Oxic microbial ferrihydrite reduction rates of Shewanella oneidensis and the potential for Fe mobilization in oxic sediments

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Berg This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6726027/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Microbially-mediated reduction of ferric iron, Fe(III), often found in the natural environment as ferrihydrite, plays a crucial role in Fe cycling, and hence nutrient and contaminant cycling, in subsurface environments. Traditionally, microbial ferrihydrite reduction has been considered an anaerobic process relegated to anoxic microsites within oxic subsurface environments. However, recent findings suggest that microbes can mediate Fe(III) reduction also under oxic conditions, although rates and environmental impact of this process are still unknown. Here, we quantified cell-specific rates of ferrihydrite reduction by the model organism Shewanella oneidensis MR-1 under oxic and anoxic conditions. Based on our experimental results, we assessed the relative contribution of oxic and anoxic pore spaces to Fe(II) mobilization in a laboratory analog of oxic aquifer sediments presented in the literature. Our results show that oxic Fe(III) reduction can significantly contribute to Fe(II) mobilization in oxic subsurface environments where anoxic microsites occupy a minority of the pore space, conditions that can be found in, e.g., shallow aquifers, well-drained soils, and capillary fringes. Despite the lower cell-specific rates of oxic Fe(III) reduction, it remains a persistent background process, playing a previously underestimated role in Fe cycling within oxic subsurface environments. Earth and environmental sciences/Biogeochemistry/Element cycles Biological sciences/Microbiology/Environmental microbiology/Water microbiology Biological sciences/Microbiology/Environmental microbiology/Soil microbiology Earth and environmental sciences/Hydrology Physical sciences/Physics/Fluid dynamics microbial iron reduction oxygen Shewanella oneidensis anoxic microsites oxic sediments ferrihydrite Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction The microbial reduction of ferric iron, Fe(III), is a key anaerobic process driving natural subsurface biogeochemistry and releasing mobile ferrous iron, Fe(II) to the environment [ 1 ]. The occurrence and the extent of microbial mobilization of Fe(II) depend on the ability of microorganisms to access and reduce solid Fe(III) minerals, the most reactive of which are considered to be amorphous oxides like ferrihydrite (Fe₂O₃·nH₂O). These minerals represent one of the main reservoirs of microbially accessible Fe(III) in soils and natural sediments. Ferrihydrite exhibits a high and non-selective adsorptive capacity [ 2 , 3 ] for contaminants such as arsenic and chromium, as well as nutrients like phosphate and nitrate [ 3 – 6 ]. Consequently, the spatial distribution of ferrihydrite and the extent of its reduction in soils and sediments are closely linked to the rate of organic carbon mineralization, the mobility of toxic compounds and micronutrients as Fe(II), and overall soil fertility in agricultural systems [ 7 – 9 ]. As Fe(III) is a less thermodynamically favorable electron acceptor than oxygen (O 2 ) [ 10 ], its microbial reduction has been considered confined in anoxic or O 2 -limited environments, like deep groundwater systems, flooded soils, ferruginous lakes, and wetlands. Nevertheless, the detection of organic matter-stabilized Fe(II) in some oxic subsurface environments indicates that ferrihydrite reduction occurs in oxygenated waters [ 8 , 11 – 14 ]. This is typically attributed to the formation of O 2 -depleted microscopic zones, referred to as anoxic microsites or microenvironments, that remain undetected in averagely well-oxygenated sediments and soils [ 11 , 14 , 15 ]. Such anoxic microsites are expected to form where O 2 diffusion is hindered by the sediment porous structure, heterogeneous pore water flow, and outcompeted by the aerobic growth of thick biofilm layers [ 12 , 15 – 19 ], as illustrated in Fig. 1 . Though transient and small in scale, these anoxic microsites enable localized ferrihydrite reduction and anaerobic respiration even in well-oxygenated bulk systems [ 8 , 13 , 17 , 19 ]. An alternative explanation for the unexpected presence of Fe(II) in oxic, circumneutral subsurface is the metabolic versatility of certain Fe-reducing microorganisms. As early as 1990, Shewanella putrefaciens [ 20 ] was shown to reduce soluble Fe(III) complexes while respiring O 2, suggesting a hybrid aerobic and anaerobic lifestyle. Though initially overlooked, this idea resurfaced in 2019 when diverse actinobacteria were found to reduce both soluble and solid Fe(III) [ 21 ] in oxic batch incubations. These studies, however, measured O 2 only at the bulk scale, leaving unresolved whether Fe(III) reduction occurs in the presence of O 2 or in undetected anoxic microsites. Recent microfluidic experiments using O 2 sensors confirmed that the facultative Fe reducer, Shewanella oneidensis MR-1 ( S. oneidensis ) can reduce Fe(III) in persistently oxic conditions in the microenvironment surrounding the cells [ 16 ]. This observation supports that microbial Fe(III) reduction in oxic sediments and soils is not confined solely to anoxic microsites, as previously assumed. Similar hybrid behavior has been observed for facultative denitrifiers [ 22 ], sulfate reducers [ 23 ], and Escherichia coli [ 24 ], suggesting this hybrid metabolism may be more widespread than previously recognized. Nonetheless, the rates and environmental importance of oxic Fe(III) reduction remain unknown. Scaling the rates of oxic Fe(III) reduction is critical to understanding iron and contaminant mobility in natural and engineered systems. The quantification of Fe(III) reduction rates under oxic conditions poses methodological challenges because Fe(II), produced by microbial ferrihydrite reduction, can be rapidly re-oxidized and re-precipitated as secondary minerals [ 3 ]. As a result, Fe(II) may not accumulate in oxic porewaters, explaining why oxic Fe(III) reduction has so far been difficult to detect and rendering dissolved Fe(II) concentrations a poor indicator of microbial reduction rates in such environments. In this study, we quantify Fe(III) reduction rates by S. oneidensis under oxic and anoxic conditions using a ferrozine-based method that stabilizes Fe(II) and prevents re-oxidation [ 25 , 26 ]. Applying these rates to a representative sediment model, we reveal the overlooked contribution of oxic microbial iron reduction to Fe(II) mobilization in natural environments. 2 Results 2.1 Overall oxic and anoxic Fe(II) production rates in batch. S. oneidensis grown in the dark in 10% v/v Luria Bertani broth, supplemented with 2 mM ferrihydrite and 1 mM ferrozine (FZ), mediated iron reduction under both oxic and anoxic conditions. Fe(II) progressively accumulated in the liquid medium under both conditions (Fig. 2 A), in the form of stable FZ-Fe(II) complexes [ 20 , 25 , 26 ]. Under anoxic conditions, Fe(II) concentrations (Fig. 3 a) exceeded 300 µM after 72 hours, indicating efficient ferrihydrite respiration by S. oneidensis . These Fe(II) levels surpassed the chelating capacity of 1 mM ferrozine (Supplementary Information, SI, Section S1 for further details). Fe(II) measurements are therefore reported only for up to 72 hours, during which soluble iron increased linearly. Under oxic conditions, Fe(II) concentrations also increased linearly over time, reaching 92.5 ± 28.2 µM within 144h (Fig. 2 a). In other words, ferrihydrite reduction occurred at a constant rate, although slower than under anoxic conditions. Ongoing aerobic respiration of S. oneidensis after the addition of ferrihydrite and ferrozine is evidenced by the lower O 2 concentrations in live S. oneidensis incubations compared to killed controls under the same stirring conditions (Fig. 2 b). The slope of the linear trend quantified the overall Fe(II) production rate for S. oneidensis incubations, with rates of 3.8 ± 0.43 µM h⁻¹ (mean ± standard deviation, R² = 0.99) under anoxic conditions and 0.62 ± 0.084 µM h⁻¹ (R² = 0.94) under oxic conditions. Remarkably, the overall oxic Fe(II) mobilization rates were only ~ 6 times lower than those observed in anoxic conditions. Killed (Fig. 2 a) and negative (SI, Section S2) controls exhibited small increases in soluble Fe(II), ranging from 4–6 µM for anoxic conditions and 9–12 µM under oxic conditions after 144h of incubation. These results suggest that abiotic ferrihydrite reduction, possibly driven by reducing groups contained in organic matter [ 20 , 26 ], and reactions involving dead cell material (biomass) contributed minimally to the reduction of Fe(III) in Fe-oxides, as observed under both anoxic [ 20 , 26 ] and oxic conditions [ 27 ]. Linear fitting of the controls estimated the mean abiotic Fe(II) production rates to be 0.078 ± 0.0048 µM h⁻¹ in oxic conditions and 0.034 ± 0.0043 µM h⁻¹ in anoxic conditions. After subtracting the calculated abiotic contribution [ 26 ], the overall Fe(II) production rates attributed to microbial activity were determined to be 0.54 ± 0.089 µM h⁻¹ under oxic ( R O ) and 3.8 ± 0.43 µM h⁻¹ under anoxic ( R A ) conditions. 2.2 Oxic and anoxic Fe (III) reduction rate per cell To normalize Fe(III) reduction rates to possible differences in growth rates under oxic and anoxic conditions, a cell-specific rate was calculated. For this, cell concentrations in batches were estimated in the stationary growth phase, just before the addition of ferrihydrite and ferrozine. The cell concentration increased from the initial inoculum under both anoxic and oxic conditions (Fig. 3 a), consistent with protein concentration measurements (Figure S2, SI, Section S3). As expected, bacteria under aerobic conditions grow faster, resulting in cell concentrations one order of magnitude higher (2.1 x 10 9 ± 2.3 x 10 9 cell mL − 1 ) than in anoxic conditions (1.2 x 10 8 ± 5.7 x 10 7 cell mL − 1 ). By normalizing the overall Fe(II) production rate in batches computed in Section 2.1 to the cell concentration, we estimated the ferrihydrite reduction rate per cell (Fig. 3 b) under oxic (2.6 x 10 − 10 ± 0.5 x 10 − 10 µM h − 1 cell − 1 ) and anoxic conditions ( 3.2 x 10 − 8 ± 1.6 x 10 − 8 µM h − 1 cell − 1 ). As a result, cells reduced ferrihydrite ≈ 120 times faster under anoxic than under oxic conditions. 2.3 Modeling of Fe(II) mobilization in oxic sediments. To scale the potential impact of oxic Fe(III) reduction in sediments and soils, we modeled microbially-mediated Fe(II) mobilized from ferrihydrite reduction in a 27 mm-long laboratory analog of sandy aquifer sediment presented in the literature [ 15 ]. This laboratory system was used in [ 15 ] for real-time mapping of biomass colonization, O 2 concentrations, and anoxic microsite formation in the pore space for 45 hours, proving a level of insight into microscale O 2 and biomass dynamics that is not achievable in natural systems to date. According to the results presented in [ 15 ], the microbial biomass progressively colonized the sediment grain surfaces, eventually occupying up to 1.2 mm 3 of available pore space at 45 hours in the laboratory analog (Fig. 4 A), corresponding to 35% of the total pore volume. The system remained oxic at the centimeter scale. Still, anoxic microsites formed transiently, peaking at 26 hours with a maximum volume of 0.05 mm 3 (Fig. 4 A), i.e., 1.42% (± 0.99%) of the pore space, and almost disappeared at t > 35 h. In our model, we assumed a homogeneous distribution of ferrihydrite. Therefore, Fe(II) mobilization was modeled as a ubiquitous process, occurring at every point of the pore space colonized by biomass. However, the rate of iron reduction varied according to local O 2 levels, between oxic conditions ( r o = 2.6 x 10 − 10 µM h − 1 cell − 1 ) and anoxic microsites ( r a = 3.2 x 10 − 8 µM h − 1 cell − 1 , Eqs. (1–3), Section 4.5). The contribution of anoxic microsites to Fe(II) mobilization ( c A , Fig. 4 B) mirrored their temporal dynamics. Initially, when the system was fully oxic ( t < 10 hours), only oxic Fe reduction occurred in the biomass-colonized pore space. Once anoxic microsites are formed, while occupying only 1–2% of the total pore space, they contribute up to 79–94% to the total Fe(II) mobilization rate. Since oxic Fe(II) mobilization persisted throughout the experiment, the cumulative contribution of oxic biomass ( C O , Fig. 4 C) accounted for 21%-42% of the total Fe(II) mobilized over 45 hours. 3 Discussion Comparing the temporal dynamics of Fe(II) concentrations in oxic and anoxic batches, we found that a S. oneidensis population grown under oxic conditions reduces ferrihydrite at a rate less than one order of magnitude slower than under anoxic conditions. Our observations indicated that Fe(II) concentrations reached 58.6 ± 7.8 µM after 72 hours of oxic S. oneidensis batch incubations. These results align with previous findings obtained during the growth of S. oneidensis on ferrihydrite with 10% v/v LB in an O₂-sensing microfluidic device, where Fe(II) accumulated up to 88.4 ± 19.3 µM over 72 hours, while persistent oxic conditions were observed at the microscale [ 16 ]. Thus, we rule out the formation of anoxic microsites in our setup and attribute Fe(II) mobilization to oxic, microbially mediated ferrihydrite reduction. The slightly lower range of Fe(II) concentrations observed in this study compared to [ 16 ] may be due to the formation of soluble FeOH + . At the imposed pH [ 28 ] and ferrozine concentrations [ 29 ], this process could compete with Fe(II) chelation by ferrozine, meaning a small portion of Fe(II) might not have been captured in our measurements. In contrast, samples in [ 16 ] were acidified, releasing Fe(II) from organic complexes and hydroxyl ions before ferrozine addition and assay. Previous studies [ 25 , 26 ] have shown that the formation of ferrozine-Fe(II) complex can enhance the bioreduction of Fe-oxides, such as hematite, by maintaining low Fe(II) geochemical activity. However, the Fe(II) concentration measured at 72 hours is similar to the one found in the previous study [ 16 ], without a complexing agent, suggesting that ferrozine addition did not artifactually enhance Fe(III) reduction in our setup. Our results also align with previous studies by Arnold et al. (1990) [ 20 ], which examined another Shewanella strain, S. putrefaciens , grown on dissolved Fe(III) and beef extract. In S . putrefaciens batch incubations, the Fe(III) reduction rate in the presence of O 2 was about one order of magnitude lower (12 µM h − 1 ) than under anoxic conditions (240 µM h − 1 ). Notably, S. oneidensis exhibits significantly slower Fe(III) reduction rates (~ 100 times) compared to S. putrefaciens [ 20 ] under both oxic and anoxic conditions. This discrepancy can be attributed to several factors. First, Arnold et al. [ 20 ] used dissolved Fe(III), which is likely more bioavailable than solid Fe(III) in ferrihydrite. This means that the mineral ferrihydrite used in our study, while being more representative of Fe(III) form found in natural systems (e.g., rocks [ 30 ], sediments [ 31 ], and soils [ 7 ]), likely reduces Fe(III) accessibility to bacteria, slowing down Fe(II) mobilization. Additionally, strain-specific traits, medium composition and concentration, and incubation temperature may have contributed to the slower Fe(III) reduction rate of S. oneidensis compared to S. putrefaciens . Indeed, S. putrefaciens has one of the highest dissimilatory Fe(III) reduction rates per cell reported in the literature [ 20 ]. Moreover, the nutrient conditions and temperature in our study (10% v/v diluted Luria-Bertani broth, equivalent to 2 g L⁻¹ LB; 23°C) were less favorable than those in S. putrefaciens incubations (8 g L⁻¹ Difco broth; 30°C) [ 20 , 32 ]. It is worth noting that a minor abiotic contribution to ferrihydrite reduction was observed in both oxic and anoxic control incubations, though it was significantly lower than the microbially mediated reduction. This aligns with previous studies where Fe-oxides (hematite or ferrihydrite) were observed to undergo abiotic reduction under both anoxic [ 25 , 26 ] and oxic [ 27 ] incubations. This process is attributed to the complex composition of natural organic matter, which contains reduced functional groups capable of acting as electron shuttles for Fe(III) under anoxic conditions while retaining their reducing capacity in the presence of O 2 [ 27 ]. In our incubations, the yeast extract, the major constituent of LB broth, was used to mimic the complex composition of natural organic matter [ 33 ], likely contributing to abiotic ferrihydrite reduction. The Fe(III)-ferrihydrite reduction rate per cell under anoxic conditions (3.2 × 10⁻⁸ ± 1.6 × 10⁻⁸ µM h⁻¹ cell⁻¹ at a cell concentration of 1.2 × 10⁸ ± 5.7 × 10⁷ cells mL⁻¹) aligns with previous findings for the same strain at a similar cell concentration (1.1 × 10⁻⁸ ± 8.1 × 10⁻¹⁰ µM h⁻¹ cell⁻¹ at 6.7 × 10⁸ cells mL⁻¹) [ 34 ], reinforcing the reliability of our results. Interestingly, although the cell-specific Fe(III) reduction rate under oxic conditions is two orders of magnitude lower than under anoxic conditions, the total amount of Fe(II) mobilized in oxic conditions is less than one order of magnitude slower than in anoxic incubations. This discrepancy is due to the significantly higher cell density attained under oxic conditions, emphasizing the need to consider the collective activity of the population rather than just individual cell efficiency in Fe(III) reduction in large-scale environmental systems. Our study was not designed to elucidate the physiological mechanisms by which S. oneidensis mediates Fe(III) reduction in the presence of O₂, which would require a dedicated genetic investigation. However, if Fe(III) uptake serves an energetic purpose in parallel to O 2 , as suggested by Arnold et al. [ 20 ], the significantly lower Fe(III) reduction per cell under oxic conditions supports the idea that O₂ remains the preferred electron acceptor for this facultative Fe(III) reducer. This quantitative difference could also result from distinct Fe(III) reduction mechanisms, such as indirect electron shuttling mediated by organic matter and radicals [ 10 ]. Additionally, the observed variations in cell shape and size between oxic and anoxic conditions (Figure S3, SI, Section S3) further suggest potential differences in the ecophysiology of S. oneidensis . Although oxic microbially mediated Fe(III)-ferrihydrite reduction occurs ~ 100 times slower than under anoxic conditions, it could play a crucial role in environments where oxic conditions dominate and anoxic microsites constitute a minority of the pore space [ 12 , 14 ]. Our model simulation revealed that oxic ferrihydrite reduction can contribute approximately one-third of Fe(II) mobilized in the lab sediment analog within just a few days. This unexpectedly large contribution is explained by the fact that the cells exposed to oxic conditions constitute the majority of the biomass growing in the pore space of the oxic aquifer lab analog. Despite its slower instantaneous rate, Fe(III) reduction under oxic conditions acts as a continuous background process, gradually affecting an increasing portion of the pore space as biomass colonizes the porous medium. Similar conditions are likely to occur in certain subsurface environments, including well-drained and unsaturated soils, capillary fringes, and shallow aquifers, among the most microbially active environmental systems. Despite their limited spatial extent and temporal duration, anoxic microsites host most of the Fe(II) mobilization in oxic sediments. Their contribution is inherently dynamic and halts immediately upon microsite dissipation. This transient behavior is expected in natural sediments and soils [ 14 , 35 ], and is likely driven by poorly constrained factors such as water saturation and nutrient distribution [ 19 ]. Our assessment reveals that the main uncertainty in quantifying Fe(II) mobilization rates stems from the lack of high-resolution O₂ measurements. This limitation likely leads to a systematic underestimation of oxic Fe(III) reduction, underscoring the need for improved microscale oxygen mapping in future studies. Note that our approach does not aim to predict absolute Fe(II) concentrations in natural soil and sediment porewater, as it does not include a comprehensive description of Fe(II) dynamics. For example, it overlooks key microscale environmental factors—like pH gradients, shear stress, and nutrient availability—that change with biofilm thickness and microbial community structure [ 36 – 38 ], physiological differences in Fe(III) reduction under oxic vs. anoxic conditions, and ignores Fe(II) reactions in pore spaces, such as complexation, oxidation, or interactions with oxygen radicals. Nonetheless, it offers an interesting framework for quantifying Fe(II) mobilization rates and advancing our understanding of Fe cycling in oxic subsurface environments. 4 Conclusions Our study shows that S. oneidensis mediates ferrihydrite reduction under oxic conditions, driving Fe(II) mobilization even in sediments with limited or transient anoxic pore space volume. Anoxic microsites remain the dominant Fe(II) source, but their contribution is short-lived and spatially limited, underscoring the need to account for microscale O₂ gradients and biomass distribution in biogeochemical models. The impact of different environmental factors (e.g., type and concentration of nutrients and ferrihydrite, pH, temperature, etc.) on oxic Fe(III) reduction rate is yet to be investigated. However, our results highlight the need to integrate ferrihydrite reduction into iron cycle conceptualizations across oxic subsurface environments, such as shallow aquifers, capillary fringes, soils, and coastal and lake sediments. Even if Fe(II) remains undetected, oxic Fe(III) reduction might still occur, hidden by rapid Fe(II) re-oxidation by O 2 . Accurate ferrihydrite reduction rates are critical for predicting contaminant and nutrient release. Oxic microbial ferrihydrite reduction may explain elevated arsenate, chromate, and other metal levels in well-oxygenated systems [ 39 ] [ 8 , 40 ]. Moreover, oxic microbial ferrihydrite reduction may have interesting applications in bioremediation [ 41 ]. Fe(III) reducers generate Fe(II) [ 42 ], a strong reductant, capable of transforming pollutants like chlorinated solvents [ 42 , 43 ] in oxic subsurface environments. 5 Methods 5.1 Experimental medium A liquid medium was prepared with deionized water, 10% v/v Luria Bertani broth (LB, Sigma Aldrich), and 20 mM PIPES (piperazine-N,N′-bis(2-ethanesulfonic) acid, Thermo Scientific). After adjusting the pH to ~ 7.1 using HCl (37%, Sigma Aldrich), the medium was sterilized by autoclaving. 5.2 Incubation setups The same experimental medium was used in oxic and anoxic incubations. The oxic incubation (Fig. 5 A) was performed in a 50 mL sterile Erlenmeyer flask, where a magnetic stirrer continuously stirred 25 mL of medium to maintain air-saturation conditions in the liquid phase. The flask was equipped with a 5 mm spot sensor (OXSP5 supplied by PyroScience) to monitor real-time bulk oxygen (O 2 ) concentrations. A sterile porous sponge lid sealed the vial to avoid air-borne contamination. For anoxic incubations (Fig. 5 B) 25 mL of sterile medium was placed in a 60 mL sterile serum vial crimped with a black butyl rubber lid and purged with 0.22 µm-filtered N 2 . To keep anoxic and oxic setups consistent, stirring was imposed also under anoxic conditions. Both oxic and anoxic incubations were inoculated by the same aliquot (1:50 v/v) of an aerobic culture of Shewanella oneidensis MR-1 ( S. oneidensis ), grown overnight in LB liquid broth at 30°C and incubated in an orbital shaker (180 rpm). S. oneidensis is a facultative Fe reducer commonly used to study Fe reduction under anoxic conditions [ 44 , 45 ], and it was recently found capable of mediating ferrihydrite reduction under oxic conditions [ 16 ]. After 48 hours of inoculation, corresponding to the early stationary phase of S. oneidensis growth in both oxic and anoxic incubations (see SI, Section S3), 2 mM of ferrihydrite (synthesized in the laboratory [ 46 ] and sterilized under UV light for 20 minutes) was added to triplicate cultures. This ferrihydrite concentration was chosen to reflect typical levels found in soils [ 3 ]. Mineral addition in the anoxic incubations was handled in a glove box (N 2 atmosphere, Jacomex) to avoid O 2 contamination. S. oneidensis was then incubated with ferrihydrite for six days (144 hours) under anoxic and oxic conditions. Uninoculated sterile medium and inoculated medium fixed with 4% formaldehyde (each in triplicate) were also incubated as negative and killed controls, respectively, under oxic and anoxic conditions. All the vials were wrapped in aluminum foil to protect them from light. 5.3 Cell count. At the time of inoculation and at 48 hours, before adding ferrihydrite to the culture, 1 mL aliquots of each incubation were sampled and fixed adding 36% formaldehyde to a final dilution of 4%. Cells were stained with DAPI (4',6-diamidino-2-phenylindole, final concentration 1µg mL − 1 ) and, after 15 minutes of reaction time, the sample was pipetted into a microfluidic device and imaged using an inverted microscope in the DAPI fluorescence channel. Images were post-processed to count cells and estimate cell concentrations during the stationary phase. Details on the microfluidic cell counting procedure are included in SI, Section S3. 5.4 Temporal dynamics of Fe(II) concentrations. Immediately after adding ferrihydrite, a filtered-sterilized 50 mM Ferrozine (FZ,3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt, Sigma Aldrich) stock solution was added to the incubations in a final concentration of 1 mM. Ferrozine is a well-known Fe(II) chelator that forms a magenta complex with Fe(II), which has an absorbance peak at 560 nm. The complex traps the Fe(II) potentially produced by microbially-mediated Fe(III) reduction[ 20 , 25 , 26 , 29 ], preventing its re-oxidation or re-precipitation into secondary mineral phases. Fe(II) concentration was monitored every 24 hours in each vial by measuring the absorbance of the FZ-F(II) complex via a spectrophotometric method (see SI, Section S1 for details). At the end of the incubations, the measured Fe(II) concentrations in the oxic incubations were verified by mass spectrometry (see SI, Section S4 for details and results). 5.5 Fe(III) reduction rates. The temporal evolution of Fe(II) concentration was linearly fitted using cftool MATLAB® (R2021b, version 9.11.0.1769968) to estimate the overall Fe(III) reduction rates under oxic ( R O , [µM h − 1 ]) and anoxic ( R A , [µM h − 1 ]) conditions. Microbial growth attained the stationary phase at t < 48 h. Therefore, the number of alive cells is constant. We normalized the overall Fe(III) reduction rates to the cell concentrations at 48 hours to determine the Fe(III) reduction rate per cell under oxic ( r o , [µM h − 1 cell − 1 ]) and anoxic ( r A , [µM h − 1 cell − 1 ]) conditions. 5.6 Probabilistic assessment of Fe(II) mobilization in oxic sediments Estimating the relative contribution of oxic and anoxic ferrihydrite reduction in oxic sediments to Fe(II) mobilization requires mapping the pore space colonized by biomass and the corresponding microscale distribution of O 2 concentrations. Although methodological limitations and the opacity of soils and sediments prevent direct monitoring in real samples [ 11 , 19 ] recent advancements in microfluidic approaches have allowed direct measurements of biomass and O 2 concentration distribution at the pore scale in laboratory analogs of oxic sediments and soils [ 15 , 18 , 22 , 47 , 48 ] that we used as a reference. We used the data presented in [ 15 ] who simulated the porewater flow in sandy sediment progressively colonized by an aerobic model strain Pseudomonas Putida GB1. Biomass and O 2 concentrations were mapped at the microscale hourly for 45 hours to identify the portion of pore space colonized by biomass and characterized by anoxic conditions, i.e., anoxic microsite volume. To the best of our knowledge, this is the only study where O 2 and biomass were mapped simultaneously in a heterogeneous confined environment, and no similar studies are available for S. oneidensis to date. By elaborating on this dataset, we tracked the time evolution of pore space volume occupied by oxic biomass ( V O ) and anoxic microsites ( V A ) (Fig. 4 a, see SI Section S5, for computational details). We assumed that ferrihydrite was homogeneously distributed in the pore space and that all the cells growing in the sediment could perform ferrihydrite reduction, switching between oxic ( r O ) and anoxic ( r A ) rates as a function of the microenvironment experienced by the cells. In other words, ferrihydrite reduction is feasible in every portion of the pore space colonized by biomass independently of the redox state, with rates varying in space and time, responding to the local O 2 concentration. The total Fe(II) mobilization rate ( M T [µM h − 1 ]) resulted from the sum of oxic ( M O ) and anoxic biomass ( M A ) contributions expressed as follows \(\:\left\{\begin{array}{c}{M}_{A}\left(t\right)={r}_{A}\:{\rho\:}_{cell}{\:V}_{A}\left(t\right)\\\:\:\:{M}_{o}\left(t\right)={r}_{o}{\:\rho\:}_{cell}{\:V}_{o}\left(t\right)\:\:\:\end{array}\right.\) Eq. (1) Here, r A and r O [µM cell − 1 h − 1 ] are the ferrihydrite reduction rates estimated from anoxic and oxic S. oneidensis incubations and cell counting, while ρ cell [cell mm − 3 ] is the cell density per unit of pore space. The quantities V A and V O are the pore space volume [mm 3 ] occupied by anoxic microsites and oxic biomass in the oxic sediment elaborated from [ 15 ]. (Fig. 4 A). To assess the instantaneous relative contribution of anoxic microsites ( c A ) and oxic biomass ( c O ) to Fe(II) mobilization rate, we assumed a constant and uniform cell density in the pore space occupied by biomass, and Eq. (1) is recast into \(\:\left\{\begin{array}{c}{c}_{A}\left(t\right)=\frac{{M}_{A}}{{M}_{T}}=\frac{{r}_{A}{\:V}_{A}\left(t\right)}{{r}_{A}{\:V}_{A}\left(t\right){+r}_{o}{\:V}_{o}\left(t\right)}\\\:\:\:{c}_{o}\left(t\right)=\frac{{M}_{O}}{{M}_{T}}=\frac{{r}_{O}{\:V}_{O}\left(t\right)}{{r}_{A}{\:V}_{A}\left(t\right){+r}_{o}{\:V}_{o}\left(t\right)}\:\:\:\end{array}\right.\) Eq. (2) To assess the relative impact of oxic biomass ( C O ) and anoxic microsite ( C A ) in Fe mobilization on a longer timescale, the relative contribution of Fe(II) mobilized by anoxic microsites and oxic biomass was then compared to Fe(II) mobilized cumulated over 45 hours, i.e., \(\:\left\{\begin{array}{c}{C}_{A}\left(t\right)=\frac{\sum\:_{0}^{t}{r}_{A}{\:V}_{A}\left(t\right)}{\sum\:_{0}^{T}\left[{r}_{A}{\:V}_{A}\left(t\right){+r}_{o}{\:V}_{o}\left(t\right)\right]}\\\:\:\:{C}_{o}\left(t\right)=\frac{\sum\:_{0}^{t}{r}_{O}{\:V}_{O}\left(t\right)}{\sum\:_{0}^{T}\left[{r}_{A}{\:V}_{A}\left(t\right){+r}_{o}{\:V}_{o}\left(t\right)\right]}\:\:\:\end{array}\right.\) Eq. (3) The uncertainty associated with the input parameters ( r O and r A ) and predictors ( V O and V A ) is incorporated into the analysis and propagated to the final outputs using a Monte Carlo approach (see details in SI, Section S5). Declarations Acknowledgments The authors thank Prof. Rizlan Bernier-Latmani and Dr. Ashley Brown (Environmental Microbiology Laboratory, EPFL, Lausanne, CH) for providing the Shewanella Oneidesis MR-1 strain. Giulia Ceriotti acknowledges Dr. Florence Morgenthaler Grand for the technical support at the Cellular Image Facility – UNIL, and Prof. William Burgos for the insightful discussion on ferrozine-amended live bacterial incubations. Funding Giulia Ceriotti acknowledged the financial support of the Matterhorn grant (FGSE funding) and SNSF Sinergia fund (grant no. CRSII5_213522). Alice Bosco-Santos acknowledged the financial support provided by the 2022 Agassiz Foundation (grant no. 26086987) and the Fondation pour l’Université de Lausanne. Author Contributions GC: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing, Formal analysis, Software, Visualization. AB-S: Conceptualization, Funding acquisition, Visualization, Writing – review & editing. JB: Funding acquisition, Resources, Supervision, Writing – review & editing. References Kappler, A., et al., An evolving view on biogeochemical cycling of iron. Nature Reviews Microbiology, 2021. 19 (6): p. 360-374. Pallud, C., Y. Masue-Slowey, and S. Fendorf, Aggregate-scale spatial heterogeneity in reductive transformation of ferrihydrite resulting from coupled biogeochemical and physical processes. Geochimica et Cosmochimica Acta, 2010. 74 (10): p. 2811-2825. Huang, J., et al., Fe (II) redox chemistry in the environment. Chemical Reviews, 2021. 121 (13): p. 8161-8233. Dixit, S. and J.G. Hering, Comparison of arsenic (V) and arsenic (III) sorption onto iron oxide minerals: implications for arsenic mobility. Environmental science & technology, 2003. 37 (18): p. 4182-4189. Zhao, Y., et al., The mobility and fate of Cr during aging of ferrihydrite and ferrihydrite organominerals. 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Zhang, L., et al., Iron reduction by diverse actinobacteria under oxic and pH-neutral conditions and the formation of secondary minerals. Chemical Geology, 2019. 525 : p. 390-399. Ahmerkamp, S., et al., Simultaneous visualization of flow fields and oxygen concentrations to unravel transport and metabolic processes in biological systems. Cell Reports Methods, 2022. 2 (5). Schoeffler, M., et al., Growth of an anaerobic sulfate‐reducing bacterium sustained by oxygen respiratory energy conservation after O2‐driven experimental evolution. Environmental microbiology, 2019. 21 (1): p. 360-373. Pedraz, L., N. Blanco‐Cabra, and E. Torrents, Gradual adaptation of facultative anaerobic pathogens to microaerobic and anaerobic conditions. The FASEB Journal, 2020. 34 (2): p. 2912-2928. Royer, R.A., et al., Enhancement of biological reduction of hematite by electron shuttling and Fe (II) complexation. Environmental science & technology, 2002. 36 (9): p. 1939-1946. Royer, R.A., et al., Enhancement of hematite bioreduction by natural organic matter. Environmental science & technology, 2002. 36 (13): p. 2897-2904. Bauer, I. and A. Kappler, Rates and extent of reduction of Fe (III) compounds and O2 by humic substances. Environmental science & technology, 2009. 43 (13): p. 4902-4908. Morgan, B. and O. Lahav, The effect of pH on the kinetics of spontaneous Fe (II) oxidation by O2 in aqueous solution–basic principles and a simple heuristic description. Chemosphere, 2007. 68 (11): p. 2080-2084. Smith, G.L., et al., Complexation of ferrous ions by ferrozine, 2, 2′-bipyridine and 1, 10-phenanthroline: Implication for the quantification of iron in biological systems. Journal of Inorganic Biochemistry, 2021. 220 : p. 111460. Posth, N., et al., Banded iron formations , in Encyclopedia of geobiology . 2011, Springer. Gankhurel, B., et al., Arsenic and uranium contamination of Orog Lake in the Valley of Gobi Lakes, Mongolia: Field evidence of conservative accumulation of U in an alkaline, closed-basin lake during evaporation. Journal of Hazardous Materials, 2022. 436 : p. 129017. Obuekwe, C.O., D.W. Westlake, and F.D. Cook, Effect of nitrate on reduction of ferric iron by a bacterium isolated from crude oil. Canadian journal of microbiology, 1981. 27 (7): p. 692-697. Zhang, I.H., et al., Ratio of electron donor to acceptor influences metabolic specialization and denitrification dynamics in Pseudomonas aeruginosa in a mixed carbon medium. Frontiers in Microbiology, 2021. 12 : p. 711073. Lies, D.P., et al., Shewanella oneidensis MR-1 uses overlapping pathways for iron reduction at a distance and by direct contact under conditions relevant for biofilms. Applied and environmental microbiology, 2005. 71 (8): p. 4414-4426. Kuzyakov, Y. and E. Blagodatskaya, Microbial hotspots and hot moments in soil: concept & review. Soil Biology and Biochemistry, 2015. 83 : p. 184-199. Ramasamy, P. and X. Zhang, Effects of shear stress on the secretion of extracellular polymeric substances in biofilms. Water Science and Technology, 2005. 52 (7): p. 217-223. Hegler, F., et al., Does a low-pH microenvironment around phototrophic FeII-oxidizing bacteria prevent cell encrustation by FeIII minerals? FEMS Microbiology Ecology, 2010. 74 (3): p. 592-600. Ackermann, M. and S. van Vliet, Spatial self-organization of metabolism in microbial systems: a matter of enzymes and chemicals. Cell systems, 2023. 14 (2): p. 98-108. Smedley, P.L., Sources and distribution of arsenic in groundwater and aquifers. 2008. Aftabtalab, A., et al., Review on the interactions of arsenic, iron (oxy)(hydr) oxides, and dissolved organic matter in soils, sediments, and groundwater in a ternary system. Chemosphere, 2022. 286 : p. 131790. Jiang, Y., et al., Advances in Fe (III) bioreduction and its application prospect for groundwater remediation: A review. Frontiers of Environmental Science & Engineering, 2019. 13 : p. 1-11. Tosco, T., et al., Transport of ferrihydrite nanoparticles in saturated porous media: role of ionic strength and flow rate. Environmental science & technology, 2012. 46 (7): p. 4008-4015. Watts, M.P. and J.R. Lloyd, Bioremediation via microbial metal reduction. Microbial metal respiration: from geochemistry to potential applications, 2012: p. 161-201. Shi, L., et al., Molecular underpinnings of Fe (III) oxide reduction by Shewanella oneidensis MR-1. Frontiers in Microbiology, 2012. 3 : p. 50. Abboud, R., et al., Low-temperature growth of Shewanella oneidensis MR-1. Applied and environmental microbiology, 2005. 71 (2): p. 811-816. Cornell, R.M. and U. Schwertmann, The iron oxides: structure, properties, reactions, occurrences, and uses . Vol. 664. 2003: Wiley-vch Weinheim. Borer, B., R. Tecon, and D. Or, Spatial organization of bacterial populations in response to oxygen and carbon counter-gradients in pore networks. Nature communications, 2018. 9 (1): p. 769. Borer, B., et al., Reduced gravity promotes bacterially mediated anoxic hotspots in unsaturated porous media. Scientific reports, 2020. 10 (1): p. 8614. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6726027","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471325853,"identity":"62d4be9c-68d6-49b6-800d-b8c183c7d4dd","order_by":0,"name":"Giulia Ceriotti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie3NMQrCMBQG4BcKuoiuAcFcIcXFQXD0GsliJ8HRoUik4CS66i30BpFCpx5A0KFFcNZFnMTXKihC09Uh/5A83uPjB7Cx+cc4QLTIJ6LwoS0cphpAmwh8iBa0nQ1mAt9XxFK9NsWE9WtcJz6wRjOcnq73jrc5BNjiHwuJGyIREbjrhQxwosPNcYckOheTICMVEDwmM5qTvVSaqLCEPED03sTjZYQ5SOQMW2ovIkoJdyojLRfUXcUk4PGAuuuMiMjQsgy36f3WZY15NU3G3Qmr7700ufiGFp1/9GctCgG2KMPRxsbGxibPE02iXurQmyCoAAAAAElFTkSuQmCC","orcid":"","institution":"University of Lausanne","correspondingAuthor":true,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Ceriotti","suffix":""},{"id":471325854,"identity":"1a3a9f76-3945-4d60-996a-cc869c19b9b3","order_by":1,"name":"Alice Bosco-Santos","email":"","orcid":"","institution":"University of Lausanne","correspondingAuthor":false,"prefix":"","firstName":"Alice","middleName":"","lastName":"Bosco-Santos","suffix":""},{"id":471325855,"identity":"b6ab6557-df87-43b9-9dcc-df267f1e16f0","order_by":2,"name":"Jasmine S. Berg","email":"","orcid":"","institution":"University of Lausanne","correspondingAuthor":false,"prefix":"","firstName":"Jasmine","middleName":"S.","lastName":"Berg","suffix":""}],"badges":[],"createdAt":"2025-05-22 14:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6726027/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6726027/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-16963-w","type":"published","date":"2025-08-26T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84691096,"identity":"48cfe6b0-2950-43ac-9efd-9658eaeec967","added_by":"auto","created_at":"2025-06-16 09:44:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":314122,"visible":true,"origin":"","legend":"\u003cp\u003ea) Representation of a porous oxic subsurface environment. b) Zoom in of the pore space and solid grains with attached biofilms of variable thicknesses controlled by the solid matrix structure and pore water flow. Depending on the thickness of the biofilm that mostly controls the microscale balance between O\u003csub\u003e2\u003c/sub\u003e transport and the intensity of aerobic respiration, the biofilm may remain permanently oxygenated (c) or host the formation of an anoxic microsite (d).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6726027/v1/cc9c80127199d8a49edb24d5.png"},{"id":84691092,"identity":"383c7e8f-e9b5-4e4d-94bd-810df27ff4a4","added_by":"auto","created_at":"2025-06-16 09:44:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":131032,"visible":true,"origin":"","legend":"\u003cp\u003ea) Evolution of Fe(II) concentrations in oxic and anoxic S. oneidensis incubations and the corresponding killed controls every 24 hours for 144 hours. A vertical bar indicates the standard deviation computed over triplicates. Results for negative controls are reported in SI (Section S2). Dashed lines indicate the linear interpolation of Fe(II) concentrations. b) Evolution of O\u003csub\u003e2\u003c/sub\u003e concentrations measured every 10 minutes by an optical spot sensor in oxic S. oneidensis incubations, reported as the average over the 3 replicates. The shaded area indicates the standard deviation around the average. For comparison, O\u003csub\u003e2\u003c/sub\u003e concentrations measured in one replicate of killed control are reported (black dots). Time t = 0 h corresponds to ferrihydrite and ferrozine addition to S. oneidensis cultures.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6726027/v1/5551bb6a1ea5eddb2c2c1015.png"},{"id":84691093,"identity":"c72d8cd7-86b4-40c6-a6bf-bb766de65d03","added_by":"auto","created_at":"2025-06-16 09:44:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":93766,"visible":true,"origin":"","legend":"\u003cp\u003ea) Cell concentrations (±standard deviation) of S. oneidensis computed for the initial inoculum and after 48 hours of incubation under oxic and anoxic conditions. b) Ferrihydrite, Fe(III), reduction rate per cell (±standard deviation) under oxic and anoxic conditions.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6726027/v1/e76267378eb198d8dc41372a.png"},{"id":84691095,"identity":"686de9e4-b43c-4ac3-85b7-a8d3825a633d","added_by":"auto","created_at":"2025-06-16 09:44:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":107213,"visible":true,"origin":"","legend":"\u003cp\u003ea) Pore space volume occupied by oxic biomass (V\u003csub\u003eO\u003c/sub\u003e) and anoxic microsites (V\u003csub\u003eA\u003c/sub\u003e) as a function of time computed from data in [15]. b) Temporal trend of the relative contribution of oxic biomass (c\u003csub\u003eO\u003c/sub\u003e) and anoxic microsites (c\u003csub\u003eA\u003c/sub\u003e) to the overall Fe(II) mobilization rate during the experiment (Eq. (2), Section 4.6). c) Cumulative relative contribution of Fe(II) mobilized by anoxic microsites and by oxic biomass during the entire duration of the experiment (45h), i.e., c\u003csub\u003eo\u003c/sub\u003e and c\u003csub\u003ea\u003c/sub\u003e integrated over time for 45 hours (Eq.(3), Section 4.6).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6726027/v1/ca19bc4e72efb66e86ec75b1.png"},{"id":84691409,"identity":"3ac59c86-3f23-4a25-88eb-8cf57747187b","added_by":"auto","created_at":"2025-06-16 09:52:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":117052,"visible":true,"origin":"","legend":"\u003cp\u003eSketches of S. oneidensis incubation setups under oxic conditions(a) and anoxic conditions (b).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6726027/v1/a3b4af701bc53ea533f62c9a.png"},{"id":90344855,"identity":"6513579a-75d6-4f83-a19b-31d4f326a8a1","added_by":"auto","created_at":"2025-09-01 16:06:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1377611,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6726027/v1/52ac1323-655a-46db-96b5-2e74257566e1.pdf"},{"id":84691102,"identity":"a36f1afe-c19d-4301-8a38-1f868fe849d0","added_by":"auto","created_at":"2025-06-16 09:44:47","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1243558,"visible":true,"origin":"","legend":"","description":"","filename":"SImodified.docx","url":"https://assets-eu.researchsquare.com/files/rs-6726027/v1/56bf10f8fe8b71969f5b7e92.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOxic microbial ferrihydrite reduction rates of \u003cem\u003eShewanella oneidensis\u003c/em\u003e and the potential for Fe mobilization in oxic sediments\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe microbial reduction of ferric iron, Fe(III), is a key anaerobic process driving natural subsurface biogeochemistry and releasing mobile ferrous iron, Fe(II) to the environment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The occurrence and the extent of microbial mobilization of Fe(II) depend on the ability of microorganisms to access and reduce solid Fe(III) minerals, the most reactive of which are considered to be amorphous oxides like ferrihydrite (Fe₂O₃\u0026middot;nH₂O). These minerals represent one of the main reservoirs of microbially accessible Fe(III) in soils and natural sediments.\u003c/p\u003e \u003cp\u003eFerrihydrite exhibits a high and non-selective adsorptive capacity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] for contaminants such as arsenic and chromium, as well as nutrients like phosphate and nitrate [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequently, the spatial distribution of ferrihydrite and the extent of its reduction in soils and sediments are closely linked to the rate of organic carbon mineralization, the mobility of toxic compounds and micronutrients as Fe(II), and overall soil fertility in agricultural systems [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs Fe(III) is a less thermodynamically favorable electron acceptor than oxygen (O\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], its microbial reduction has been considered confined in anoxic or O\u003csub\u003e2\u003c/sub\u003e-limited environments, like deep groundwater systems, flooded soils, ferruginous lakes, and wetlands. Nevertheless, the detection of organic matter-stabilized Fe(II) in some oxic subsurface environments indicates that ferrihydrite reduction occurs in oxygenated waters [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This is typically attributed to the formation of O\u003csub\u003e2\u003c/sub\u003e-depleted microscopic zones, referred to as anoxic microsites or microenvironments, that remain undetected in averagely well-oxygenated sediments and soils [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Such anoxic microsites are expected to form where O\u003csub\u003e2\u003c/sub\u003e diffusion is hindered by the sediment porous structure, heterogeneous pore water flow, and outcompeted by the aerobic growth of thick biofilm layers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Though transient and small in scale, these anoxic microsites enable localized ferrihydrite reduction and anaerobic respiration even in well-oxygenated bulk systems [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn alternative explanation for the unexpected presence of Fe(II) in oxic, circumneutral subsurface is the metabolic versatility of certain Fe-reducing microorganisms. As early as 1990, \u003cem\u003eShewanella putrefaciens\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was shown to reduce soluble Fe(III) complexes while respiring O\u003csub\u003e2,\u003c/sub\u003e suggesting a hybrid aerobic and anaerobic lifestyle. Though initially overlooked, this idea resurfaced in 2019 when diverse actinobacteria were found to reduce both soluble and solid Fe(III) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] in oxic batch incubations. These studies, however, measured O\u003csub\u003e2\u003c/sub\u003e only at the bulk scale, leaving unresolved whether Fe(III) reduction occurs in the presence of O\u003csub\u003e2\u003c/sub\u003e or in undetected anoxic microsites. Recent microfluidic experiments using O\u003csub\u003e2\u003c/sub\u003e sensors confirmed that the facultative Fe reducer, \u003cem\u003eShewanella oneidensis\u003c/em\u003e MR-1 (\u003cem\u003eS. oneidensis\u003c/em\u003e) can reduce Fe(III) in persistently oxic conditions in the microenvironment surrounding the cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This observation supports that microbial Fe(III) reduction in oxic sediments and soils is not confined solely to anoxic microsites, as previously assumed. Similar hybrid behavior has been observed for facultative denitrifiers [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], sulfate reducers [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and \u003cem\u003eEscherichia coli\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], suggesting this hybrid metabolism may be more widespread than previously recognized.\u003c/p\u003e \u003cp\u003eNonetheless, the rates and environmental importance of oxic Fe(III) reduction remain unknown. Scaling the rates of oxic Fe(III) reduction is critical to understanding iron and contaminant mobility in natural and engineered systems. The quantification of Fe(III) reduction rates under oxic conditions poses methodological challenges because Fe(II), produced by microbial ferrihydrite reduction, can be rapidly re-oxidized and re-precipitated as secondary minerals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As a result, Fe(II) may not accumulate in oxic porewaters, explaining why oxic Fe(III) reduction has so far been difficult to detect and rendering dissolved Fe(II) concentrations a poor indicator of microbial reduction rates in such environments.\u003c/p\u003e \u003cp\u003eIn this study, we quantify Fe(III) reduction rates by \u003cem\u003eS. oneidensis\u003c/em\u003e under oxic and anoxic conditions using a ferrozine-based method that stabilizes Fe(II) and prevents re-oxidation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Applying these rates to a representative sediment model, we reveal the overlooked contribution of oxic microbial iron reduction to Fe(II) mobilization in natural environments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Overall oxic and anoxic Fe(II) production rates in batch.\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. oneidensis\u003c/em\u003e grown in the dark in 10% v/v Luria Bertani broth, supplemented with 2 mM ferrihydrite and 1 mM ferrozine (FZ), mediated iron reduction under both oxic and anoxic conditions. Fe(II) progressively accumulated in the liquid medium under both conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), in the form of stable FZ-Fe(II) complexes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder anoxic conditions, Fe(II) concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) exceeded 300 \u0026micro;M after 72 hours, indicating efficient ferrihydrite respiration by \u003cem\u003eS. oneidensis\u003c/em\u003e. These Fe(II) levels surpassed the chelating capacity of 1 mM ferrozine (Supplementary Information, SI, Section S1 for further details). Fe(II) measurements are therefore reported only for up to 72 hours, during which soluble iron increased linearly. Under oxic conditions, Fe(II) concentrations also increased linearly over time, reaching 92.5\u0026thinsp;\u0026plusmn;\u0026thinsp;28.2 \u0026micro;M within 144h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In other words, ferrihydrite reduction occurred at a constant rate, although slower than under anoxic conditions. Ongoing aerobic respiration of \u003cem\u003eS. oneidensis\u003c/em\u003e after the addition of ferrihydrite and ferrozine is evidenced by the lower O\u003csub\u003e2\u003c/sub\u003e concentrations in live \u003cem\u003eS. oneidensis\u003c/em\u003e incubations compared to killed controls under the same stirring conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe slope of the linear trend quantified the overall Fe(II) production rate for \u003cem\u003eS. oneidensis\u003c/em\u003e incubations, with rates of 3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 \u0026micro;M h⁻\u0026sup1; (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, R\u0026sup2; = 0.99) under anoxic conditions and 0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.084 \u0026micro;M h⁻\u0026sup1; (R\u0026sup2; = 0.94) under oxic conditions. Remarkably, the overall oxic Fe(II) mobilization rates were only\u0026thinsp;~\u0026thinsp;6 times lower than those observed in anoxic conditions.\u003c/p\u003e \u003cp\u003eKilled (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and negative (SI, Section S2) controls exhibited small increases in soluble Fe(II), ranging from 4\u0026ndash;6 \u0026micro;M for anoxic conditions and 9\u0026ndash;12 \u0026micro;M under oxic conditions after 144h of incubation. These results suggest that abiotic ferrihydrite reduction, possibly driven by reducing groups contained in organic matter [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and reactions involving dead cell material (biomass) contributed minimally to the reduction of Fe(III) in Fe-oxides, as observed under both anoxic [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and oxic conditions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Linear fitting of the controls estimated the mean abiotic Fe(II) production rates to be 0.078\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0048 \u0026micro;M h⁻\u0026sup1; in oxic conditions and 0.034\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0043 \u0026micro;M h⁻\u0026sup1; in anoxic conditions. After subtracting the calculated abiotic contribution [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the overall Fe(II) production rates attributed to microbial activity were determined to be 0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.089 \u0026micro;M h⁻\u0026sup1; under oxic (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e) and 3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 \u0026micro;M h⁻\u0026sup1; under anoxic (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Oxic and anoxic Fe (III) reduction rate per cell\u003c/h2\u003e \u003cp\u003eTo normalize Fe(III) reduction rates to possible differences in growth rates under oxic and anoxic conditions, a cell-specific rate was calculated. For this, cell concentrations in batches were estimated in the stationary growth phase, just before the addition of ferrihydrite and ferrozine.\u003c/p\u003e \u003cp\u003eThe cell concentration increased from the initial inoculum under both anoxic and oxic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), consistent with protein concentration measurements (Figure S2, SI, Section S3). As expected, bacteria under aerobic conditions grow faster, resulting in cell concentrations one order of magnitude higher (2.1 x 10\u003csup\u003e9\u003c/sup\u003e \u0026plusmn; 2.3 x 10\u003csup\u003e9\u003c/sup\u003e cell mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than in anoxic conditions (1.2 x 10\u003csup\u003e8\u003c/sup\u003e \u0026plusmn; 5.7 x 10\u003csup\u003e7\u003c/sup\u003e cell mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eBy normalizing the overall Fe(II) production rate in batches computed in Section 2.1 to the cell concentration, we estimated the ferrihydrite reduction rate per cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) under oxic (2.6 x 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e \u0026plusmn; 0.5 x 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e \u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and anoxic conditions ( 3.2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e \u0026plusmn; 1.6 x 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e \u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). As a result, cells reduced ferrihydrite\u0026thinsp;\u0026asymp;\u0026thinsp;120 times faster under anoxic than under oxic conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Modeling of Fe(II) mobilization in oxic sediments.\u003c/h2\u003e \u003cp\u003eTo scale the potential impact of oxic Fe(III) reduction in sediments and soils, we modeled microbially-mediated Fe(II) mobilized from ferrihydrite reduction in a 27 mm-long laboratory analog of sandy aquifer sediment presented in the literature [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This laboratory system was used in [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] for real-time mapping of biomass colonization, O\u003csub\u003e2\u003c/sub\u003e concentrations, and anoxic microsite formation in the pore space for 45 hours, proving a level of insight into microscale O\u003csub\u003e2\u003c/sub\u003e and biomass dynamics that is not achievable in natural systems to date.\u003c/p\u003e \u003cp\u003eAccording to the results presented in [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the microbial biomass progressively colonized the sediment grain surfaces, eventually occupying up to 1.2 mm\u003csup\u003e3\u003c/sup\u003e of available pore space at 45 hours in the laboratory analog (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), corresponding to 35% of the total pore volume. The system remained oxic at the centimeter scale. Still, anoxic microsites formed transiently, peaking at 26 hours with a maximum volume of 0.05 mm\u003csup\u003e3\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), i.e., 1.42% (\u0026plusmn;\u0026thinsp;0.99%) of the pore space, and almost disappeared at t\u0026thinsp;\u0026gt;\u0026thinsp;35 h.\u003c/p\u003e \u003cp\u003eIn our model, we assumed a homogeneous distribution of ferrihydrite. Therefore, Fe(II) mobilization was modeled as a ubiquitous process, occurring at every point of the pore space colonized by biomass. However, the rate of iron reduction varied according to local O\u003csub\u003e2\u003c/sub\u003e levels, between oxic conditions (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e = 2.6 x 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e \u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and anoxic microsites (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e = 3.2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e \u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Eqs.\u0026nbsp;(1\u0026ndash;3), Section 4.5). The contribution of anoxic microsites to Fe(II) mobilization (\u003cem\u003ec\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) mirrored their temporal dynamics. Initially, when the system was fully oxic (\u003cem\u003et\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;10 hours), only oxic Fe reduction occurred in the biomass-colonized pore space. Once anoxic microsites are formed, while occupying only 1\u0026ndash;2% of the total pore space, they contribute up to 79\u0026ndash;94% to the total Fe(II) mobilization rate. Since oxic Fe(II) mobilization persisted throughout the experiment, the cumulative contribution of oxic biomass (\u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) accounted for 21%-42% of the total Fe(II) mobilized over 45 hours.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eComparing the temporal dynamics of Fe(II) concentrations in oxic and anoxic batches, we found that a \u003cem\u003eS. oneidensis\u003c/em\u003e population grown under oxic conditions reduces ferrihydrite at a rate less than one order of magnitude slower than under anoxic conditions.\u003c/p\u003e \u003cp\u003eOur observations indicated that Fe(II) concentrations reached 58.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8 \u0026micro;M after 72 hours of oxic \u003cem\u003eS. oneidensis\u003c/em\u003e batch incubations. These results align with previous findings obtained during the growth of \u003cem\u003eS. oneidensis\u003c/em\u003e on ferrihydrite with 10% v/v LB in an O₂-sensing microfluidic device, where Fe(II) accumulated up to 88.4\u0026thinsp;\u0026plusmn;\u0026thinsp;19.3 \u0026micro;M over 72 hours, while persistent oxic conditions were observed at the microscale [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Thus, we rule out the formation of anoxic microsites in our setup and attribute Fe(II) mobilization to oxic, microbially mediated ferrihydrite reduction.\u003c/p\u003e \u003cp\u003eThe slightly lower range of Fe(II) concentrations observed in this study compared to [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] may be due to the formation of soluble FeOH\u003csup\u003e+\u003c/sup\u003e. At the imposed pH [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and ferrozine concentrations [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], this process could compete with Fe(II) chelation by ferrozine, meaning a small portion of Fe(II) might not have been captured in our measurements. In contrast, samples in [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] were acidified, releasing Fe(II) from organic complexes and hydroxyl ions before ferrozine addition and assay.\u003c/p\u003e \u003cp\u003ePrevious studies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] have shown that the formation of ferrozine-Fe(II) complex can enhance the bioreduction of Fe-oxides, such as hematite, by maintaining low Fe(II) geochemical activity. However, the Fe(II) concentration measured at 72 hours is similar to the one found in the previous study [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], without a complexing agent, suggesting that ferrozine addition did not artifactually enhance Fe(III) reduction in our setup.\u003c/p\u003e \u003cp\u003eOur results also align with previous studies by Arnold et al. (1990) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], which examined another \u003cem\u003eShewanella\u003c/em\u003e strain, \u003cem\u003eS. putrefaciens\u003c/em\u003e, grown on dissolved Fe(III) and beef extract. In \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eputrefaciens\u003c/em\u003e batch incubations, the Fe(III) reduction rate in the presence of O\u003csub\u003e2\u003c/sub\u003e was about one order of magnitude lower (12 \u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than under anoxic conditions (240 \u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Notably, \u003cem\u003eS. oneidensis\u003c/em\u003e exhibits significantly slower Fe(III) reduction rates (~\u0026thinsp;100 times) compared to \u003cem\u003eS. putrefaciens\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] under both oxic and anoxic conditions. This discrepancy can be attributed to several factors. First, Arnold et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] used dissolved Fe(III), which is likely more bioavailable than solid Fe(III) in ferrihydrite. This means that the mineral ferrihydrite used in our study, while being more representative of Fe(III) form found in natural systems (e.g., rocks [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], sediments [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and soils [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]), likely reduces Fe(III) accessibility to bacteria, slowing down Fe(II) mobilization. Additionally, strain-specific traits, medium composition and concentration, and incubation temperature may have contributed to the slower Fe(III) reduction rate of \u003cem\u003eS. oneidensis\u003c/em\u003e compared to \u003cem\u003eS. putrefaciens\u003c/em\u003e. Indeed, \u003cem\u003eS. putrefaciens\u003c/em\u003e has one of the highest dissimilatory Fe(III) reduction rates per cell reported in the literature [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, the nutrient conditions and temperature in our study (10% v/v diluted Luria-Bertani broth, equivalent to 2 g L⁻\u0026sup1; LB; 23\u0026deg;C) were less favorable than those in \u003cem\u003eS. putrefaciens\u003c/em\u003e incubations (8 g L⁻\u0026sup1; Difco broth; 30\u0026deg;C) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is worth noting that a minor abiotic contribution to ferrihydrite reduction was observed in both oxic and anoxic control incubations, though it was significantly lower than the microbially mediated reduction. This aligns with previous studies where Fe-oxides (hematite or ferrihydrite) were observed to undergo abiotic reduction under both anoxic [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and oxic [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] incubations. This process is attributed to the complex composition of natural organic matter, which contains reduced functional groups capable of acting as electron shuttles for Fe(III) under anoxic conditions while retaining their reducing capacity in the presence of O\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In our incubations, the yeast extract, the major constituent of LB broth, was used to mimic the complex composition of natural organic matter [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], likely contributing to abiotic ferrihydrite reduction.\u003c/p\u003e \u003cp\u003eThe Fe(III)-ferrihydrite reduction rate per cell under anoxic conditions (3.2 \u0026times; 10⁻⁸ \u0026plusmn; 1.6 \u0026times; 10⁻⁸ \u0026micro;M h⁻\u0026sup1; cell⁻\u0026sup1; at a cell concentration of 1.2 \u0026times; 10⁸ \u0026plusmn; 5.7 \u0026times; 10⁷ cells mL⁻\u0026sup1;) aligns with previous findings for the same strain at a similar cell concentration (1.1 \u0026times; 10⁻⁸ \u0026plusmn; 8.1 \u0026times; 10⁻\u0026sup1;⁰ \u0026micro;M h⁻\u0026sup1; cell⁻\u0026sup1; at 6.7 \u0026times; 10⁸ cells mL⁻\u0026sup1;) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], reinforcing the reliability of our results. Interestingly, although the cell-specific Fe(III) reduction rate under oxic conditions is two orders of magnitude lower than under anoxic conditions, the total amount of Fe(II) mobilized in oxic conditions is less than one order of magnitude slower than in anoxic incubations. This discrepancy is due to the significantly higher cell density attained under oxic conditions, emphasizing the need to consider the collective activity of the population rather than just individual cell efficiency in Fe(III) reduction in large-scale environmental systems.\u003c/p\u003e \u003cp\u003eOur study was not designed to elucidate the physiological mechanisms by which \u003cem\u003eS. oneidensis\u003c/em\u003e mediates Fe(III) reduction in the presence of O₂, which would require a dedicated genetic investigation. However, if Fe(III) uptake serves an energetic purpose in parallel to O\u003csub\u003e2\u003c/sub\u003e, as suggested by Arnold et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the significantly lower Fe(III) reduction per cell under oxic conditions supports the idea that O₂ remains the preferred electron acceptor for this facultative Fe(III) reducer. This quantitative difference could also result from distinct Fe(III) reduction mechanisms, such as indirect electron shuttling mediated by organic matter and radicals [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, the observed variations in cell shape and size between oxic and anoxic conditions (Figure S3, SI, Section S3) further suggest potential differences in the ecophysiology of \u003cem\u003eS. oneidensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAlthough oxic microbially mediated Fe(III)-ferrihydrite reduction occurs\u0026thinsp;~\u0026thinsp;100 times slower than under anoxic conditions, it could play a crucial role in environments where oxic conditions dominate and anoxic microsites constitute a minority of the pore space [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our model simulation revealed that oxic ferrihydrite reduction can contribute approximately one-third of Fe(II) mobilized in the lab sediment analog within just a few days. This unexpectedly large contribution is explained by the fact that the cells exposed to oxic conditions constitute the majority of the biomass growing in the pore space of the oxic aquifer lab analog. Despite its slower instantaneous rate, Fe(III) reduction under oxic conditions acts as a continuous background process, gradually affecting an increasing portion of the pore space as biomass colonizes the porous medium. Similar conditions are likely to occur in certain subsurface environments, including well-drained and unsaturated soils, capillary fringes, and shallow aquifers, among the most microbially active environmental systems.\u003c/p\u003e \u003cp\u003eDespite their limited spatial extent and temporal duration, anoxic microsites host most of the Fe(II) mobilization in oxic sediments. Their contribution is inherently dynamic and halts immediately upon microsite dissipation. This transient behavior is expected in natural sediments and soils [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and is likely driven by poorly constrained factors such as water saturation and nutrient distribution [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our assessment reveals that the main uncertainty in quantifying Fe(II) mobilization rates stems from the lack of high-resolution O₂ measurements. This limitation likely leads to a systematic underestimation of oxic Fe(III) reduction, underscoring the need for improved microscale oxygen mapping in future studies.\u003c/p\u003e \u003cp\u003eNote that our approach does not aim to predict absolute Fe(II) concentrations in natural soil and sediment porewater, as it does not include a comprehensive description of Fe(II) dynamics. For example, it overlooks key microscale environmental factors\u0026mdash;like pH gradients, shear stress, and nutrient availability\u0026mdash;that change with biofilm thickness and microbial community structure [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], physiological differences in Fe(III) reduction under oxic vs. anoxic conditions, and ignores Fe(II) reactions in pore spaces, such as complexation, oxidation, or interactions with oxygen radicals. Nonetheless, it offers an interesting framework for quantifying Fe(II) mobilization rates and advancing our understanding of Fe cycling in oxic subsurface environments.\u003c/p\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eOur study shows that \u003cem\u003eS. oneidensis\u003c/em\u003e mediates ferrihydrite reduction under oxic conditions, driving Fe(II) mobilization even in sediments with limited or transient anoxic pore space volume. Anoxic microsites remain the dominant Fe(II) source, but their contribution is short-lived and spatially limited, underscoring the need to account for microscale O₂ gradients and biomass distribution in biogeochemical models.\u003c/p\u003e \u003cp\u003eThe impact of different environmental factors (e.g., type and concentration of nutrients and ferrihydrite, pH, temperature, etc.) on oxic Fe(III) reduction rate is yet to be investigated. However, our results highlight the need to integrate ferrihydrite reduction into iron cycle conceptualizations across oxic subsurface environments, such as shallow aquifers, capillary fringes, soils, and coastal and lake sediments. Even if Fe(II) remains undetected, oxic Fe(III) reduction might still occur, hidden by rapid Fe(II) re-oxidation by O\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eAccurate ferrihydrite reduction rates are critical for predicting contaminant and nutrient release. Oxic microbial ferrihydrite reduction may explain elevated arsenate, chromate, and other metal levels in well-oxygenated systems [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, oxic microbial ferrihydrite reduction may have interesting applications in bioremediation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Fe(III) reducers generate Fe(II) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], a strong reductant, capable of transforming pollutants like chlorinated solvents [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] in oxic subsurface environments.\u003c/p\u003e"},{"header":"5 Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e5.1 Experimental medium\u003c/h2\u003e \u003cp\u003eA liquid medium was prepared with deionized water, 10% v/v Luria Bertani broth (LB, Sigma Aldrich), and 20 mM PIPES (piperazine-N,N\u0026prime;-bis(2-ethanesulfonic) acid, Thermo Scientific). After adjusting the pH to ~\u0026thinsp;7.1 using HCl (37%, Sigma Aldrich), the medium was sterilized by autoclaving.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e5.2 Incubation setups\u003c/h2\u003e \u003cp\u003eThe same experimental medium was used in oxic and anoxic incubations. The oxic incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) was performed in a 50 mL sterile Erlenmeyer flask, where a magnetic stirrer continuously stirred 25 mL of medium to maintain air-saturation conditions in the liquid phase. The flask was equipped with a 5 mm spot sensor (OXSP5 supplied by PyroScience) to monitor real-time bulk oxygen (O\u003csub\u003e2\u003c/sub\u003e) concentrations. A sterile porous sponge lid sealed the vial to avoid air-borne contamination. For anoxic incubations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) 25 mL of sterile medium was placed in a 60 mL sterile serum vial crimped with a black butyl rubber lid and purged with 0.22 \u0026micro;m-filtered N\u003csub\u003e2\u003c/sub\u003e. To keep anoxic and oxic setups consistent, stirring was imposed also under anoxic conditions.\u003c/p\u003e \u003cp\u003eBoth oxic and anoxic incubations were inoculated by the same aliquot (1:50 v/v) of an aerobic culture of \u003cem\u003eShewanella oneidensis\u003c/em\u003e MR-1 (\u003cem\u003eS. oneidensis\u003c/em\u003e), grown overnight in LB liquid broth at 30\u0026deg;C and incubated in an orbital shaker (180 rpm). \u003cem\u003eS. oneidensis\u003c/em\u003e is a facultative Fe reducer commonly used to study Fe reduction under anoxic conditions [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], and it was recently found capable of mediating ferrihydrite reduction under oxic conditions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter 48 hours of inoculation, corresponding to the early stationary phase of \u003cem\u003eS. oneidensis\u003c/em\u003e growth in both oxic and anoxic incubations (see SI, Section S3), 2 mM of ferrihydrite (synthesized in the laboratory [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and sterilized under UV light for 20 minutes) was added to triplicate cultures. This ferrihydrite concentration was chosen to reflect typical levels found in soils [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mineral addition in the anoxic incubations was handled in a glove box (N\u003csub\u003e2\u003c/sub\u003e atmosphere, Jacomex) to avoid O\u003csub\u003e2\u003c/sub\u003e contamination. \u003cem\u003eS. oneidensis\u003c/em\u003e was then incubated with ferrihydrite for six days (144 hours) under anoxic and oxic conditions. Uninoculated sterile medium and inoculated medium fixed with 4% formaldehyde (each in triplicate) were also incubated as negative and killed controls, respectively, under oxic and anoxic conditions. All the vials were wrapped in aluminum foil to protect them from light.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e5.3 Cell count.\u003c/h2\u003e \u003cp\u003eAt the time of inoculation and at 48 hours, before adding ferrihydrite to the culture, 1 mL aliquots of each incubation were sampled and fixed adding 36% formaldehyde to a final dilution of 4%. Cells were stained with DAPI (4',6-diamidino-2-phenylindole, final concentration 1\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and, after 15 minutes of reaction time, the sample was pipetted into a microfluidic device and imaged using an inverted microscope in the DAPI fluorescence channel. Images were post-processed to count cells and estimate cell concentrations during the stationary phase. Details on the microfluidic cell counting procedure are included in SI, Section S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e5.4 Temporal dynamics of Fe(II) concentrations.\u003c/h2\u003e \u003cp\u003eImmediately after adding ferrihydrite, a filtered-sterilized 50 mM Ferrozine (FZ,3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt, Sigma Aldrich) stock solution was added to the incubations in a final concentration of 1 mM. Ferrozine is a well-known Fe(II) chelator that forms a magenta complex with Fe(II), which has an absorbance peak at 560 nm. The complex traps the Fe(II) potentially produced by microbially-mediated Fe(III) reduction[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], preventing its re-oxidation or re-precipitation into secondary mineral phases. Fe(II) concentration was monitored every 24 hours in each vial by measuring the absorbance of the FZ-F(II) complex via a spectrophotometric method (see SI, Section S1 for details). At the end of the incubations, the measured Fe(II) concentrations in the oxic incubations were verified by mass spectrometry (see SI, Section S4 for details and results).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5.5 Fe(III) reduction rates.\u003c/h2\u003e \u003cp\u003eThe temporal evolution of Fe(II) concentration was linearly fitted using \u003cem\u003ecftool\u003c/em\u003e MATLAB\u0026reg; (R2021b, version 9.11.0.1769968) to estimate the overall Fe(III) reduction rates under oxic (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e, [\u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]) and anoxic (\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e, [\u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]) conditions. Microbial growth attained the stationary phase at t\u0026thinsp;\u0026lt;\u0026thinsp;48 h. Therefore, the number of alive cells is constant. We normalized the overall Fe(III) reduction rates to the cell concentrations at 48 hours to determine the Fe(III) reduction rate per cell under oxic (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e, [\u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]) and anoxic (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e, [\u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]) conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5.6 Probabilistic assessment of Fe(II) mobilization in oxic sediments\u003c/h2\u003e \u003cp\u003eEstimating the relative contribution of oxic and anoxic ferrihydrite reduction in oxic sediments to Fe(II) mobilization requires mapping the pore space colonized by biomass and the corresponding microscale distribution of O\u003csub\u003e2\u003c/sub\u003e concentrations. Although methodological limitations and the opacity of soils and sediments prevent direct monitoring in real samples [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] recent advancements in microfluidic approaches have allowed direct measurements of biomass and O\u003csub\u003e2\u003c/sub\u003e concentration distribution at the pore scale in laboratory analogs of oxic sediments and soils [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] that we used as a reference.\u003c/p\u003e \u003cp\u003eWe used the data presented in [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] who simulated the porewater flow in sandy sediment progressively colonized by an aerobic model strain \u003cem\u003ePseudomonas Putida\u003c/em\u003e GB1. Biomass and O\u003csub\u003e2\u003c/sub\u003e concentrations were mapped at the microscale hourly for 45 hours to identify the portion of pore space colonized by biomass and characterized by anoxic conditions, i.e., anoxic microsite volume. To the best of our knowledge, this is the only study where O\u003csub\u003e2\u003c/sub\u003e and biomass were mapped simultaneously in a heterogeneous confined environment, and no similar studies are available for \u003cem\u003eS. oneidensis\u003c/em\u003e to date. By elaborating on this dataset, we tracked the time evolution of pore space volume occupied by oxic biomass (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e) and anoxic microsites (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, see SI Section S5, for computational details).\u003c/p\u003e \u003cp\u003eWe assumed that ferrihydrite was homogeneously distributed in the pore space and that all the cells growing in the sediment could perform ferrihydrite reduction, switching between oxic (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e) and anoxic (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) rates as a function of the microenvironment experienced by the cells. In other words, ferrihydrite reduction is feasible in every portion of the pore space colonized by biomass independently of the redox state, with rates varying in space and time, responding to the local O\u003csub\u003e2\u003c/sub\u003e concentration.\u003c/p\u003e \u003cp\u003eThe total Fe(II) mobilization rate (\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e [\u0026micro;M h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e]) resulted from the sum of oxic (\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e) and anoxic biomass (\u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) contributions expressed as follows\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\left\\{\\begin{array}{c}{M}_{A}\\left(t\\right)={r}_{A}\\:{\\rho\\:}_{cell}{\\:V}_{A}\\left(t\\right)\\\\\\:\\:\\:{M}_{o}\\left(t\\right)={r}_{o}{\\:\\rho\\:}_{cell}{\\:V}_{o}\\left(t\\right)\\:\\:\\:\\end{array}\\right.\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(1)\u003c/p\u003e \u003cp\u003eHere, \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e [\u0026micro;M cell\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e] are the ferrihydrite reduction rates estimated from anoxic and oxic \u003cem\u003eS. oneidensis\u003c/em\u003e incubations and cell counting, while \u003cem\u003eρ\u003c/em\u003e\u003csub\u003e\u003cem\u003ecell\u003c/em\u003e\u003c/sub\u003e [cell mm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e] is the cell density per unit of pore space. The quantities \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e are the pore space volume [mm\u003csup\u003e3\u003c/sup\u003e] occupied by anoxic microsites and oxic biomass in the oxic sediment elaborated from [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo assess the instantaneous relative contribution of anoxic microsites (\u003cem\u003ec\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) and oxic biomass (\u003cem\u003ec\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e) to Fe(II) mobilization rate, we assumed a constant and uniform cell density in the pore space occupied by biomass, and Eq.\u0026nbsp;(1) is recast into\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\left\\{\\begin{array}{c}{c}_{A}\\left(t\\right)=\\frac{{M}_{A}}{{M}_{T}}=\\frac{{r}_{A}{\\:V}_{A}\\left(t\\right)}{{r}_{A}{\\:V}_{A}\\left(t\\right){+r}_{o}{\\:V}_{o}\\left(t\\right)}\\\\\\:\\:\\:{c}_{o}\\left(t\\right)=\\frac{{M}_{O}}{{M}_{T}}=\\frac{{r}_{O}{\\:V}_{O}\\left(t\\right)}{{r}_{A}{\\:V}_{A}\\left(t\\right){+r}_{o}{\\:V}_{o}\\left(t\\right)}\\:\\:\\:\\end{array}\\right.\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(2)\u003c/p\u003e \u003cp\u003eTo assess the relative impact of oxic biomass (\u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e) and anoxic microsite (\u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) in Fe mobilization on a longer timescale, the relative contribution of Fe(II) mobilized by anoxic microsites and oxic biomass was then compared to Fe(II) mobilized cumulated over 45 hours, i.e.,\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\left\\{\\begin{array}{c}{C}_{A}\\left(t\\right)=\\frac{\\sum\\:_{0}^{t}{r}_{A}{\\:V}_{A}\\left(t\\right)}{\\sum\\:_{0}^{T}\\left[{r}_{A}{\\:V}_{A}\\left(t\\right){+r}_{o}{\\:V}_{o}\\left(t\\right)\\right]}\\\\\\:\\:\\:{C}_{o}\\left(t\\right)=\\frac{\\sum\\:_{0}^{t}{r}_{O}{\\:V}_{O}\\left(t\\right)}{\\sum\\:_{0}^{T}\\left[{r}_{A}{\\:V}_{A}\\left(t\\right){+r}_{o}{\\:V}_{o}\\left(t\\right)\\right]}\\:\\:\\:\\end{array}\\right.\\)\u003c/span\u003e \u003c/span\u003e Eq.\u0026nbsp;(3)\u003c/p\u003e \u003cp\u003eThe uncertainty associated with the input parameters (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) and predictors (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eO\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e\u003c/sub\u003e) is incorporated into the analysis and propagated to the final outputs using a Monte Carlo approach (see details in SI, Section S5).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors thank Prof. Rizlan Bernier-Latmani and Dr. Ashley Brown (Environmental Microbiology Laboratory, EPFL, Lausanne, CH) for providing the \u003cem\u003eShewanella Oneidesis\u0026nbsp;\u003c/em\u003eMR-1 strain. Giulia Ceriotti acknowledges Dr. Florence Morgenthaler Grand for the technical support at the Cellular Image Facility \u0026ndash; UNIL, and Prof. William Burgos for the insightful discussion on ferrozine-amended live bacterial incubations.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eGiulia Ceriotti acknowledged the financial support of the Matterhorn grant (FGSE funding) and SNSF Sinergia fund (grant no. CRSII5_213522). Alice Bosco-Santos acknowledged the financial support provided by the 2022 Agassiz Foundation (grant no. 26086987) and the Fondation pour l\u0026rsquo;Universit\u0026eacute; de Lausanne.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eGC: Conceptualization, Data curation, Investigation, Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Formal analysis, Software, Visualization. AB-S: Conceptualization, Funding acquisition, Visualization, Writing \u0026ndash; review \u0026amp; editing. JB: Funding acquisition, Resources, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKappler, A., et al., \u003cem\u003eAn evolving view on biogeochemical cycling of iron.\u003c/em\u003e Nature Reviews Microbiology, 2021. \u003cstrong\u003e19\u003c/strong\u003e(6): p. 360-374.\u003c/li\u003e\n\u003cli\u003ePallud, C., Y. Masue-Slowey, and S. 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Or, \u003cem\u003eSpatial organization of bacterial populations in response to oxygen and carbon counter-gradients in pore networks.\u003c/em\u003e Nature communications, 2018. \u003cstrong\u003e9\u003c/strong\u003e(1): p. 769.\u003c/li\u003e\n\u003cli\u003eBorer, B., et al., \u003cem\u003eReduced gravity promotes bacterially mediated anoxic hotspots in unsaturated porous media.\u003c/em\u003e Scientific reports, 2020. \u003cstrong\u003e10\u003c/strong\u003e(1): p. 8614.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"microbial iron reduction, oxygen, Shewanella oneidensis, anoxic microsites, oxic sediments, ferrihydrite","lastPublishedDoi":"10.21203/rs.3.rs-6726027/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6726027/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobially-mediated reduction of ferric iron, Fe(III), often found in the natural environment as ferrihydrite, plays a crucial role in Fe cycling, and hence nutrient and contaminant cycling, in subsurface environments. Traditionally, microbial ferrihydrite reduction has been considered an anaerobic process relegated to anoxic microsites within oxic subsurface environments. However, recent findings suggest that microbes can mediate Fe(III) reduction also under oxic conditions, although rates and environmental impact of this process are still unknown. Here, we quantified cell-specific rates of ferrihydrite reduction by the model organism \u003cem\u003eShewanella oneidensis\u003c/em\u003e MR-1 under oxic and anoxic conditions. Based on our experimental results, we assessed the relative contribution of oxic and anoxic pore spaces to Fe(II) mobilization in a laboratory analog of oxic aquifer sediments presented in the literature. Our results show that oxic Fe(III) reduction can significantly contribute to Fe(II) mobilization in oxic subsurface environments where anoxic microsites occupy a minority of the pore space, conditions that can be found in, e.g., shallow aquifers, well-drained soils, and capillary fringes. Despite the lower cell-specific rates of oxic Fe(III) reduction, it remains a persistent background process, playing a previously underestimated role in Fe cycling within oxic subsurface environments.\u003c/p\u003e","manuscriptTitle":"Oxic microbial ferrihydrite reduction rates of Shewanella oneidensis and the potential for Fe mobilization in oxic sediments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 09:44:42","doi":"10.21203/rs.3.rs-6726027/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-07T17:16:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-03T11:57:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-03T05:50:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197713460226900141476039637710853293124","date":"2025-07-03T02:47:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273354609989210123993573339321511368436","date":"2025-06-19T01:13:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283967281395676877717351585576346719451","date":"2025-06-16T08:20:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-12T14:57:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-12T14:56:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-12T08:50:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-05T10:44:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-05T09:45:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a316cb3-5847-442b-aea6-db2b1650c57e","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50105856,"name":"Earth and environmental sciences/Biogeochemistry/Element cycles"},{"id":50105857,"name":"Biological sciences/Microbiology/Environmental microbiology/Water microbiology"},{"id":50105858,"name":"Biological sciences/Microbiology/Environmental microbiology/Soil microbiology"},{"id":50105859,"name":"Earth and environmental sciences/Hydrology"},{"id":50105860,"name":"Physical sciences/Physics/Fluid dynamics"}],"tags":[],"updatedAt":"2025-09-01T16:00:13+00:00","versionOfRecord":{"articleIdentity":"rs-6726027","link":"https://doi.org/10.1038/s41598-025-16963-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-26 15:56:57","publishedOnDateReadable":"August 26th, 2025"},"versionCreatedAt":"2025-06-16 09:44:42","video":"","vorDoi":"10.1038/s41598-025-16963-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-16963-w","workflowStages":[]},"version":"v1","identity":"rs-6726027","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6726027","identity":"rs-6726027","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}