Environment
Microbial Leaching of Iron from Hematite: Direct or Indirect Elution
2020 Volume 61 Issue 2 Pages 396-401
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2020 Volume 61 Issue 2 Pages 396-401
Exiguobacterium oxidotolerans was found to be effective for dissolving Fe2+ from hematite via reduction under alkaline conditions. However, the possible mechanism for bacterial reduction of hematite in seawater is still unclear. The present work has investigated the reductive dissolution of iron by the bacteria in two elution systems, namely direct and indirect elution systems. Greater than 30 mg L−1 of Fe was dissolved and measured in a direct elution system. Fourier transform infrared (FTIR) and field-emission scanning electron microscopy (FE-SEM) revealed the surface oxidation and particle aggregation in our direct elution system. The obtained results suggest that direct interaction of bacterial cells with hematite facilitates iron elution, probably due to electron transfer to hematite via the cell membrane, resulting in reductive elution of hematite.
Soluble iron is essential for the marine/coastal environment. A small amount of eluted iron could assist seaweed growth in seaweed-depleted areas.1,2) Therefore, supplying soluble iron to the ocean would facilitate the growth of seaweed and support the preservation of the marine environment. As seaweed growth is essential for supporting the coastal community, the eluted iron should be supplied to the ocean in sufficient quantities (normally 5.88–27.9 mg L−1) for maintaining the health of the marine system.3,4) However, coastal seawater condition (pH 7.9–8.2, oxic) limits the possibility of iron dissolution since these conditions induce the precipitation of hydroxide.5,6) Fe-fertilizer which contains steal slag and bark compost is used to supply iron to the coastal area. Our previous study showed that bacteria isolated from Fe-fertilizer are important for iron elution at a pH range of 6–7.7) We reported that Exiguobacterium oxidotolerans plays a crucial role in the dissolution of Fe(III) oxide (Fe2O3, hematite). The interaction between E. oxidotolerans and hematite was monitored, and 23 mg L−1 of eluted Fe species was detected after a few days of incubation. Dissolution of hematite would be correlated with organic acids (e.g. oxalic acids and citric acids) production in the system. However, the possible mechanism of iron elution via bacterial activity was still unclear.
To clarify this mechanism during the incubation period, we have investigated two systems for iron elution, especially focusing on the contact conditions between hematite and bacteria. One system is based on the previous study, where E. oxidotolerans cells can directly contact the hematite surface. In this system (Fig. 1(a)), E. oxidotolerans bacteria could exchange electrons directly with Fe(III) oxide via surface contact, thereby resulting in the reduction of Fe(III) to soluble Fe(II). Figure 1(b) illustrates another system, indirect elution, where hematite particles were segregated from bacteria cells by a dialysis membrane. In the indirect elution system, metabolites such as oxalic acid from bacteria can pass through the dialysis membrane to provide electrons to Fe(III) oxide indirectly. This is based on the hypothesis that Fe(III) dissolution mainly results from interactions between organic acid and hematite. Oxalic acid could dissolve Fe(III) oxide by complexation.8,9) However, they reported that the observed iron oxide dissolution via oxalate complexation was optimum at a pH of 2.5–3.0.9) Therefore, low pH is more suitable and preferable for the elution of Fe(III) oxide compared to higher pH, especially greater than 7. Therefore, we investigated the effects of additional organic acids, including oxalic, acetic, citric and lactic acids on a hematite surface as compared with organic acids produced from bacteria.
Examined iron elution system; (a) direct elution: bacteria directly give electron to Fe(III) oxide, (b) indirect elution: organic acids pass through dialysis membrane to give electron to Fe(III) oxide.
We monitored the elution of iron, chemical properties of the culture medium, organic acids production, and hematite surface alteration during 10 days of incubation. Our study could lead to the clarification of the mechanism that affects the elution of iron oxide by bacteria under seawater conditions.
Artificial seawater was prepared by dissolving the following salts in 1 L of distilled water: NaCl 28 g; MgSO4·7H2O 7.0 g; MgCl2·6H2O 4.0 g; CaCl2·2H2O 1.47 g; and KCl 0.7 g. Modified Postgate’s B medium was prepared by dissolving the following components in a 1 L mixture of distilled water/artificial seawater (1:1, v/v): NaCl 26 g; KH2PO4 0.5 g; NH4Cl 1.0 g; Na2SO4 1.0 g; CaCl2·2H2O 0.1 g; MgSO4·7H2O 2.0 g; sodium lactate (60–70%) 5 mL; yeast extract 1.0 g; L-ascorbic acid 0.1 g and FeSO4·7H2O 0.5 g; the pH was 8.0 ± 0.2. The medium was sterilized by autoclaving at 121°C for 20 min. Plate Count Agar (PCA) medium was prepared by dissolving the following components in 1 L of distilled water: bacteriological peptone 5.0 g; protease peptone 5.0 g; L-cysteine 0.25 g; NaCl 5.0 g; ammonium iron (III) citrate 1.0 g; K2HPO4 0.3 g; and agar 15.0 g. The pH of the PCA medium was adjusted to 7.4 ± 0.2, and was sterilized by autoclave (121°C, 20 min) before use. Glycerol stocks of E. oxidotolerans were enriched with Postgate’s B medium at 20.0 ± 0.5°C in an incubator with agitation at 120 rpm for 5 days to obtain a cell density of 108 cell mL−1. Then, 50 µL aliquots were plated into PCA agar plates for colony isolation. After 5 days of incubation, orange-pale colonies were selected for enrichment in Postgate’s B medium as the main culture for the elution test.
2.2 Direct or indirect elution of Fe ions from hematiteThe 30 mL of Postgate’s B media were transferred to a 50 mL centrifuge tube. The 150 µL of culture solution had been added to the tube, followed by 0.15 g of hematite powder, for direct elution. For indirect elution, hematite powder was introduced inside a dialysis membrane (MWCO: 12000–14000 Da, Japan Medical Science, Japan) instead of directly exposed to the medium and bacteria cells. The investigation interval was 10 days. In order to confirm the effect of metabolites on the dissolution of hematite, similar experiments were conducted using a mixture of organic acids which were detected during the incubation period. The concentrations of organic acids were chosen based on the highest concentration of organic acids produced, as measured on the same day of incubation as the detection of the highest eluted iron concentration, as previously reported (Table 1).7) Hematite elution with only medium was investigated as a negative control. The reaction mixtures of the 4 systems were incubated at 20 ± 0.5°C in a shaking incubator at 160 rpm for 10 days.
After 0, 1, 3, 5, 7 and 10 days of incubation, a 1.2 mL aliquot of culture suspension was transferred to a 2 mL centrifugal tube and then centrifuged at 6500 rpm for 10 min. After the supernatant was filtered with a 0.45 µm membrane filter, a 1 mL aliquot of the filtrate was transferred to a polyethylene tube and diluted to 10 mL with 0.01 M HCl to preserve Fe in the solution. The total iron concentration was analyzed with an ICPE-9000 type ICP-AES (Shimadzu, Japan). The concentrations of Fe(II) species in the media were spectroscopically determined with ferrozine as an indicator of Fe(II). A 0.05 mL aliquot of 0.05 mM ferrozine was added to a 1 mL aliquot of media which was centrifuged and filtered (0.45 µm). After 30 min of incubation, the concentrations of Fe(II) species were determined based on the concentration of Fe(II)-ferrozine complexes by measuring the absorbance at 562 nm.
The dynamics of the oxidation-reduction potential (ORP) of the media was monitored with an ORP meter (TOA-DKK, Japan) during 10 days of incubation. The concentration of oxalic acid in the media was analyzed by IC20-type Ion Chromatography (Thermo Fisher Scientific, USA) equipped with a Dionex™ IonPac ICE-AS6 column (Thermo Fisher Scientific, USA), using 1 mM aqueous heptafluorobutyric acid solution as a mobile phase.
2.4 Surface analysis of hematiteThe hematite slurry after 10 days of culture was transferred into a visking tube and dialyzed against ultrapure water. After dialysis, the slurry was freeze-dried to obtain powdered samples. The Fourier transform infrared (FTIR) spectroscopy, 6200HFV (Jasco, Japan) equipped with attenuated total reflectance (ATR) extension ATR-pro one (Jasco, Japan) with view-through diamond prism was introduced to observe the hematite surface alteration. Spectra were obtained via the spectra manager program (Jasco, Japan).
For hematite surface investigation, a field-emission scanning electron microscope (FE-SEM, JSM-6500F, JEOL Ltd., Japan) was used. The samples were sputtered with Pt ion at an ion current of 10 mA by a JFC-110E ion sputtering machine (JEOL, Japan) for 60 seconds before observation. The observation was done under 5–10 kV accelerating voltage, at 10,000x magnification.
Figure 2 shows iron concentration in the cultured media in four systems. The total iron concentration was almost zero at the beginning of incubation and increased up to 30.6 mg L−1 at the end of incubation in direct elution (Fig. 2(a)). Negligible amounts of iron concentration was found with indirect elution. The significant difference in total iron concentration between direct and indirect elution indicates that direct contact of bacteria cells with hematite particles is crucial for iron elution. The bacteria was found to produce several organic acids such as oxalic acid, which could possibly dissolve hematite and form a complex with Fe ions to give a soluble complex Fe(C2O4)33− or Fe(C2O4)22−.9) To elucidate the effect of organic acids, we tested iron elution from hematite by adding the organic acids listed in Table 1 to a hematite suspension in the absence of bacteria cells. The addition of organic acids to hematite did not induce iron elution, indicating that bacteria cells are necessary for iron elution. The reduction of Fe(III) oxide by microorganisms is a natural process for producing energy in anaerobic respiration, and hematite or goethite can be reduced to soluble Fe ions.10,11) Nevin and Lovly12) demonstrated that dissimilatory Fe(III)-reducing bacteria Geobacter metallireducens does not release an extracellular electron-shuttling compounds to reduce Fe(III) oxide when bacterial cells were separated from Fe(III) oxide by a semipermeable membrane. The Fe2+ concentration profile (Fig. 2(b)) was similar to the trend of total iron concentration, with the final Fe(II) concentration at 7.39 mg L−1, which is much lower than that of Fe(III). This could be due to the possibility that iron species were mainly present as Fe(III)-oxalate complex.
Comparison in hematite elution between direct and indirect elution; (a) total iron and (b) Fe(II) species.
The chemical properties of the cultured solution were monitored daily. The decrease in pH value might affect the amounts of iron elution (Fig. 3(a)). The reductive phase of ORPs less than 0 also support the reduction of iron (Fig. 3(b)). During the 10 days of incubation, the pH value in the direct and indirect system was lowered in two days to a pH around 7.4. In the added organic acids system, the pH was the lowest of all samples at around 6.8–7, and almost constant during the entire investigation. In the hematite only system, the pH was the highest at 7.8 and also mostly stable during the investigation. The ORP of direct elution showed a more significant decrease compared to that of indirect elution. This could be due to the amount of ferrous species eluted into the culture solution. Figure 3(b) showed that in the added organic acids system, the ORP was lower than in the direct or indirect elution tests at the beginning of incubation. However, the ORP was mostly stable during the entire monitoring time in the added organic acids system. In the hematite only system (negative control), the ORP was the highest at the oxidative phase and remained mostly stable. The ORPs monitoring showed that microorganism activities might influence the decrease of ORPs in both direct and indirect elution. The reduction in both chemical profiles supported the iron elution data. The decrease of pH and ORP were preferable to the dissolution of iron by E. oxidotolerans, especially in direct elution where ORP reached −300 mV.
Physicochemical parameter during cultivation; (a) pH and (b) ORPs.
Oxalic acid production was monitored during 10 days of incubation (Fig. 4). We reported that oxalic acid was detected at the highest concentration compared to other organic acids such as acetic, citric and lactic acids.7) Both direct and indirect elution systems could provide information on the activities of bacteria. However, bacteria in the direct elution system produced higher amounts of oxalic acid, reaching a maximum at 2199 mg L−1 while indirect elution produced a concentration of 1986 mg L−1 (Fig. 4). The differing amounts of oxalic acid production between direct and indirect elution might depend on the exposure of bacteria cells to the hematite surface. There is evidence showing that iron oxide-containing conditions could help increase of bacterial growth and metabolism.10,13–16) The trace amount of eluted Fe(II) could act as a nutrient to the bacteria and might also increase the production of bacterial metabolites such as oxalic acid.10,14) Oxalic acid plays a key role in dissolving iron oxides.9,17–20) Direct contact would induce reductive elution of iron from hematite, and trace amounts of eluted iron could be assimilated and used by the bacteria to produce oxalic acid, which can act as a chelating agent for eluted iron. This also might be one of the reasons for the high amount of eluted iron shown in direct elution.
Oxalic acid production in direct and indirect systems.
Comparison of the ATR-FTIR spectra of hematite between direct and indirect elution is shown in Fig. 5. In direct elution, a specific peak appeared around 1220–1250 (cm−1), indicating P=O asymmetric stretching vibration and P–OH bending vibration.19) The attributed peak for Fe–P–O complexation/interaction which indicated bacteria cell attached to hematite also appeared around the group frequency at 1033–1085 (cm−1).21,22) The formation of iron with phosphate might be one of the evidence to detect the eluted of iron in the direct elution system.22–24) The appearance of amino acid functional group around 1590–1680 (cm−1) for primary amine (NH bend) and group frequency around 1550 (cm−1) for secondary amine (NH-bend),21–23,25) also detected in direct elution system. These two amino acids-related peaks might suggest to the bacteria cell adsorb/attach to the hematite surface which might corresponded to bacteria cell interaction with iron.25) No clear peak was observed in indirect elution.
ATR-FTIR analysis of hematite in direct and indirect systems.
Figure 6 shows FE-SEM image of hematite surface after 10 days incubation in direct and indirect elution system. Although we could not distinguish bacteria cells and hematite particles in direct elution, the appearance in direct elution was obviously different from that in indirect and untreated system. The surface of the particles in direct elution was much smoother, and particles seemed to be aggregated or fused with each other. It would suggest that iron was dissolved and reprecipitated again on the hematite surface. The surface of hematite in indirect elution is similar to that of untreated hematite. The difference in appearance of hematite surface between direct and indirect elution would support the assumption that the bacteria cells are directly interacting with hematite particle, where electron transfer via cytochrome would take place.26–31)
FE-SEM image of hematite surface; (a) direct elution, (b) indirect elution and (c) untreated hematite.
The amount of iron concentration during the incubation showed significantly different between two elution systems. Direct interaction between hematite and cell surface would have a major contribution to elution of iron rather than indirect hematite surface interaction. The effect of direct interaction-reduction between hematite and microbial cells via electron transfer has been reported.28,29,31) The microbial electron transfer between bacteria cells and solid materials, as has been reported and called extracellular electron transfer (EET).26–32) EET is a type of microbial metabolic process which requires electron transfer between microbial cells and extracellular solid materials.30,33) The model organisms have been investigated in direct EET, in which microorganisms attach to solid surfaces and then directly transfer electrons.30,34–36) The c-type cytochrome plays an important role in direct EET. The electron is transferred from inner to outer membrane via electron hopping through multiple redox-active proteins (cytochrome) that connect microbial respiratory chains and external surfaces.30) The possible mechanism in direct elution might occur via bacteria cell attachment and directly transferring electrons to the Fe(III) oxide surface, which acts as terminal electron acceptor in the system. Here, oxalic acid could form a complex with Fe ions to give a soluble complex Fe(C2O4)33− or Fe(C2O4)22− (Fig. 7). Higher amount of iron dissolution in direct elution would be related to the higher production of oxalic acid. Based on this study, when bacteria cells can not interact directly with the Fe(III) oxide surface, the reduction of Fe(III) oxide might not occur. Therefore, the reduction of Fe(III) oxide in the indirect system would depend solely on the bacterial metabolites, which are produced at lower concentrations than in direct elution. Bacterial activity also reduced when the bacteria could not utilize Fe(III)/Fe(II) as a metabolism source.10,15,28,29) However, a long-period study should be done to confirm the final concentration of iron elution in the two systems.
Hypothesis of Fe(III) oxide elution in direct system.
The interaction between bacteria cells and hematite surfaces facilitate the elution of hematite. Iron elution was only detected with direct elution, and the monitoring of oxalic acid production also showed a higher concentration for direct elution. In addition, a chemical profile which is preferable for iron elution was observed mainly in the direct elution system. Surface analysis also showed the alteration and scattering of hematite particles only in direct elution. From this combined evidence, we propose the hypothesis which is summarized in Fig. 7, namely that direct interaction between bacteria cells and the surface of hematite performs a major role in the reduction of insoluble Fe(III) to eluted-Fe(II).
This work was partly supported by JSPS KAKENHI grant number JP18H03395. We thank Dr. Hisanori Iwai and Dr. Mitsuo Yamamoto for their useful comments on the utilization of iron in seaweed-depleted areas. The authors also thank Prof. Naoki Hiroyoshi (Hokkaido University) for the analysis by ATR-FTIR.
