Process for microbial induced stable iron precipitation to prevent and mitigate corrosion

Abstract

A method of treating corroded rebar in a reinforced concrete structure having one or more cracks, including steps of delivering a composition through the one or more cracks to the corroded rebar, and contacting the composition with the corroded rebar to form an iron precipitate providing a biogenic coating on the corroded rebar. The delivered composition includes at least one or more microbes, selected from bacteria and fungi, and combinations thereof; and one or more electron donors.

Description

[0001] DREX- 1244 WO Application

[0002] PROCESS FOR MICROBIAL INDUCED STABLE IRON PRECIPITATION TO PREVENT AND MITIGATE CORROSION

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 703,233, filed on October 4, 2024, the entire disclosure of which is hereby incorporated by reference in its entirety as if set forth fully herein.

[0005] STATEMENT OF GOVERNMENT INTEREST

[0006] This invention was made with government support under Contract Number D23AC00043-00sPlea awarded by the US Department of Interior (DOI) / Defense Advanced Research Programs Agency (DARPA). The Government has certain rights in this invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Exposure of steel to carbon dioxide (CO2) in moist environments can lead to extreme corrosion and if this is not properly managed, it can cause degradation, expansion and damage to the steel structure and the surrounding environment.

[0009] Corrosion of iron-based materials poses a significant challenge to infrastructure and industrial systems, often leading to substantial economic and safety concerns1 3. Corrosion in reinforced concrete occurs when the steel reinforcement reacts with electron acceptors available in the matrix of concrete such as oxygen, moisture, and chloride ions, diminishing the structural integrity and durability4,5. As a result of consequential oxidative reactions, the passive oxide layers covering the steel reinforcement can break down, initiating localized corrosion such as pitting or crevice corrosion6. Additionally, fluctuations in the water chemistry of pore spaces, temperature of the matrix of concrete, and flow conditions of the liquids present in the surrounding environment, may accelerate the corrosion process by destabilizing the protective film on the reinforcement or the pipe surface6.

[0010] Current methods to mitigate steel corrosion include the use of chemical corrosion inhibitors which often impose operational constraints, increase costs, and may cause environmental issues due to the presence of harmful ions such as chlorides and sulfur species in the corrosion inhibitors. Other measures include using low-permeability concrete, applying protective coatings to the reinforcement, maintaining adequate concrete cover, and using non- corrosive reinforcement materials such as stainless steel or fiber-reinforced polymers4,7. These conventional approaches to corrosion mitigation often have a negative environmental DREX- 1244 WO Application impact due to the cumulative release of toxic substances7. In addition, conventional methods such as mechanical cleaning, electrochemical reduction, and alkaline sulfate treatment are labor intensive and time consuming, and therefore, incur high costs for implementation8.

[0011] In recent years, biomineralization processes have emerged as promising alternatives for corrosion control as they are non-toxic, environmentally friendly, can thrive in low- oxygen conditions, and have the potential for self-healing / activation which makes them cost- effective7,9 15. For instance, Fang et al. demonstrated that facultative anaerobic denitrifying bacteria can facilitate carbonate precipitation which forms dense, adherent calcium carbonate layers on steel surfaces, effectively acting as a barrier against corrosive agents12. Moreover, it was reported that microbial-induced biomineralization can result in the production of stable iron minerals16. These stable iron minerals can help mitigate corrosion by forming protective layers on corroded iron surfaces11. As shown in Error! Reference source not found., these biogenically formed mineral layers can act as passivating barriers, shielding the reinforcement core from further corrosion. Certain iron-reducing bacteria, such as Shewanella loihica and S. oneidensis, convert ferric iron (Fe3+) to ferrous iron (Fe2+) in corroded layers which subsequently leads to the biogenic precipitation of stable minerals including magnetite ([Fe2+][Fe3+]2[O2‘]4), siderite ([Fe2+][CC>32']), and vivianite ([Fe2+]s[PO43']2). These minerals can reduce surface reactivity, limit oxygen and moisture penetration, and ultimately slow corrosion progression11 12 16’17.

[0012] It was previously demonstrated that S. loihica strain PV-4 is able to reduce Fe3+to Fe2+using organic compounds such as lactate, formate, and acetate, as well as inorganic compounds such as hydrogen as electron donors18,19. PV-4 reduces ferric iron via extracellular electron transfer mechanisms involving cytochromes like MtrC and OmcA, which are part of its electron transport chain18. These proteins are expressed on the cell's outer membrane and mediate electron transfer to solid-phase Fe3+oxides.

[0013] Similar to S. loihica, S. oneidensis employs multi-heme cytochromes (e.g., MtrC, MtrA, OmcA) for extracellular electron transfer, and therefore, are capable of inducing iron reduction20. It has been shown that S. oneidensis possesses a remarkable metabolic versatility and can consume both soluble Fe3complexes (e.g.. feme citrate) and insoluble iron minerals21. On the other hand, Tessaracoccus lapidicaptus is a relatively less studied iron- reducing bacterium compared to the Shewanella species22 24. Literature on T. lapidicaptus suggests that it employs a different pathway for iron reduction, likely involving direct contact with iron minerals or non-cytochrome-based electron shuttling mechanisms23,24. DREX- 1244 WO Application

[0014] This microbial-induced biomineralization / stabilization approach offers a promising bio-inspired method for stabilizing and preserving iron containing structures. For instance, siderite (FeCOs), a stable ferrous carbonate mineral, has demonstrated potential for protecting iron surfaces by forming a passive layer that inhibits further corrosion11,17’23. Production of biogenic siderite includes three steps as outlined in the reactions below26: (a) bacterial reduction of ferric iron through electron transfer, (b) oxidation of an electron donor (e.g. lactate) and carbonate ion generation, and (c) reaction of ferrous iron and carbonate ion.

[0015] (a) Fe3++ e Fe2+

[0016] (b) CHsCHOHCOO" + 2H2O CH3COO‘ + 6H++ CO32’

[0017] (c) Fe2++ CO32' FeCO3

[0018] Previous studies have underscored the critical role of iron-reducing bacteria, particularly those of the Shewanella genus, in influencing the corrosion of metallic surfaces17,27. While some studies suggest iron-reducing bacteria exacerbate corrosion by destabilizing passive layers, other studies investigated their potential to form protective biofilms or biominerals that may inhibit further corrosion progression28,29. For instance, Wehkala showed that Shewanella putrefaciens develops biofilms on carbon steel that can potentially protect the surface of the metal against corrosion17. Additionally, research has mostly focused on understanding the biochemical pathways and environmental conditions employed by the iron-reducing bacteria20,23,10. Nonetheless, a few recent studies have also aimed to assess the morphological and microstructural characteristics of the resulting biogenic iron minerals, as well as their in situ chemical properties31. By using a combination of advanced microscopic and spectroscopic techniques, Han et al. provided a more detailed understanding of the factors controlling the formation and stability7of biogenic siderite, which is crucial for both environmental and engineering applications31.

[0019] While there has been limited research on microbial-induced iron biomineralization (MIIB), key gaps remain in understanding and optimizing microbe-driven production of stable iron minerals for corrosion mitigation. In particular, there is little known about how MIIB can help mitigate corrosion in reinforced concrete where specific microenvironmental conditions such as low pH and an abundance of chloride ions persist32,33. For that purpose, it is essential to investigate the effectiveness of MIIB in conjunction with particular corrosion microenvironments in reinforced concrete. In addition, previous studies have largely focused on iron-reducing bacteria in the context of biogeochemical cycling and environmental remediation. However, their potential for forming protective ferrous mineral layers to mitigate corrosion in reinforced concrete remains underexplored. Additionally, limited DREX- 1244 WO Application research has compared the effectiveness of different iron-reducing bacterial species under varying conditions, such as electron donor availability and oxygen levels, to optimize biomineralization efficiency. A comprehensive quantitative comparison of the performance of the selected bacterial strains has yet to be presented in the existing literature. Furthermore, since the ty pe of iron reduction mechanism used by the bacterial species can govern the necessity of soluble iron availability as opposed to direct contact with solid reinforcement, it is crucial to compare bacterial species with different mechanisms to assess the applicability of this biomineralization approach in real-life scenarios.

[0020] SUMMARY OF THE INVENTION

[0021] The following sentences may be used to describe the invention:

[0022] 1 . In a first aspect, the present invention relates to a method of treating corroded rebar in a reinforced concrete structure comprising one or more cracks, comprising steps of: delivering a composition through the one or more cracks to the corroded rebar, wherein the composition comprises one or more microbes, selected from bacteria and fungi, and combinations thereof; and one or more electron donors; and contacting the composition with the corroded rebar, wherein the step of contacting forms an iron precipitate providing a biogenic coating on the corroded rebar.

[0023] 2. The method of sentence 1, wherein the one or more electron donors is a carbon source, optionally selected from the group consisting of acetate, lactate, glucose, ethanol, pyruvate, formate, methanol, propionate, butyrate, succinate, glycerol, mannitol, sucrose, malate, fructose, galactose, xylose, ribose, fumarate, cellobiose, and combinations thereof.

[0024] 3. The method of any one of sentences 1 - 2, wherein the one or more microbes are present in an initial cell concentration range between OD600 = 0. 1 to 1, based on the amount of the one or more electron donors.

[0025] 4. The method of any one of sentences 1 - 3, wherein the iron precipitate forming composition comprises two or more microbes. DREX- 1244 WO Application

[0026] 5. The method of any one of sentences 1 - 4, wherein the bacteria may be a strain selected from the group consisting of Shewanella, Tessaracoccus. Magnetospirillum. Geobacter, Desulfuromonas, Priestia, Lysinibacillus, Bacillus, Halomonas. Deinococcus, Sporosarcina, Lysinibacillus. Bacillusmiscanthi , and combinations thereof.

[0027] 6. The method of any one of sentences 1 - 5, wherein the one or more microbes comprises a carbonic anhydrase expressing bacteria or a ureolytic bacteria, and optionally, the ureolytic bacteria is selected from the group consisting of Priestia megaterium ATCC 14581, Lysinibacillus sphaericus LMG 22257, Bacillus subtilis ATCC 6051, Halomonas halodesnitrificans ATCC 13511, Deinococcus radiodurans MTCC 4465, Sporosarcina pasteurii ATCC 11859, Lysinibacillus sphaericus ATCC 13805, and combinations thereof.

[0028] 7. The method of any one of sentences 1 - 6, wherein the one or more microbes comprises a non-ureolytic bacteria, optionally selected from the group consisting of Bacillus subtilis ATCC 11774 and Bacillus miscanthi AK13.

[0029] 8. The method of any one of sentences 1 - 7, wherein the microbes comprise a fungus.

[0030] 9. The method of sentence 8, wherein the fungus is selected from a genus selected from the group consisting of Trichoderma, Neurospora. Pestalotiopsis, Myrothecium, and Fusarium, or optionally, the fungus is a strain selected from the group consisting of Trichoderma reesei ATCC 13631, Neurospora crassa, Pestalotiopsis sp.,Myrthecium gramineum, and Fusarium oxysporum ATCC MYA-1198.

[0031] 10. The method of any one of sentences 1 - 9, wherein the reduction matrix further comprises one or more enzymes, optionally, the one or more enzy mes are selected from the group consisting of carbonic anhydrase and urease. Urease and carbonic anhydrase may be employed, for example, in a concentration range of 300-500 mg / L and, more preferably, 417 mg / L.

[0032] 11. The method of any one of sentences 1 - 10, wherein the composition further comprises a grow th media for the bacteria or fungi, or a buffer. DREX- 1244 WO Application

[0033] 12. The method of any one of sentences 1 - 11, wherein the one or more microbes comprises an iron-reducing bacteria.

[0034] 13. The method of any one of sentences 1 - 12, wherein the one or more microbes comprises a carbonate producing microbe or an anhydrase producing microbe, or the composition further comprises a carbonate producing enzyme.

[0035] 14. The method of any one of sentences 1 - 13, wherein the iron precipitate is selected from the group consisting of siderite (iron carbonate), magnetite (FesC ) and vivianite (iron phosphate).

[0036] 15. The method of any one of sentences 1 - 14, wherein the one or more microbes is a species selected from Shewcmella loihica (PV-4), Shewanella oneidensis (MR-1), and Tessaracoccus lapidicaptus (IPBSL-7).

[0037] 16. The method of sentence 14, wherein the iron precipitate comprises siderite in a concentration of from about 20% to about 75%, or from about 34% to about 50%, based on the total of mineral phases.

[0038] 17. The method of any one of sentences 1 - 16, wherein the method is carried out at a pH of from about 5 to 8 pH, or from about 6 to 7 pH.

[0039] 18. The method of any one of sentences 1 - 17, wherein the one or more electron donors is present in a concentration of from 0.01 M to about 1 M, or from about 0.025 M to about 0.5 M.

[0040] 19. The method of any one of sentences 1 - 18, wherein the composition further comprises a growth media selected from M9, M63, Vogel-Bonner Medium, Basal salts medium, and synthetic defined medium.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows a schematic of a corrosion layer before and after bacterial treatment, demonstrating the biogenic protective layer. DREX- 1244 WO Application

[0043] Figure 2 shows a schematic of a solid-based experimental procedure for treatment and characterization of iron coupons (from left to right: matrix preparation and sterilization, inoculation, and incubation) according to an embodiment of the invention.

[0044] Figure 3 shows a chart of Fe2+concentration and the corresponding PV4 bacterial grow th over time for abiotic and bacterially treated samples (± standard deviation data is shown for three replicates).

[0045] Figure 4A shows X-ray diffraction (XRD) spectra of precipitates collected from samples treated under aerobic (AC) and anaerobic (BC) conditions with calcium lactate (CaL2).

[0046] Figure 4B shows XRD spectra of precipitates collected from samples treated under aerobic (AN), anaerobic (BN), and hypoxic (CN) conditions with sodium lactate (NaL), denoting siderite detection (‘‘cps’’ denotes the corresponding X-ray photon counts per second according to the provided scale).

[0047] Figure 5 show s a chart of mineral phase ratio using quantitative XRD analysis for precipitates collected after 14 days from bacterially treated reduction matrices with calcium lactate (CaL2) and sodium lactate (NaL) inoculated with PV-4 bacterial cells under aerobic, hypoxic, and anaerobic conditions demonstrating the ratio of mineral phases detected.

[0048] Figure 1 show s a chart of mineral phase ratio for an abiotic control and samples treated with different bacterial species after 14 days.

[0049] Figure 2 shows macroscopic images of a) untreated iron coupon, b) iron coupon after 28 days in abiotic control, and c) iron coupon after 28 days of treatment in a reduction matrix with IPBSL-7. The circles in image b) and the circles on the righthand side of image c) indicate a corrosion layer after 28 days. The circles on the lefthand side of image c) show' the discolored substrate, and the squares indicate white precipitate formation due to bacterial activity after treatment.

[0050] Figure 3 shows scanning electron microscopy (SEM) images from the surface of iron coupons after 28 days. Images a) & b) show coupons exposed to abiotic conditions, and images c) & d) show coupons exposed to bacterial reduction matrices inoculated with IPBSL- 7 bacterial cells. The smaller square in image b) represents the substrate layer of the coupon. The larger square in image b) indicates abiotic precipitates over corrosion scales. The circles in images (c) and (d) highlight spherical particles associated with biogenic siderite. The squares in image (d) represent the stacked fonnation of new flat microcrystals which developed further to form the spherical particles. DREX- 1244 WO Application

[0051] Figure 4 shows XRD spectra of precipitates collected from pre-corroded iron coupons in an abiotic control (ACC), a pre-passivated iron coupon in bacterial treatment (BTP). and a pre-corroded iron coupon in bacterial treatment (BTC) C‘cps” denotes the corresponding X- ray photon counts per second according to the provided scale).

[0052] Figure 10 shows a chart of mineral phase distribution from a precipitate layer collected from pre-corroded iron coupons in an abiotic control (ACC), a pre-passivated iron coupon in bacterial treatment (BTP), and a pre-corroded iron coupon in bacterial treatment (BTC).

[0053] Figure 5 shows a chart of mass over surface area (area density) of different Fe2+and Fe?+mineral phases obtained from the precipitate layer of pre-corroded iron coupons in an abiotic control (ACC), a pre-passivated iron coupon in bacterial treatment (BTP), and a precorroded iron coupon in bacterial treatment (BTC) (± standard deviation data is shown for three replicates).

[0054] Figure 12 shows a graph of ODeoo absorbance values for PV-4 bacterium at vary ing salt levels, e.g., 0%, 1%, and 2%, within their first 10 days of incubation (± standard deviation data is shown for three replicates; absorbance values at day 0 are predicted via calculations based on the inoculated volume).

[0055] Figure 13 shows a graph of ODeoo absorbance values for MR-1 bacterium at vary ing salt levels, e.g., 0%, 1%, and 2%, within their first 10 days of incubation (± standard deviation data is shown for three replicates).

[0056] Figure 14 shows a graph of ODeoo absorbance values for IPBSL-7 bacterium at varying salt levels, e.g., 0%, 1%, and 2%, within their first 10 days of incubation (± standard deviation data is shown for three replicates).

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention relates to compositions prepared from one or more microbes and one or more electron donors, wherein the electron donor may also function as a carbon source. More specifically, the invention relates to methods of treating pre-existing corrosion in aged reinforced concrete elements, made from iron and iron alloys in reinforced concrete structures having existing cracks to reduce corrosion or prevent further corrosion by bringing the compositions in contact with the corroded rebar to form a biogenic coating of an iron- containing precipitate. Figure 1 illustrates via a schematic the method of the present invention. DREX- 1244 WO Application

[0059] When the composition comprising the one or more microbes and the one or more electron donors contacts an electron acceptor, such as the Fe3+ions generated by the corroded rebar, the composition reacts to form an iron-containing precipitate which functions as a protective biogenic coating on the corroded rebar.

[0060] The production of stable iron precipitates (including siderite) can be done under two main conditions:

[0061] 1) solution-based; where, for example, siderite production, its crystalline form and kinetics, and the effect of concrete pore solution chemistry is carried out and studied in A solution matrix, and

[0062] 2) solid-based; where, for example, siderite production, its crystalline form and kinetics, and the effect of concrete pore solution chemistry’ is carried out and studied on corroded iron coupons.

[0063] Both abiotic and biogenic formation of siderite and / or other stable iron minerals are possible for use in the solution-based and the solid-based approaches. Six bacterial strains capable of MISP activity can be used for biogenic siderite formation. Additionally, abiotic and biogenic siderite production, its crystalline form and kinetics can be used inside reinforced concrete (in situ approach).

[0064] The present invention is directed to mitigating corrosion in reinforced concrete and other iron-based materials by microbial-induced production of siderite (ferrous carbonate) and vivianite (ferrous phosphate), as stable forms of iron compounds. The capability of iron- reducing bacteria, including Shewanella loihica (PV-4), Shewanella oneidensis (MR-1), and Tessaracoccus lapidicaptus (IPBSL-7), to reduce ferric iron (Fe3+) as the electron acceptor and consequently, to produce stable iron minerals was demonstrated in both solution-based (soluble source of ferric iron) and solid-based (insoluble source of ferric iron) matrices. While the bacterial-induced reduction of iron, and consequently, the biogenic production of stable iron minerals was confirmed using X-ray diffraction (XRD), results indicated that the quantity’ of biogenic ferrous minerals produced is greatly influenced by the ty pe of electron donor and environmental oxygen levels.

[0065] For instance, using sodium lactate as the electron donor under hypoxic conditions resulted in the highest biogenic siderite ratio (34%). IPBSL-7 exhibited the highest siderite- to-total precipitate ratio after treatment (50%) compared to the other two bacterial species. Additionally, examining the microstructure of the biogenic layer precipitated on corroded iron coupons using scanning electron microscopy (SEM) supported the formation of ferrous minerals after bacterial treatment in the form of spherical as well as flat microcrystals. DREX- 1244 WO Application

[0066] Gravimetric analysis of the biogenic precipitate suggested that it can potentially function as a comparatively dense (with a mass over surface area of 12 mg / cm2) protective layer against further corrosion. Accordingly, the present method is suitable for forming an iron precipitate resulting in a final ferrous carbonate (siderite) phase ranging from about 1% to about 75%, or from about 34% to about 50%, based on the total of the mineral phases.

[0067] A novel approach to promote the precipitation of siderite (also known as iron (II) carbonate) and other stable iron precipitates to prevent or mitigate corrosion exploits microbial (including bacterial and fungal) metabolism that induces iron reduction. This invention employs microbial induced stable iron precipitation (MiSIP), such as microbial induced siderite production (MISP), to prevent or mitigate corrosion in steel-reinforced concrete. For that purpose, a process to cultivate, apply, engineer, and use microbial strains for MiSIP to prevent or mitigate corrosion of iron products such as rebar in cementitious materials has been developed.

[0068] Stable corrosion products, such as siderite, can naturally precipitate in specific thermodynamic conditions including high temperature, pressure and pH and once precipitated, can act as a protective layer to mitigate electrochemical corrosion. Microbial induced stabilization of iron, in contrast, is a sustainable, environmentally friendly, and continuous remediation method that can ensure durability and mechanical integrity' of an iron structure by producing a naturally growing protective layer on the surface of iron or steel.

[0069] The present invention is advantageous since it removes possible health concerns associated with using chemical corrosion inhibitors, and since it is a bio-based treatment. Also, the present invention is more time and cost efficient when compared with conventional corrosion mitigation methods.

[0070] The present method includes delivering the composition to pre-existing cracks, or may include micro-dnlling and / or injection utilizing high pressure systems, to penetrate further into the pre-existing concrete elements.

[0071] EXAMPLES

[0072] Materials

[0073] Error! Reference source not found, outlines the experiments, matrices and conditions employed in the Examples. The experimental methodology involved two main phases: (1) solution-based experiments where a soluble ferric iron source was used and (2) solid-based experiments where a solid non-soluble ferric iron source was used, to investigate siderite production. Three bacterial species including Shewanella loihica strain PV-4, Shewanella DREX- 1244 WO Application oneidensis strain MR-1, and Tessaracoccus lapidicaptus strain IPBSL-7 were selected as the microorganisms for this study since they were shown to reduce ferric iron under appropriate environmental conditions, such as favorable oxygen levels, electron donor type and concentration, and salinity11,16’24. Additionally, four different organic electron donors including sodium acetate, calcium acetate, calcium lactate, and sodium lactate were studied. Moreover, three different oxygen conditions including aerobic, hypoxic, and anaerobic conditions were investigated.

[0074] DREX-1244WO Application

[0075] Table 1

[0076] Summary of experimental design and conditions

[0077] *Denotes matrices that did not produce sufficient amounts of precipitate for further analysis.

[0078] DREX- 1244 WO Application

[0079] Strains and Medium

[0080] To compare the iron-reducing performance of various microorganisms, three bacterial species that use different mechanisms and require varying conditions for oxygen availability were studied: 1) Shewanella loihica, a facultative anaerobe which uses a cytochrome-based mechanism, 2) Shewanella oneidensis, an obligate aerobe which uses a cytochrome-based mechanism, and 3) Tessaracoccus lapidicaptus , a facultative anaerobe which uses a noncytochrome-based mechanism.

[0081] Shewanella loihica PV-4 (DSM 17748) used in this study was obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). Routine cultivation was carried out aerobically in Luria-Bertani (LB) medium containing 1% (w / v) NaCl at pH 7 and at 30°C, with agitation at 125 rpm. For experiments, bacterial cells were cultured overnight, and cell biomass was harvested by centrifugation at 7500 rpm for 5 minutes and discarding the supernatant. Finally, the pellet was resuspended in fresh medium without NaCl.

[0082] Shewanella oneidensis MR-1 (ATCC 700550) was obtained from ATCC (American Type Culture Collection). It was cultured aerobically in Trypto-casein soy medium containing 1% (w / v) NaCl at pH 7.3. After overnight incubation at 30°C and 125 rpm. cells were harvested by centrifugation at 7500 rpm for 5 min.

[0083] Tessaracoccus lapidicaptus IPBSL-7 (DSM 27266) was obtained from the DSMZ. It was grown anaerobically in Trypticase Soy Yeast Extract medium with 1% (w / v) NaCl at pH 7.1, at a temperature of 30°C and 125 rpm. Following overnight cultivation, the cells were harvested by centrifugation at 7500 rpm for 5 min and resuspended in fresh medium without NaCl. Initial growth studies were conducted with varying NaCl concentrations (0%, 1%, and 2%) to investigate bacterial growth and viability with NaCl concentrations lower (in case of passivated iron) or higher (in case of corroded iron) than the recommended optimal media, see Figure , Figure , and 14. All three bacterial species exhibited an exponential increase in ODeoo (Optical Density at 600 nm) absorbance values across all tested media with salt concentrations ranging from 0% to 2%, indicating the adaptability' of the bacteria to vary ing salinity' levels. This finding was critical for subsequent corrosion studies involving chloride-rich conditions for inducing accelerated corrosion, as well as conditions with no chloride to avoid corrosion in control samples. DREX- 1244 WO Application

[0084] Solution-based matrix preparation

[0085] Reduction matrices for solution-based experiments in-vitro were prepared in capped culture media bottles by mixing the following components: (1) an electron acceptor, e.g., ferric chloride (FeCL) with a concentration of 0.01 M as the ferric iron source, (2) an electron donor, e.g., sodium acetate (NaA) with a concentration of 0.5 M, calcium acetate (CaA2) with a concentration of 0.25 M. calcium lactate (CaL?) with different concentrations of 0.025 M and 0.25 M, or sodium lactate (NaL) with different concentrations of 0.05 M and 0.5 M, (3) a minimal growth media, e g., M9 salts containing 0.1 102 M of KH2PO4, 0.04277 M of NaCl, 0.2387 M of Na2HPO4, and 0.09347 M of NFLC1, and (4) a microorganism, e.g., PV-4, MR-1 or IPBSL-7.

[0086] Preliminary examples using NaA and CaA2 as electron donors did not produce sufficient precipitates for further analysis after 14 days. This was possibly due to the fact that Shewanella species have been reported to favor denitrification over iron reduction when using acetate as their electron donor19. Therefore, subsequent experiments and the corresponding results were only carried out and presented for CaL and NaL2 as the electron donors. Reduction matrices for studying the effect of CaL2 and NaL were prepared under aerobic conditions with a dissolved oxygen concentration (DO) of approx. 8.6 mg / L, as well as under anaerobic condition with a DO of < 0.05 mg / L. Anaerobic conditions were achieved by heating the reduction matrices up to 70-80°C, followed by degassing with nitrogen gas34. Additionally, a hypoxic condition was added for matrices containing NaL as an intermediary condition for investigating the effect of oxygen levels. To prepare the hypoxic cultures, the headspace of the degassed bottles was injected with 25 ml of atmospheric air to achieve a DO of approx. 2.9 mg / L. DO was calculated using Henry's law35. The hypoxic condition was not added to the experiments for matrices containing CaL2, after preliminary experiments predominantly showed better iron reduction for matrices containing NaL. The prepared matrices were inoculated wdth overnight cultivated cell biomass at an initial concentration corresponding to ODeoo = 0.5 and incubated at 30 °C with agitation at 125 rpm. This last step was excluded for abiotic controls.

[0087] Subsequently, reduction matrices for studying the effect of microorganisms were prepared with NaL under hypoxic conditions and ere inoculated with either PV-4, MR-1 or IPBSL-7. DREX- 1244 WO Application

[0088] Solid-based matrix preparation

[0089] Reduction matrices for solid-based experiments were prepared in capped culture media bottles using a rendition of the solution-based experiments including NaL under hypoxic conditions and inoculated with IPBSL-7, with the inclusion of iron coupons as the electron acceptor instead of ferric chloride. Iron coupons were prepared by cutting sheets of A36 steel carbon into 10 mm x 20 mm x 5 mm coupons. Subsequently, to simulate the microenvironmental conditions of passivated or corroded reinforcement in concrete, iron coupons were pre-submerged for 14 days in either a highly alkaline concrete pore solution (pH =13) for passivation, or a solution with high chloride concentration (3% NaCl) for accelerated corrosion36, respectively. While the purpose for pre-conditioning in high alkaline pore solution was to induce passive layer formation similar to that of reinforcement in concrete, it is important to note that subsequent experiments were carried out at a lower pH (between 6 to 7) which represents the lower local pH at corroding sites and is optimal for bacterial activity22. When de-passivation occurs as a result of chloride attack, release of metal ions and production of H+via hydrolysis reactions can drop local pH at corroding sites below neutral pH. and in extreme scenarios, as low as 3 to 637. Accordingly, the present method consists of treating the iron coupons at a pH below neutral point, i.e., 6 - 7 pH, after passivation or de-passivation.

[0090] Concrete pore solution was synthesized by preparing a solution containing 0.28 M of NaOH and 0.57 M of KOH with a final pH equal to 13, according to ASTM C15038. Iron coupons were then washed with isopropyl alcohol and sterilized by 15 min exposure to UV light to eliminate any unwanted microorganisms. Thereafter, the preconditioned iron coupons were transferred to serum bottles where a solution containing NaL, M9 salts, and IPBSL-7 bacteria was added under sterile and hypoxic conditions. The matrices were inoculated by injecting overnight cultivated cell biomass at an initial concentration corresponding to ODeoo = 0.5 and subsequently, incubated at 30°C with agitation at 125 rpm. This last step was omitted for abiotic controls. Error! Reference source not found, schematically demonstrates the instill procedure of solid-based experiments for the treatment of prepared iron coupons.

[0091] Fe2concentration measurements

[0092] A modified method of Kooli et al.11was used to measure the Fe2+concentration over time. 100 pL of 5 M HC1 was immediately added to 900 pL of the collected sample after sampling at 0, 24h, 48h, and 120h after inoculation. Samples were centrifuged for 1 minute at 7500 rpm to separate the supernatant. A 100 pL aliquot of the supernatant was combined with 900 pL of ferrozine reagent, i.e., 0.1% ferrozine was prepared in a 100 mM HEPES buffer, pH DREX- 1244 WO Application

[0093] 7). The mixture was thoroughly homogenized. The absorbance of the solution was measured at 562 nm using a UV-Vis Thermo Scientific™ GENESYS 20 spectrophotometer. A calibration curve was prepared using serial dilutions of a 1 mM ferrous ammonium sulfate solution in acidic MilliQ water (adjusted to pEI 2). Absorbance values were plotted against known Fe2+concentrations to establish a standard curve. The Fe2+concentration in the samples was determined by comparing the measured absorbance to the calibration curve.

[0094] XRD and microscopic experiments

[0095] For solution-based experiments, samples were collected after 14 days, centrifuged at 7500 rpm for 25 min, and oven-dried at 105 °C overnight. The precipitates were ground into fine powders for characterization using X-ray diffraction (XRD) analysis. A Rigaku-MiniFlex 600 equipped with Cu Ka ( = 1.387 A) was used for XRD analysis at Bragg angle 20 in the range of 15°-70° with a step of 0.02°. An open source Profex software was used to identify the mineral phases. For quantitative XRD analysis, 15% w / w of rutile was added to the powdered samples as the reference crystalline phase. Subsequently, the ratio of each of the mineral phases that were detected was quantified via the software.

[0096] For solid-based experiments, iron coupons were collected after 28 days (longer incubation time to accumulate more precipitates), washed wi th DI water and air-dried overnight before scanning electron microscopy (SEM) imaging using a Thermo Fisher Apreo 2S Lo Vac. Subsequently, the precipitate layer on the surface of the iron coupons was scraped off using a razor blade and was ground into a powder for quantitative XRD analysis following the same procedure as the solution-based experiments.

[0097] 1. Results And Discussion

[0098] Solution-based experiments

[0099] Error! Reference source not found, shows the concentration of Fe2+measured using the ferrozine assay for abiotic and bacterially treated samples, as well as the corresponding bacterial growth over time. The results indicated that while there is no remarkable change observed in Fe2+concentration during the first 48 hours in both abiotic control and bacterial treatment samples, an exponential increase in Fe2+in samples treated with PV-4 bacteria was observed. Comparably, there was negligible Fe2+detected in abiotic samples with no major increase in concentration over time. This observation confirms that the iron reduction observed in samples treated with bacteria has a biogenic nature. The slight decline observed in treatment samples during the first two days may be attributed to the inherent margin of error in DREX- 1244 WO Application spectrophotometric measurements, particularly at such low concentration levels. Given the sensitivity of the method, minor fluctuations were expected and did not necessarily indicate a significant change in the actual concentration. The absorbance measurements showed a significant increase in bacterial growth within the first day of incubation. However, considering the corresponding Fe2+concentration, iron reduction appeared to have a lag phase during the first 48 hours of bacterial growth, after which the bacteria effectively utilize ferric ions as electron acceptors and the Fe2+concentration exponentially increased. After only 5 days of bacterial activity, Fe2+was detected at a concentration of 0.43 mM for solutions with an initial FeCh concentration of 10 mM, which accounted for a successful reduction of over 4% in total Fe?+.

[0100] Error! Reference source not found.A and 4B show the XRD spectra of precipitates collected from matrices containing 0.25 M of CaL2 (See Figure 4A) and 0.5 M of NaL (See Figure 4B) under various conditions. In case of abiotic control and low-concentration conditions (0.025 M of CaL2 and 0.05 M of NaL), a zero-to-negligible amount of precipitate was observed after incubation; therefore, XRD was not conducted on these samples. As shown in Error! Reference source not found., qualitative XRD analysis confirmed the formation of siderite (S) in high concentration biotic matrices treated with both CaL2 in Figure 4A and NaL in Figure 4B as electron donors, implying successful iron reduction and mineral stabilization. Since no precipitate was detected in abiotic matrices, it w as inferred that the siderite production in bacterially treated samples has a biogenic nature. Higher concentrations of electron donors (0.25 M CaL2 and 0.5 M NaL) promoted precipitation, indicating a concentration-dependent biogenic siderite mineralization. It was interpreted that there is a threshold for electron donor concentration below' which the bacterial activity7will not result in insoluble mineral precipitation. Further analysis on matrices with various electron donor concentrations may be useful in determining that threshold.

[0101] In comparison to CaL2, NaL (0.5 M) demonstrated superior performance as an electron donor, with its hypoxic matrix resulting in the highest siderite production according to the XRD data, underscoring the critical role of oxygen availability in modulating bioreduction pathways. While siderite was the main mineral detected where NaL was used as the electron donor, CaL2 showed a significant presence of hydroxyapatite (HA) and hematite (H).

[0102] Figure shows the ratio of mineral phases detected in biotic reduction matrices containing either 0.25 M CaL2or 0.5 M NaL as electron donors under various levels calculated using quantitative XRD analysis. In biotic samples with CaL2, aerobic conditions resulted in a siderite production equal to 3% of the total precipitate mass, with no hematite detected as DREX- 1244 WO Application opposed to abiotic controls. This suggests that oxygen availability allows for partial bioreduction of iron, favoring siderite precipitation over hematite. Biotic CaL? matrices under the anaerobic condition resulted in a slightly lower siderite formation (2%) compared to the aerobic condition (3%).

[0103] Additionally, calcium carbonate (calcite) and halite were also detected in all CaL2 matrices as secondary minerals. Formation of calcium carbonate can be explained by possible oxidation of CaL2. A similar transformation has been previously reported as a result of bacterial -induced organic carbon oxidation39. The consumption of organic carbon to form calcium carbonate can potentially have a negative effect on the production of siderite, as it can deplete the carbonate present in the system which is a required component in the process. However, XRD analysis indicated that the presence of calcium carbonate was remarkably higher under the aerobic condition, indicating the effect of oxygen on the oxidation of organic carbon.

[0104] Another notable observation was the detection of a large ratio of calcium phosphate in the form of hydroxyapatite (-76% of total precipitate mass) in all samples containing CaL2. This can be explained as being a result of a hydrothermal reaction between Ca2+from CaL2 and phosphate that is present in the system from the M9 media40. Interestingly, this mineral which is formed as a side product of the reduction matrix used has the potential to provide additional protection against corrosion41. Hydroxyapatite is known to displace chloride ions present in the corrosive environment, forming a stable chlorapatite phase and decreasing the corrosion rate42.

[0105] Similarly, siderite formation was detected in all biotic reduction matrices with NaL via XRD analysis. The siderite formation proved to be of a biogenic nature as there was no detection in the corresponding sterile controls. As shown in Figure, results demonstrated that NaL was the more effective electron donor for siderite production compared to CaL2, particularly under hypoxic conditions, with a siderite to total precipitate ratio of 34%. This observation suggests that optimization of oxygen concentration can enhance the iron reduction induced by facultative anaerobic bacteria. Under aerobic and anaerobic conditions, siderite formation was relatively lower, with analogous ratios of 15% in both cases.

[0106] Similar to matrices with CaL2, halite was also detected in matrices with NaL; however, in the absence of Ca2+, various forms of potassium phosphate (KPO) emerged as the dominant secondary mineral phases instead of HA as observed previously with CaL2. Another contrasting observation was the detection of hematite, but it was found to have a lower ratio than siderite in all samples with different oxygen conditions, confirming that the reactions successfully favored the reduction of iron. Moreover, a notable observation was the detection of magnetite DREX- 1244 WO Application production (4%) in NaL matrices under hypoxic conditions. It can be hypothesized that low concentrations of oxygen within the system lead to the formation of magnetite, a mixed-valent oxide which contains both Fe2+and Fe3+. This can potentially be a biogenic outcome of FeC , and / or hematite reduction2’.

[0107] Based on the results of the aforementioned study on electron donors and oxygen concentration, NaL, particularly under hypoxic conditions, proved to be the most effective matrix for siderite formation. Subsequently, for investigating the bioreduction / mineralization capacity of the three selected bacterial species, solution-based experiments were conducted using the most optimal reduction matrix (i.e., 0.5 MNaL in hypoxic condition). Figure 1 shows the ratio of mineral phases for samples treated with the three selected bacterial species as well as the abiotic control. The screening of bacterial species revealed that S. loihica (PV4), . oneidensis (MR-1), and T. lapidicaptus (IPBSL-7) were all capable of producing siderite under the most optimal condition. XRD analysis showed that bacterial treatments consistently produced siderite in comparison to abiotic controls, with vary ing siderite ratios depending on the species. IPBSL-7 exhibited the highest ratio (50%), indicating its superior ability to reduce ferric ions to ferrous ions, a key step in stable mineral formation and combating oxidation of iron. For that purpose, IPBSL-7 was selected for further investigations of iron reduction in solid-based experiments where corroded iron coupons were used as the source of ferric iron / electron acceptor.

[0108] Analy sis of mineral phase ratios in PV-4. MR-1, and IPBSL-7 bacterial strains further demonstrated the variability in mineralization pathways among microbial species. The strainspecific ability' to reduce Fe3+and the metabolic utilization of lactate electron donors significantly influenced the relative abundance of siderite versus other mineral phases. These findings highlight the importance of bacterial physiology in driving the mineralization of iron- bearing phases under varying environmental conditions. The lower ratio of siderite observed in samples treated using PV-4 can be due to the fact that S. loihica has previously shown high activity' in reducing poorly crystalline Fe3+oxides, such as ferrihydrite, but lower activity' on more crystalline forms like hematite or magnetite43. Similarly, while it has been shown that S'. oneidensis is effective at reducing amorphous Fe3+oxides, lower efficiency with more crystalline forms such as hematite has been reported44. However, studies have highlighted S. oneidensis’’ s ability' to reduce iron even in the presence of competing electron acceptors such as nitrate and sulfate, indicating its competence for iron reduction compared to S. loihica^-*6. In contrast to Shewanella species which thrive in fresh or brackish water systems and are less tolerant of high salinity47, most experiments on T. lapidicaptus have focused on its role in niche DREX- 1244 WO Application environments, such as subsurface sediments or extreme environments including hypersaline or hydrothermal systems. Compared to Shewanella species, T. lapidicaptus's metabolic plasticity and its ability to thrive under extreme conditions (e.g., high salinity, low oxygen) have been highlighted previously24, which can further explain the higher siderite production observed in the present invention.

[0109] Solid-Based Experiments

[0110] Figure 2 shows macroscopic images of iron coupons before (see image a) and after 28 days of conditioning in an abiotic matrix (see image b) and the bacterially treated matrix (see image c). Distinct biogenic precipitate layers on iron coupons treated with bacterial reduction matrices can be observed in Figure 7, image c). which were absent in the untreated reference and the abiotic control (Figure 2, respectively). These distinct layers included light brown deposits and sparse white powder-like precipitates, as shown by the right circles and the left circles, respectively, in Figure 2, image c), likely indicating biogenic minerals and organic matter. In contrast, a red-brown color typical of that of a corrosion layer can be observed in the abiotic control (as shown by the circles in image b) of Figure 7. Additionally, a dark discoloration covering the substrate layer was observed in the bacteria-treated sample compared to that of the abiotic control (shown by the squares in Figure 2, image c).

[0111] Figure 3 demonstrates the microstructure of the iron coupons’ surfaces after 28 days of conditioning in abiotic and bacterially treated matrices. SEM imaging using secondary electrons revealed distinctive surface morphologies for both the abiotic control and the bacterially treated iron coupons. The bacterially treated coupons exhibited crystalline formations that densely and uniformly covered the original corrosion layers, suggesting a potential corrosion-protective role for the precipitates (as shown in Figure 3, image c). This uniform layer consisted of spherical particles with diameters ranging from 4 pm to 4.5 pm that were each developed by multi-layer stacking of flat microcrystals (as shown in Figure 3, image d). These flat microcrystals have a range of diameters that start as small as 0.4 pm and grow to form crystals larger than 2 pm (highlighted using squares in Figure 3, image d). The distinctive morphology observed here closely represents that of previously reported biogenic siderite particles31. In contrast, abiotic controls demonstrated exposed and irregular scale-like surface morphologies typical of corrosion layers48(as shown in Figure 3, images a)-b). No visually notable cry stalline formation was observed in the control samples. These observations demonstrated the microbial influence on precipitate development, as bacteria facilitated the DREX- 1244 WO Application mineralization process leading to more compact and continuous layers of a more stable crystalline iron mineral (i.e. siderite).

[0112] Subsequently, quantitative XRD analysis was conducted on precipitate collected from the coupons by scraping off the surface using a surgical scalpel. Figure 4 shows XRD diffractograms associated with precipitates collected from pre-corroded iron coupons in abiotic control (ACC), pre-passivated iron coupon in bacterial treatment (BTP), and pre-corroded iron coupon in bacterial treatment (BTC). It was observed that while a wide range of various iron minerals were detected in all samples, precipitates collected from BTP and BTC primarily contain ferrous minerals including vivianite and siderite. On the other hand, while there was magnetite detected in ACC, a notable presence of ferric minerals including wuestite and lepidocrocite was observed. Figure shows the mineral phase distribution of precipitate layers across abiotic and bacterially treated iron coupons based on quantitative analysis of the XRD results. Quantitative XRD analysis confirmed that the biogenic precipitate layers in bacterial- treated samples were composed primarily of siderite and vivianite, with a significant reduction in corrosion products such as hematite, lepidocrocite, and wuestite found in the abiotic control. Therefore, the bacterial treatment facilitated the reduction of ferric iron oxides, promoting the formation of more stable ferrous minerals. The predominant presence of vivianite and siderite in the bacterial-treated samples highlights the role of microbial activity in reducing ferric iron and facilitating the precipitation of protective minerals. The pre-corroded samples yielded a higher ratio of ferrous minerals (up to 80%) compared to iron coupons that were passivated. It can be inferred that the presence of active corrosion products can facilitate the access of bacteria to electron acceptors and consequently, can yield a higher iron reduction activity7. In contrast, abiotic controls exhibited higher concentrations of ferric iron minerals with no siderite and vivianite detected in their precipitate layer, indicating the presence of less stable iron minerals. These findings align with existing research11,17, demonstrating that microbial- induced mineralization can enhance the stability of corroded surfaces by promoting the precipitation of stable mineral phases.

[0113] Figure 5 shows the area density (i.e., mass / area) of the precipitates obtained from the abiotic control and the bacterial treatments. According to Figure 5, quantitative analysis revealed that the biogenic precipitate layers formed on bacteria-treated samples had a higher area density compared to the abiotic controls. The increased area density is attributed to the enhanced microbial reduction processes that promote the precipitation of more dense mineral layers. These layers can effectively cover the underlying corroded surfaces, providing a barrier against further corrosion. As shown in Figure 5, the solid-based experiments with pre-corroded DREX- 1244 WO Application iron coupons with the highest area density of Fe2+minerals further confirmed the protective role of these precipitates, as the biogenic layers reduced the exposure of the iron surface to corrosive agents. These layers reduce the presence of feme iron corrosion products and enhance the stability of the corroded surfaces. Hence, the XRD analysis backed by the SEM imaging, highlights the effectiveness of microbial-induced mineralization in transforming and stabilizing corrosion products, suggesting a viable approach for corrosion mitigation in metal structures or reinforcement.

[0114] The present invention demonstrated the potential of microbial-induced iron biomineralization (MIIB) as a method for corrosion mitigation in reinforced concrete. By simulating concrete-reinforcement environments, the research showed that iron-reducing bacteria — Tessaracoccus lapidicaptus IPBSL-7, Shewanella loihica PV-4, and Shewanella oneidensis MR-1 — can effectively reduce ferric iron (Fe3+) to ferrous iron (Fe2+), resulting in the precipitation of stable biogenic minerals. Quantitative XRD analysis confirmed siderite (ferrous carbonate) as the predominant biogenic mineral formed in the solution-based bacterial- treated samples, particularly under hypoxic conditions using sodium lactate as the electron donor. This mineralization process significantly reduced the presence of ferric iron corrosion products, such as hematite, lepidocrocite, and wuestite, and promoted the formation of stable ferrous minerals, including siderite and vivianite.

[0115] Solid-based experiments further validated the protective role of these biogenic precipitate layers. SEM imaging revealed compact and uniform microcrystalline layers on the bacterial-treated iron coupons, which were absent in abiotic controls. Additionally, it was show n that the bacterial treatments resulted in higher area densities of the biogenic layers that can potentially act as a barrier against further corrosion by reducing the exposure to oxygen and moisture. The results illustrate that electron donor concentration, oxygen availability, and bacterial strain specificity influence the production of siderite during microbial iron reduction. These results showcase the successful formation of stable iron minerals on corroded iron species as a proof of concept for the application of this biomineralization method in stabilizing corroded iron structures.

Claims

DREX- 1244 WO ApplicationWhat is claimed is;1. A method of treating corroded rebar in a reinforced concrete structure comprising one or more cracks, comprising steps of delivering a composition through the one or more cracks to the corroded rebar, wherein the composition comprises one or more microbes, selected from bacteria and fungi, and combinations thereof; and one or more electron donors; and contacting the composition with the corroded rebar to form an iron precipitate providing a biogenic coating on the corroded rebar.

2. The method of claim 1, wherein the one or more electron donors is a carbon source, optionally selected from the group consisting of acetate, lactate, glucose, ethanol, pyruvate, formate, methanol, propionate, butyrate, succinate, glycerol, mannitol, sucrose, malate, fructose, galactose, xylose, ribose, fumarate, cellobiose, and combinations thereof.

3. The method of any one of claims 1 - 2, wherein the one or more microbes are present in an initial cell concentration range between OD600 = 0. 1 to 1 , based on an amount of the one or more electron donors.

4. The method of any one of claims 1 - 3, wherein the composition comprises two or more different microbes.

5. The method of any one of claims 1 - 4, wherein the bacteria is a strain selected from the group consisting of Shewanella, Tessaracoccus. Magnetospirillum, Geobacter,Desulfur omonas, Priestia, Lysinibacillus, Bacillus, Halomonas, Deinococcus, Sporosarcina, Lysinibacillus, Bacillusmiscanthi, and combinations thereof.

6. The method of any one of claims 1 - 5, wherein the one or more microbes comprises a carbonic anhydrase expressing bacteria or a ureolytic bacteria.

7. The method of any one of claims 1 - 6, wherein the one or more microbes comprises a ureolytic bacteria and is selected from the group consisting of Priestia megaterium ATCC 14581, Lysinibacillus sphaericus LMG 22257, Bacillus subtilis ATCC 6051, HalomonasDREX- 1244 WO Application halodesnitrificans ATCC 13511, Deinococcus radiodurans MTCC 4465, Sporosarcina pasteurii ATCC 11859, Lysinibacillus sphaericus ATCC 13805, and combinations thereof.

8. The method of any one of claims 1 - 7, wherein the one or more microbes comprises a non-ureolytic bacteria, optionally selected from the group consisting of Bacillus subtilis ATCC 11774 and Bacillus miscanthi AK13.

9. The method of any one of claims 1 - 8, wherein the one or more microbes comprise a fungus.

10. The method of claim 9, wherein the fungus is selected from a genus selected from the group consisting of Trichoderma, Neurospora. Pestalotiopsis, Myrothecium, and Fusarium, or optionally, the fungi is a strain selected from the group consisting of Trichoderma reesei ATCC 13631, Neurospora crassa, Pestalotiopsis sp.,Myrthecium gramineum, and Fusarium oxysporum ATCC MYA-1198.

11. The method of any of any one of claims 1 - 10, wherein the one or more microbes is a species selected from Tessaracoccus lapidicaptus. Shewanella loihica, and Shewanella oneidensis.

12. The method of any one of claims 1 - 11, wherein the composition further comprises one or more enzymes, and. optionally, the one or more enzymes are selected from the group consisting of carbonic anhydrase and urease.

13. The method of any one of claims 1 - 12, wherein the composition further comprises a growth media for the bacteria or fungi, or a buffer.

14. The method of any one of claims 1 - 13. wherein the one or more microbes comprises an iron-reducing bacteria.

15. The method of any one of claims 1 - 14. wherein the one or more microbes comprises a carbonate producing microbe, or an anhydrase producing microbe, or the composition further comprises a carbonate producing enzyme.DREX- 1244 WO Application16. The method of any one of claims 1 - 15, wherein the iron precipitate is selected from the group consisting of siderite (iron carbonate), magnetite (FesCh) and vivianite (iron phosphate).

17. The method of claim 16, wherein the iron precipitate comprises siderite in a concentration of from about 20% to about 75%, or from about 34% to about 50%, based on a mineral phase ratio.

18. The method of any one of claims 1 - 17, wherein the method is carried out at a pH of from about 5 to 8 pH, or from about 6 to 7 pH.

19. The method of any one of claims 1 - 18, wherein the one or more electron donors is present in a concentration of from 0.01 M to about 1 M, or from about 0.025 M to about 0.5 M.

20. The method of any one of claims 1 - 19, wherein the composition further comprises a growth media selected from M9. M63, Vogel-Bonner Medium, Basal salts medium, and synthetic defined medium.

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