Reductive Transformation of Fe(III) (oxyhydr)Oxides by Mesophilic Homoacetogens in the Genus Sporomusa

Abstract

Microbial reduction of iron contributes to the dissolution and transformation of iron-containing minerals in nature. Diverse groups of homoacetogenic bacteria (homoacetogens) have been reported to reduce insoluble Fe(III) oxides, such as hydrous ferric oxide (HFO), an Fe(III) mineral commonly found in soils and sediments. Several members of genus Sporomusa reportedly oxidize Fe(0), indicating the presence of an extracellular electron-uptake mechanism. However, the ability of the genus to reduce insoluble Fe(III) oxides is limited, and the underlying reduction mechanism remains to be elucidated. In this study, the HFO reduction ability of three Sporomusa spp. (Sporomusa sp. strain GT1, Sporomusa sphaeroides, and Sporomusa ovata) and a homoacetogen of a different genus (Acetobacterium woodii) were assayed under organotrophic (ethanol) and lithotrophic (H₂ + CO₂) conditions without a chelator or reducing reagent. All tested homoacetogens showed acetogenic growth and concomitant reduction of HFO under both organotrophic and lithotrophic conditions. Analysis of the growth stoichiometry showed that Fe(III) reduction does not support direct energy conservation, thereby indicating that Fe(III) reduction is a side reaction of acetogenesis to dissipate the excess reducing power. HFO was reduced to a soluble Fe(II) form by microbial activity. In addition, we observed that strain GT1, S. sphaeroides, and S. ovata reduced crystalline Fe(III) oxides, and HFO was reductively transformed into magnetite (Fe₃O₄) under phosphate-limiting conditions. Separation of HFO by a dialysis membrane still permitted Fe(II) production, although the reduction rate was decreased, suggesting that Fe(III) reduction is at least partially mediated by soluble redox compound(s) secreted from the cells. Finally, culture experiments and comparative genomic analysis suggested that electron transfer by flavins and multiheme c-type cytochrome were not directly correlated with Fe(III) reduction activity. This study reveals the capability of Sporomusa spp. in the reductive transformation of iron mineral and indicates the potential involvement of these organisms in iron and other mineral cycles in nature.

Keywords: element cycle; extracellular electron transfer; homoacetogenic bacteria; iron reduction; mineral transformation.

FIGURE 1

Appearance of homoacetogen cultures in the presence of HFO. Homoacetogens were cultivated on ethanol (20 mM) in the presence of HFO.

FIGURE 2

Growth-dependent HFO reduction by strain GT1 under organotrophic conditions. Strain GT1 was cultivated on ethanol (20 mM) in the presence or absence of HFO. (A) Cell density, (B) concentration of ethanol (triangles) and acetate (diamonds), and (C) Fe(II) concentration were periodically determined during cultivation. Closed symbols, cultures with HFO. Open symbols, cultures without HFO. Data are presented as the means of three independent cultures, and error bars represent standard deviations.

FIGURE 3

Difference in HFO reduction activity among homoacetogens under organotrophic growth conditions. Homoacetogens were cultivated on ethanol (20 mM) in the presence of HFO. (A) Fe(II) production rate in the exponential phase. (B) Fe(II) accumulation in the stationary phase. Data are presented as the means of three independent cultures, and error bars represent standard deviations. ***p < 0.001, **p < 0.01, determined by one-way analysis of variance with Tukey’s honest significance difference test.

FIGURE 4

Growth dependent HFO reduction by strain GT1 under lithotrophic condition. Strain GT1 was cultivated on H₂ + CO₂ (80:20) in the presence or absence of HFO. (A) Cell density, (B) concentration of acetate, and (C) Fe(II) concentration were periodically determined during cultivation. Closed symbols, cultures with HFO. Open symbols, cultures without HFO. Data are presented as the means of three independent cultures, and error bars represent standard deviations.

FIGURE 5

Difference in HFO reduction activity among homoacetogens under lithotrophic growth conditions. Homoacetogens were cultivated on H₂ + CO₂ (80:20) in the presence of HFO. (A) Fe(II) production rate in the exponential phase. (B) Fe(II) accumulation in the stationary phase. Data are presented as the means of three independent cultures, and error bars represent standard deviations. ***p < 0.001, determined by one-way analysis of variance with Tukey’s honest significance difference test.

FIGURE 6

Difference in crystalline Fe(III) oxide reduction activity among homoacetogens under organotrophic growth conditions. Homoacetogens were cultivated on ethanol (20 mM) in the presence of goethite [α-FeO(OH), 9 mmol/L] or hematite (α-Fe₂O₃, 4.5 mmol/L). The white and black bars indicate Fe(II) contents in the supernatant and sediment, respectively. Data are presented as the means of three independent cultures, and error bars represent standard deviations.

FIGURE 7

Transformation of HFO to magnetite by ethanol-grown culture of strain GT1. Strain GT1 was cultivated on ethanol (20 mM) in the presence of HFO under phosphate-limiting conditions. (A) Appearance culture bottles. Asterisk indicates a magnet. Arrow indicates magnet-attracted sediment. (B) Powder X-ray diffraction patterns of the sediments from the ethanol-grown culture of strain GT1. Open triangle, peaks from magnetite.

FIGURE 8

Effects of physical separation of HFO from the cells on reduction. Homoacetogens were cultivated with HFO on ethanol. Cultivation was conducted under the same conditions as described in section “Microorganisms and cultivations,” except that the culture medium volume was increased to 120 mL and HFO was placed inside the dialysis device. (A) Cell density, (B) acetate concentration, and (C) Fe(II) concentration were determined in the stationary phase. ***p < 0.001, **p < 0.01, determined by one-way analysis of variance with Tukey’s honest significance difference test.