Heme d formation in a Shewanella benthica hemoglobin.

In our continued investigations of microbial globins, we solved the structure of a truncated hemoglobin from Shewanella benthica, an obligate psychropiezophilic bacterium. The distal side of the heme active site is lined mostly with hydrophobic residues, with the exception of a tyrosine, Tyr34 (CD1) and a histidine, His24 (B13). We found that purified SbHbN, when crystallized in the ferric form with polyethylene glycol as precipitant, turned into a green color over weeks. The electron density obtained from the green crystals accommodated a trans heme d, a chlorin-type derivative featuring a γ-spirolactone and a vicinal hydroxyl group on a pyrroline ring. In solution, exposure of the protein to one equivalent of hydrogen peroxide resulted in a similar green color change, but caused by the formation of multiple products. These were oxidation species released on protein denaturation, likely including heme d, and a species with heme covalently attached to the polypeptide. The Tyr34Phe replacement prevented the formation of both heme d and the covalent linkage. The ready modification of heme b by SbHbN expands the range of chemistries supported by the globin fold and offers a route to a novel heme cofactor.

Engineering bionanoreactor in bacteria for efficient hydrogen production.

Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 µmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis.

Combined metabolomics and proteomics to reveal the mechanism of S. oneidensis MR-1 degradation malathion enhanced by FeO/C.

Iron oxide @ biochar (FeO/C) promotes bacterial growth and facilitates electron transfer, thereby effectively promoting malathion degradation by Shewanella oneidensis MR-1 (S. oneidensis MR-1). This study elucidated the underlying mechanism of FeO/C-enhanced malathion degradation by S. oneidensis MR-1 through a combination of metabolomics and proteomics analysis. The kinetic fitting results from the degradation experiment indicated that 0.1 g/L FeO/C exerted the most significant enhancement effect on malathion degradation by S. oneidensis MR-1. Observations from Scanning Electron Microscopy and Laser Scanning Confocal Microscopy, along with physiological and biochemical analysis, showed that FeO/C enhanced the growth and oxidative response of S. oneidensis MR-1 under malathion stress. In addition, metabolomics and proteomics analysis revealed an increase in certain electron transfer related metabolites, such as coenzymes, and the upregulation of proteins, including coenzyme A, sdhD, and petC. Overall, spectroscopic analysis suggested that Fe2+, which was reduced from Fe3+ by S. oneidensis MR-1 in FeO/C, promoted electron transfer in S. oneidensis MR-1 to enhance the degradation of malathion. This study offers enhanced strategies for efficient removal of malathion contaminants.

Mechanistic insights into sulfadimethoxine degradation via microbially driven Fenton reactions.

Biodegradation, while cost-effective, is hindered by the requirement for specialized microorganisms and co-contaminants. Innovative biological technologies like the microbially driven Fenton reaction, hold promise for enhancing degradation efficiency. However, the intricate biochemical processes and essential steps for effective degradation in such systems have remained unclear. In this study, we harnessed the potential of the microbially driven Fenton reaction by employing Shewanella oneidensis MR-1 (MR-1). Our approach showcased remarkable efficacy in degrading a range of contaminants, including sulfadimethoxine (SDM), 4,4'-dibromodiphenyl ether (BDE-15) and atrazine (ATZ). Using SDM as a model contaminant of emergent contaminants (ECs), we unveiled that biodegradation relied on the generation of hydroxyl radicals (•OH) and involvement of oxidoreductases. Transcriptomic analysis shed light on the pivotal components of extracellular electron transfer (EET) during both anaerobic and aerobic periods. The presence of reactive oxidizing species induced cellular damage and impeded DNA repair, thereby affecting the Mtr pathway of EET. Moreover, the formation of vivianite hindered SDM degradation, underscoring the necessity of maintaining iron ions in the solution to ensure sustainable and efficient degradation. Overall, this study offers valuable insights into microbial technique for ECs degradation, providing a comprehensive understanding of degradation mechanisms during aerobic/anaerobic cycling.

Review of cathodic electroactive bacteria: Species, properties, applications and electron transfer mechanisms.

Cathodic electroactive bacteria (C-EAB) which are capable of accepting electrons from solid electrodes provide fresh avenues for pollutant removal, biosensor design, and electrosynthesis. This review systematically summarized the burgeoning applications of the C-EAB over the past decade, including 1) removal of nitrate, aromatic derivatives, and metal ions; 2) biosensing based on biocathode; 3) electrosynthesis of CH4, H2, organic carbon, NH3, and protein. In addition, the mechanisms of electron transfer by the C-EAB are also classified and summarized. Extracellular electron transfer and interspecies electron transfer have been introduced, and the electron transport mechanism of typical C-EAB, such as Shewanella oneidensis MR-1, has been combed in detail. By bringing to light this cutting-edge area of the C-EAB, this review aims to stimulate more interest and research on not only exploring great potential applications of these electron-accepting bacteria, but also developing steady and scalable processes harnessing biocathodes.

Mixed Conducting Polymers Alter Electron Transfer Thermodynamics to Boost Current Generation from Electroactive Microbes.

Electroactive microbes that can release or take up electrons are essential components of nearly every ecological niche and are powerful tools for the development of alternative energy technologies. Small-molecule mediators are critical for this electron transfer but remain difficult to study and engineer because they perform concerted two-electron transfer in native systems but only individual, one-electron transfers in electrochemical studies. Here, we report that electrode modification with ion- and electron-conductive polymers yields biosimilar, concerted two-electron transfer from Shewanella oneidensis via flavin mediators. S. oneidensis biofilms on these polymers show significantly improved per-microbe current generation and morphologies that more closely resemble native systems, setting a new paradigm for the study and optimization of these electron transfer processes. The unprecedented concerted electron transfer was found to be due to altered mediator electron transfer thermodynamics, enabling biologically relevant studies of electroactive biofilms in the lab for the first time. These important findings pave the way for a complete understanding of the ecological role of electroactive microbes and their broad application in sustainable technologies.

Distinct influence of model electron shuttles on anaerobic mononitrophenols reduction in aquatic environments by Shewanella oneidensis MR-1.

The environmental fate and risks of mononitrophenols (mono-NPs), the simplest nitrophenols (NPs) often found in aquatic environments, are profoundly influenced by anaerobic bioreduction and co-existing electron shuttles (ESs), but little is known about the underlying mechanisms. Here, we elucidate the pathways of anaerobic mono-NPs bioreduction by Shewanella oneidensis MR-1 and assess the effect of model ESs on these processes. We found that all three mono-NPs isomers could be readily reduced to their corresponding aminophenols by S. oneidensis MR-1 under anaerobic conditions. CymA, a core component of the Mtr respiratory pathway, performs a dynamic role in these bioreduction, which is highly dependent on the bioreduction kinetics. The exogenous addition of quinones was found to accelerate the mono-NPs bioreduction through interactions with key outer-membrane proteins (e.g., OmcA and MtrC), and all these processes matched well to linear free energy relationships (LFERs). Surprisingly, adding riboflavin did not influence the bioreduction of all three mono-NPs isomers, which may be due to the contribution of OmcA and MtrC to these bioreduction processes and their downregulated expression. This study enhances our understanding of the environmental fate of mono-NPs and their bioconversion processes, providing valuable insights for the bioremediation of nitrophenol-contaminated sites.

Fabrication of bio-abiotic hybrid living hydrogel for bifunctional electrochemical conversion.

Design and intelligent use renewable natural bioenergy is an important challenge. Electric microorganism-based materials are being serve as an important part of bioenergy devices for energy release and collection, calling for suitable skeleton materials to anchor live microbes. Herein we verified the feasibility of constructing bio-abiotic hybrid living materials based on the combination of gelatin, Li-ions and exoelectrogenic bacteria Shewanella oneidensis manganese-reducing-1 (MR-1). The gelatin-based mesh contains abundant pores, allowing microbes to dock and small molecules to diffuse. The hybrid materials hold plentiful electronegative groups, which effectively anchor Li-ions and facilitate their transition. Moreover, the electrochemical characteristics of the materials can be modulated through changing the ratios of gelatin, bacteria and Li-ions. Based on the gelatin-Li-ion-microorganism hybrid materials, a bifunctional device was fabricated, which could play dual roles alternatively, generation of electricity as a microbial fuel cell and energy storage as a pseudocapacitor. The capacitance and the maximum voltage output of the device reaches 68 F g-1 and 0.67 V, respectively. This system is a new platform and fresh start to fabricate bio-abiotic living materials for microbial electron storage and transfer. We expect the setup will extend to other living systems and devices for synthetic biological energy conversion.

Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

Elucidation of high-pressure processing toward microbial inhibition, physicochemical properties, collagen fiber and muscle structure of blood clam edible portion.

Impact of high-pressure processing (HP-P) on microbial inactivation, protein oxidation, collagen fiber, and muscle structure of the edible portion (EP) of blood clams (BC) was investigated. Aerobic plate count, Vibrio parahaemolyticus, V. vulnificus, other Vibrio spp. and Shewanella algae counts were not detectable when HP-P pressure of ≥300 MPa was applied. Carbonyl, disulphide bond content, and surface hydrophobicity upsurged as HP-P with augmenting pressure was employed. Protein with ∼53 kDa appeared when HP-P at 100 and 200 MPa was implemented. Increased pressure enhanced gap formation and abnormal muscle cell structure arrangements. HP-P also affected connective tissue, causing size reduction and disruption of the collagen filament fibers. However, firmness and toughness of BC-EP with HP-P ≤ 300 MPa were comparable to those of the control. HP-P at 300 MPa was therefore appropriate for treatment of BC with maintained textural properties, while less protein oxidation, collagen fiber and muscle structure disruption occurred.

The dmsEFABGH operon encodes an essential and modular electron transfer pathway for extracellular iodate reduction by Shewanella oneidensis MR-1.

Extracellular iodate reduction by Shewanella spp. contributes to iodide generation in the biogeochemical cycling of iodine. However, there is a disagreement on whether Shewanella spp. use different extracellular electron transfer pathways with dependence on electron donors in iodate reduction. In this study, a series of gene deletion mutants of Shewanella oneidensis MR-1 were created to investigate the roles of dmsEFABGH, mtrCAB, and so4357-so4362 operons in iodate reduction. The iodate-reducing activity of the mutants was tested with lactate, formate, and H2 as the sole electron donors, respectively. In the absence of single-dms gene, iodate reduction efficiency of the mutants was only 12.9%-84.0% with lactate at 24 hours, 22.1%-85.9% with formate at 20 hours, and 19.6%-57.7% with H2 at 42 hours in comparison to complete reduction by the wild type. Progressive inhibition of iodate reduction was observed when the dms homolog from the so4357-so4362 operon was deleted in the single-dms gene mutants. This result revealed complementation of dmsEFABGH by so4357-so4362 at the single-gene level, indicating modularity of the extracellular electron transfer pathway encoded by dmsEFABGH operon. Under the conditions of all electron donors, significant inhibition of iodate reduction and accumulation of H2O2 were detected for ΔmtrCAB. Collectively, these results demonstrated that the dmsEFABGH operon encodes an essential and modular iodate-reducing pathway without electron donor dependence in S. oneidensis MR-1. The mtrCAB operon was involved in H2O2 elimination with all electron donors. The findings in this study improved the understanding of molecular mechanisms underlying extracellular iodate reduction.IMPORTANCEIodine is an essential trace element for human and animals. Recent studies revealed the contribution of microbial extracellular reduction of iodate in biogeochemical cycling of iodine. Multiple reduced substances can be utilized by microorganisms as energy source for iodate reduction. However, varied electron transfer pathways were proposed for iodate reduction with different electron donors in the model strain Shewanella oneidensis MR-1. Here, through a series of gene deletion and iodate reduction experiments, we discovered that the dmsEFABGH operon was essential for iodate reduction with at least three electron donors, including lactate, formate, and H2. The so4357-so4362 operon was first demonstrated to be capable of complementing the function of dmsEFABGH at single-gene level.

Chlorophyll a acts as a natural photosensitizer to drive nitrate reduction in nonphotosynthetic microorganisms.

With the death and decomposition of widely distributed photosynthetic organisms, free natural pigments are often detected in surface water, sediment and soil. Whether free pigments can act as photosensitizers to drive biophotoelectrochemical metabolism in nonphotosynthetic microorganisms has not been reported. In this work, we provide direct evidence for the photoelectrophic relationship between extracellular chlorophyll a (Chl a) and nonphotosynthetic microorganisms. The results show that 10 µg of Chl a can produce significant photoelectrons (∼0.34 A/cm2) upon irradiation to drive nitrate reduction in Shewanella oneidensis. Chl a undergoes structural changes during the photoelectric process, thus the ability of Chl a to generate a photocurrent decreases gradually with increasing illumination time. These changes are greater in the presence of microorganisms than in the absence of microorganisms. Photoelectron transport from Chl a to S. oneidensis occurs through a direct pathway involving the cytochromes MtrA, MtrB, MtrC and CymA but not through an indirect pathway involving riboflavin. These findings reveal a novel photoelectrotrophic linkage between natural photosynthetic pigments and nonphototrophic microorganisms, which has important implications for the biogeochemical cycle of nitrogen in various natural environments where Chl a is distributed.

Evolution of paralogous multicomponent systems for site-specific O-sialylation of flagellin in Gram-negative and Gram-positive bacteria.

Many bacteria glycosylate flagellin on serine or threonine residues using pseudaminic acid (Pse) or other sialic acid-like donor sugars. Successful reconstitution of Pse-dependent sialylation by the conserved Maf-type flagellin glycosyltransferase (fGT) may require (a) missing component(s). Here, we characterize both Maf paralogs in the Gram-negative bacterium Shewanella oneidensis MR-1 and reconstitute Pse-dependent glycosylation in heterologous hosts. Remarkably, we uncovered distinct acceptor determinants and target specificities for each Maf. Whereas Maf-1 uses its C-terminal tetratricopeptide repeat (TPR) domain to confer flagellin acceptor and O-glycosylation specificity, Maf-2 requires the newly identified conserved specificity factor, glycosylation factor for Maf (GlfM), to form a ternary complex with flagellin. GlfM orthologs are co-encoded with Maf-2 in Gram-negative and Gram-positive bacteria and require an invariant aspartate in their four-helix bundle to function with Maf-2. Thus, convergent fGT evolution underlies distinct flagellin-binding modes in tripartite versus bipartite systems and, consequently, distinct O-glycosylation preferences of acceptor serine residues with Pse.

Modularized Engineering of Shewanella oneidensis MR-1 for Efficient and Directional Synthesis of 5-Aminolevulinic Acid.

Shewanella oneidensis MR-1 has found widespread applications in pollutant transformation and bioenergy production, closely tied to its outstanding heme synthesis capabilities. However, this significant biosynthetic potential is still unexploited so far. Here, we turned this bacterium into a highly-efficient bio-factory for green synthesis of 5-Aminolevulinic Acid (5-ALA), an important chemical for broad applications in agriculture, medicine, and the food industries. The native C5 pathway genes of S. oneidensis was employed, together with the introduction of foreign anti-oxidation module, to establish the 5-ALA production module, resulting 87-fold higher 5-ALA yield and drastically enhanced tolerance than the wild type. Furthermore, the metabolic flux was regulated by using CRISPR interference and base editing techniques to suppress the competitive pathways to further improve the 5-ALA titer. The engineered strain exhibited 123-fold higher 5-ALA production capability than the wild type. This study not only provides an appealing new route for 5-ALA biosynthesis, but also presents a multi-dimensional modularized engineering strategy to broaden the application scope of S. oneidensis.

Dual-mode harvest solar energy for photothermal Cu2-xSe biomineralization and seawater desalination by biotic-abiotic hybrid.

Biotic-abiotic hybrid photocatalytic system is an innovative strategy to capture solar energy. Diversifying solar energy conversion products and balancing photoelectron generation and transduction are critical to unravel the potential of hybrid photocatalysis. Here, we harvest solar energy in a dual mode for Cu2-xSe nanoparticles biomineralization and seawater desalination by integrating the merits of Shewanella oneidensis MR-1 and biogenic nanoparticles. Photoelectrons generated by extracellular Se0 nanoparticles power Cu2-xSe synthesis through two pathways that either cross the outer membrane to activate periplasmic Cu(II) reduction or are directly delivered into the extracellular space for Cu(I) evolution. Meanwhile, photoelectrons drive periplasmic Cu(II) reduction by reversing MtrABC complexes in S. oneidensis. Moreover, the unique photothermal feature of the as-prepared Cu2-xSe nanoparticles, the natural hydrophilicity, and the linking properties of bacterium offer a convenient way to tailor photothermal membranes for solar water production. This study provides a paradigm for balancing the source and sink of photoelectrons and diversifying solar energy conversion products in biotic-abiotic hybrid platforms.

Impact of Condition Variations on Bioelectrochemical System Performance: An Experimental Investigation of Sulfamethoxazole Degradation.

Bioelectrochemical systems (BESs) are an innovative technology for the efficient degradation of antibiotics. Shewanella oneidensis (S. oneidensis) MR-1 plays a pivotal role in degrading sulfamethoxazole (SMX) in BESs. Our study investigated the effect of BES conditions on SMX degradation, focusing on microbial activity. The results revealed that BESs operating with a 0.05 M electrolyte concentration and 2 mA/cm2 current density outperformed electrolysis cells (ECs). Additionally, higher electrolyte concentrations and elevated current density reduced SMX degradation efficiency. The presence of nutrients had minimal effect on the growth of S. oneidensis MR-1 in BESs; it indicates that S. oneidensis MR-1 can degrade SMX without nutrients in a short period of time. We also highlighted the significance of mass transfer between the cathode and anode. Limiting mass transfer at a 10 cm electrode distance enhanced S. oneidensis MR-1 activity and BES performance. In summary, this study reveals the complex interaction of factors affecting the efficiency of BES degradation of antibiotics and provides support for environmental pollution control.

A biocompatible electrode/exoelectrogens interface augments bidirectional electron transfer and bioelectrochemical reactions.

Bidirectional electron transfer is about that exoelectrogens produce bioelectricity via extracellular electron transfer at anode and drive cytoplasmic biochemical reactions via extracellular electron uptake at cathode. The key factor to determine above bioelectrochemical performances is the electron transfer efficiency under biocompatible abiotic/biotic interface. Here, a graphene/polyaniline (GO/PANI) nanocomposite electrode specially interfacing exoelectrogens (Shewanella loihica) and augmenting bidirectional electron transfer was conducted by in-situ electrochemical modification on carbon paper (CP). Impressively, the GO/PANI@CP electrode tremendously improved the performance of exoelectrogens at anode for wastewater treatment and bioelectricity generation (about 54 folds increase of power density compared to blank CP electrode). The bacteria on electrode surface not only showed fast electron release but also exhibited high electricity density of extracellular electron uptake through the proposed direct electron transfer pathway. Thus, the cathode applications of microbial electrosynthesis and bio-denitrification were developed via GO/PANI@CP electrode, which assisted the close contact between microbial outer-membrane cytochromes and nanocomposite electrode for efficient nitrate removal (0.333 mM/h). Overall, nanocomposite modified electrode with biocompatible interfaces has great potential to enhance bioelectrochemical reactions with exoelectrogens.

Efficient Enhancement of Extracellular Electron Transfer in Shewanella oneidensis MR-1 via CRISPR-Mediated Transposase Technology.

Electroactive bacteria, exemplified by Shewanella oneidensis MR-1, have garnered significant attention due to their unique extracellular electron-transfer (EET) capabilities, which are crucial for energy recovery and pollutant conversion. However, the practical application of MR-1 is constrained by its EET efficiency, a key limiting factor, due to the complexity of research methodologies and the challenges associated with the practical use of gene editing tools. To address this challenge, a novel gene integration system, INTEGRATE, was developed, utilizing CRISPR-mediated transposase technologies for precise genomic insertion within the S. oneidensis MR-1 genome. This system facilitated the insertion of extensive gene segments at different sites of the Shewanella genome with an efficiency approaching 100%. The inserted cargo genes could be kept stable on the genome after continuous cultivation. The enhancement of the organism's EET efficiency was realized through two primary strategies: the integration of the phenazine-1-carboxylic acid synthesis gene cluster to augment EET efficiency and the targeted disruption of the SO3350 gene to promote anodic biofilm development. Collectively, our findings highlight the potential of utilizing the INTEGRATE system for strategic genomic alterations, presenting a synergistic approach to augment the functionality of electroactive bacteria within bioelectrochemical systems.

Red-Light-Induced Genetic System for Control of Extracellular Electron Transfer.

Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.

Morphological, Microstructural, and In Situ Chemical Characteristics of Siderite Produced by Iron-Reducing Bacteria.

Dissimilatory iron-reducing bacteria (DIRB) oxidize organic matter or hydrogen and reduce ferric iron to form Fe(II)-bearing minerals, such as magnetite and siderite. However, compared with magnetite, which was extensively studied, the mineralization process and mechanisms of siderite remain unclear. Here, with the combination of advanced electron microscopy and synchrotron-based scanning transmission X-ray microscopy (STXM) approaches, we studied in detail the morphological, structural, and chemical features of biogenic siderite via a growth experiment with Shewanella oneidensis MR-4. Results showed that along with the growth of cells, Fe(II) ions were increasingly released into solution and reacted with CO32- to form micrometer-sized siderite minerals with spindle, rod, peanut, dumbbell, and sphere shapes. They are composed of many single-crystal siderite plates that are fanned out from the center of the particles. Additionally, STXM revealed Fh and organic molecules inside siderite. This suggests that the siderite crystals might assemble around a Fh-organic molecule core and then continue to grow radially. This study illustrates the biomineralization and assembly of siderite by a successive multistep growth process induced by DIRB, also provides evidences that the distinctive shapes and the presence of organic molecules inside might be morphological and chemical features for biogenic siderite.