used for the experiments? Or a freshwater solution? This information might be important enough to merit mention here, in the main text.\nLine 203: This intro sentence foreshadows some discussion of ROS in the paragraph. But the ROS discussion never arrives. Is this intentional? Should the authors consider a different opening to this paragraph?\nLine 282: Has anyone estimated these upwelling velocities for the Archean?\nLine 287: How is salinity treated in this calculation? The solution is salinity-dependent.\nLine 302: Consider stating the dissolved O2 abundances in the surface waters for this set of calculations (you did it for the previous ones, and reading on also the subsequent ones).\nLine 330: Have the authors considered calculating how much O2 is released to the atmosphere in each scenario? Thereafter, they may also consider thinking about whether the calculation is consistent with pO2 estimates for the Archean (e.g., those derived from S-MIF, or detrital redox-sensitive grains).\nLine 336: What are those maximum O2 concentrations? Please state them here.\nLine 343: Please cite the “earlier models”.\nLine 367: An end-parentheses is missing."}, "author_response": "Response to Reviews\nResponse to Reviewer #1:\n(Remarks to the Author): The authors inves@gate the hypothesis that elevated Fe(II) concentra@ons in the Archaean oceans may have limited cyanobacterial growth, poten@ally explaining the ~500 million-year gap between the emergence of oxygenic photosynthesis and the Great Oxida@on Event. Through a series of experiments in which a cyanobacterial strain was inoculated in media with varying Fe(II) and SiO₂ concentra@ons, the authors conclude that SiO₂ could have mi@gated Fe toxicity in the early oceans. This study follows up on Swanner et al. (2015a, 2015b) and adopts much of their experimental design and modeling framework. Consequently, its main novelty lies in introducing SiO₂ as an addi@onal variable in the system. Overall, I find the study insufficiently novel. Moreover, the presenta@on of the results and discussion could be streamlined to improve clarity and flow, as the current structure is difficult to follow. In its present form, I cannot recommend publica@on in Nature Communica@ons.\nWe thank reviewer 1 for the helpful feedback and have revised our manuscript accordingly, see detailed comments below.\n1. While I understand the authors support of their model on oxygenic photosynthesis driving the origin of banded iron forma@ons. The abstract and introduc@on include strong claims such as “Banded iron forma@ons (BIF) were deposited… with cyanobacterial oxygen (O2) as the main oxidant for Fe(II)(aq) oxida@on” which could be nuanced to include the possibility of abio@c processes contribu@ng to the forma@on of BIF deposits (e.g. photooxida@on of Fe(II) or greenalite precursor models)\nWe changed the abstract and introduc@on following the reviewer’s recommenda@on to make sure other deposi@on pathways are sufficiently credited.\nLines 24-26: “Banded iron forma@ons (BIF) were deposited abundantly between 2.7-2.4 Ga from iron- and silica-rich oceans, with cyanobacterial oxygen (O ) as a possible oxidant for Fe(II) oxida@on and 2 (aq) Fe(III) oxyhydroxide precipita@on.”\nLines 55-58: “While the deposi@on of the oldest BIF was probably linked to anoxygenic phototrophic Fe(II)-oxidizers 22,23, photochemical Fe(II) oxida@on 24,25 or abio@c deposi@on processes such as chemical greenalite deposi@on 26,27, the prolifera@on of cyanobacteria would have accelerated Fe(II) oxida@on and thus BIF deposi@on 12,28.”\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\n2. The hypothesis regarding elevated SiO₂ condi@ons and their poten@al mi@ga@ng effects is not well mo@vated in the introduc@on, leaving the reader uncertain about why this variable is important to inves@gate in the first place. Much of the content in lines 456–475 does not directly discuss the results but instead introduces a relevant body of background work that should be presented earlier in the manuscript to establish a stronger ra@onale for the study.\nThe authors agree that the mo@va@on of using SiO should be men@oned earlier in the introduc@on 2 and its role in mi@ga@ng poten@al Fe(II)-related toxicity emphasized. We, therefore, revised the introduc@on as follows:\nLines 108-110: “Dreher and colleagues 34 reported increasing Fe(II) toxicity with increasing Fe(II) (aq) (aq) concentra@ons, but observed markedly enhanced bacterial growth compared in the presence of silica. Based on these results, we hypothesize that silica reacts with Fe(II) thus dis@nctly lowering (aq), Fe(II) reac@vity and preven@ng Fe(II)-induced ROS forma@on. To test this hypothesis, we experimentally examined the combined effects of Fe(II) and SiO on cyanobacterial O (aq) 2(aq) 2 produc@on, Fe(II) oxida@on, and poten@al ROS-related toxicity.”\n3.-The authors tested mul@ple condi@ons, which increases the robustness of the study. However, the results are presented in a manner that makes it difficult for the reader to discern the major conclusions and differences between the various condi@ons. Since the main focus of the paper is on the effects of SiO₂ on Fe(II) toxicity, it may be more effec@ve to first present the overall trends associated with SiO₂ presence or absence, followed by a more detailed analysis of the specific Fe(II) condi@ons. This approach would help the reader bePer appreciate the key paPerns in the data.\nWe appreciate the reviewer’s sugges@on to restructure the Results sec@on and note that we explored this approach by beginning directly away with the effect of Si on Fe(II) toxicity. However, this organiza@on immediately raises a fundamental ques@on for the reader: “what is the baseline level of O produc@on under “healthy” condi@ons, i.e., in the absence of both Fe(II) and Si, against which all 2 other treatments are compared?. For this reason, we believe it is clearer to retain the current structure and to present the results systema@cally, star@ng with the unamended control experiments (no Fe(II), no Si), followed by experiments with increasing Fe(II) concentra@ons in the absence of Si, and finally those with Fe(II) in the presence of Si. This progression allows the rescuing effect of Si on Fe(II) toxicity to be demonstrated unambiguously. We hope that this ra@onale is acceptable to the editor and reviewer.\n3.- Following the same reasoning, the presenta@on of the figures could be improved to bePer facilitate comparisons between the different experimental condi@ons. For example, lines 145–146 state that “...This O₂ accumula@on peaked earlier in the ‘no silica’ setup (aper 50 days) compared to the ‘high silica’ treatment (aper 60–70 days) (Fig. 1E),” yet Fig. 1E only shows the first ~10 days of data, while the complete dataset is actually presented in Fig. 2. Similar issues occur throughout the remainder of the sec@on, making it difficult to link the authors’ claims to the data shown in the figures, as each Fe condi@on is ploPed on a different @me scale.\nSimilarly, the water-column modeling sec@on presents three figures (Figures 5–7) comprising a total of 24 panels, which makes data comparison and iden@fica@on of the main conclusions challenging for the reader.\nWe agree with the reviewer that plo9ng datasets on different @mescales may ini@ally appear counterintui@ve. However, if all experiments were displayed on a uniform 90-day x-axis (as in the 5000 µM setup), the differences in Fe(II) oxida@on rates would be obscured. The @mescale most relevant for the reader is the interval of ac@ve Fe(II) oxida@on by the cyanobacteria, and we therefore focus on (i.e., zoom into) this period. To avoid confusion, we have clarified this ra@onale in the figure cap@on and, following the reviewer’s sugges@on, explicitly refer to Fig. 2, where the longer-term effects on cell counts and oxygen produc@on are presented.\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\nLines 145-150: “Fig. 1: Geochemical data of Fe(II) oxida@on experiments containing Synechococcus sp. PCC 7002. Panels A to C show the results of setups without amendment of Fe(II)(aq). Panels D to F show the results of setups with amendment of 500 µM Fe(II)(aq), panels G to I with 2500 µM Fe(II)(aq), and panels J to L with 5000 µM Fe(II)(aq). Filled symbols indicate setups with 2200 µM silica (‘high silica’), empty symbols indicate setups without silica (‘no silica’). Fe(II) (grey), accumulated oxygen (blue), cell numbers (green) are ploPed as average values in three triplicates with the standard devia@on as error bars of the experiments. Please note that the @me scale (x-axis) varies for the different plots because of the differences in the dura@on of ac@ve Fe(II) oxida@on.”\nLines 132-135: “The experiments were ploPed in the respec@ve @mescale of the ac@ve Fe(II) oxida@on, as this sets the focus on the respec@ve period with Fe(II)(aq) in the system, which is the most relevant for determining toxicity effects and the laPer modelling.”\nHere are some sugges@ons that I believe would improve the figure presenta@on:\n- Figures 1 and 2 could be combined into a single figure. It seems the authors intended to highlight the @mescales at which Fe(II) concentra@ons approached zero in solu@on. I suggest using a consistent @me scale for each Fe condi@on (e.g., displaying the full 150-day dataset) and including insets of the Fe(II) concentra@on curves to show the early stages in more detail (e.g., the first 10, 30, or 90 days).\n- Figures 5, 6, and 7 could also be combined into a single figure, as most panels share similar Fe(II) and O₂ scales for the 10⁶–10⁷ cells/ml. Incorpora@ng colored curves and clear legends would greatly improve the visual comparability of the data.\nWe understand the reviewer’s point and generally agree that using iden@cal @mescales across plots is, in principle, desirable. Accordingly, we prepared the figure following the reviewer’s sugges@on (see figure provided below this response). However, our assessment is that the revised version is less effec@ve, because when all experiments are shown on the same @mescale the key dynamics occurring over very short intervals (par@cularly in the 500 and 2500 µM Fe(II) treatments) become visually indis@nguishable. As a result, the main panels occupy substan@al space while conveying liPle informa@on, and the reader is effec@vely forced to rely on the inset plots to see where the relevant processes occur. For this reason, we believe it is clearer to retain Figures 1 and 2 in their original form, while more explicitly explaining the ra@onale for the different @mescales in the cap@ons and improving cross-referencing to specific figures and panels in the main text. We note that Figures 1 and 2 were designed to address dis@nct aspects of the study: Figure 1 focuses on the period of ac@ve Fe(II) oxida@on, whereas Figure 2 illustrates the longer-term toxicity effects. Nevertheless, if the editor prefers the revised figure with uniform @mescales, we are of course happy to adopt that format. Finally, following the reviewer’s sugges@on, we have combined figures 5, 6 and 7.\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\n3.- The discussion sec@on presents mixed results. In par@cular, lines 382–394 introduce a new dataset on day-night cycles well aper the main results and several paragraphs of discussion, making it difficult to connect these findings to the broader implica@ons for the system.\nWe would like to thank the reviewer for this comment and are happy to clarify this point. The dataset referred by the reviewer actually stems from a separate study. In the mean@me, since submission of the present paper to Nature Communica@ons, the paper containing the men@oned dataset has been published and is now cited in the text (Dreher et al., 2025).\nLines 389-391: “In these con@nuous light experiments, the presence of 2200 µM silica resulted in remarkably higher cell counts and oxygen concentra@ons compared to the silica-free equivalents 34.”\nMinor comments/sugges@ons\n1. Line 129.- Should be Fig S1 A, not Fig 1A\nChanged, thank you for no@cing.\n2. In Fig 3. The presented intensi@es rela@ve to the abio@c and anoxic 5000 uM Fe condi@on is crea@ng nega@ve intensi@es/counts for the 2500 and 5000 uM Fe condi@ons, which can be confusing to the reader and at first glance might be interpreted as if there is something interfering with the\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\nspectrophotometric measurements. I would suggest normalizing the results instead to remove this ar@fact.\nWe would like to thank the reviewer for this comment. As noted, all data presented are already normalized: specifically, all counts were normalized to an abio@c, anoxic reference setup containing 5000 µM Fe(II) and 2200 µM of SiO . Consequently, the nega@ve values iden@fied by the reviewer 2 indicate that counts measured in the bio@c setup (i.e., those including cyanobacterial cells) were lower than those in the abio@c setup. Since the data are normalized, it is important to show the nega@ve values. However, we agree that this might ini@ally be confusing for the reader, and we have therefore explained this in more detail in the figure cap@on, as follows:\nLines 247-249: “and presence of 2200 µM SiO (orange). Please note that the normaliza@on of all 2(aq) measurements to the abio@c control can result in nega@ve values. Error bars show the standard….”.\n3. For the ROS measurements, why was a metasilicate solu@on used instead of the monomeric silica applied in the long-term experiments?\nThe original mixture consisted of a metasilicate solu@on in MQ water, which was then heated in a water bath to 90°C to create monomeric silica. We now clarified this in the method sec@on.\nLines 572-575: “FeCl was added from anoxic stock solu@ons to achieve final concentra@ons of 0, 500, 2 2500 and 5000 µM. We then added pH-adjusted silica from a metasilicate stock solu@on preheated to 90°C in a water bath to monomerize the silica, leading to a final concentra@ons of 0 and 2200 µM in duplicates in two different experimental runs.”\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\nResponse to Reviewer #2\n(Remarks to the Author): This study seeks to understand the chemical factors that may have influenced the prolifera@on of cyanobacteria and the Great Oxida@on Event, topics of broad interest to the geoscience community. The study specifically focuses on the poten@al connec@ons among iron toxicity, oxida@ve stress, and elevated Fe(II) concentra@ons in Archean oceans as factors that may have limited cyanobacterial prolifera@on and the impact of high SiO2 concentra@ons in Archean seawater as a poten@al mi@ga@ng factor in iron toxicity and ROS genera@on.\nThe study uses an experimental approach to test the impact of Fe(II) concentra@ons, oxida@ve stress, and SiO2 concentra@ons on cyanobacterial growth and O2 produc@on. The authors measure O2 concentra@ons, Fe(II) and SiO2 concentra@ons, Fetotal, cell numbers, and ROS produc@on to characterize the interac@ve effects of Fe(II) and SiO2 on cyanobacterial physiology, oxygen produc@on, and growth. These experimental findings are paired with geochemical modeling to characterize total O2 produc@on under Archean-type condi@ons and scale the laboratory results to early oceans. Together, the experimental and modeling results demonstrate how day/night cycles and elevated SiO2 concentra@ons in Archean oceans could have mi@gated ROS stress and Fe(II) toxicity, playing instrumental roles in the prolifera@on of cyanobacteria and the GOE.\nOverall, the manuscript is well wriPen, the experiments are well designed, and the results are compelling and novel. This study provides important new experimentally derived insights into the ways in which Fe(II) toxicity and mi@ga@on of toxicity and ROS genera@on by SiO2 may have impacted cyanobacterial prolifera@on, oxygen accumula@on, and the GOE. The experimental results convincingly demonstrate that Fe(II) toxicity nega@vely impacts cyanobacterial growth and O2 produc@on. The experiments also clearly demonstrate that under elevated concentra@ons of SiO2 (similar to es@mates of Archean oceans), the impact of Fe(II) toxicity and ROS stress is diminished, evidenced by higher growth and O2 produc@on and diminished ROS in high SiO2 cultures. Using these experimental findings, the study refines models that predict O2 accumula@on and ocean Fe(II) concentra@ons during the Archean and demonstrates that elevated SiO2 concentra@ons in Archean oceans may have played a key role in cyanobacterial prolifera@on and the GOE.\nThe findings are novel, and the results will be of broad interest. I support publica@on of the manuscript, but I have several comments and sugges@ons outlined below and in the aPached document regarding details of experimental design, presenta@on of the data, and poten@al discussion points that would strengthen the manuscript. Broadly, 1) the introduc@on and discussion could be strengthened by posing a possible mechanism to explain *how* silica is mi@ga@ng the impacts of Fe(II) toxicity and ROS, 2) some details of the experimental design should be men@oned in the main text (see below), 3) the data in figure 1 should be ploPed on the same @mescales to more robustly show trends highlighted in the text, 4) there are some men@ons of the precipitates but no data characterizing what these precipitates are – if these data were collected, it would be useful to show them. These comments and other minor sugges@ons are outlined in the aPached document with line references.\nWe thank reviewer #2 for the posi@ve and detailed feedback. Please find below our responses to all comments.\n1. Line 106: The introduc@on does a great job of pain@ng a picture both of the connec@on between Fe(II) and oxida@ve stress, the poten@al mi@ga@on of these impacts by elevated SiO2 concentra@ons, and rela@onship between Fe and SiO2 in BIFs indica@ng iron-silica interac@ons in Archean oceans. However, the specific connec@on between Fe(II) stress and SiO2 mi@ga@on and the poten@al underlying mechanism is not clear. What is the specific hypothesis or hypothesized mechanism being tested? That Si may remove FeII? Or that it may directly interact with the cells in some way? Men@oning the hypothesized mechanism in the introduc@on and the discussion (see later comments) would be of interest to the community and strengthen the findings from the study.\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\nThe authors agree with this comment. Following the reviewer’s sugges@on, we now explicitly state the hypothesis that silica and Fe(II) interact with each other, therefore partly preven@ng or at least aq dis@nctly slowing down the Fenton’s reac@on of Fe(II) with O . aq 2\nLine 108-114: “Dreher and colleagues 34 reported increasing Fe(II) toxicity with increasing Fe(II) (aq) (aq) concentra@ons, but observed markedly enhanced bacterial growth compared in the presence of silica. Based on these results, we hypothesize that silica reacts with Fe(II) thus dis@nctly lowering (aq), Fe(II) reac@vity and preven@ng Fe(II)-induced ROS forma@on. To test this hypothesis, we experimentally examined the combined effects of Fe(II) and SiO on cyanobacterial O (aq) 2(aq) 2 produc@on, Fe(II) oxida@on, and poten@al ROS-related toxicity.”\n2. Line 120: Although the experimental design is outlined in the results sec@on, it would be useful to include a brief note at the beginning of the results sec@on to help the reader follow the results. Just a sentence or two no@ng the basic setup, number of replicates, and variables being tested.\nWe agree that this would be useful and therefore added two sentences in the beginning of the results sec@on. Addi@onally, we added the informa@on that the experiments were performed in triplicates to the figure cap@ons of Fig. 1 and 2.\nLines 126-129: “We conducted Fe(II) oxida@on experiments, under ini@ally anoxic condi@ons, using Fe(II) concentra@ons ranging from 0.5 to 5 mM. Following inocula@on with the cyanobacterial (aq) strain Synechococcus PCC 7002, we monitored Fe(II), Fe(tot), cyanobacterially produced O , dissolved 2 Si ,and cell counts over @me (for sterile controls see Fig. S3).”\nLines 147-149 +230-241-232: “Filled symbols indicate setups with 2200 µM silica (‘high silica’), while empty symbols indicate setups without silica (‘no silica’). Fe(II) (grey), accumulated oxygen (blue), cell numbers (green) are ploPed as average values from triplicates with the standard devia@ons shown as error bars.”\n3. Results/Methods: Related to this, it is not clear between the ini@al growth condi@ons and experimental condi@ons whether the experiments were conducted under anoxic condi@ons. It would be helpful to explicitly state this at the beginning of the results sec@on as this is a key parameter. Whether the experiments were conducted under oxic or anoxic condi@ons has strong implica@ons for Fe oxida@on both at @me 0 and over the course of the experiments as well as the changes in O2 observed.\nThe authors agree that it is important to point out that all oxygen measured during the experiment is the result of cyanobacterial ac@vity. We now explicitly men@on this in text.\nLines 126-129: “We conducted Fe(II) oxida@on experiments, under ini@ally anoxic condi@ons, using Fe(II) concentra@ons ranging from 0.5 to 5 mM. Following inocula@on with the cyanobacterial (aq) strain Synechococcus PCC 7002, we monitored Fe(II), Fe(tot), cyanobacterially produced O , dissolved 2 Si ,and cell counts over @me (for sterile controls see Fig. S3).”\nLines 524-526: “The experiments were ini@ally fully anoxic, so all oxygen detected during the course of the experiments was produced solely by the inoculated cyanobacteria.”\n4. Results/Methods: Also related to the experimental design, were sterile controls used to confirm that Fe(II) was not oxidized in the absence of cyanobacteria?\nThanks for this excellent ques@on. We added plots of the sterile (abio@c) control setups to the SI. We ran two anoxic controls in parallel, one containing 5000 µM Fe(II) and 2200 µM silica, the other (aq) one containing no addi@onal Fe(II) and no silica. (aq)\nFig.S3: “Sterile controls of the Fe(II) oxida@on experiments. Panel A shows the results of setups without amendment of Fe(II) or Si. Panel B shows the results of setups with amendment of (aq) 5000 µM Fe(II) and 2200 µM Si.” (aq)\nLines 127-129. “Following inocula@on with the cyanobacterial strain Synechococcus sp. PCC 7002, we monitored Fe(II), Fe(total), cyanobacterially produced O , dissolved silica, and cell counts over @me 2 (for sterile controls see Fig. S3).”\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\n5. Line 126 and Figure 1A: Why does Fe(II) increase in the control condi@on by the end of the experiment?\nThank you for this comment. The total Fe(II) indeed increased from ca. 40 µM to approximately 60 µM. Since in these experiments no Fe(II) was added like in the 500 mM, 2500 µM and 5000 µM Fe(II) experiments, the detected Fe(II) exclusively stems from the trace metal solu@on of the growth medium. We assume the fluctua@on in detected concentra@ons is related to precipita@on (and remobiliza@on) of Fe(II) with phosphate or silica at the glass wall. We have detected this before (No@ni et al., 2019).\n6. Figure 1: Why are the plots in Fig 1 ploPed on different @mescales? It would be more useful to directly compare the experiments over the same @mescales (x axis) and then re-scale the Y axes so that small changes (like those in A, E, F, and I) can be more clearly seen.\nSee also response to a similar comment by reviewer #1: We agree that it might seem a bit counterintui@ve to plot all the datasets on different @mescales. However, if every setup would be ploPed on an x-axis @mescale of 90 days (like the 5000 µM setup), the differences in the Fe(II) oxida@on rates would disappear completely. The @mescale that is relevant for the reader, however, is the @meframe of ac@ve Fe(II) oxida@on by the cyanobacteria - thus is why we zoomed in into this period. To clarify this and avoid confusion, we have now clarified this in the figure cap@on and also directly refer to “Fig. 2”, were the long-term effect on the cell counts and oxygen produc@on are described.\nLines 145-150: “Fig. 1: Geochemical data of Fe(II) oxida@on experiments containing Synechococcus sp. PCC 7002. Panels A to C show the results of setups without amendment of Fe(II) . Panels D to F (aq) show the results of setups with amendment of 500 µM Fe(II) , panels G to I with 2500 µM Fe(II) , (aq) (aq) and panels J to L with 5000 µM Fe(II) . Filled symbols indicate setups with 2200 µM silica (‘high (aq) silica’), empty symbols indicate setups without silica (‘no silica’). Fe(II) (grey), accumulated oxygen (blue), cell numbers (green) are ploPed as average values in three triplicates with the standard devia@on as error bars of the experiments. Please note that the @me scale (x-axis) varies for the different plots because of the differences in the dura@on of ac@ve Fe(II) oxida@on.”\n7. Line 148: Although error is stated in the table, it would be helpful to state in the text to make it clear that the oxida@on rates at this concentra@on of silica and Fe(II) were similar within error. As wriPen, it seems to imply that there is a difference in the oxida@on rates with and without silica, but these values are similar within error at the 500 μM Fe(II) concentra@on based on the table.\nAgreed, we added this informa@on.\nLines 159-160: “With and without silica, the Fe(II) oxida@on rates were similar within the calculated error (Fehler! Verweisquelle konnte nicht gefunden werden.).”\n8. Line 163: The color changes do not seem to be the key result, especially given the poten@al confounding variables and mechanisms underpinning the color changes that the authors note. These \"color change\" results could be moved to the supplemental document so that the results of the main text could more clearly focus on the key quan@fiable trends.\nWe would like to thank the reviewer for this feedback and understand that the color changes are not a quan@ta@ve variable. However, in the incuba@ons with cyanobacteria and Fe(II)/Si, the color changes are very obvious and clearly indicate cyanobacterial growth and ac@vity (O produc@on 2 leading to Fe(II) oxida@on). Because the main goal of the study was to demonstrate how Si rescues the Fe(II)-influenced ac@vity of the cyanobacteria, we consider the color changes as an easy but very illustra@ve way of following the cyanobacterial ac@vity. We would therefore prefer to keep this informa@on in the text, as it might be very useful for other scien@sts who would like to do similar work.\n9. Line 182: Is Fe s@ll ge9ng oxidized in the high silica experiments? If so, why were Fe(III) precipitates (i.e., orange colors) not observed? Or is the Fe(II) being complexed by the silica? Do you have any data (e.g., SEM/EDS) characterizing the nature of the precipitates? This could be helpful in\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\nunderstanding the underlying mechanism by which silica may be buffering against Fe(II) toxicity.\nYes, the Fe(II) was s@ll ge9ng oxidized in the high Si experiments (the observed green color just indicates that the cyanobacteria grew very well even in the presence of high Fe(II) . This is (aq) described in the following lines where we wrote:\nLines 207-211: “The Fe(II) oxida@on rates were similar between the ‘no silica’ and ‘high silica’ setups—77 and 72 µM/day, respec@vely (Fehler! Verweisquelle konnte nicht gefunden werden. J; Fehler! Verweisquelle konnte nicht gefunden werden.) —but notably lower than the rates observed in experiments with 2500 µM Fe(II) . In the ‘high silica’ setups, the ini@al SiO concentra@on of (aq) 2(aq) 1500 µM declined to 500 µM by day 31 (Fehler! Verweisquelle konnte nicht gefunden werden. D), aper which it either stabilized or con@nued to precipitate, reaching final concentra@ons between 100 and 200 µM. “\nIn the present study, we unfortunately did not use SEM/EDS for analysis of the precipitates. However, the mineral composi@on should be comparable to what we found in our previous study (Dreher et al. 2025, GCA), where we found evidence for the co-precipita@on of Si and Fe in aggregates. We now cite this study in the text.\nLines 360-364: “Our working hypothesis is that silica binds Fe(II) , forming Fe-Si-aggregates that slow aq Fe(II) oxida@on and consequently inhibit ROS forma@on. Previous studies have shown that slower Fe(II) oxida@on rates reduce ROS forma@on 38. Suppor@ng this, Dreher and colleagues 34 showed, using SEM/EDS, that Fe-Si aggregates precipitate under similar experimental condi@ons, further strengthening our hypothesis.”\n10. Line 189: Do you have a hypothesis to explain this variability?\nWe thank the reviewer for this comment. We aPribute the observed variability in O accumula@on 2 between different boPles to minor differences in setup composi@on, such as small varia@ons in inoculum cell numbers or in glass surface proper@es affec@ng trace metal sorp@on and precipita@on. Such variability is common in microbial experiments that include minerals and metals. However, we believe adding that discussing this in detail would detract from the main focus of our study, so we have chosen not to include it in our manuscript.\n11. Line 231: Why was a different star@ng concentra@on of silica used in these experiments compared to the growth experiments (1600 μM compared to 2200 μM)?\nThank you for poin@ng this out. We understand that this is confusing. However, in both experiments 2200 µM Si was added. The missing frac@on of the silica was in the solid frac@on. We filtered the samples; therefore, we name the measured Si frac@on “dissolved silica”. This is now explained in the method sec@on.\nLines 549-551: “Due to the filtra@on of the samples (0.22 µm) and removal of Si precipitates, the measured remaining silica frac@on is named ‘dissolved silica’.”\n12. Line 363: Here, as in the introduc@on, you could consider posing a poten@al mechanism to explain *how* silica mi@gates the impact of Fe(II) toxicity and ROS. The results of the study are very compelling and demonstrate that there is a correla@on and that SiO2 may have been a key component to Fe (II) toxicity and ROS mi@ga@on, but these results would be even more impac€ul if you outlined some poten@al mechanisms.\nWe agree with the reviewer and have therefore added a paragraph to explain the underlying hypothesis bePer.\nLines 360-364: “Our working hypothesis is that silica binds Fe(II) , forming Fe-Si-aggregates that slow aq Fe(II) oxida@on and consequently inhibit ROS forma@on. Previous studies have shown that slower Fe(II) oxida@on rates reduce ROS forma@on36. Suppor@ng this, Dreher and colleagues 32 showed, using SEM/EDS, that Fe-Si aggregates precipitate under similar experimental condi@ons, further strengthening our hypothesis.”\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\n13. Line 414: Do you think that the length of the day maPers?\nThis is an excellent ques@on. As we did not vary different day lengths in our experiments, we cannot draw any defini@ve conclusion regarding this ques@on. However, other research suggests that there most likely is a day-night-op@mum for cyanobacteria (e.g. Muhetaer et al.2020). We discuss research regarding the influence of day-night-cycles on photoferrotrophic bacteria and cyanobacteria in lines 403-427.\n14. Line 458: Do you have any data aside from observa@ons of cloudiness in the boPles to characterize the precipitates?\nWe did not analyze the precipitates in this study. However, in our previous study (Dreher et al. 2025, GCA), we analyzed the precipitates during iron cycling experiments in detail and found the precipitates to contain iron and silica. We therefore added this important informa@on and cite Dreher et al. (2025).\nLines 464-469: “The protec@on effect of SiO against ROS forma@on can be explained by the ability 2(aq) of Si to bind Fe(II) , yielding Fe(II)-Si complexes27,34 that lower Fe(II) reac@vity (with O ) and thus (aq) 2 inhibit/reduce Fenton-type reac@ons and ROS produc@on. The forma@on of amorphous Fe(II)-Si aggregates hypothesized in our experiments correlates well with previous studies simula@ng Archean ocean condi@ons (1 mM SiO , 1.1 mM Fe(II) , pH of 6.5–7.5; 60,61).”\n2(aq) (aq)\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\nResponse to Reviewer #3\n(Remarks to the Author): Dreher et al. report new experimental data tes@ng the effects of dissolved Fe(II) and SiO2 on Synechococcus O2 produc@on rates. The study is a more detailed and improved version of earlier ones (e.g., Swanner et al. 2015). The study results are neat and novel and could have important implica@ons for Archean biogeochemical cycles. The results could also shed some light on enigma@c Archean iron forma@on deposi@onal condi@ons.\nI am not an experimentalist by training; I hope at least one other reviewer is. But I do study Archean Earth. My personal opinion is that the findings are probably novel enough to merit publica@on in Nature Communica@ons. Many knowledge gaps remain unfilled in our understanding of Archean Fe cycling—and many scien@sts are ac@ve in this area. This paper could provide new perspec@ve.\nThe authors thank reviewer #3 for the posi@ve feedback.\nMy most impac€ul comments are:\n1. The authors need to dedicate at least some text in the introduc@on to explaining why high Archean dissolved Si concentra@ons are important. Why do we think this was the case, and why is that informa@on important? This is incredibly important because it is the true novelty of these new experiments.\nWe agree and have therefore added the requested informa@on to the introduc@on.\nLines 107-114: “However, the poten@al mi@ga@ng impact of high SiO concentra@ons on Fe(II) 2(aq) (aq) toxicity has not been previously considered. Dreher and colleagues 34 reported increasing Fe(II) (aq) toxicity with increasing Fe(II) concentra@ons, but observed markedly enhanced bacterial growth (aq) compared in the presence of silica. Based on these results, we hypothesize that silica reacts with Fe(II) thus dis@nctly lowering Fe(II) reac@vity and preven@ng Fe(II)-induced ROS forma@on. To test (aq), this hypothesis, we experimentally examined the combined effects of Fe(II) and SiO on (aq) 2(aq) cyanobacterial O produc@on, Fe(II) oxida@on, and poten@al ROS-related toxicity.”\n2\n2. Salinity does not seem to have been considered in the O2 solubility calcula@ons. I think this should change.\nWhat is typically meant by solubility of oxygen in water is the aqueous-phase concentra@on of oxygen in equilibrium with the atmosphere. This equilibrium is affected by salinity (some@mes referred to as “sal@ng out”). However, our model assumes that the atmospheric oxygen concentra@on is zero, so that this equilibrium is irrelevant. There could be an effect at very high oxygen concentra@ons: If the sum of par@al pressures of all vola@le compounds exceeds the fluid pressure (which increases linearly with depth), the water is supersaturated with respect to dissolved gases, and bubbles can form. These bubbles rise. With a con@nuous produc@on of oxygen, this would lead to a layer with oxygen concentra@ons that are in local equilibrium with the bubbles. Excess oxygen ul@mately vents to the atmosphere. We have not included this mechanism in our model.\nLines 300-304:”Fig. 5: Modelled oxygen (lep, blue) and Fe(II) (right, grey) concentra@ons in the ancient ocean water column for an upwelling velocity of 4 (panels A-D), 95 (panels E-H) and 473 m/yr (panels I-L). Panels A, C, E, G, I and K are calculated for hypothesized 105 cells/mL in the early ocean, B, D, F, H, J, L for 106 cells/mL. Panels A, B, E, F, I, and J are modeled based on the photosynthe@c rate (r ) calculated from the lowest cell count we measured in our experiments at d0; C, C, G, H, K and L photo are based on the highest cell count we measured at d0.”\n3. How might strictly benthic Archean cyanobacteria impact their findings? Some researchers would argue that all Archean cyanos were benthic and stuck to the seafloor (i.e., not planktonic and free-floa@ng; Sanchez-Baracaldo, 2015). How might this assump@on change their hypotheses, if at all?\nIn our lab experiments, it likely makes liPle difference whether benthic or planktonic cyanobacteria were used, as the small boPle volumes allow oxygen to rapidly diffuse throughout the liquid and headspace. By contrast, the 1-D steady-state model of the ocean cannot handle benthic organisms as\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\nit does not reach down to the seafloor. In this model the pho@c zone does not reach the seafloor (no sunlight present at the seafloor). As a consequence, restric@ng Archean photosynthesis to benthos implies restric@ng it to very shallow shelf regions.\nSome line-by-line comments:\n4. Line 106: Why are high silica contents important for the problem? I know why, but the average reader may not. A few sentences explaining the mo@va@on for exploring high silica seem necessary.\nThe authors agree that poin@ng out the importance of the silica in the system is necessary.\nLines 107-114: “However, the poten@al mi@ga@ng impact of high SiO concentra@ons on Fe(II) 2(aq) (aq) toxicity has not been previously considered. Dreher and colleagues 34 reported increasing Fe(II) (aq) toxicity with increasing Fe(II) concentra@ons, but observed markedly enhanced bacterial growth (aq) compared in the presence of silica. Based on these results, we hypothesize that silica reacts with Fe(II) thus dis@nctly lowering Fe(II) reac@vity and preven@ng Fe(II)-induced ROS forma@on. To test (aq), this hypothesis,, we experimentally examined the combined effects of Fe(II) and SiO on (aq) 2(aq) cyanobacterial O produc@on, Fe(II) oxida@on, and poten@al ROS-related toxicity”\n2\n5. Line 109: Was a synthe@c seawater-like solu@on used for the experiments? Or a freshwater solu@on? This informa@on might be important enough to merit men@on here, in the main text.\nWe conducted the experiments in ar@ficial seawater medium and added the informa@on accordingly.\nLines 114-118: “Specifically, we incubated Synechococcus sp. PCC 7002 under alterna@ng day-night- cycles (16 h light; 8 h dark) in ar@ficial seawater medium, in the absence of SiO (0 µM, which we 2(aq) refer to as ‘no-silica’) and in the presence of SiO (2200 µM, which we refer to as ‘high-silica’) and 2(aq) varying ini@al Fe(II) concentra@ons (0, 500, 2500, 5000 µM).” (aq)\n6. Line 203: This intro sentence foreshadows some discussion of ROS in the paragraph. But the ROS discussion never arrives. Is this inten@onal? Should the authors consider a different opening to this paragraph?\nIt was not inten@onal to foreshadow into a discussion at this point of the manuscript, as results and discussion are strictly separated. We changed the sentence.\nLines 213-216: “In addi@on to experiments focusing on cyanobacterial growth and ac@vity (i.e., cell numbers, O produc@on, resul@ng Fe(II) oxida@on and dissolved silica (see Fig. S1), we explored the 2 role of silica in promo@ng long-term cell viability experiments by observing poten@ally mi@ga@on effects of the harmful effects of ROS generated in the presence of both Fe(II) and O .” (aq) 2\n7. Line 282: Has anyone es@mated these upwelling veloci@es for the Archean?\nThe authors are not aware of trustworthy calcula@ons, that is why variables of modern oceans (like the black sea, or the Atlan@c ocean) were used (See Table S4).\n8. Line 287: How is salinity treated in this calcula@on? The solu@on is salinity-dependent.\nSee answer to ques@on 2 (by the same reviewer).\n9. Line 302: Consider sta@ng the dissolved O2 abundances in the surface waters for this set of calcula@ons (you did it for the previous ones, and reading on also the subsequent ones).\nWe agree and changed the text accordingly to include the respec@ve oxygen concentra@ons.\nLines 306-313: “By increasing the upwelling rate to 95 m yr⁻¹, our model predicts that, at a cyanobacterial abundance of 10⁵ cells mL⁻¹ (Fig. 6 A, C), Fe(II) would have reached the ocean (aq) surface. Oxygen concentra@ons at the surface would have reached between 0.04 to 0.4 µM maximum concentra@on. At a higher theore@cal abundance of 10⁶ cells mL⁻¹ (Fig. 6 B, D), however, the pho@c zone remained Fe(II) -free down to depths of ~200 m or ~125 m, respec@vely. In these high cell (aq) density scenarios, O accumulated to much higher concentra@ons within the upper 0–50 m of the 2 water column, with modelled values reaching (200 µM) or exceeding (800 µM) maximum O 2 satura@on.”\nBlack: Reviewer comments\nGreen: Author comments\nBlue: Changes made in manuscript\nLine numbers refer to the clean, revised version of the manuscript\n10. Line 330: Have the authors considered calcula@ng how much O2 is released to the atmosphere in each scenario? Thereaper, they may also consider thinking about whether the calcula@on is consistent with pO2 es@mates for the Archean (e.g., those derived from S-MIF, or detrital redox-sensi@ve grains).\nThe oxygen release to the atmosphere is a direct outcome of the model (effec@ve diffusion coefficient @mes the ver@cal concentra@on gradient at the ocean surface. As stated above (and in the manuscript) the model assumes that the atmosphere is free of oxygen. Evalua@ng the evolu@on of 𝑝 would require coupling our model to an atmosphere model that includes terrestrial oxygen sinks. !! This is beyond the scope of our study.\n11. Line 336: What are those maximum O2 concentra@ons? Please state them here.\nWe added the requested informa@on.\nLines 333-337: “Incorpora@ng the upwelling rates and Fe(II) concentra@ons applied in the studies (aq) of Swanner and colleagues 11 into our models (see Fig. S4-S6), and applying our most conserva@ve O 2 produc@on rate (r of 0.122 µM/day), yields maximum O concentra@ons at least an order of photo 2 magnitude greater than previously es@mated (33 µM of maximum oxygen compared to 2-4 µM of Swanner and colleagues11)”\n12. Line 343: Please cite the “earlier models”.\nChanged.\nLine 342: Cita@on: “Swanner and colleagues (2015): Modula@on of oxygen produc@on in Archaean oceans by episodes of Fe(II) toxicity”.\n13. Line 367: An end-parentheses is missing.\nCorrected.\nResponse to Reviewer #1:\n(Remarks to the Author): While I agree with the other reviewers that the study is based on a robust dataset, and regardless of its poten@al suitability for publica@on in Nature Communica@ons, I respecDully disagree with the assessment that the manuscript is well wriEen. In its revised form, I cannot recommend the manuscript for publica@on. Substan@al restructuring of the data presenta@on and the Discussion should be required before it can be considered for publica@on. This is not a maEer of stylis@c preference; rather, the study’s content is not being communicated efficiently to a general readership, regardless of the quality of the underlying science. Below, I provide specific details and sugges@ons to improve readability.\nWe thank reviewer #1 for the construc@ve feedback and the effort to revise our manuscript.\nFirst, I thank the authors for upda@ng Figure 1 and for merging Figures 5, 6, and 7 into a new Figure 5. The revised figures are much easier to read, and the interpreta@on of the data are more readily extracted. Regarding Figure 1, I understand the authors’ concerns about displaying smaller insets; however, I find that comparing datasets on iden@cal @mescales is far more effec@ve. This approach also allows visualiza@on of the full (long-term) Fe(II) oxida@on profiles, which were not even shown in the original figure. I have several comments to further improve the readability of Figure 1. First, the updated insets appear to be downscaled versions of the original plots. As a result, the font size is extremely small and difficult to read. I recommend re-ploYng these panels with increased font sizes, symbol sizes, and line thicknesses to enhance clarity. In addi@on, for the dissolved O₂ and cell count insets, the y-axis ranges extend well beyond what is necessary to highlight differences between condi@ons. For example, the original panels 1E and 1F span ranges of 1–600 and 0–3 × 10⁸, respec@vely, whereas adjus@ng these to 0–200 and 0–5 × 10⁷ would make the differences more apparent. Finally, the revised figure is missing panel labels (A through L), which should be added for clarity and ease of reference.\nWe appreciate the opinion of the reviewer, and we also agree that consistent x-axes, on the first sight, would appear useful. We therefore visualize the data on the same x-axes (@me) in Figure 2 where we have now added the iron oxida@on panels over the en@re course of the experiment. However, we prefer to keep Figure 1 as it is in the manuscript, because our aim in this figure was to shig the reader’s aEen@on to the first days of ac@ve Fe(II) oxida@on. As we decided to adapt the x-axes to the respec@ve Fe(II) oxida@on period, for consistency and comparability, we set the y-axes maxima consistently over all plots in figures 1 and 2 (e.g., all oxygen plots from 0 to 600 µM). We hope that this is also fine with the editor and reviewer. Since we prefer keeping the figure as it is (without the inserts), there is no issue with the smaller font size and therefore we did not change it in Fig. 1.\nDespite the revisions made by the authors, I s@ll find the main text very difficult to read. I previously suggested modifying how the results are presented in the sec@on “Effects of different silica concentra@ons on Fe(II) oxida@on by cyanobacterial O₂ under alterna@ng day–night cycles.” The authors indicated that they prefer to retain the current structure. While I understand their ra@onale for organizing the results by Fe(II) condi@on and then by the presence or absence of silica, the sec@on remains overly verbose and repe@@ve. As currently structured, the authors sequen@ally describe each Fe(II) condi@on, followed by color changes, cell growth, O₂ accumula@on, Fe(II) oxida@on, and silica precipita@on, repea@ng this format for both silica-free and silica-amended experiments. This results in an en@rely descrip@ve sec@on spanning roughly ten paragraphs. Although I appreciate the authors’ effort to be thorough, I feel that the key findings are obscured by the level of detail provided. In addi@on, most numerical values reported ogen add noise rather than insight, par@cularly when the implica@ons of these differences are not explicitly discussed. For example, in lines 134–138, the\n1\nreported differences in final cell densi@es between the “no silica” and “high silica” setups are rela@vely small, and it is unclear whether these differences are meaningful in the context of the study. Similar examples occur throughout much of the Results sec@on. I therefore recommend restructuring this sec@on by (1) summarizing observa@ons more concisely and (2) presen@ng key quan@ta@ve results in a table, similar to Table 1, instead of lis@ng every single number in the main text. Such a table could report rates of cell growth and dissolved O₂ accumula@on, poten@ally over both short and long @mescales (as this @mescale difference is relevant for the study). This approach would substan@ally improve readability while ensuring that the most important results are clearly communicated.\nAs requested by the reviewer, we have added more geochemical informa@on to Table 1 and now refer to it in the text. We acknowledge that the increased level of detail might be distrac@ng to some readers and as suggested, we therefore have shortened the text slightly. However, we believe that presen@ng these results systema@cally provides a clearer overall picture of the experiments. By organizing the data star@ng from the respec@ve Fe(II) condi@ons, followed by presen@ng the results on color changes, cell growth, O₂ accumula@on, Fe(II) oxida@on, and silica precipita@on, we maintain a structured and comprehensive overview of the dataset. We hope the reviewer and editor appreciate this ra@onale and the authors’ approach to presen@ng the experimental results.\nAlong similar lines, the logic of the Discussion is very difficult to follow in its current form. For example, the authors begin by sta@ng that “oxygenic photosynthesis on the early Earth proceeded without harmful ROS stress” before even explaining the postulated mechanisms by which ROS forma@on is mi@gated by the presence of silica or by day–night cycles. The Discussion should be structured as a stepwise logical argument that progresses from specific experimental results to broader implica@ons, which is not the case in its current form. For instance, it would be clearer first it is described the specific mechanisms through which silica and day–night cycling aEenuate ROS forma@on in the authors’ experiments. This could then be followed by a broader discussion of the implica@ons for how oxygenic photosynthesis may have proceeded on the early Earth without ROS toxicity, and how this influenced water-column redox structure and O₂ distribu@on in seawater.\nWe agree with the reviewer and therefore have re-organized the discussion accordingly. The new order now is:\n- ROS forma@on and the mechanis@c func@on of Si for ROS preven@on\n- Day-night-cycles reduce ROS forma@on compared to con@nuous light and sta@c condi@ons\n- Implica@ons of the oxygen distribu@on in the seawater\n- Oxygenic photosynthesis on early Earth proceeded without harmful ROS stress to cyanobacteria\nThe very conclusive paragraph has been kept at the end.\nMinor comments:\n1. Line 3.- Would be relevant to cite “Fournier, G. P., Moore, K. R., Rangel, L. T., PayeEe, J. G., Momper, L., & Bosak, T. (2021). The Archean origin of oxygenic photosynthesis and extant cyanobacterial lineages. Proceedings of the Royal Society B, 288(1959), 20210675.” where they also support an Archean origin of total group cyanobacteria.\nWe added the cita@on to lines 39-40.\n2. Line 74-75. Missing a reference.\nWe added Kappler et al. (2021) “An evolving view on biogeochemical cycling of iron” as reference.\nLines 76-78: “While the Fe3+ drives the forma@on of Fe(III) minerals, the generated radicals can undergo further reac@ons, forming addi@onal ROS like ozone (O ) and hydroperoxyl radicals (OOH•) 3 (for a review see 33).”\n3. Line 130-133. Refer to Figure 1 in this sentence.\nDone as suggested.\n4. Lines 360-364. This is the mo@va@on and hypothesis of the study, and it was not incorporated effec@vely in the introduc@on. I would suggest exchanging these lines (360-364) to the ones in the introduc@on which are a summary of what is shown here: “Dreher and colleagues reported increasing Fe(II)(aq) toxicity with increasing Fe(II)(aq) concentra@ons, but observed markedly enhanced bacterial growth compared in the presence of silica.”\nAs suggested by the reviewer, we have switched the paragraphs accordingly.\nLines 111-115: “Our working hypothesis is that silica binds Fe(II) , forming Fe-Si-aggregates that slow aq Fe(II) oxida@on and consequently inhibit ROS forma@on. Previous studies have shown that reduced Fe(II) oxida@on rates lead to lower ROS forma@on 36. Consistent with this, Dreher and colleagues 37 showed, using SEM/EDS, that Fe-Si aggregates precipitate under similar experimental condi@ons, further suppor@ng our hypothesis.”\nLines 447-450: “Dreher and colleagues 36 reported increasing Fe(II) toxicity with increasing Fe(II) (aq) (aq) concentra@ons, but observed markedly enhanced bacterial growth compared in the presence of silica. Based on these results, we hypothesize that silica reacts with Fe(II) thus dis@nctly lowering (aq), Fe(II) reac@vity and preven@ng Fe(II)-induced ROS forma@on.”\n5. Line 380 .- “Taken together, our lab coupled with the modeling results…” The sentence is missing “experiments” ager “lab”.\nChanged as suggested.\nResponse to Reviewer #2:\n(Remarks to the Author): I thank the authors for their detailed responses to comments and sugges@ons. I think that the authors integrated all key sugges@ons very well and strengthened an already excellent manuscript. I have no addi@onal comments beyond those addressed in my review and those of the other reviewers. The manuscript is well wriEen, the results and interpreta@ons are novel, and the findings contribute significantly to our understanding of interplays between early cyanobacteria and environmental condi@ons on the early Earth. I support publica@on of this manuscript.\nWe thank reviewer #2 for the nice feedback and the effort taken in reviewing our manuscript again.\nResponse to Reviewer #3:\n(Remarks to the Author): I am sa@sfied with all but one of the authors' responses to my comments. My single objec@on is a small one (but s@ll important, I think). When I recommended the authors add a few lines of text stressing the importance of high Silica in Archean seawater, I was hoping to see a sentence describing the suppor@ng data for Silica-rich Archean seawater. Archean seawater WAS silica rich; this was the point I wanted them to make clearer. My apologies for the unclear direc@ons.\nWe would like to thank reviewer #3 for the posi@ve feedback and the clarifica@on. We now added a small paragraph about the evidence for high silica concentra@ons in the Archean seawater.\nLines 51-57: “Prior to the emergence of silica-precipita@ng microorganisms (e.g., diatoms), silica inputs from chemical weathering of early con@nental crust and hydrothermal vents led to elevated dissolved silica concentra@ons in the seawater. These concentra@ons were constrained chiefly by satura@on with respect to cristobalite or amorphous silica, on the order of 0.67-2.2 mM 18,19. Recent studies further suggest that under these ‘high silica’ concentra@ons, the primary precipitate may have been a gel-like composite of ferric oxyhydroxide and silica 20–22.”\nThis study seeks to understand the chemical factors that may have influenced the proliferation of cyanobacteria and the Great Oxidation Event, topics of broad interest to the geoscience community. The study specifically focuses on the potential connections among iron toxicity, oxidative stress, and elevated Fe(II) concentrations in Archean oceans as factors that may have limited cyanobacterial proliferation and the impact of high SiO concentrations in Archean 2 seawater as a potential mitigating factor in iron toxicity and ROS generation.\nThe study uses an experimental approach to test the impact of Fe(II) concentrations, oxidative stress, and SiO concentrations on cyanobacterial growth and O production. The authors 2 2 measure O concentrations, Fe(II) and SiO concentrations, Fe , cell numbers, and ROS 2 2 total production to characterize the interactive effects of Fe(II) and SiO on cyanobacterial physiology, 2 oxygen production, and growth. These experimental findings are paired with geochemical modeling to characterize total O production under Archean-type conditions and scale the 2 laboratory results to early oceans. Together, the experimental and modeling results demonstrate how day/night cycles and elevated SiO concentrations in Archean oceans could have mitigated 2 ROS stress and Fe(II) toxicity, playing instrumental roles in the proliferation of cyanobacteria and the GOE.\nOverall, the manuscript is well written, the experiments are well designed, and the results are compelling and novel. This study provides important new experimentally derived insights into the ways in which Fe(II) toxicity and mitigation of toxicity and ROS generation by SiO may 2 have impacted cyanobacterial proliferation, oxygen accumulation, and the GOE. The experimental results convincingly demonstrate that Fe(II) toxicity negatively impacts cyanobacterial growth and O production. The experiments also clearly demonstrate that under 2 elevated concentrations of SiO (similar to estimates of Archean oceans), the impact of Fe(II) 2 toxicity and ROS stress is diminished, evidenced by higher growth and O production and 2 diminished ROS in high SiO cultures. Using these experimental findings, the study refines 2 models that predict O accumulation and ocean Fe(II) concentrations during the Archean and 2 demonstrates that elevated SiO concentrations in Archean oceans may have played a key role in 2 cyanobacterial proliferation and the GOE.\nThe findings are novel, and the results will be of broad interest. I support publication of the manuscript, but I have several comments and suggestions outlined below regarding details of experimental design, presentation of the data, and potential discussion points that would strengthen the manuscript. Broadly, 1) the introduction and discussion could be strengthened by posing a possible mechanism to explain how silica is mitigating the impacts of Fe(II) toxicity and ROS, 2) some details of the experimental design should be mentioned in the main text (see below), 3) the data in figure 1 should be plotted on the same timescales to more robustly show trends highlighted in the text, 4) there are some mentions of the precipitates but no data characterizing what these precipitates are – if these data were collected, it would be useful to show them. These comments and other minor suggestions are outlined in the attached document with line references.\nDetailed suggestions:\n Line 106: The introduction does a great job of painting a picture both of the connection between Fe(II) and oxidative stress, the potential mitigation of these impacts by elevated SiO concentrations, and relationship between Fe and SiO in BIFs indicating iron-silica 2 2 interactions in Archean oceans. However, the specific connection between Fe(II) stress and SiO mitigation and the potential underlying mechanism is not clear. What is the 2 specific hypothesis or hypothesized mechanism being tested? That Si may remove FeII? Or that it may directly interact with the cells in some way? Mentioning the hypothesized mechanism in the introduction and the discussion (see later comments) would be of interest to the community and strengthen the findings from the study.\n Line 120: Although the experimental design is outlined in the results section, it would be useful to include a brief note at the beginning of the results section to help the reader follow the results. Just a sentence or two noting the basic setup, number of replicates, and variables being tested.\n Results/Methods: Related to this, it is not clear between the initial growth conditions and experimental conditions whether the experiments were conducted under anoxic conditions. It would be helpful to explicitly state this at the beginning of the results section as this is a key parameter. Whether the experiments were conducted under oxic or anoxic conditions has strong implications for Fe oxidation both at time 0 and over the course of the experiments as well as the changes in O observed.\n2\n Results/Methods: Also related to the experimental design, were sterile controls used to confirm that Fe(II) was not oxidized in the absence of cyanobacteria?\n Line 126 and Figure 1A: Why does Fe(II) increase in the control condition by the end of the experiment?\n Figure 1: Why are the plots in Fig 1 plotted on different timescales? It would be more useful to directly compare the experiments over the same timescales (x axis) and then re- scale the Y axes so that small changes (like those in A, E, F, and I) can be more clearly seen.\n Line 148: Although error is stated in the table, it would be helpful to state in the text to make it clear that the oxidation rates at this concentration of silica and Fe(II) were similar within error. As written, it seems to imply that there is a difference in the oxidation rates with and without silica, but these values are similar within error at the 500 µM Fe(II) concentration based on the table.\n Line 163: The color changes do not seem to be the key result, especially given the potential confounding variables and mechanisms underpinning the color changes that the authors note. These \"color change\" results could be moved to the supplemental document so that the results of the main text could more clearly focus on the key quantifiable trends.\n Line 182: Is Fe still getting oxidized in the high silica experiments? If so, why were Fe(III) precipitates (i.e., orange colors) not observed? Or is the Fe(II) being complexed by the silica? Do you have any data (e.g., SEM/EDS) characterizing the nature of the precipitates? This could be helpful in understanding the underlying mechanism by which silica may be buffering against Fe(II) toxicity.\n Line 189: Do you have a hypothesis to explain this variability?\n Line 231: Why was a different starting concentration of silica used in these experiments compared to the growth experiments (1600 µM compared to 2200 µM)?\n Line 363: Here, as in the introduction, you could consider posing a potential mechanism to explain *how* silica mitigates the impact of Fe(II) toxicity and ROS. The results of the study are very compelling and demonstrate that there is a correlation and that SiO may 2 have been a key component to Fe (II) toxicity and ROS mitigation, but these results would be even more impactful if you outlined some potential mechanisms.\n Line 414: Do you think that the length of the day matters?\n Line 458: Do you have any data aside from observations of cloudiness in the bottles to characterize the precipitates?"}]

Decision letter