Iron reduction under oxic conditions by Microbacterium deferre sp. nov. A1-JK^T

Domain: earth_science
Published: 2025-07-04
Editorial outcome: minor_revision

Abstract
Microbacterium deferre sp. nov. A1-JKT is a metabolically versatile Gram-positive bacterium isolated from the oxic-anoxic interface of freshwater sediments colonised by cable bacteria. Here, we report the metabolic ability of M. deferre A1-JKT to simultaneously reduce oxygen and soluble Fe(III), challenging the long-standing paradigm that microbial Fe(III) reduction is restricted to anaerobic conditions. Electrochemical analyses demonstrate that M. deferre A1-JKT is capable of extracellular electron transfer (EET) mediated by secreted flavins. It retains electroactivity under mildly alkaline and halophilic conditions, reflecting its broad environmental tolerance. Genomic analyses reveal a non-canonical system involving cytochrome FccA and flavin reductase FmnA, without the involvement of conventional flavin-based extracellular electron transfer (FLEET) components. The ability to couple oxygen and Fe(III) reduction under oxic conditions demonstrates respiratory flexibility, enabling M. deferre A1-JKT to utilize both aerobic and anaerobic processes simultaneously in fluctuating environments. These findings offer insights into microbial adaptation to dynamic geochemical gradients and potentially explain strategies used by bacterial life during oxygenation of Earth’s atmosphere.

Introduction
Extracellular electron transfer (EET) is a process where microbes exchange electrons with extracellular acceptors and donors for energy generation and growth. EET is often used to access distant or scarce electron acceptors when soluble alternatives are unavailable1, but can also facilitate the utilization of soluble extracellular substrates2. Electroactive microbes broadly employ two different mechanisms to perform EET. Direct electron transfer relies on conductive nanowires and outer membrane cytochromes to perform EET via surface contact3,4. Mediated electron transfer, on the other hand, uses soluble redox shuttles that diffuse across the cell to ultimately reduce an extracellular electron acceptor5–7.

In recent years, electroactivity has expanded beyond the model organisms Shewanella oneidensis and Geobacter sulfurreducens8. Several other microbes, which were originally not viewed through the lens of electroactivity, have since been discovered to be electroactive. These include several Gram-positive bacteria like Enterococcus faecalis9,10, Listeria monocytogenes11, Lactiplantibacillus plantarum12 and filamentous Lysinibacillus varians GY3213. Electroactive microbes have been discovered in all kinds of ecosystems14, as electroactivity plays a key role in adapting to nutrient/oxygen limited zones and provides a survival advantage in fluctuating redox conditions15.

Sulphidic freshwater and marine sediments populated by cable bacteria are examples of such redox dynamic environments. Cable bacteria are filamentous, sulphide-oxidising bacteria, ubiquitously present in aquatic sediments16–18. Cable bacteria spatially segregate sulphide oxidation from oxygen reduction but link these half-reactions via long-distance electron transport through periplasmic, conductive fibres over centimetre distances18. Distinct biogeochemical gradients of oxygen, sulphide and pH are created in sediments due to the bioelectrochemical activity of cable bacteria, which consume oxygen at a record-breaking rate19. In such sediments, microaerophilic bacteria create a dynamic veil at the oxic-anoxic interface, where oxygen concentrations fluctuate. Many of these bacteria are facultative anaerobes20,21. Cable bacteria actively transporting electrons towards the oxic surface attract flocks of other bacteria22. This intriguing phenomenon was proposed to happen through interspecies EET and raises the possibility of studying the role of electroactivity in flocking bacteria in this dynamic environment22,23.

In this work, a novel Gram-positive bacterium, Microbacterium deferre A1-JKT, was isolated from the cable bacteria-transversed microaerophilic veil and studied for its electroactive metabolism. Unlike Gram-negative bacteria such as S. oneidensis and G. sulfurreducens, Gram-positive bacteria lack an outer membrane and instead possess a thick peptidoglycan cell wall, which limits direct electron transfer across the cell envelope24. Consequently, Gram-positive bacteria typically rely on soluble redox shuttles, such as flavins, to mediate EET11. Direct interspecies electron transfer via outer membrane cytochromes is rare in Gram-positives, with Thermincola potens JR being one notable exception25.

Microbacterium species are found in diverse environmental conditions, including a high-altitude desert26, human clinical specimens27 and even a radioactive waste site28. As polyextremophiles, members of Microbacterium can grow as psychrophiles29, halophiles30, both acido- and alkaliphiles under a wide range of pH conditions (pH 5–11) and as facultative anaerobes31. In pure cultures, M. deferre A1-JKT displays metabolic flexibility and can utilise several carbon sources, as most others of this genus32. Our experiments demonstrate that it can anaerobically respire Fe(III) and electrodes through flavin-mediated EET. We present the mechanism of EET in M. deferre A1-JKT along with a potential model of its electron transfer pathway, supported by physiological and genomic analyses. Further, we demonstrate the reduction of Fe(III) by M. deferre A1-JKT under aerobic conditions, contrary to the widely accepted notion of Fe(III) reduction occurring only anaerobically33. This discovery has implications for understanding microbial biogeochemical cycling on modern as well as early Earth transitioning towards the Great Oxidation Event. Besides, it offers opportunities to study bifurcations in microbial metabolism, which bacteria may use to optimise growth and survival in dynamic environments.

Results
Current generation by M. deferre A1-JKT in electrochemical reactors
The ability of M. deferre A1-JKT to use insoluble and soluble electron acceptors was initially evaluated electrochemically. In electrochemical three-electrode cells, carbon felt electrode constituted the insoluble electron acceptor. All three-electrode cells inoculated with M. deferre A1-JKT, demonstrated a similar trend generating electric current over time: a lag phase of ~20 h while the cells acclimatised to the electrode as the electron acceptor; a rising-current phase, leading to a peak; and finally, a decrease in current (Fig. 1a). Current levels increased steadily from ~10 µA at 24 h and peaked at ~27 µA after 60 h. The subsequent decrease in current likely corresponded to a depletion of nutrients and cell death, as fresh medium injection into the reactors increased the current to previous levels similar to the log phase (Fig. 1a). Sterile nutrient medium, heat-killed M. deferre A1-JKT cells and M. deferre A1-JKT cells without an electron donor did not generate any current, implying that the microbe oxidised the provided electron donor (lactate) and subsequently reduced the electrode.

Soluble iron reduction under anoxic and oxic conditions
M. deferre A1-JKT also demonstrated an ability to reduce soluble Fe(III) to Fe(II) under anoxic conditions, which was determined by the ferrozine assay with ferric citrate (30 mM final concentration) provided as the electron acceptor (Fig. 1b). Fe(III)-reduction activity was evident as Fe(II) accumulated over time in incubations containing M. deferre A1-JKT.

The ability to reduce Fe(III) in the presence of oxygen was then evaluated, since both these electron acceptors are present at the oxic-anoxic interface of the sediment from which M. deferre A1-JKT was isolated. In two parallel experiments, oxygen concentration was measured in the headspace of the culture tubes subjected to constant stirring to prevent biofilm formation. In the first set of experiments, after the initial flush of atmospheric air, oxygen concentrations in M. deferre A1-JKT cultures containing 2 mM Fe(III)-citrate exhibited a distinct decline (Fig. 1c, top panel, red lines). In contrast, oxygen levels in the sterile nutrient medium control remained stable throughout the experiment (Fig. 1c, top panel, black lines). The decrease in oxygen concentration in the M. deferre A1-JKT cultures indicated active microbial oxygen consumption. In parallel, Fe(III) reduction, measured as Fe(II) accumulation, was assessed under these conditions (Fig. 1c, bottom panel). Fe(II) accumulation was evident in the presence of M. deferre A1-JKT, with the rate dependent on oxygen availability, seen as a change in the slope of the Fe(III) reduction rate after oxygen depletion (Fig. 1c, bottom panel, blue line). Under aerobic conditions, Fe(II) accumulation was moderate, indicating that Fe(III) reduction occurred alongside oxygen consumption. In anaerobic conditions, using the same experimental setup, Fe(II) production was higher than in the presence of oxygen (Fig. 1c, bottom panel, green line vs. blue line), reaching a maximum concentration of ~1.2 mM after six days. In sterile medium controls, minimal Fe(II) accumulation was observed under both aerobic and anaerobic conditions.

Figure 1d presents the results of the second set of Fe(III)-reduction experiments conducted under oxic conditions, now with oxygen concentrations maintained at atmospheric levels (~21%). Notably, Fe(II) production occurred consistently even under stable atmospheric conditions. Since the cultures were constantly stirred at 200 rpm and bubbled with air during the course of these experiments, no biofilm was formed and cells remained planktonic34. Under these conditions, no oxygen microgradients were expected to form, which are usually associated with biofilms35. Therefore, planktonic cells of M. deferre A1-JKT were the principal drivers of Fe(III) reduction under oxygen-saturated atmospheric conditions. Our experimental data challenge the long-held notion that Fe(III) reduction is a strictly anaerobic process and support similar evidence that facultative microorganisms can mediate Fe(III) reduction in oxic environments35.

The prioritization of oxygen over iron as an electron acceptor is expected due to its higher redox potential but aerobic conditions appear to only partially suppress Fe(III) reduction (Fig. 1c). This metabolic flexibility has significant implications for redox-stratified environments such as sediments, marshes, and soils, where oxygen and Fe(III) gradients coexist. By simultaneously reducing oxygen and Fe(III), M. deferre A1-JKT could contribute to the generation of a specific niche for iron cycling and thus influence geochemical processes, the microbial community composition and species distribution in these habitats. Additionally, the resilience of M. deferre A1-JKT to oxygen fluctuations makes it a promising candidate for applications where oxygen intrusion cannot be fully controlled, for example, in bioremediation strategies targeting metal contaminants in dynamic redox settings.

Mechanism of extracellular electron transfer
We investigated the mechanism of EET in M. deferre A1-JKT through electrochemical analyses. Cyclic Voltammetry (CV) of M. deferre A1-JKT cultures with a carbon felt working electrode produced a distinct redox couple at −0.302 V (vs Ag/AgCl) (Fig. 2a), suggesting involvement of soluble redox shuttles mediating electron transfer6. The CV shows a second oxidation peak, occurring at +0.18 V (Fig. 2a). This peak was, however, inconsistent in differential pulse voltammetry (DPV) (Fig. 2b), suggesting a smaller role in EET. Based on the potential, this peak was identified as soluble iron as demonstrated previously for iron chloride36.

The peak heights at negative potentials increased in the CV and DPV over time (Fig. 2b). This indicated that M. deferre A1-JKT synthesised potential redox shuttles, with concentrations correlated to cell growth. Randles–Ševčík plots (Fig. 2c) based on scan rate analysis produced linear curves proportional to the square root of the scan rates, confirming that electron transfer occurred by diffusion and that mediator concentration was the limiting factor in current generation.

Electrochemical Impedance Spectroscopy (EIS) analysis revealed increasing charge transfer resistance over time, as seen in Fig. 2d. This increase suggests that electron transfer became progressively more sluggish, possibly due to the accumulation of extracellular material on the electrode surface. The initial low impedance at 0 and 24 h reflects more favourable electron transfer conditions. Biofilm assays and Scanning Electron Microscopy (SEM) imaging revealed weak biofilm formation by M. deferre A1-JKT (Figs. S1 and S2), suggesting that electron transfer likely remained mediator-dependent and not biofilm-driven37,38.

Media swap and HPLC confirm flavin-mediated EET
By separating the electrolyte (spent medium without M. deferre A1-JKT cells) and the carbon-felt electrodes from the three-electrode cells, we determined the degree of direct and mediated electron transfer by M. deferre A1-JKT. A schematic is provided in Fig. S3 to clarify the media swap experimental setup. DPV on reactors with fresh medium and old electrodes containing attached cells of M. deferre A1-JKT showed no redox peaks, implying an absence of components necessary for performing direct electron transfer (Fig. 3a). Redox peaks were observed with the M. deferre A1-JKT electrolyte previously used in a three-electrode cell, confirming the presence of secreted mediators.

High-performance liquid chromatography (HPLC) analyses of the cell-free spent medium confirmed the presence of riboflavin. This is consistent with reports of flavins occurring at a similar negative potential in other studies6,39,40. Riboflavin concentrations in M. deferre A1-JKT samples increased from 200 nM after 24 h to 1.6 µM after 6 days at peak current generation (Fig. S4). A test run of S. oneidensis MR-1 in the same electrochemical setup yielded riboflavin at a concentration of 2.2 µM. Further, in reactors containing fresh electrodes in old electrolytes, riboflavin concentration was estimated at 1.4 µM. In reactors with new nutrient medium and old electrodes containing attached cells of M. deferre A1-JKT, riboflavin concentration after three days was only 100 nM, supporting that redox shuttles were secreted in the medium and direct electron transfer was minimal. When riboflavin (2 µM) was injected into reactors containing old electrodes in new media, current generation increased from 5 µA to 35 µA (Fig. 3b), confirming mediated electron transfer as the specific mode of EET.

Further, following the end of the experiments, the electrolyte was carefully collected and centrifuged. The supernatant was filtered, and the resulting spent media was analysed via DPV (Fig. 3c). Similar peaks were seen in the culture and the spent medium, validating the above results. Filter-sterilised spent media, supplemented with 20 mM lactate, was then inoculated with M. deferre A1-JKT cells in sterile electrochemical reactors with fresh electrodes (Fig. 3d). Current increased rapidly with no lag phase and reached a peak value around ~20 h, as the spent media already had a high concentration of flavins. This contrasts with Fig. 1a, where a longer lag phase was observed due to the absence of flavins in a fresh medium. These results further confirm riboflavin-mediated EET. In addition, flavins were secreted even under aerobic conditions, with riboflavin concentrations of 0.5 µM (Fig. S4). Riboflavin secretion in the presence of oxygen is likely the mechanism used by M. deferre A1-JKT for the reduction of Fe(III) under oxic conditions, as described above.

Versatility to perform EET in alkaline and halophilic conditions
Because M. deferre A1-JKT encounters varying pH conditions in its natural environment, its capability to grow at different pH (5–10) was tested. Growth curves under aerobic conditions at different pH showed that M. deferre A1-JKT was readily adaptable to pH changes as long as oxygen was present (Fig. S5). In the oxic zone of aquatic sediments, where iron oxides are formed, the pH can be alkaline (up to pH 9.0) due to cable bacteria activity41. Chronoamperometric plots of M. deferre A1-JKT cultures at pH 9 and 10 showed increasing currents, indicating weak electroactivity (Fig. S6).

M. deferre A1-JKT also demonstrated weak electroactivity under halophilic (4% NaCl) conditions (Fig. S6). Specific reasons behind the functionality of performing EET under high salt concentrations remain unknown. M. deferre A1-JKT was able to grow at 4% NaCl concentrations but did not grow at 10% NaCl (Table S1). The closest phylogenetic relative to M. deferre A1-JKT, Microbacterium sp. Gd4–13 (ANI 98.98%, making it M. deferre Gd4–13), demonstrated the ability to grow at salt concentrations of 10% NaCl (Fig. S7 and Table S1)30. Other Microbacterium species, amongst which is the second closest relative M. hatanonis JCM 14558 (ANI 91.47%, 5% NaCl), also showed tolerance to high salt concentrations ranging 5–10% (Table S1 and Supplementary data 1).

Potential EET pathways
Potential EET pathways of M. deferre A1-JKT based on genomic data are highlighted in Fig. 4. Its flavin production was reflected in the genome by genes for the flavin synthesis cascade: RibBADE1E2 (Table S2). A partial route using the proteins DmkA, DmkB, FmnA and Ndh3 might control electron flux towards aerobic respiration and EET42 as previously seen for other Gram-positive electroactive bacteria like L. monocytogenes, L. lactis, L. plantarum, E. faecalis, and Gram-positive bacteria with flavin-based extracellular electron transfer (FLEET)11,43,44. However, in contrast to conventional FLEET, the genes for PplA, FmnB, EetA and EetB, were absent in M. deferre A1-JKT. This is noteworthy because it suggests a non-canonical flavin-based EET pathway distinct from the FLEET system observed in Listeria monocytogenes11,45 and Enterococcus faecalis9,44, where knock-out mutants missing only one of these genes showed extremely low or no electroactivity11. The presence of FccA and FmnA, but absence of PplA, EetA and EetB components point to the possibility of an alternative mechanism adapted to Gram-positive bacterial physiology. To the best of our knowledge, only one other hybrid system has been described to date—in Vibrio natriegens, a Gram-negative bacterium that incorporates elements of two Gram-negative systems (Shewanella oneidensis and Aeromonas hydrophila)46. M. deferre A1-JKT represents the first example of a cross-Gram hybrid EET pathway, combining EET genes typically found in Gram-negatives (Shewanella, Geobacter) and Gram-positives (Enterococcus, Listeria) in a Gram-positive cellular architecture, expanding the evolutionary and mechanistic landscape of microbial EET.

Reduced flavins are exported to transfer electrons towards extracellular electron acceptors. Outside of flavins, a partial synthesis pathway for phenazines, PhzDEG was detected (Table S2). Potentially, both flavins and phenazines could be used as mediators in EET. Yet, only flavin signature peaks were detected in voltammetry measurements. The complex production and energetically expensive costs of phenazines likely prevented their synthesis in the minimal nutrient medium that was used for the electrochemical cells, or the absence of the PhzABF components. Another reason for the lack of phenazines could be that some of the genes from the phenazine synthesis operon were used in alternative biosynthetic pathways rather than serving solely in phenazine production, as has been proposed in previous studies49.

Interestingly, the outer membrane cytochrome ExtH, discovered in G. sulfurreducens, was also encountered in the genome of M. deferre A1-JKT (Supplementary Data 1). The finding of a Gram-negative outer membrane protein that forms an EET-related protein complex in a Gram-positive bacterium is notable. In G. sulfurreducens, ExtH is involved in selenium uptake50,51. ExtH-like proteins in M. deferre A1-JKT are predicted to be localised to the cytoplasm (Supplementary Data 1), and we hypothesise that these cytochrome homologs support electron transfer to Ndh3 and to DmkA and DmkB. However, the specific role in EET of these cytochromes in M. deferre A1-JKT remains unclear. We propose the existence of a periplasmic protein that helps to facilitate fast internal electron transport, although no clear evidence has been identified. This hypothesis could explain the absence of the signature peak in DPV. Further studies that involve knock-out mutations are required to elucidate the role of the ExtH-homologs, the presence of a periplasmic transport protein, and any other associated proteins.

Genomic prevalence of hybrid EET traits across the Microbacterium genus
To investigate the prevalence of hybrid EET systems within the Microbacterium genus, we analysed 667 publicly available Microbacterium genomes, including those of lower quality (<90% completeness and/or >5% contamination). None of the genomes encoded the canonical FLEET components EetA, EetB, PplA, or FmnB, reinforcing the absence of a complete FLEET pathway in this genus (Fig. 5 and Supplementary Data 2). However, various genes associated with alternative EET pathways were found across the genus. For instance, PhzB, Bfe and DmsA were detected in 3, 7 and 46 genomes, respectively. PhzA was completely absent, while PhzF, involved in phenazine biosynthesis, was present in 408 genomes. Most other phenazine and flavin biosynthetic genes were broadly distributed (Supplementary Data 2).

Key EET-associated proteins ExtH, YeeO, and FccA were detected in 314, 582, and 664 strains, respectively, indicating that hybrid EET systems—comprising both Gram-negative and Gram-positive elements—may be common in Microbacterium. Furthermore, genes encoding OmpB and OmpC, two multicopper outer membrane cytochromes involved in Fe(III) reduction in Geobacter52, were identified in 33 and 97 genomes. Other FLEET genes—dmkA, dmkB, and fmnA were also highly prevalent and found in 507, 660 and 612 genomes (Supplementary Data 2), suggesting a conserved potential for flavin-based electron transfer.

Phylogenetic analysis showed that the closest relatives of M. deferre A1-JKT shared a similar repertoire of EET-related genes, with M. hatanonis JCM 14558 being the only exception, having gained ompC and phzF (Figs. 5 and S7). Interestingly, both M. hatanonis and M. deferre A1-JKT were the only species amongst this group with dmkA. Although speculative, the presence of dmkA in M. hatanonis—originally isolated from hairspray—raises questions about potential metabolic versatility linked to unusual environments. Additionally, three of the second-closest relatives encoded dmsA, a gene used by S. oneidensis for extracellular dimethyl sulphoxide respiration53, and all but one of the closest relatives carried coxAB, with M. testaceum TPD7010 lacking coxA, confirming that they were aerobes. Given the proposed evolutionary link between cox genes and iron oxidation54, their presence further supports the association between these strains and redox-active environments.

From an ecological perspective, the distribution of these EET-associated genes does not appear confined to a single ecological niche. While some species, such as M. testaceum and M. proteolyticum are typically associated with plant hosts55,56, others—including M. enclense, M. hydrothermale, M. deferre Gd4–13, and M. deferre A1-JKT—have been isolated from marine or freshwater sediments57,58, where high rates of oxygen consumption and redox gradients are common. Even transient anaerobic conditions in such environments could provide a selective advantage to microbes capable of EET. This metabolic flexibility may help explain the ecological success and persistence of Microbacterium spp. across diverse niches, including as opportunistic pathogens in humans59.

Metabolic potential of M. deferre A1-JKT
As stated, M. deferre A1-JKT has several potential pathways to interact with extracellular electron acceptors that were discovered in the 3.5 Mb-large genome, consisting of 5 contigs with a coverage of 334, and GC% of 69.5. With the large metabolic versatility documented in the Microbacterium genus, we investigated the genomic potential of M. deferre A1-JKT towards drug degradation. Genomic analyses revealed the ability to degrade several different types of drugs, polystyrene, DMSO (dimethylsulphone or methylsulphonylmethane; MSM) and formaldehyde (Fig. S8). Further, the capability of breaking down (poly)sugars, which it likely uses for assimilation, was also present, though growth on L-oriented amino acids is likely not possible. Assimilation of single or dual-chain sugars occurs only after 48–72 h, while fermentation of such sugars could occur after 24 h. M. deferre A1-JKT was not capable of producing H2S but may produce very small amounts of N2 (Table S1).

The ability to degrade various drugs could have implications for environmental and biotechnological applications. The ability to break down complex organic compounds highlights the potential role of M. deferre A1-JKT in bioremediation, particularly in the degradation of persistent pollutants. Additionally, its capacity to assimilate and ferment (poly)sugars, may support its use in waste treatment processes where diverse organic substrates are present. Further exploration of its metabolic pathways could advance our understanding of its ecological niche and its utility in industrial processes.

Implications for early Earth’s redox evolution
The ability of M. deferre A1-JKT to simultaneously reduce both Fe(III) and oxygen under atmospheric oxygen conditions provides a modern example of microbial flexibility in redox-stratified environments. During Earth’s Great Oxidation Event (GOE), rising atmospheric oxygen levels would have imposed selective pressures on anaerobic microorganisms, many of which relied on Fe(III) as a terminal electron acceptor60. While the evolutionary origin of the EET traits observed in M. deferre A1-JKT is unknown, oxygen-tolerant EET mechanisms may represent one of several strategies that have independently evolved in microbes to enable survival under fluctuating redox conditions.

Recent studies have suggested that EET systems can mitigate the harmful effects of oxygen by dissipating excess electrons and minimising reactive oxygen species (ROS) formation61. In S. oneidensis, for instance, the redox-active disulphide motif in the extracellular cytochrome MtrC acts as an oxygen-sensitive switch, regulating flavin binding and suppressing ROS formation under aerobic conditions61. Although M. deferre A1-JKT lacks the Mtr-complex, its ability to sustain flavin-mediated EET under oxic conditions suggests the presence of alternative mechanisms to mitigate ROS toxicity. It was positive for the catalase enzyme that breaks down peroxidase, removing toxic ROS species. Based on these observations, we speculate that oxygen-tolerant EET pathways—such as those demonstrated in M. deferre A1-JKT—may reflect general microbial strategies to adapt to redox transitions. While their specific relevance to microbial evolution during the GOE remain speculative, such pathways illustrate how EET mechanisms can support microbial persistence in dynamic redox environments both past and present.

An unconventional role for EET
In addition to supporting respiration under variable redox conditions, EET may serve a previously underappreciated role as an adaptive strategy for oxygen reduction60. The outer-surface localisation of EET components such as cytochromes and flavin shuttles enables direct interaction with extracellular electron acceptors, including oxygen24. This configuration could facilitate oxygen reduction at the cell surface, mitigating intracellular ROS formation and protecting redox-sensitive cytoplasmic machinery. In S. oneidensis, improper regulation of flavin-bound EET proteins in oxic conditions results in cytotoxic peroxide production, suggesting that EET pathways must be tightly regulated to manage oxidative stress61. In M. deferre A1-JKT, the coexistence of EET with aerobic respiration suggests a strategy in which EET serves not only for energy conservation via Fe(III) reduction, but also potentially as a mechanism to offload excess electrons to oxygen via riboflavin. This perspective challenges the prevailing view of EET as an exclusively anaerobic metabolism and proposes oxygen reduction as a functional role for EET systems under oxic conditions62.

The ability of M. deferre A1-JKT to perform EET under fully oxic conditions challenges the traditional paradigm that restricts microbial Fe(III) reduction to anoxic environments. This dogma originated from early studies in strict anaerobes such as Geobacter and facultative anaerobes like Shewanella, where Fe(III) reduction was observed in the absence of oxygen, and where abiotic Fe(II) reoxidation presumably obscured microbial Fe(III) reduction under oxic conditions. However, recent work has demonstrated that Fe(III) reduction can proceed in fully oxic microenvironments where oxygen is not always the dominant electron acceptor35,63. Our findings extend this emerging view by demonstrating that the Gram-positive M. deferre A1-JKT, lacking the canonical FLEET pathway, can maintain flavin-based EET under oxic conditions.

In sediments where cable bacteria are found, unique selection pressures like pH and oxygen fluctuations arise because of cable bacteria activity. M. deferre A1-JKT has the metabolic potential to thrive in these sediments by performing EET towards minerals available as electron acceptors, as well as reducing oxygen to optimise its growth rate. To better understand the molecular mechanisms enabling simultaneous oxygen and Fe(III) reduction, studies using gene knockouts and transcriptomics are critical. Together, these findings broaden our understanding of microbial electroactivity under oxic conditions and open new avenues for exploring how facultative microbes metabolically adapt to dynamic environments.

Methods
Isolation
A trench slide was used to inoculate sediment containing freshwater cable bacterium Ca. E. aureum GS64 as per Bjerg et al.18. Microaerophilic veils are characteristically formed in trench slides as oxygen diffuses into the trench slide. After cable bacteria were observed stretching out from the trench, visualised capillary suction was employed to draw out the liquid from the microaerophilic veil, as described earlier by Lustermans et al.65. Following plating on Reasoners 2-A agar plates, M. deferre A1-JKT colonies were identified and transferred under laminar airflow to sterile agar plates to obtain pure cultures. The agar plates were incubated aerobically in dark conditions at 15 °C.

Physiological testing
API 20E and 20NE test strips (Biomérieux) were inoculated and read according to the manual. Strips were incubated in the dark at room temperature for 24 h initially, and subsequently for 48 and 72 h. Growth was determined at different temperatures: 4 °C, 15 °C, 18–22 °C (room temperature), and 37.5 °C, by inoculating and incubating Nutrient Broth (NB), VWR, R2A (Millipore) and Luria-Bertani (VWR) agar plates in the dark. Similarly, growth in incremental salt concentrations 1–4%, and 10% NaCl in LB plates were tested. Since LB contains 1% NaCl, R2A plates were used for 0% NaCl.

Nutrient medium composition for electrochemical cells
A defined nutrient medium was prepared for electrochemical studies. The nutrient medium was modified Shewanella basal medium (SBM), consisting of the following components (per litre): K2HPO4, 0.225 g; KH2PO4, 0.225 g; NaCl, 0.46 g; (NH4)2SO4, 0.225 g; MgSO4·7H2O, 0.117 g; HEPES, 23.83 g. Sodium lactate (20 mM) was provided as the carbon source. Mineral and vitamin mix (5 ml each) was added. The mineral mix consisted of nitrilotriacetic acid, 1.5 g; MnCl2·4H2O, 0.1 g; FeSO4·7H2O, 0.3 g; CoCl2·6H2O, 0.17 g; ZnCl2, 0.1 g; CuSO4·5H2O, 0.04 g; AlK(SO4)2·12H2O, 0.005 g; H3BO3, 0.005 g; Na2MoO4, 0.09 g; NiCl2, 0.12 g; Na2WO4·2H2O, 0.02 g and Na2SeO3, 0.1 g (per litre), while the vitamin mix consisted of folic acid, 0.002 g; biotin, 0.002 g; pyridoxine HCl, 0.02 g; thiamine, 0.005 g; nicotinic acid, 0.005 g; pantothenic acid, 0.005 g; cyanocobalamin, 0.0001 g; p-aminobenzoic acid, 0.005 g and thioctic acid, 0.005 g.

Iron reduction assay (Ferrozine assay)
Fe(III) reduction was estimated using ferrozine assay as described earlier66. To summarise, M. deferre A1-JKT was grown overnight in LB medium and washed and resuspended in the amended SBM to an OD of 1. Thirty microlitres of this suspension was added to 270 µl of SBM containing vitamins, minerals, 30 mM lactate and 30 mM Fe(III) citrate in 96-well plates. The plates were flushed with nitrogen for 20 min. Fifty microlitres of 5 M HCl was added to prevent oxidation of Fe(II). Thirty microlitres were taken from this well and added to 270 µl of 0.5 M HCl. Fifty microlitres of this diluted sample were mixed with 300 µl of ferrozine reagent. Absorbance was measured at 562 nm. Standard curves were made from FeSO4 dissolved in 0.5 M HCl. Fe(III) reduction was compared with S. oneidensis MR-1 grown in SBM with 30 mM lactate as the electron donor.

Electrochemical cell construction
Three-electrode cells fabricated from serum glass bottles (30 ml working volume) were used as electrochemical reactors. The working electrode consisted of carbon felt (Thermo Fisher, dimensions 2×2×0.3 cm), Ag/AgCl (REF201 Radiometer Analytical, Denmark) constituted the reference electrode and a Pt wire was used as the counter electrode. The cells were inoculated with 3 ml (10% inoculum) of M. deferre A1-JKT culture (OD 0.4), followed by addition of 27 ml nutrient medium. After inoculation, nitrogen was sparged for 20 min to make the reactors anoxic, following which electrochemical experiments were conducted. The reactors were operated at 25 °C.

Electrochemical measurements
A potential of +0.2 V was applied using a MultiPalmSens4 with the MultiTrace v4.5 software (PalmSens) potentiostat for chronoamperometry (CA). Additionally, chronoamperometric measurements were performed under alkaliphilic and halophilic conditions (pH 8–10; 4% NaCl). CV was performed at a potential window from −0.6 V to +0.6 V at a scan rate of 1 mV/s. For Randles–Sevcik plots, CV was done at varying scan rates of 1, 5, 10 and 25 mV/s. DPV was performed with the same scan window as CV, using 1 s period, 0.3 s pulse time, 50 mV pulse size and 1 mV step size. EIS was done in potentiostatic mode based on midpoint potential of the cyclic voltammogram (−0.384 V). The potential amplitude was 30 mV.

At the end of the experiments, spent medium was analysed via CV and DPV. The electrolyte from the three-electrode cells was collected and centrifuged at 6000×g for 15 min, followed by collecting the supernatant and repeating the centrifugation for 30 min. The resulting supernatant was filtered using a 0.22 µm membrane to obtain cell-free spent medium. The spent medium was transferred to new three-electrode cells with fresh electrodes for electrochemical studies. CA, CV, DPV, Randles–Sevcik plots and EIS were all conducted in triplicates.

Media swap experiments
After operating the three-electrode cells for a period of 7 days, the electrodes and electrolyte were separated into new reactors to ascertain redox-active components and elucidate the EET mechanism. The old electrodes were carefully transferred to electrochemical reactors with new nutrient medium, while new electrodes were introduced into the old electrolyte.

Aerobic Fe(III) reduction experiments
M. deferre A1-JKT was inoculated in the above-described nutrient medium in Hungate tubes (final volume 10 ml) with lactate as the electron donor. Fe(III) citrate (2 mM) was introduced in the medium as an electron acceptor. The starting OD of the culture after inoculation was 0.4. The culture and headspace in the Hungate tubes were sparged with air for 20 min through a 0.2 µm filter to make it oxygen-saturated. To prevent biofilm formation, the tubes were subjected to stirring (200 rpm) with a magnetic bead and stirrer. The headspace of the Hungate had optical oxygen spot sensors that were connected to a FirestingO2 device (Pyroscience). Data was logged using Pyroscience Workbench v1.5.3.2466. O2 concentrations were continuously measured in the headspace by oxygen spot sensors, while Fe(III) reduction was estimated by the ferrozine assay. In a separate experiment, air was continuously sparged using an aquarium pump to keep oxygen levels constant at atmospheric levels. Fe(III) reduction was estimated by ferrozine assay as described earlier.

High-performance liquid chromatography (HPLC)
HPLC was done to quantify redox shuttles. Based on the redox peaks in cyclic voltammograms, flavins were targeted for quantification by HPLC. Filtered spent medium from three-electrode cells was used. HPLC was done on a Dionex UltiMate 3000 UPLC system (Thermo Scientific) equipped with a C18 analytical column. Fluorescence of riboflavin was monitored (excitation 440 nm; emission 525 nm). Final riboflavin concentration was determined by eluting a series of riboflavin standards (100 nM, 200 nM, 400 nM, 800 nM, 1 µM, 2 µM and 3 µM) and preparing a standard curve. Samples were run in triplicates.

Microtiter dish biofilm formation assay
The ability of M. deferre A1-JKT to form biofilms was estimated by the microtiter dish biofilm formation assay, as described previously37,38. Briefly, the cells from the three-electrode reactors were diluted 1:100. One hundred microlitres of this culture was added in 96-well plates, followed by addition of 125 µl of 0.1% crystal violet solution. Following incubation at room temperature for 15 min, the plates were washed with distilled water. After drying, 125 µl of 30% acetic acid was added in the wells. Absorbance was measured at 550 nm.

Gram staining
Cells of M. deferre A1-JKT were deposited onto a drop of milliQ water, air dried and heat-fixated on a microscopy slide by moving through a flame. The sample was stained with crystal violet for 1 min and thoroughly rinsed with MilliQ water, followed by iodine solution flooding for 1 min and rinsing. Decolouring was done for 2 s with 100% ethanol and rinsing. Safranine solution was used for counterstaining for 30 s and rinsing. The slide was air dried and inspected with a light microscope as described below.

Light and scanning electron microscopy (SEM)
Samples of M. deferre A1-JKT were put on glass microscopy slides, anoxic tap water was added and covered with a glass coverslip. Light microscopy images were made with phase-contrast at 1000× with an oil lens (Zeiss Axioplan 2), black and white camera (Exi Blue) with Image-Pro Insight software. SEM samples were prepared by putting a carbon sticker on an aluminium SEM pin in a dust free environment. Carbon felt electrodes (after concluding the electrochemical cell measurements) were deposited on the carbon sticker and dried for 24 h, followed by vacuum gold-sputtering for 40 s (Agar Sputter Coater). Images were made with a Phenom ProX SEM (Phenom-World BV) using a backscattered electron detector with a 10 kV electron beam. Images were used to determine motility, spore formation, and measure cell size. Sizes were measured using the measure tool in Fiji’s ImageJ v1.54p67.

DNA extraction, 16S gene- and genome sequencing
As described in Lustermans et al.65, 16S rRNA-based taxonomy was determined through a colony PCR on a single colony of M. deferre A1-JKT, which was Sanger sequenced (Macrogen). A second sample of multiple colonies of M. deferre A1-JKT (also from an agar plate) was used for DNA extraction with the DNeasy PowerSoil kit (Qiagen). Concentration and length quality checks were done using Qubit (Fischer Scientific) and an Implen Spectrophotometer (Fischer Scientific) according to the manual. Genomic DNA was sent for Illumina NovaSeq sequencing 2×150 bp (Eurofins, Germany).

Genome assembly, annotation, analyses and phylogeny
Trimming, size filtering and adapter removal was done with Trimmomatic v0.3968 crop 125 and headcrop 20. Trimmed paired-end reads were de novo assembled with SPAdes v3.15.569 with parameters isolate, default coverage cut-off and default k-mer values. Statistics for completeness, contamination, quality, G+C content and coverage were calculated with Quast v5.2.070, fastq-stats v1.0171 and CheckM2 v1.1.070,72.

All available Microbacterium genomes were downloaded from NCBI GenBank and used as database for gene identification with PROKKA v1.14.573. Before use, the database was deduplicated with CD-HIT v4.8.1 with a length cut-off of 0.8, clustered into the most similar cluster, and 0.9 sequence identity threshold74,75.

Metabolic pathways were determined with BlastKOALA v3.0 and KEGG mapper76,77. EET- and electron shuttle synthesis genes were identified through blastp v2.11.0+78, using a manually, in-house curated, protein database of EET-related genes with an E-value cut-off of 1E-6. Cellular location predictions were done on the amino acid sequences with positive blastp results with psortb v3.0.379.

Phylogeny and taxonomic trees were determined with GTDB-Tk2 infer and align80, FastANI v1.3381 and iTOL82 v1.7 on all Microbacterium genomes from NCBI. As outgroup, Mesorhizobium loti LMG 6125 was used. ANI was used to determine differences with the two closest relatives M. hatanonis JCM 14558 and M. sp. Gd 4–13.

The Microbacterium genomes from NCBI, including M. deferre A1-JKT were checked for completeness, size, G+C content, contamination (Checkm2 v1.1.0) and average nucleotide identity (FastANI v1.33), translated to proteins (PROKKA v1.14.5), and blastp 2.11.0+ was performed with an E-value cut-off of 1E-6, protein identity cut-off at 25% (included in positive results) to determine prevalence of the EET pathway and flavin or phenazine production.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Comments to Lustermans et al.\nThe manuscript entitled “Flavin-based extracellular electron transfer under oxic conditions by Gram-positive Microbacterium\ndeferre sp. nov. A1-JK in redox-graded environments” by Lustermans et al. reported a new Gram-positive bacterium that can\nreduce Fe(III) in the presence of oxygen via extracellular electron transfer (EET). It involves isolating a novel bacterium,\ncharacterizing its physiological features, sequencing the genome, and deciphering the key genes for multiple important\nmetabolic routes. The paper is overall well-written and presents plausible results to me that this new bacterium harbors the\ndescribed flavin-based EET (although some clarifications are needed). I have a couple of suggestions to hopefully make the\nfindings more understandable.\nMajor comments\nIt is essential to know whether the cells use oxygen when they reduce Fe(III). Because they can form biofilm, what is the\nproportion of biofilm-forming vs. free-living cells in the culture experiments? If it is pretty high, the A1-JK cells may reduce\nFe(III) in anoxic microniches, and the resulting Fe(II) can be oxidized abiotically and draw down the oxygen concentration. In\nthis case, it is hard to attribute the oxygen consumption to microbial utilization.\nIt would be great to see more detailed explanations/hypotheses on (i) why this EET (or all EETs) is not inhibited by oxygen\nand (ii) why people have the widely-held notion that Fe reduction is an anaerobic process. I can see that Riboflavin\nexcretion seems to be one of the keys to this EET. Some explanations on why riboflavin excretion is not affected by\noxygenated conditions could help readers better understand this oxygen-tolerant Fe reduction process.\nIn addition to the biotechnological application potential, more data about the abundance and prevalence of this bacterium in\nthe environment would help us better understand the environmental relevance of this bacterium and the flavin-based EET.\nAccording to Fig. S7, some species related to A1-JK in the genus Microbacterium have also been cultured. I am curious to\nknow whether they have the flavin-based EET and other metabolic pathways described in the manuscript. A (culture- or\ngenome-based) assessment like this could also help us better understand the environmental relevance and\nbiotechnological application potential of this bacterium (and related others). The results can be put together with Fig. S7,\nwhich could be moved into the main text.\nMinor comments\nRef. 31 looks like the following peer-reviewed publication.\nCeriotti, G., Bosco-Santos, A., Borisov, S.M., and Berg, J.S. (2025) Decoupling microbial iron reduction from anoxic microsite\nformation in oxic sediments: a microscale investigation through microfluidic models. Frontiers in Microbiology 16.", "reviewer_2": "Lustermans et al. use a combination of electrochemical, chemical, genomic, and culture-based experiments to demonstrate\nthat a strain of Gram-positive Microbacterium, newly isolated from a microaerophilic veil surrounding freshwater cable\nbacteria, reduces soluble Fe(III) or a poised electrode not only in anoxic but also in oxic conditions via endogenous flavin as\nan electron shuttle. Additionally, they propose a new species of the genus represented by the strain. The findings are\nrelevant to the potential survival strategy of the “flocking bacteria” in the sediments inhabited by cable bacteria. However, my\nenthusiasm for the findings is dampened by several key issues.\n1. Novelty of the endogenous flavin-based EET mechanism under oxic conditions:\nAlthough the experimental design and data interpretation regarding the EET are well-executed, the novelty of this study is\nsomewhat limited. The authors present evidence for an endogenous flavin-based EET under oxic conditions in a Gram-\npositive bacterium, yet similar mechanisms have already been documented in Gram-negative Shewanella species (e.g., von\nCanstein et al. 2008, Appl. Environ. Microbiol.; Biffinger et al. 2009, Biotechnol. Bioeng.; Rosenbaum et al. 2010, Biotechnol.\nBioeng.; TarAvest et al. 2014, Biotechnol. Bioeng. —although the latter was under microaerobic conditions). Given that both\nmicrobial groups share similar EET mechanisms despite differences in membrane structure, the findings largely align with\nexisting literature rather than presenting a fundamentally new concept.\n2. Justification for the creation of a new species:\nThe authors propose the creation of a new species represented by the strain they isolated. However, the justification for this\nclassification is insufficient. Key comparative analyses, such as DNA-DNA hybridization and/or Average Nucleotide Identity\n(ANI) values, as well as physiological comparisons with other strains in the genus, are not provided. Without these standard\ncomparisons, the claim of a novel species remains unsubstantiated. Additionally, it is strongly recommended that the strain\nbe deposited in at least two international culture collections to ensure accessibility and reproducibility.\nMinor comments:\nTable S2 is challenging to interpret due to the lack of full names for the proteins listed. Including this additional information\nwould help in understanding the context and relevance of the data presented."}, "author_response": "We sincerely thank the editor and the reviewers for their insightful feedback which\nhas helped in improving the quality of the manuscript. Our responses to the\nreviewers’ questions are provided below in blue text. We have also revised the\nmanuscript suitably as per the reviewers’ feedback.\nReviewer #1 (Remarks to the Author):\nComments to Lustermans et al.\nThe manuscript entitled “Flavin-based extracellular electron transfer under oxic conditions by\nGram-positive Microbacterium deferre sp. nov. A1-JK in redox-graded environments” by\nLustermans et al. reported a new Gram-positive bacterium that can reduce Fe(III) in the\npresence of oxygen via extracellular electron transfer (EET). It involves isolating a novel\nbacterium, characterizing its physiological features, sequencing the genome, and\ndeciphering the key genes for multiple important metabolic routes. The paper is overall well-\nwritten and presents plausible results to me that this new bacterium harbors the described\nflavin-based EET (although some clarifications are needed). I have a couple of suggestions\nto hopefully make the findings more understandable.\nMajor comments\nIt is essential to know whether the cells use oxygen when they reduce Fe(III). Because they\ncan form biofilm, what is the proportion of biofilm-forming vs. free-living cells in the culture\nexperiments? If it is pretty high, the A1-JK cells may reduce Fe(III) in anoxic microniches,\nand the resulting Fe(II) can be oxidized abiotically and draw down the oxygen concentration.\nIn this case, it is hard to attribute the oxygen consumption to microbial utilization.\nResponse: We thank the reviewer for this insightful comment. We fully agree that\ndistinguishing between true aerobic Fe(III) reduction and potential reduction occurring in\nanoxic microniches within biofilms is essential for interpreting our findings. To address this,\nwe conducted additional experiments where we specifically suppressed biofilm formation by\ncontinuously stirring the cultures at 200 rpm during co-reduction of Fe(III) and O₂ (data\npresented in revised Figure 1c,d). Under these conditions, the cultures remained\nhomogeneously mixed and consisted solely of planktonic cells. We observed a concurrent\ndecrease in both oxygen and Fe(III) concentrations, consistent with active microbial\nreduction of Fe(III) in the presence of oxygen. These results confirm that the observed Fe(III)\nreduction is not dependent on biofilm-associated microniches and can proceed under fully\noxic, planktonic conditions.\nFurthermore, other complementary experiments in the manuscript independently support the\nfact that M. deferre A1-JK exhibits minimal biofilm formation:\n Microtiter plate-based biofilm assays (Figure S2) indicated weak biofilm formation.\n Scanning electron microscopy (Figure S1) revealed only sparse surface attachment\nand no thick biofilm layers.\n Electrochemical impedance spectroscopy (Figure 2d) showed low resistance at the\nelectrode surface, consistent with minimal biofilm development.\nTaken together, these observations strongly suggest that Fe(III) reduction in M. deferre A1-\nJK predominantly occurs in a planktonic state and under fully oxygenated conditions.\nLastly, the amount of Fe(II) produced via reduction of Fe (III) is too low to explain the\ndecrease in oxygen concentration presented in Figure 1c. At approximately 200-210mM\nconcentration of O in atmospheric air, the rate of iron reduction by M. deferre A1-JK is not\n2\nfast enough to bring oxygen concentration down to 0 via reoxidation of Fe (II). Even under\nanaerobic conditions, the amount of produced Fe (II) did not exceed 2mM within 6 days.\nLine 91-92: In two parallel experiments, oxygen concentration was measured in the\nheadspace of the culture tubes subjected to constant stirring to prevent biofilm formation.\nLine 111-118: Since the cultures were constantly stirred (200 rpm) and bubbled with air during\nthe course of these experiments, no biofilm was formed, thus cells remained planktonic 34.\nUnder these conditions, no oxygen microgradients were expected to from, which are usually\nassociated with biofilms35. Therefore, planktonic cells of M. deferre A1-JKT were the principal\ndrivers of Fe(III) reduction under oxygen saturated atmospheric conditions. Our experimental\ndata challenge the long-held notion that Fe(III) reduction is strictly anaerobic and support\nsimilar evidence that facultative microorganisms can mediate Fe(III) reduction in oxic\nenvironments.35\nIt would be great to see more detailed explanations/hypotheses on (i) why this EET (or all\nEETs) is not inhibited by oxygen and (ii) why people have the widely-held notion that Fe\nreduction is an anaerobic process. I can see that Riboflavin excretion seems to be one of the\nkeys to this EET. Some explanations on why riboflavin excretion is not affected by\noxygenated conditions could help readers better understand this oxygen-tolerant Fe\nreduction process.\nResponse: We appreciate the opportunity to clarify these important points. We hypothesize\nthat EET itself can serve as a metabolic adaptation to reduce Fe(III) and/or oxygen\nsimultaneously, based on the prevalent environmental conditions. We further elaborate this\nbased on data demonstrating the role of riboflavin in enabling oxygen-tolerant EET.\nRiboflavin concentrations up to 0.5 µM were observed even under aerobic conditions (Figure\nS4), and the redox peaks corresponding to flavins were unchanged under oxic growth.\nThis suggests that riboflavin secretion is regulated independently of oxygen levels. Furthermore,\nwe have added a discussion on how respiratory flexibility, including possible bifurcation\nmechanisms, may allow simultaneous oxygen and Fe(III) reduction, consistent with studies\non Shewanella oneidensis. We also now cite recent work that challenges the traditional view\nof Fe(III) reduction as strictly anaerobic (Ceriotti et al., 2025 and Keller et al., 2025).\nWe thank the reviewer for highlighting this key conceptual point, which we now elaborate\nfurther in the revised manuscript.\nLine 392-397: In M. deferre A1-JKT, the coexistence of EET with aerobic respiration suggests\na strategy where EET serves not only energy conservation via Fe(III) reduction but also\npotentially as a mechanism to offload excess electrons to oxygen via riboflavin. This\nperspective challenges the prevailing view of EET as an exclusively anaerobic metabolism\nand proposes oxygen reduction as a functional role for EET systems under oxic conditions.\n(i) Why is EET not inhibited by oxygen?\nTraditionally, extracellular electron transfer (EET) has been studied under anoxic conditions\ndue to the high redox potential of oxygen and the assumption that EET evolved primarily to\naccess alternative terminal electron acceptors. However, our results suggest that EET may\nalso function under oxic conditions as a form of respiratory flexibility, where cells\nsimultaneously reduce both Fe(III) and oxygen, depending on their availability. In M. deferre\nA1-JK, the co-reduction of Fe(III) and oxygen, even under stirred, planktonic conditions,\nsupports this hypothesis. We therefore hypothesize that EET itself can serve as a metabolic\nadaptation to reduce Fe(III) and/or oxygen simultaneously, based on the prevalent\nenvironmental conditions.\nIt is also possible that bifurcation—a mechanism known from some anaerobes and\nfacultative organisms—may contribute to this dual electron flow. Although speculative, this is\nconsistent with analogous findings in Shewanella oneidensis, where co-reduction of oxygen\nand Fe(III) has been observed and attributed to metabolic adaptability rather than strict\npathway separation. We have added a discussion of this hypothesis in the revised Results &\nDiscussion section, highlighting possible redox balancing mechanisms that permit\nsimultaneous electron flux to multiple acceptors.\nLine 384-397: An unconventional role for EET\nIn addition to supporting respiration under variable redox conditions, EET may serve a\npreviously underappreciated role as an adaptive strategy for oxygen reduction. The outer-\nsurface localization of EET components such as cytochromes and flavin shuttles enables\ndirect interaction with extracellular electron acceptors, including oxygen24. This configuration\ncould facilitate oxygen reduction at the cell surface, mitigating intracellular ROS formation and\nprotecting redox-sensitive cytoplasmic machinery. In S. oneidensis, dysregulation of flavin-\nbound EET proteins in oxic conditions results in cytotoxic peroxide production, suggesting that\nEET pathways must be tightly regulated to manage oxidative stress61. In M. deferre A1-JKT,\nthe coexistence of EET with aerobic respiration suggests a strategy where EET serves not\nonly energy conservation via Fe(III) reduction but also potentially as a mechanism to offload\nexcess electrons to oxygen via riboflavin. This perspective challenges the prevailing view of\nEET as an exclusively anaerobic metabolism and proposes oxygen reduction as a functional\nrole for EET systems under oxic conditions.\n(ii) Why is Fe(III) reduction widely viewed as an anaerobic process?\nThe perception of Fe(III) reduction as strictly anaerobic stems largely from early model\nsystems (e.g., Geobacter and Shewanella spp.) where Fe(III) reduction was primarily\nobserved under anoxic conditions. Furthermore, abiotic reoxidation of Fe(II) in the presence\nof oxygen often obscured the detection of biologically driven Fe(III) reduction in oxic\nenvironments. Only recently have studies (e.g., Ceriotti et al., 2025; Keller et al., 2025 and\nthe current study) begun to revise this view by showing microbial Fe(III) reduction under\nmicroaerobic or even aerobic conditions in natural systems.\nRiboflavin-mediated EET and oxygen:\nOur data suggest that riboflavin excretion, a critical component of this EET pathway, is not\nrepressed by oxygen. Specifically, riboflavin was secreted at concentrations up to 0.5 µM\nunder fully aerobic conditions (Figure S4), and characteristic redox peaks of extracellular\nflavins remained stable across oxic and anoxic growth. These observations indicate that\nriboflavin export and function may be regulated independently of oxygen levels.\nTogether, these results contribute to a growing recognition that EET is not an exclusively\nanaerobic strategy but may instead serve as a metabolic bridge across fluctuating redox\nregimes. We have now clarified and expanded this discussion in the revised manuscript to\nimprove conceptual accessibility for the reader.\nIn addition to the biotechnological application potential, more data about the abundance and\nprevalence of this bacterium in the environment would help us better understand the\nenvironmental relevance of this bacterium and the flavin-based EET.\nResponse: We thank the reviewer for raising this important point regarding the ecological\ncontext and prevalence of M. deferre A1-JK and related flavin-based EET mechanisms.\nWhile we unfortunately currently lack metagenomic or field-based data to directly assess the\nenvironmental abundance and prevalence of M. deferre A1-JK, we can provide relevant\ncontextual information. In our previous study (Lustermans et al., 2021), three out of\napproximately twenty isolates obtained from the same environmental inoculum were\nclassified within the Microbacterium genus (and suspected to be the same strain even),\nindicating that members of this group were naturally present and culturable from the original\nredox-stratified cable bacteria environment.\nMoreover, Microbacterium is a taxonomically and ecologically diverse genus, currently\ncomprising nearly 700 sequenced genomes and known to inhabit a wide range of\necosystems—including soil, sediments, plant-associated environments, and extreme\nhabitats such as high-salinity or cold environments. We now cite additional literature in the\nrevised manuscript demonstrating the ubiquity and ecological versatility of Microbacterium\nspp., which supports the plausibility of their broader environmental relevance.\nRegarding the prevalence of flavin-based EET within the genus, we have further analyzed\npublicly available Microbacterium genomes for the presence of putative EET-related genes,\nand these findings are discussed in figure 5. This genomic evidence suggests that flavin-\nmediated electron transfer capability may not be unique to M. deferre A1-JK, and could be\nmore widespread within the genus than previously appreciated.\nWe acknowledge that a more comprehensive environmental survey would strengthen this\naspect of the study and have now noted this as a future research direction in the revised\nDiscussion.\nLine 304-346 Genomic prevalence of hybrid EET traits across the Microbacterium genus\nM. deferre A1-JKT is not unique in possessing a hybrid EET system. To investigate the\nprevalence of such systems, we analyzed 667 publicly available Microbacterium genomes,\nincluding those of lower quality (<90% completeness and/or >5% contamination). None of the\ngenomes encoded the canonical FLEET components EetA, EetB, PplA, or FmnB, reinforcing\nthe absence of a complete FLEET pathway in this genus (Figure 5, Table S4). However,\nvarious genes associated with alternative EET pathways were found across the genus. For\ninstance, phzB, bfe, and dmsA were detected in 3, 7, and 46 genomes respectively. phzA was\ncompletely absent, while phzF, a gene involved in phenazine biosynthesis, was present in 408\ngenomes. Most other phenazine and flavin biosynthetic genes were broadly distributed (Table\nS4).\nKey EET-associated proteins ExtH, YeeO, and FccA were detected in 314, 582, and 664\nstrains respectively, indicating that hybrid EET systems—comprising both Gram-negative and\nGram-positive elements—may be common in Microbacterium. Furthermore, genes encoding\nOmpB and OmpC, two multicopper outer membrane cytochromes involved in Fe(III) reduction\nin Geobacter52 , were identified in 33 and 97 genomes. Other FLEET genes: dmkA, dmkB,\nand fmnA were also highly prevalent and found in 507, 660, and 612 genomes (Table S4),\nsuggesting a conserved potential for flavin-based electron transfer.\nPhylogenetic analysis showed that the closest relatives of M. deferre A1-JKT shared a similar\nrepertoire of EET-related genes, with M. hatanonis JCM14558 being the only exception,\nhaving gained ompC and phzF (Figure 5). Interestingly, both M. hatanonis and M. deferre A1-\nJKT were the only species amongst this group with dmkA. Although speculative, the presence\nof dmkA in M. hatanonis—originally isolated from hairspray—raises questions about potential\nmetabolic versatility linked to unusual environments. Additionally, three of the second-closest\nrelatives encoded dmsA, a gene used by S. oneidensis for extracellular dimethyl sulfoxide\nrespiration53, and all but one of the closest relatives carried coxAB, with M. testaceum\nTPD7010 lacking coxA, confirming that they were aerobes. Given the proposed evolutionary\nlink between cox genes and iron oxidation54, their presence further supports the association\nbetween these strains and redox-active environments.\nFrom an ecological perspective, the distribution of these EET-associated genes does not\nappear confined to a single habitat type. While some species such as M. testaceum and M.\nproteolyticum are typically associated with plant hosts55,56, others—including M. enclense, M.\nhydrothermale, M. Gd 4-13, and M. deferre A1-JKT—have been isolated from marine or\nfreshwater sediments57,58 , where high rates of oxygen consumption and redox gradients are\ncommon. Even transient anaerobic conditions in such environments could provide a selective\nadvantage to microbes capable of EET. This metabolic flexibility may help explain the\necological success and persistence of Microbacterium spp. across diverse niches, including\nas opportunistic pathogens in humans59.\nFigure 5 Phylogenetic dendrogram of M. deferre A1-JKT with closest (green) and second-\nclosest relatives (grey), combined with a heatmap of the amount of gene copies of relevant\nextracellular electron transfer proteins. Only named species with a completeness of >99% and\ncontamination <2.5% were selected from the second closest relatives.\nAccording to Fig. S7, some species related to A1-JK in the genus Microbacterium have also\nbeen cultured. I am curious to know whether they have the flavin-based EET and other\nmetabolic pathways described in the manuscript. A (culture- or genome-based) assessment\nlike this could also help us better understand the environmental relevance and\nbiotechnological application potential of this bacterium (and related others). The results can\nbe put together with Fig. S7, which could be moved into the main text.\nResponse:\nWe thank the reviewer for this valuable suggestion, which has prompted us to expand our\nphylogenomic and functional analysis of the Microbacterium genus.\nIn response, we have reconstructed and significantly extended the phylogenetic tree\n(originally S7) to include all 668 publicly available Microbacterium genomes. Each genome\nwas translated and screened via blastp against a curated database of known EET-\nassociated proteins, using the same criteria applied to our analysis of strain A1-JK. The\nresults of this comparative genomic assessment are now summarized in a new dataset\n(Table S3).\nTo improve accessibility and highlight the broader significance of this finding, we have\nmoved a subset of the phylogenetic tree—now annotated with EET gene\npresence/absence—into the main manuscript as a new figure (figure 5). We also expanded\nthe Results and Discussion sections to explore how the genomic capacity for flavin-based\nEET in other Microbacterium species may reflect ecological adaptation to redox-dynamic or\nelectron acceptor-limited environments.\nThis genome-level analysis not only supports the environmental relevance of flavin-mediated\nEET beyond strain A1-JK but also opens new avenues for identifying and harnessing similar\ncapabilities in closely related strains for potential biotechnological applications.\nMinor comments\nRef. 31 looks like the following peer-reviewed publication.\nCeriotti, G., Bosco-Santos, A., Borisov, S.M., and Berg, J.S. (2025) Decoupling microbial iron\nreduction from anoxic microsite formation in oxic sediments: a microscale investigation\nthrough microfluidic models. Frontiers in Microbiology 16.\nResponse: Thank you for pointing this out. We have updated Ref. 31 to correctly cite the\npeer-reviewed publication by Ceriotti et al. in Frontiers in Microbiology (2025).\nReviewer #2 (Remarks to the Author):\nLustermans et al. use a combination of electrochemical, chemical, genomic, and culture-\nbased experiments to demonstrate that a strain of Gram-positive Microbacterium, newly\nisolated from a microaerophilic veil surrounding freshwater cable bacteria, reduces soluble\nFe(III) or a poised electrode not only in anoxic but also in oxic conditions via endogenous\nflavin as an electron shuttle. Additionally, they propose a new species of the genus\nrepresented by the strain. The findings are relevant to the potential survival strategy of the\n“flocking bacteria” in the sediments inhabited by cable bacteria. However, my enthusiasm for\nthe findings is dampened by several key issues.\n1. Novelty of the endogenous flavin-based EET mechanism under oxic conditions:\nAlthough the experimental design and data interpretation regarding the EET are well-\nexecuted, the novelty of this study is somewhat limited. The authors present evidence for an\nendogenous flavin-based EET under oxic conditions in a Gram-positive bacterium, yet\nsimilar mechanisms have already been documented in Gram-negative Shewanella species\n(e.g., von Canstein et al. 2008, Appl. Environ. Microbiol.; Biffinger et al. 2009, Biotechnol.\nBioeng.; Rosenbaum et al. 2010, Biotechnol. Bioeng.; TarAvest et al. 2014, Biotechnol.\nBioeng. —although the latter was under microaerobic conditions). Given that both microbial\ngroups share similar EET mechanisms despite differences in membrane structure, the\nfindings largely align with existing literature rather than presenting a fundamentally new\nconcept.\nResponse: We appreciate the reviewer’s thoughtful comments regarding the novelty of our\nstudy. While flavin-based extracellular electron transfer (FLEET) mechanisms have indeed\nbeen well-documented in Gram-negative bacteria such as Shewanella spp. (e.g., von\nCanstein et al., 2008; Biffinger et al., 2009; Rosenbaum et al., 2010; TarAvest et al., 2014),\nour work represents the first report of such a mechanism in a Gram-positive Microbacterium\nspecies operating under fully oxic conditions.\nThis distinction is critical for several reasons. First, Microbacterium spp. lack an outer\nmembrane, which fundamentally alters the architecture required for EET. Despite this\nstructural difference, our isolate demonstrates robust Fe(III) reduction in fully oxygenated\nsystems, including under active aeration and stirring—an ability previously unreported for\nany Gram-positive bacterium. Notably, our genomic analysis revealed that canonical Gram-\npositive FLEET genes such as pplA, eetA/B, and fmnB are entirely absent in this strain.\nBased on the work of Light et al. (2018), deletion of even one of these genes typically\nabolishes EET activity. Yet, our strain performs FLEET effectively, implying the presence of a\npreviously undescribed EET architecture.\nFurthermore, we discovered the presence of genes typically associated with Shewanella and\nGeobacter pathways, suggesting a horizontal gene transfer event and making this only the\nsecond known case of a Gram-negative-derived hybrid EET system (following Conley et al.,\n2020 with Vibrio natriegens). Crucially, ours is the first such hybrid reported in a Gram-\npositive bacterium, representing a novel evolutionary and functional configuration of EET\ncomponents across the bacterial cell envelope.\nFinally, while aerobic electrochemical activity has been observed in Shewanella, the\nconcurrent presence of oxygen and effective Fe(III) reduction in a Gram-positive context\nchallenges the current paradigm that restricts Gram-positive EET to anoxic conditions. We\nhave revised the manuscript to better emphasize these novel aspects, and we hope that the\nexpanded discussion clarifies the unique contributions of our findings to the broader\nunderstanding of microbial EET under fluctuating redox regimes.\n2. Justification for the creation of a new species:\nThe authors propose the creation of a new species represented by the strain they isolated.\nHowever, the justification for this classification is insufficient. Key comparative analyses,\nsuch as DNA-DNA hybridization and/or Average Nucleotide Identity (ANI) values, as well as\nphysiological comparisons with other strains in the genus, are not provided. Without these\nstandard comparisons, the claim of a novel species remains unsubstantiated. Additionally, it\nis strongly recommended that the strain be deposited in at least two international culture\ncollections to ensure accessibility and reproducibility.\nResponse: We thank the reviewer for this constructive comment and fully agree that robust\ntaxonomic classification requires both genomic and physiological support. In response, we\nhave substantially strengthened the evidence supporting our proposal of a novel species.\nTo address the genomic criteria, we performed comprehensive comparative analyses using\nboth pairwise ANI and all-to-all fastANI approaches across the Microbacterium genus. Our\nisolate, strain A1-JK, shows ANI values of 91.47% with M. hatanonis, 94.10% with\nMicrobacterium sp. AG1240, and 98.98% with Microbacterium sp. Gd 4-13. While the latter\nvalue suggests close relatedness, M. sp. Gd 4-13 remains an unnamed strain and exhibits\ndistinct physiological traits. Specifically, it is described to grow optimally at 10% NaCl,\nwhereas A1-JK displays very poor growth under these conditions. Based on this genomic\nand phenotypic divergence, we propose that Gd 4-13 represents a strain of Microbacterium\ndeferre. We have added these comparative values to the revised manuscript, along with a\ndetailed physiological comparison involving three other Microbacterium species (see\nupdated Table S1).\nRegarding strain availability, we appreciate the reviewer’s recommendation and have taken\nthe necessary steps to ensure accessibility and reproducibility. Strain A1-JK has been\naccepted for deposition in two international culture collections—DSMZ and BCCM/LMG.\nWhile final processing is ongoing and we are waiting for the catalogue numbers, we have\nattached the forms for deposit from both institutes.\nWe have revised the manuscript to include these data and clarifications, and we thank the\nreviewer again for helping improve the scientific rigor and transparency of our taxonomic\nclaims.\nMinor comments:\nTable S2 is challenging to interpret due to the lack of full names for the proteins listed.\nIncluding this additional information would help in understanding the context and relevance\nof the data presented.\nResponse: We thank the reviewer for this suggestion. The proteins have been clarified in\nthe description of table S2.\n8"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have beautifully addressed my comments/concerns. Acceptance is recommended.", "reviewer_2": "Lustermans et al. employ a combination of electrochemical, chemical, genomic, and culture-based approaches to show that\na newly isolated Gram-positive strain of Microbacterium, obtained from the microaerophilic veil surrounding freshwater cable\nbacteria, is capable of reducing soluble Fe(III) or a poised electrode not only under anoxic but also oxic conditions,\nusing endogenously produced flavins as electron shuttles. The authors also propose this strain as a novel species within the\ngenus. These findings provide valuable insights into potential survival strategies of \"flocking bacteria\" in sediments\ninhabited by cable bacteria.\nThe authors have addressed my previous concerns thoroughly and made thoughtful revisions supported by new data.\nOverall, I am pleased with the improvements and find the manuscript much strengthened. Only a few minor points remain to\nbe addressed:\n1. The newly added discussion on implications for the Great Oxidation Event is interesting, but should be moderated. As it is\nunclear when this microbe evolved or acquired the ability to perform EET under oxic conditions, the conclusions regarding\nearly Earth redox transitions may currently be too speculative.\nMinor comments:\n• Please report the average nucleotide identity (ANI) value between strain A1-JK and Microbacterium hatanonis JCM 14558\nin the main text.\n• Please include the genome or assembly accession numbers used in the phylogenetic tree and ANI analysis. This\ninformation is important for ensuring reproducibility and transparency.\n• The reviewer appreciates the effort the authors have made to construct a comprehensive phylogenetic tree of the available\nMicrobacterium genomes. However, Fig. S7 is now difficult to read due to the number of strains included, particularly in the\nprinted version. It would be helpful to add an inset showing a close-up of the clade containing the strain of interest and its\nclosest relatives.\n• Please carefully check the manuscript for incorrect figure and table references. E.g., line 236."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe authors have beautifully addressed my comments/concerns. Acceptance is\nrecommended.\nResponse: We are extremely pleased with the reviewer’s constructive feedback that greatly\nhelped in improving the manuscript. Thank you very much for your time and efforts.\nReviewer #2 (Remarks to the Author):\nLustermans et al. employ a combination of electrochemical, chemical, genomic, and culture-\nbased approaches to show that a newly isolated Gram-positive strain of Microbacterium,\nobtained from the microaerophilic veil surrounding freshwater cable bacteria, is capable of\nreducing soluble Fe(III) or a poised electrode not only under anoxic but also oxic conditions,\nusing endogenously produced flavins as electron shuttles. The authors also propose this\nstrain as a novel species within the genus. These findings provide valuable insights into\npotential survival strategies of \"flocking bacteria\" in sediments inhabited by cable bacteria.\nThe authors have addressed my previous concerns thoroughly and made thoughtful\nrevisions supported by new data. Overall, I am pleased with the improvements and find the\nmanuscript much strengthened. Only a few minor points remain to be addressed.\nResponse: We sincerely thank the reviewer for their evaluation and positive comments on\nour revised manuscript. We are pleased that the improvements and new data have\nstrengthened the manuscript. We have carefully addressed the remaining points as outlined\nbelow.\n1. The newly added discussion on implications for the Great Oxidation Event is interesting,\nbut should be moderated. As it is unclear when this microbe evolved or acquired the ability to\nperform EET under oxic conditions, the conclusions regarding early Earth redox transitions\nmay currently be too speculative.\nResponse: We agree with the reviewer that the evolutionary timing of M. deferre A1-JKT and\nits EET traits is unknown, and that the current discussion could appear too speculative. We\nhave accordingly revised the relevant paragraph in the Implications for early Earth redox\nevolution section to clearly state that while M. deferre A1-JKT provides a modern example of\nmicrobial flexibility in redox-stratified environments, its relevance to the Great Oxidation\nEvent remains speculative. The revised text now reads:\n“The ability of M. deferre A1-JKT to simultaneously reduce both Fe(III) and oxygen under\natmospheric oxygen conditions provides a modern example of microbial flexibility in redox-\nstratified environments. During Earth's Great Oxidation Event (GOE), rising atmospheric\noxygen levels would have imposed selective pressures on anaerobic microorganisms, many\nof which relied on Fe(III) as a terminal electron acceptor⁶⁰. While the evolutionary origin of the\nEET traits observed in M. deferre A1-JKT is unknown, oxygen-tolerant EET mechanisms may\nrepresent one of several strategies that have independently evolved in microbes to enable\nsurvival under fluctuating redox conditions.\nRecent studies have suggested that EET systems can mitigate the harmful effects of oxygen\nby dissipating excess electrons and minimizing reactive oxygen species (ROS) formation⁶¹.\nIn S. oneidensis, for instance, the redox-active disulfide motif in the extracellular cytochrome\nMtrC acts as an oxygen-sensitive switch, regulating flavin binding and suppressing ROS\nformation under aerobic conditions⁶¹. Although M. deferre A1-JKT lacks the Mtr-complex, its\nability to sustain flavin-mediated EET under oxic conditions suggests the presence of\nalternative mechanisms to mitigate ROS toxicity. It was positive for the catalase enzyme that\nbreaks down peroxidase, removing toxic ROS species. Based on these observations, we\nspeculate that oxygen-tolerant EET pathways – such as those demonstrated in M. deferre A1-\nJKT – may reflect general microbial strategies to adapt to redox transitions. While their specific\nrelevance to microbial evolution during the GOE remains speculative, such pathways illustrate\nhow EET mechanisms can support microbial persistence in dynamic redox environments both\npast and present.”\nMinor comments:\n• Please report the average nucleotide identity (ANI) value between strain A1-JK and\nMicrobacterium hatanonis JCM 14558 in the main text.\nResponse: We thank the reviewer for this suggestion. We have now included the ANI value\nbetween M. deferre A1-JKT and M. hatanonis JCM 14558 in the Results and Discussion\nsection in lines 239-241 (new text in green).\n”Other Microbacterium species, amongst which is the second closest relative M. hatanonis\nJCM14558 (ANI 91.47%, 5% NaCl), also showed tolerance to high salt concentrations\nranging 5-10% (Table S2).”\n• Please include the genome or assembly accession numbers used in the phylogenetic tree\nand ANI analysis. This information is important for ensuring reproducibility and transparency.\nResponse: We fully agree with the reviewer on the importance of transparency and\nreproducibility. We have clarified in the legend of Figure 5 and in the Methods section\n(Genome assembly, annotation, analyses and phylogeny) that the ncbi accession numbers\nof the genomes used for the phylogenetic tree and ANI analysis are listed in Supplementary\nTable S5. The project number for M. deferre A1-JKT (PRJNA1125322) was already provided,\nis now clearly referenced and is also part of Table S5.\n• The reviewer appreciates the effort the authors have made to construct a comprehensive\nphylogenetic tree of the available Microbacterium genomes. However, Fig. S7 is now difficult\nto read due to the number of strains included, particularly in the printed version. It would be\nhelpful to add an inset showing a close-up of the clade containing the strain of interest and\nits closest relatives.\nResponse: We appreciate this helpful suggestion. We have now added an inset to Figure S7\nthat highlights the clade containing M. deferre A1-JKT and its closest relatives and second\nclosest relatives, since the closest relatives comprise only 3 other Microbacterium species.\nThe updated figure improves readability both on-screen and in the printed version. We have\nupdated the legend of Figure S7 to reflect this change.\n• Please carefully check the manuscript for incorrect figure and table references. E.g., line\n236.\nResponse: We thank the reviewer for this attention to detail. We have now carefully checked\nthe entire manuscript for incorrect figure and table references and have corrected any\ninconsistencies. The example at line 236 and other minor references have been verified and\nupdated as needed, which resulted in Table S1 and S2 switching places."}]

Decision letter