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Dependence of Secondary Mineral Formation on Fe(II) Production from Ferrihydrite Reduction by Shewanella oneidensis MR-1 Rui Han, Tongxu Liu, Fangbai Li, Xiaomin Li, Dandan Chen, and Yundang Wu ACS Earth Space Chem., Just Accepted Manuscript • DOI: 10.1021/ acsearthspacechem.7b00132 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018
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Dependence of Secondary Mineral Formation on Fe(II) Production from
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Ferrihydrite Reduction by Shewanella oneidensis MR-1
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Rui Han,a,b Tongxu Liu,a,* Fangbai Li,a Xiaomin Li,a,c Dandan Chena, and Yundang Wua
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a
Guangdong Institute of Eco-environmental Science & Technology, Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and Management, Guangzhou 510650, China
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b
College of Marine Technology and Environment, Dalian Ocean University, Dalian 116023, China
c
The Environmental Research Institute, MOE Key Laboratory of Theoretical Chemistry of Environment,
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South China Normal University, Guangzhou 510006, China
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*
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E-mail address:
[email protected] (T.X.Liu)
Corresponding author. Tel.: +86 20 37021396.
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ACS Earth and Space Chemistry
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(Re-submitted March, 2018)
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1
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ABSTRACT: Although dissimilatory iron reduction and secondary mineral formation by
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Shewanella oneidensis MR-1 have been widely recognized, questions remain about the effects
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of biogenic Fe(II) on the rate and extent of secondary mineral formation and the importance
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of biogenic Fe(II)-induced crystallization processes. In this study, we investigated the effects
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of different mutants of MR-1 on the bioreduction and mineralization of ferrihydrite. The
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results indicate that while the reduction rates of ferrihydrite by ∆mtrD, ∆mtrF, and ∆omcA are
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similar to that of the wild type (WT), the capacity to reduce ferrihydrite decreased
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dramatically in the mutants ∆cymA and ∆mtrA. The order for Fe(III) reduction by MR-1 WT
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and mutants was ranked as follows: WT ≈ ∆mtrD ≈ ∆mtrF >∆omcA > ∆mtrC > ∆cymA >
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∆mtrA. Secondary minerals of ferrihydrite were characterized using X-ray diffraction, Fourier
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transform infrared spectra, and scanning electron microscopy. The results show that goethite
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and hematite were the main secondary minerals formed during the first two days in all
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treatments, and then magnetite appeared in the WT, ∆mtrD, ∆mtrF and ∆omcA treatments,
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while magnetite began to appear from the sixth day onwards in the ∆mtrC treatment.
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However, no magnetite was observed during the six days in the ∆mtrA and ∆cymA incubation
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treatments. The plausible electron transfer pathways of bioreduction and phase transformation
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were also verified using thermodynamic calculations of elementary reactions. This study
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clarified the importance of Fe(II) production in secondary mineral formation processes and
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highlighted the significance of biogenic Fe(II)-catalyzed crystallization. This information may,
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in turn, help us to better understand natural microbe−mineral interaction processes.
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KEYWORDS: Biotransformation; ferrihydrite; mutant; Shewanella oneidensis MR-1;
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extracellular electron transfer.
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1. INTRODUCTION
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Iron is the most abundant redox-active metal in the Earth’s crust, and iron-bearing minerals
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are ubiquitous reactive constituents of aquifers, soils, and sediments. Dissimilatory Fe(III)
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reduction is widely observed in natural systems (aquatic sediments, soils, and subsurface
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environments) and has major impacts on iron geochemistry, the fate of trace metals and
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nutrients, and the degradation of organic matter.1 Dissimilatory iron-reducing bacteria (DIRB)
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can reduce Fe(III) to Fe(II) under anoxic conditions relying on hydrogen or the oxidation of
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organic matter to obtain energy for maintenance and cell growth.2,3 A wide phylogenetic
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diversity of microorganisms, including archaea and bacteria, are capable of dissimilatory
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Fe(III) reduction. Of all the identified strains of DIRB, Geobacter and Shewanella are by far
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the most extensively investigated.4
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Iron exists predominantly in an insoluble solid phase (e.g., ferrihydrite, goethite, and
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hematite) in the environment under circumneutral pH conditions.4 Shewanella oneidensis
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MR-1 (MR-1) has two main pathways of extracellular electron transfer (EET): (i) to directly
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transfer electrons from outer membrane (OM)-bound cytochromes and porin-cytochrome
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complexes toward iron oxides, or along organic appendages named “nanowires,” which are
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extensions of the outer membrane and periplasm produced by MR-1 5; (ii) to use endogenous
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organic molecules (e.g., flavin or riboflavin mononucleotides) as electron shuttles that diffuse
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in the medium and mediate EET; thus, DIRB must pump electrons from the internal
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cytoplasmic membrane to iron oxides located outside the cell.6 To overcome this physical
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barrier, MR-1 developed a pathway (i.e., Mtr) that requires multihaem c-type cytochromes
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(c-Cyts) for transferring electrons from the inner membrane via the periplasm and OM to
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external minerals.7,8 Electron transport proteins of the Mtr pathway include multihaem c-Cyts
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(CymA, MtrA, MtrC, OmcA, and a porin-like OM protein MtrB).7,8 The roles of these
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proteins in the electron transfer process have been documented as follows: CymA is an inner 3
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membrane-anchored quinol dehydrogenase that oxidizes quinol in the inner membrane and
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transfers the released electrons to the periplasmic MtrA; MtrA delivers electrons to the
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OM-anchored MtrC and OmcA, which can directly reduce solid metal (hydr)oxides.9-12 For
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most Shewanella members, the EET process is generally performed by an MtrCAB complex.
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Biochemical characterization of this complex indicates that MtrB functions as a
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trans-membrane sheath into which both MtrC and MtrA are partially inserted. 8,13,14 MtrF,
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MtrD, and MtrE are the homologues of MtrC, MtrA, and MtrB, respectively. MtrF, MtrD, and
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MtrE are thought to form an OM-spanning complex, namely MtrFDE, which has the same
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overall structure as MtrCAB but only makes a minor contribution to the process of iron
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reduction.15 Despite progress in understanding the key proteins in the Mtr pathway via
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mutants and purified proteins,10,15-18 there is a critical need to quantify the roles of these
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proteins in EET to iron minerals owing to their implications for biogeochemical redox
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processes. Therefore, it is necessary to further investigate the effects of key proteins and their
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homologues on the kinetics of iron reduction by wild type (WT) and mutants of MR-1.
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Poorly crystalline hydrous ferric oxides (e.g., ferrihydrite), which commonly exist in soils
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and sediments, are thermodynamically unstable and, with time, transform to more crystalline
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Fe(III)-oxides, e.g., goethite and/or hematite,19 resulting in a loss of ability to scavenge other
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trace metals from solution.20 Ferrihydrite is considered the most (bio)available Fe(III)
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(hydr)oxide for DIRB, and electron transfer proteins from Shewanella play an important role
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in the extracellular reduction of ferrihydrite, followed by biomineralization. Previous studies
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have focused on the factors influencing secondary mineralization, such as iron reduction rates,
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electron donor/acceptor concentrations, cell density, pH, and redox potential.19,21,22
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Although the roles of some OM c-Cyts (MtrC and OmcA) have been documented with regard
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to biomineralization of ferrihydrite by MR-1,23 questions still remain about the relationship
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between the rate of iron bioreduction and the extent of secondary mineral formation, and the 4
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importance of Fe(II)-compounds as catalysts in these chemical processes.24
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In this study, MR-1 was selected because it is a DIRB that is well known for its abilities to
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use a variety of iron oxides and oxyhydroxides as terminal electron acceptors.25 The
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objectives of this study were to examine the rates of Fe(III) reduction and the concomitant
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secondary mineral formation by wild type and mutants of MR-1, with implications for
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quantifying the effects of biogenic Fe(II) production on the rates and extents of secondary
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mineral formation in the dissimilatory iron reduction processes of ferrihydrite.
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2. MATERIALS AND METHODS
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2.1. Materials. MR-1 wild type (WT) that had been isolated from anoxic sediments from
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Lake Oneida, NY26 was purchased from MCCC (Marine Culture Collection of China, China).
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All mutant-deletion strains ∆mtrA, ∆mtrC, ∆mtrD, ∆mtrF, ∆omcA, and ∆cymA were provided
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by Professor Gao Haichun from Zhejiang University, as documented in previous studies.
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8,15,27,28
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98%). Piperazine-N–N-bis-2-ethanesulfonic acid (PIPES) was purchased from J&K Chemical
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(Beijing, China). Lactate was purchased from Sinopharm Chemical Reagent Co., Ltd.
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(Shanghai, China). Other chemicals were purchased from Guangzhou Chemical Reagent
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Factory (Guangzhou, China). All solutions were prepared using Milli-Q deoxygenated
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ultrapure water (18 MΩ cm, EASYpure II RF/UV, USA).
All chemicals were of analytical reagent grade and the highest available purity (≥
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2.2. Preparation of ferrihydrite. Ferrihydrite was synthesized by neutralizing 0.4 mM
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FeCl3·6H2O with 1 mM NaOH in a high-density polyethylene container.29 Firstly, aqueous
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solutions of 0.4 mol L-1 FeCl3·6H2O and 1 mol L-1 NaOH were prepared. Then the FeCl3
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solution was stirred intensely and evenly while the NaOH solution was trickled steadily into it.
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As the circumneutral pH approached, the NaOH solution was added more slowly, and
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a thick red slurry of ferrihydrite formed. At pH 7.0, the slurry was allowed to ripen for 2–6 h, 5
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with a slight decrease in pH (usually < 1 pH unit). The slurry was adjusted back to pH 7.0 and
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centrifuged at 3 600 g for 20 min at 4 °C. After decanting the supernatant, the solid was
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re-suspended, washed with Milli-Q water, and centrifuged again. This procedure was repeated
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seven times to reduce the residual chloride content to less than 1 mM. The slurry was then
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kept in Milli-Q water prior to use.30
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2.3. Biotransformation of ferrihydrite by wild type and mutants of MR-1. MR-1 WT
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and various mutant cells (∆mtrA, ∆mtrC, ∆mtrD, ∆mtrF, ∆omcA, and ∆cymA) were grown
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aerobically in a Luria-Bertani medium (10 g L-1 NaCl, 5 g L-1 yeast extract, 10 g L-1 tryptone)
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at 30 °C with shaking at 180 rpm. When approaching the exponential phase, cells were
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harvested by centrifuging at 7 000 g at 4 °C for 10 min. The pellets were then washed three
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times and re-suspended in 30 mM PIPES buffer at pH 7.0 ± 0.2. The Fe(III) reduction
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medium contained 30 mM PIPES (pH 7.0 ± 0.2) as a buffer, 50 mM ferrihydrite as the sole
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electron acceptor, and 20 mM lactate as the sole electron donor. The bacterial concentration in
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each treatment was OD600nm = 1.0, and the number of bacterial cells (wild and mutants) for an
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OD600nm = 1.0 were measured using the DAPI staining method.31 Growth curves of the
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bacterial cells (wild and mutants) were plotted using the method outlined in Supporting
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Information, and the total protein contents of wild type and mutants at OD600nm = 1.0 were
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measured. An experimental group without bacteria was used as a control. The pH was also
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maintained at approximately 7.0 ± 0.2 using 30 mM PIPES. The medium was dispensed into
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100-mL serum bottles purged with O2-free N2 for at least 30 min, capped with butyl rubber
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closures, and crimp sealed. All anaerobic media were sterilized by autoclaving at 121 °C for
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20 min and then cooled to room temperature in a Bactron Anaerobic/Environmental Chamber
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II (Shellab, Sheldon Manufacturing Inc., Cornelius, OR, USA) before use. Inoculation and
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sampling were conducted using sterile syringes and needles. All experiments were incubated
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in an anaerobic chamber in the dark at 30 °C. Three reactor bottles per treatment were taken 6
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out for analysis at the sampling intervals of 0, 1, 2, 3, and 6 d. 2.4. Analytical methods. The total Fe(II) concentration was determined by extracting Fe(II)
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from the suspensions using 0.5 M HCl for 1.5 h32 and assaying the extract using the
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1,10-phenanthroline colorimetric method at 510 nm on a UV−Vis spectrophotometer
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(TU-1810PC, Beijing Purkinje General Instruments, China). Standard curves were made
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using ammonium ferrous sulfate dissolved in 0.5 M hydrochloric acid. The mineral samples
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were collected from the incubations by filtration inside the anaerobic chamber. Samples were
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filtered onto 0.22 µm filters (VCTP Millipore Isopore), washed twice with deoxygenated
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double deionized (DDI) water, and dried in the anaerobic chamber. The dry minerals were
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then characterized. The X-ray diffraction (XRD) patterns of powder samples before and after
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reactions were recorded on a Bruker Advance Diffractometer (Bruker Co., USA) at room
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temperature, operating at 40 kV and 40 mA, using Cu Kα radiation (λ = 0.15418 nm). Data
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were acquired in the range of 15° to 70°, using a step of 0.04°. The phases were identified by
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comparing diffraction patterns with those on standard powder XRD cards compiled by the
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Joint Committee on Powder Diffraction Standards (JCPDS).33 Fourier transform infrared
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spectroscopy (FTIR) was recorded using an FTIR spectrometer (Perkin−Elmer, model Vector
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33, Bruker Co., USA) at room temperature. The morphology was investigated by scanning
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electron microscopy, and the samples were dispersed onto carbon-coated Cu grids in the
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anaerobic chamber. SEM images were obtained using field-emission scanning electron
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microscopy (LEO1530VP, Zeiss, Germany).
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3. RESULTS AND DISCUSSION
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3.1. Effect of mutants on bioreduction of ferrihydrite. The kinetics of Fe(II) generation
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during ferrihydrite reduction by WT and mutants are shown in Figure 1a. While no Fe(II) was
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generated in the ferrihydrite control without MR-1, the presence of MR-1 WT and mutants 7
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obviously stimulated Fe(II) generation, indicating that the reduction of ferrihydrite to Fe(II)
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occurred via a biological reaction. Fe(II) generation by WT increased quickly to ~ 4 mM in
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the first two days and then decreased gradually to ~2.8 mM at the end of incubation (day 6).
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The Fe(II) generation by ∆mtrD and ∆mtrF was very close to that of WT. Fe(II) generation by
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∆omcA also increased at the beginning and then decreased, but the Fe(II) concentration was
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lower than that generated by WT, ∆mtrD, or ∆mtrF. Fe(II) generation by ∆mtrC, ∆cymA, and
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∆mtrA increased over time during the six days of incubation. To clearly illustrate Fe(II)
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generation rates of all the treatments, the zero-order reduction rates of Fe(II) generation
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during the increasing stage of the kinetic curves were calculated (Figure 1b). Because the
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concentration of Fe(II) in these systems decreases after two days due to the reaction between
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Fe(II) and ferrihydrite, production rates of Fe(II) in the WT and mutants without MtrD or
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MtrF were calculated using the data of the first two days. Compared to the Fe(II) production
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rate for WT (2.05 mM d-1), a slight decrease was observed for ∆mtrD (1.80 mM d-1) and
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∆mtrF (1.75 mM d-1); the rates for other mutants decreased substantially, by 64.9%, 77.1%,
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88.3%, and 93.7% for ∆omcA, ∆mtrC, ∆cymA, and ∆mtrA, respectively. Therefore, the order
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for the capacity of reducing ferrihydrite by MR-1 WT and mutants was ranked as follows: WT
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≈ ∆mtrD ≈ ∆mtrF > ∆omcA > ∆mtrC > ∆cymA > ∆mtrA, indicating their roles in controlling
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the electron transfer pathway. The initial cell number at OD600 nm = 1.0 added in all treatment
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groups (Figure 1c) was not identical for each strain of MR-1. WT had an initial cell number of
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2.35×109 which is 2.35 times and 4.7 times more than those of the ∆mtrA and ∆cymA
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treatments, respectively. The total protein contents of WT and mutants at OD600nm = 1.0
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(Figure S2) were consistent with the cell numbers in Figure 1c, thus the initial cell numbers
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were different. To clearly differentiate the effects of gene deletion and quantitative variation
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of cells used in Fe(III) oxide reduction, the cell numbers in Figure 1c were used to calibrate
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the rates in Figure 1b. The calibrated rates in Figure S3 for various treatments with WT and 8
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mutants are consistent with the original rates for Fe(II) production in Figure 1b. The above
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results indicated that the knock outs of different electron transfer proteins influenced the cell
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numbers of bacteria, and also affected the reduction rate of ferrihydrite and the secondary
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mineral formation.
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3.2. Effect of mutants on secondary mineral formation. The mineralogy of ferrihydrite
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changed along with ferrous release during the bioreduction processes, which was indicated by
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visible color changes of the incubated suspension (inserts of Figure 2). After six days of
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incubation, the color of the ferrihydrite suspension was dark red after reacting with ∆cymA,
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∆mtrA, and ∆mtrC, while those with WT, ∆mtrD, ∆mtrF and ∆omcA had turned black. The
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different colors of the reaction suspensions might be attributed to different secondary
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minerals.34
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Morphology. The samples were examined using SEM techniques to identify the changes in
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surface morphology of the solid phases during ferrihydrite reduction by WT and mutants. The
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ferrihydrite image showed a smooth flat surface (Figure 2a), which might be attributed to an
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aggregation of the tiny ferrihydrite particles after the drying processes. After incubation with
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MR-1 WT and mutants, large particles formed with a wide range of particle sizes between 20
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nm and 200 nm (Figure 2b-h). It was apparent that the particle sizes of minerals with WT,
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∆mtrD, ∆mtrF, and ∆omcA were much smaller than those with ∆mtrC, ∆cymA and ∆mtrA.
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The difference in particle sizes was similar to that of the iron reduction rates. Since the
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particle sizes during microbial mineralization might be determined by the mineral
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components, crystallization conditions, and the location relatively to the cells, the single SEM
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images did not provide crucial information for the secondary minerals except the surface
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morphology of the overall mixtures with cells and minerals. Therefore, specific differences of
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the secondary minerals were further characterized by FTIR and XRD as discussed below.
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Crystal structures. The solid samples of ferrihydrite before and after reduction by MR-1 9
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WT and mutants were further examined by FTIR. For the control treatment with ferrihydrite
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alone, the FTIR patterns of ferrihydrite remained unchanged, as no bioreduction or
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biomineralization occurred during the six-day incubation. The major peaks at 590 cm-1, 1040
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cm-1, 1120 cm-1, and 1175 cm-1 (Figure 3a) represent the characteristic peaks of ferrihydrite.34
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After reduction of ferrihydrite by MR-1 WT and mutants, the peaks for ferrihydrite changed
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by differing amounts (Figure 3b-h). Specifically, while the peaks for ferrihydrite at 1120 cm-1
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and 1175 cm-1 for WT, ∆mtrD, and ∆mtrF nearly disappeared after six days of incubation, the
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intensity of these peaks for the other mutants were only slightly lower. Two sharp peaks at
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797 cm-1 and 887 cm-1 appeared in all the treatments with MR-1 and were ascribed to the
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Fe–OH bond of goethite.35 It can also be seen that the peak at 590 cm-1 shifted to 564 cm-1 for
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the treatments with WT, ∆mtrD, ∆mtrF, and ∆omcA, which may be attributed to the Fe–O
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bond of magnetite.36 These results indicate that ferrihydrite was gradually reduced and
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transformed by WT and mutants over time, followed by the formation of goethite and
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magnetite.
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Microbial reduction of ferrihydrite was followed by recrystallization processes and
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formation of more crystalline iron (hydr)oxides.34 The XRD patterns of minerals in the
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control treatment of ferrihydrite showed no obvious diffraction peaks owing to its amorphous
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structure that remained unchanged over the six-day incubation (Figure 4a). For MR-1 WT or
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mutants, new diffraction peaks appeared, and their relative intensities changed over time
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(Figure 4b-h). After comparing those peaks with JCPDS cards, the newly formed crystal
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structures were found to include goethite (No. 29-0713), hematite (No. 33-0664), and
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magnetite (No. 19-0629). Goethite (2θ = 33.2°) and hematite (2θ = 35.7°, 54.1°, 62.4°, 64.0°)
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were the main forms of secondary minerals in the first two days for all the treatments with
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MR-1. For WT, ∆mtrD, ∆mtrF and ∆omcA, magnetite (2θ = 33.2°, 43.2°, 57.1°, 62.5°) began
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to appear on the third day, followed by a gradual disappearance of hematite, and then 10
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magnetite became the main form on the sixth day. For ∆omcA and ∆mtrC, magnetite began to
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appear on the fourth day and the sixth day, respectively. For ∆mtrA and ∆cymA, only hematite
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and goethite were observed, with no magnetite observed during the six-day incubation.
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Equal amounts of ferrihydrite were used as the starting materials and all the samples were
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characterized at the same time. Thus, the relative contents of all the minerals can be compared
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according to the diffraction peaks and the kinetics of each mineral can be estimated from the
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changes of the peak intensities over time. The relative peak areas of goethite (2θ = 33.2o),
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magnetite (2θ = 62.5o) and hematite (2θ = 35.7o) were plotted as a function of incubation time
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(Figure 5). For goethite, the peak areas of all treatments with MR-1 WT and mutants
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increased with incubation time (Figure 5a). During the first day, the order of goethite
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formation amounts was similar to that for Fe(II) production in Figure 1b. The peak areas for
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∆mtrA, ∆mtrC and ∆cymA on the sixth day were slightly higher than those of WT, ∆mtrD,
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∆mtrF and ∆omcA. The changing trend for hematite was opposite to that for goethite with all
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the treatments. The peak intensities of hematite (2θ = 35.7°) in the treatments of WT, ∆mtrD,
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∆mtrF and ∆omcA on the sixth day were slightly higher than those of ∆mtrA, ∆mtrC and
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∆cymA (Figure 5b). Meanwhile, the peak of magnetite (2θ = 57.1°) appeared and increased as
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the reaction proceeded, indicating that the minerals were transformed into magnetite from the
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third day of reaction. Since goethite are just intermediate mineral products, the order of
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formation of goethite in the following days was not well matched with that of Fe(II)
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production. For magnetite (Figure 5c), diffraction peaks began to appear on the second day
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for WT, ∆mtrD, and ∆mtrF; the third day for ∆omcA; and the fourth day for ∆mtrC. No
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magnetite peaks were observed for ∆mtrA and ∆cymA over the six days of incubation. Hence,
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the order of formation rates of magnetite as the final mineral product was consistent with
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Fe(II) production.
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3.3. Thermodynamics of bioreduction and biomineralization. The above experimental 11
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results clearly indicate that Fe(II) generation can occur via bioreduction processes and iron
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(oxyhdr)oxides formation via re-crystallization. These bioreduction processes were identified
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as enzymatic reactions between outer membrane c-type cytochromes (OM c-Cyts) and iron
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(oxyhdr)oxides; the re-crystallization processes may be induced by biogenic Fe(II) or direct
275
enzymatic reactions. To examine the specific reactions occurring during the bioreduction and
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biomineralization processes, the thermodynamics of all possible reactions were calculated
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according to the reported values of various species and minerals involved in the
278
biotransformation processes (Table 1).
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The starting mineral, ferrihydrite, can be reduced by MR-1, as may the secondary minerals.
280
The reduced product was usually free ferrous (Fe2+); the Fe(II)-containing mineral (Fe3O4)
281
was also observed via XRD and FITR characterization. Hence, the half reactions for all
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possible bioreduction processes with Fe2+ and Fe3O4 as reduced products are summarized as
283
Rxns. 1 – 4 in Table 2. As free energy or chemical potential is the driving force of the
284
reactions, the standard free energy change ( ∆G 0r ) of the half reaction for iron reduction was
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calculated with Eq. 1 based on the total free energies of formation of the products ( ∆G 0f products )
286
and reactants ( ∆G 0f reactants ) in Table 1,
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∆G 0r = ∑ ∆G f0 products − ∑ ∆G 0f reactants
(1)
288
where ∆G 0r for the hydrogen half-cell is zero, ∆G 0r for the electrons cancels out, and the
289
unit is KJ mol-1.
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The results in Table 2 indicate that the ∆G 0r values for all the reactions with Fe2+ as
291
reduced products are ranked as α-FeOOH > am-FeOOH > α-Fe2O3 > Fe3O4. In the
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bioreduction system, more negative ∆G 0r values may reflect greater difficulty in iron
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reduction reactions with MR-1. Regarding the formation processes of secondary minerals, it
294
was noted that am-FeOOH, α-FeOOH, and α-Fe2O3 were all Fe(III) minerals, implying that 12
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non-redox reactions among them may directly induce phase transformation. In addition, the
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biogenic Fe(II) and Fe(III) minerals (am-FeOOH, α-FeOOH, and α-Fe2O3) may also combine
297
with each other via non-redox reactions by forming Fe(III)−Fe(II) mixed minerals (Fe3O4).
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These reactions are written according to the mass balance law as Rxns. 5 – 10 in Table 3. It
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has been reported that ferrihydrite is thermodynamically metastable and transforms into the
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more-crystalline products goethite and hematite via a long-term aging process.42 Therefore, it
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is essential to clarify whether the aging processes of iron (oxyhydr)oxides plays a role in
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biomineralization processes. The ∆G 0r values of the non-redox reactions for phase
303
transformation were calculated based on Eq. 1. The ∆G 0r values for Rxns. 5 and 6
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(am-FeOOH and α-FeOOH) in Table 4 were lower than zero, indicating that the reactions of
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am-FeOOH → α-FeOOH and am-FeOOH→ α-Fe2O3 may spontaneously occur, though they
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may be very slow.42 The reaction α-FeOOH→ α-Fe2O3 (Rxn. 7), and all three reactions
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between Fe2+ and iron (oxyhydr)oxides (Rxns. 8 – 10) are not thermodynamically favorable
308
via non-redox reactions, and it was supported that the transformation from goethite to
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hematite only occurred under heating at about 250 oC.43,44
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Recently, the Fe(II)-catalyzed phase transformation of iron (oxyhydr)oxides to more
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crystalline forms has attracted much attention from geochemists owing to the wide
312
implications for both geochemistry and contaminant mobilization.45 Fe(II) is generated from
313
bioreduction processes, and biogenic Fe(II) may also induce the phase transformation of iron
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(oxyhydr)oxides. Based on thermodynamic calculations (Rxns. 7 – 10), direct transformation
315
via non-redox reactions should be unfeasible, but recent observations45 imply that an
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“induction period” is associated with the reorganization of highly disordered surfaces, and
317
reactive intermediates were responsible for atom-exchange and feasible electron transfer,
318
finally resulting in the phase transformation of iron (oxyhydr)oxides. Based on this
319
mechanism, reactions involving reactive intermediates are listed in Table 4. The elementary 13
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reaction can be written as Rxns. 11 – 13. The first step (Rxn. 11) is surface complexation with
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the formation of the reactive intermediate (≡Fe(III)OFe(II)+). The second step (Rxn. 12) is the
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internal electron transfer to another Fe(III) atom in the structure, forming ≡Fe(II)OFe(III)+. In
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the third step (Rxn. 13), the “new” Fe(II) is eventually released into solution via desorption,
324
and the “new” Fe(III) in the structure becomes a more-crystalline (oxyhydr)oxide. In a similar
325
way, phase transformations to goethite, hematite, and magnetite can be proposed as Rxns. 14
326
– 17.
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3.4 Roles of proteins in ferrihydrite reduction. Based on experimental results and the
328
thermodynamics of iron reduction and mineralization, the roles of all proteins in the Mtr
329
pathway for bioreduction and biomineralization of ferrihydrite are proposed in Figure 6.
330
Because CymA is an inner-membrane quinol dehydrogenase that transfers electrons to
331
MtrA,8,16,46 the deletion of cymA may significantly lower electron output from the menaquinol
332
pool to the periplasm,9 resulting in a substantial decrease (88.3%) of ferrihydrite reduction
333
activity (Figure 1b). MtrA is a soluble decaheme c-Cyts localized in the periplasm and
334
associated with the outer membrane that accepts electrons from CymA and donates electrons
335
to OmcA, MtrC, or MtrF.47-49 Thus, a lack of MtrA could lead to a deficiency in electron
336
transfer from CymA to OM c-Cyts, resulting in the observed decrease (93.7%) in ferrihydrite
337
reduction activity (Figure 1b). These results also imply that the presence of inner-membrane
338
CymA and periplasmic MtrA is essential for electron transfer to ferrihydrite reduction by
339
MR-1.
340
Both MtrC and OmcA are lipoproteins inserted in the OM that are partially exposed to the
341
extracellular environment.50-52 They accept electrons from MtrA and donate electrons to the
342
extracellular terminal electron acceptors including iron oxides.13,47 The deletion of mtrC or
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omcA only partially inhibited the ferrihydrite reduction, indicating that MtrA could donate
344
electrons to alternative functional proteins to complete the electron transfer to ferrihydrite,27 14
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345
so it is reasonable to infer that the lack of OmcA retards, but does not completely inhibit,
346
biotransformation processes. The reduction rate by ∆mtrC was lower than that by ∆omcA
347
(Figure 1b), suggesting that MtrC is more efficient in the electron transfer pathways for
348
ferrihydrite reduction by MR-1, which may be because the level of OmcA in a whole cell of
349
MR-1 (8×10-20 moles) is lower than that of MtrC (1.2×10-19 moles).17 However, the Fe(II)
350
concentration in the ∆omcA treatment during the bioreduction of ferrihydrite may be
351
underestimated (Figure 1a and Figure 5c) since increased production of magnetite may fix
352
more Fe(II) ions during the ∆omcA treatment than the ∆mtrC treatment.
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MtrF and MtrD are homologous components of MtrC and MtrA, respectively, and the rates
354
of iron reduction and final product (magnetite) generation for ∆mtrD and ∆mtrF were close to
355
that for WT, so the deletion of mtrF and mtrD only had a minor influence on the bioreduction
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and biomineralization processes of ferrihydrite. Similar results were also found in the
357
reduction of Fe(III) minerals by Geobacter sulfurreducens.53 Although MtrD and MtrF have
358
structures that are similar to those of MtrA and MtrC, respectively,54,55 MtrD and MtrF did
359
not play a key role in ferrihydrite reduction in the presence of MtrA and MtrC.27
360
3.5 Importance of Fe(II) production to secondary mineral formation. Regarding
361
secondary mineral formation from ferrihydrite, two processes might influence this pathway.
362
Firstly, Fe(II) generated and released through dissimilatory iron reduction of ferrihydrite
363
might be adsorbed to the surface of ferrihydrite, leading to precipitation and re-crystallization
364
and resultant changes in mineral structures.36 Secondly, microorganisms can be directly
365
involved in the transformation of minerals via enzymatic iron reduction and re-crystallization
366
processes.34,56 From the current results of mineral characterization, goethite was generated
367
very quickly in the first two days as the primary mineral, which is consistent with a report
368
stating that goethite was the solid-phase product formed during the first day of dissimilatory
369
iron reduction of ferrihydrite by Shewanella putrefaciens strain CN32.21 As the reaction 15
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proceeded over time, magnetite became the primary biomineralization product. A similar
371
report investigating bioreduction of ferrihydrite by Shewanella putrefaciens strain CN32
372
demonstrated that magnetite was a stable end-product in PIPES-buffered media,57 whereas
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hematite formed through internal-dehydration and rearrangement within the ferrihydrite
374
aggregates58; thus, hematite was found in all the treatments. The formation of hematite and
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the transformation of hematite to magnetite may be due to direct effects of microorganisms on
376
minerals. The high efficiency of electron transfer from microorganism to the mineral surface
377
favored the transformation of hematite to magnetite. For ∆mtrA, ∆mtrC and ∆cymA with slow
378
iron reduction rates, goethite and hematite were the dominant secondary minerals. MtrA,
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MtrC and CymA are key components of the Mtr pathway, and the combination complex
380
(CymA-MtrCAB) was found to be the most significant electron conduit in ferrihydrite
381
reduction by biomineralization with MR-1,8,16,46. Thus, knocking out either cymA or mtrA
382
leads to serious interruption of the overall electron transport chain,9 resulting in low levels of
383
electron transfer to ferrihydrite. MtrC plays a critical role in iron reduction through either a
384
direct mechanism involving electron transport or in conjunction with OmcA.50 Knocking out
385
mtrC caused the loss of the most effective pathway for electron transmission to the outside,
386
which greatly weakened the electron transport ability. Therefore, highly efficient electron
387
transport ability is the key factor controlling the bioreduction of ferrihydrite. The results of
388
Fe(II) production presented in Figure 1 and the results of secondary mineral formation shown
389
in Figure 5 indicate that the higher Fe(II) production with WT, ∆mtrF and ∆mtrD led to
390
higher formation rates of secondary minerals (goethite, hematite, and magnetite), while those
391
treatments with lower Fe(II) production showed lower formation rates of secondary minerals.
392
Therefore, the rates and extents of secondary mineral formation appear to be strongly
393
dependent on the amounts of Fe(II) produced from the bioreduction of ferrihydrite.
394
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4. CONCLUSION
396
Various MR-1 mutants were investigated regarding ferrihydrite reduction and secondary
397
mineral formation. High reduction rates were well-correlated with the rate and extent of
398
secondary mineral formation. CymA-MtrCAB-ferrihydrite is the dominant electron transfer
399
pathway of ferrihydrite reduction and Fe(II) production, resulting in higher rates and extents
400
of secondary mineral formation. After knocking out the three key electron transfer proteins,
401
electron output capacity was weakened, resulting in a retardation of the ferrihydrite reduction
402
rate and the rate and extent of secondary mineral formation. Other proteins (i.e., MtrDEF) had
403
little contribution to the overall processes of ferrihydrite reduction and secondary mineral
404
formation. The thermodynamic feasibility of bioreduction and phase transformation was
405
calculated based upon the free energies and chemical potentials of elementary reactions and
406
redox couples. This study provides a systematic and quantitative understanding of the roles of
407
biogenic Fe(II) production in the crystal transformation of ferrihydrite during dissimilatory
408
iron reduction processes.
409 410
ACKNOWLEDGEMENTS
411
This work was funded by the National Natural Science Foundation of China (grants
412
41571130052, 41522105, and 41471216), the Guangdong Natural Science Fund for
413
Distinguished Young Scholars (grant 2017A030306010), the Excellent Talent Fund of
414
Guangdong Academy of Sciences (GDAS) (grant 2017GDASCX-0408), the SPICC
415
(Scientific Platform and Innovation Capability Construction) program of GDAS, and an
416
Australian Research Council DECRA grant (DE150100500).
417 418
Supporting Information
419
Additional data can be found in the Supporting Information including additional descriptions 17
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of experiment method, Figures S1-S3 with illustrations. This material may be found in the
421
online version of this article.
422 423
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equilibrium adsorption, SEM, FTIR and ITC. Colloids Surf. B Biointerfaces 2010,80, 79–85.
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(36) Yang, K.; Peng, H.; Wen, Y.; Ning, L. Re-examination of characteristic FTIR spectrum of secondary layer in
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nanoparticles.
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transformation of ferrihydrite and its environmental implications. Environ. Sci. Technol. 2013, 47,
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9660−9667. doi:10.1021/es4010398.
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conditions on the Fe (II)-accelerated transformation of ferrihydrite to lepidocrocite and goethite.Environ.
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membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400. J. Biol. Chem.
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cytochrome required for metal reduction in Shewanella putrefaciens MR-1. Mol. Microbiol. 2001, 39, 21
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B.H.; Fredrickson, J.K.; Nealson, K.H. 2007. Current production and metal oxide reduction by
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Shewanella oneidensis MR-1 wild type and mutants. Appl. Environ. Microbiol. 2007, 73,
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(42) Katrin, R.; Marcus, S.; Johannes, G. Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration. Appl. Environ. Microbiol. 2012, 78, 913–921. doi: 10.1128/aem.06803-11. (43) Faria D. L. A. D., Lopes F. N. Heated goethite and natural hematite: Can Raman spectroscopy be used to differentiate them? Vib. Spectrosc., 2007, 45:117-121. doi:10.1016/j.vibspec.2007.07.003.
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(44) Gualtieri A .F, Venturelli P. In situ study of the goethite-hematite phase transformation by real time
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synchrotron powder diffraction. Am. Mineral., 1999, 84:895-904. doi:10.2138/am-1999-5-625.
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(45) Shi, L.; Chen, B.; Wang, Z.; Elias, D.A.; Mayer, M.U.; Gorby, Y.A.; Ni, S.; Lower, B.H.; Kennedy, D.W.;
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J.K.; Squier, T.C. Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two
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outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1. J. Bacteriol. 2006, 188,
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4705–4714. doi:10.1128/JB.01966-05.
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(46) Myers, C.R. and Myers, J.M. Cell surface exposure of the outer membrane cytochromes of Shewanella
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lipoproteins. Lett. Appl. Microbiol. 2004, 39, 466–470. doi: 10.1111/j.1472-765X.2004.01611.x.
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(48) Cutting, R.S.; Coker, V.S.; Fellowes, J.W.; Lloyd, J.R.; Vaughan, D.J. Mineralogical and morphological
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constraints on the reduction of Fe(III) minerals by Geobacter sulfurreducens. Geochim.Cosmochimi.
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(49) Breuer M., Zarzycki P.; Shi L.; Clarke T.A.; Edwards M.J.; Butt J.N.; Richardson D.J.; Fredrickson
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MR-1: Structures, functions and opportunities. J. R. Soc. Interface 2015, 12, 1117. doi:
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(51) Liu, T.; Li, X.; Li, F.; Zhang, W.; Chen, M.; Zhou, S. Reduction of iron oxides by Klebsiella pneumoniae
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L17: Kinetics and surface properties. Colloids Surf. A Physicochem. Eng. Asp. 2011, 379, 143–150.
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Biotransformation of Ni-substituted hydrous ferric oxide by an Fe(III)-reducing bacterium. Environ. Sci.
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(53) Fischer, W.R. The formation of hematite from amorphous iron(III) hydroxide. Clays Clay Miner. 1975, 23, 33–37. doi:10.1346/ccmn.1975.0230105.
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R.L. The NBS tables of chemical thermodynamic properties: selected values for inorganic and C1 and
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(55) Hiemstra T. Formation, stability, and solubility of metal oxide nanoparticles: Surface entropy, enthalpy, and
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energy
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Geochim.
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(56) Diakonov, I.; Khodakovsky, I.; Schott, J.; Sergeeva, E. Thermodynamic properties of iron oxides and
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hydroxides. I. Surface and bulk thermodynamic properties of goethite (α-FeOOH) up to 500 K. Eur. J.
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Mineral. 1994, 6, 967–983. doi:10.1127/ejm/6/6/0967.
588 589 590 591
(57) Hemingway B.S. Thermodynamic properties for busenite, NiO, magnetite, Fe3O4, and hematite, Fe2O3, with comments on selected oxygen buffer reactions. Am. Mineral. 1990, 75, 781–790. (58) Helgeson, H.C. Thermodynamic of hydrothermal systems at elevated temperatures and pressures. Am. J. Sci. 1969, 267, 729-804. doi:10.1021/es9026248.
592
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Page 24 of 34
593 594 595
Table 1. Standard free energies of iron (oxyhydr)oxides, soluble ferrous and some other
596
species at 0.1 MPa and 298 K ∆G 0r (kJ mol-1) Source (Reference)
Ion/Solid
Fe2+ H+ H2O am-FeOOH α-FeOOH α-Fe2O3 Fe3O4
-78.8 0 -238.2 -472.8 -492.1 -744.3 -1016.1
597 598
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ACS Earth and Space Chemistry
599 600 601
Table 2. Calculated Gibbs free energies for half reactions of bioreduction processes of iron
602
(oxyhydr)oxides No
Reactions
1
C 3 H 5 O 3− + 4am -FeOOH + 7H + → C 2 H 3O −2 + HCO 3− + 6H 2 O + 4Fe 2+
∆G 0r (kJ mole-1)* -78.3
2
C 3 H 5 O 3− + 4α -FeOOH + 7H + → C 2 H 3O 2− + HCO 3− + 6H 2 O + 4Fe 2+
-59.0
3
C 3 H 5 O 3− + 2α -Fe 2 O 3 + 7H + → C 2 H 3O 2− + HCO 3− + 4H 2 O + 4Fe 2+
-59.8
− 3
+
− 2
− 3
C3 H 5 O + 2Fe3O 4 + 11H → C 2 H 3O + HCO + 6H 2 O + 6Fe
4 0 r
603
* ∆G
604
products ( ∆G 0f products ) and reactants ( ∆G 0f reactants ) in Table 1.
2+
-82.4
was calculated using Eq. 1 based on the total free energies of formation of the
605
25
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606 607 608
Table 3. Calculated Gibbs free energies of non-redox mineralization processes of iron
609
(oxyhydr)oxides
No
Reactions
∆G 0r (kJ mol-1)*
5
-19.3
7
am-FeOOH → α -FeOOH 1 1 am-FeOOH → α -Fe2O3 + H 2O 2 2 2α -FeOOH → α -Fe2O3 + H 2O
8
Fe 2+ +2am-FeOOH → Fe 3O 4 + 2H +
8.3
6
9 10 0 r
2+
Fe +2α -FeOOH → Fe3O 4 + 2H 2+
-36.9 1.7
+
Fe +α -Fe 2 O 3 +H 2 O → Fe 3O 4 + 2H
46.9 +
610
* ∆G
611
products ( ∆G 0f products ) and reactants ( ∆G 0f reactants ) in Table 1.
45.2
was calculated using Eq. 1 based on the total free energies of formation of the
612
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ACS Earth and Space Chemistry
613 614 615
Table 4. List of reactions for biogenic Fe(II)-catalyzed mineralization processes of iron
616
(oxyhydr)oxides
617
No
Reactions of Biogenic Fe(II)-Catalyzed Mineralization
11
Elementary reaction* ≡ Fe(III)OH+Fe2+ →≡ Fe(III)OFe(II)+ +H+
12
≡ Fe(III)OFe(II)+ →≡ Fe(II)OFe(III)+
13
≡ Fe(II)OFe(III) + +H + →≡ Fe(III)OH new +Fe 2+
14
Goethite formation am -FeOOH+Fe → am -FeOOH • Fe 2+ → α -FeOOH + Fe 2+
15
Hematite formation 2 am-FeOOH+2Fe → 2( am-FeOOH • Fe 2+ ) → α -Fe 2 O 3 + H 2 O + 2Fe 2+
16
Magnetite formation 2 am-FeOOH+2Fe → 2( am-FeOOH • Fe 2+ ) → Fe 3O 4 + 2H + + Fe 2+
17
2α -FeOOH+2Fe 2+ → 2(α -FeOOH • Fe 2+ ) → Fe 3O 4 + 2H + + Fe 2+
2+
2+
2+
618 619
* ≡Fe(III)OFe(II)+ is the surface complex with Fe(II) adsorbing on the hydroxyl surface of
620
iron oxides. ≡Fe(II)OFe(III)+ is the surface complex after internal electron transfer.
621
≡Fe(II)OHnew is the “new” hydroxyl surface of more-crystalline (oxyhydr)oxides.
622
am-FeOOH•Fe2+, α-FeOOH•Fe2+, and α-Fe2O3•2Fe2+ represent the reactive surface complex
623
with Fe(II) adsorbing on hydroxyl surfaces of iron oxides.
624 625
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626
Figure captions
627
Figure 1. (a) Kinetics of Fe(II) generation during ferrihydrite reduction by MR-1 wild type
628
(WT) and the six types of mutant cells. Data are presented as means ± standard deviations
629
(SD) of triplicate replicates. (b) Zero-order reduction rates for Fe(II) generation. The
630
reduction of 50 mM ferrihydrite was determined for MR-1 WT and mutants (∆mtrA, ∆mtrC,
631
∆mtrD, ∆mtrF, ∆omcA, and ∆cymA) using lactate as an electron donor. Ferrihydrite without
632
bacteria was used as a negative control. (c) The number of bacterial cells (WT and mutants)
633
with the initial cell density (OD600nm = 1.0).
634
Figure 2. SEM of ferrihydrite after six days of microbial reductions by MR-1 WT and six
635
types of mutant cells. Ferrihydrite without bacteria was used as a negative control. Inset
636
pictures show photographs of the reaction suspensions after six days of incubation. (a) No cell,
637
(b) WT, (c) ∆mtrA, (d) ∆mtrC, (e) ∆mtrD, (f) ∆mtrF, (g) ∆omcA, (h) ∆cymA.
638
Figure 3. Fourier transform infrared spectra of ferrihydrite during the six-day reduction by
639
MR-1 WT and six types of mutant cells. (a) Ferrihydrite without bacteria, (b) WT, (c) ∆mtrA,
640
(d) ∆mtrC, (e) ∆mtrD, (f) ∆mtrF, (g) ∆omcA, (h) ∆cymA.
641
Figure 4. X-ray diffraction patterns of ferrihydrite during the six-day reduction by MR-1 WT
642
and six types of mutants. (a) No cell, (b) WT, (c) ∆mtrA, (d) ∆mtrC, (e) ∆mtrD, (f) ∆mtrF, (g)
643
∆omcA, (h) ∆cymA, (i) the JCPDS cards of goethite, hematite and magnetite.
644
Figure 5. Secondary mineral generation kinetics of ferrihydrite by MR-1 WT and six types of
645
mutants based on the change in relative intensity over time (for goethite: 2θ = 33.2o, hematite:
646
2θ = 35.7o and magnetite: 2θ = 62.5o). (a) Goethite, (b) hematite, (c) magnetite.
647 648
28
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Figure 1.
649 WT ∆mtrA
(a)
∆mtrC ∆mtrD
∆mtrF ∆omcA
3
2
1 0
1
2
650
3 Time (d)
∆mtrD
WT
(b)
2.0
4
5
6
∆omcA
1.5
No cell
0.5
∆cymA
∆mtrC
1.0
∆mtrA
r (mM d-1)
∆cymA No cell
∆mtrF
HCl-extractable Fe(II) (mmol-1)
4
0.0
651
652
∆cymA
1
∆mtrA
2
∆omcA
∆mtrC
WT
3
∆mtrF
∆mtrD
(c) Bacteria number (×109) (cells mL-1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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0
653 29
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654 655
Figure 2.
656 657 658
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659
Figure 3.
660 (a)
1175 1120 1040
(b)
No cell
Transmittance
Transmittance
3d 2d 1d 0d
6d
WT
564
3d 2d 1d
0d
1200
(c)
590
797
4d
4d
661
887
1175 1120 1040
6d
590
6d
1000
1175 1120 1040
800 887
600
797
1200
400
590
(d)
∆mtrA
1000
1175 1120 1040
6d
887
800
600
797
590
800
600
400
∆mtrC
4d
Transmittance
Transmittance
4d 3d 2d 1d 0d
3d 2d 1d 0d
1200
1000
800
600
1200
400
1000
Wavenumber (cm-1)
662 (e)
887
1175 1120 1040
797
(f)
∆mtrD
Transmittance
Transmittance
∆mtrF
564
4d
3d 2d 1d
3d 2d 1d 0d
0d
1200
(g)
887 797 590
1175 1120 1040
6d
564
4d
663
400
Wavenumber (cm-1)
590
6d
1000
1175 1120 1040
887
800
600
797
590
6d
400
1200
(h)
∆omcA
1000
1175 1120 1040
887
6d
800
600
797
590
800
600
400
∆cymA
564
4d Transmittance
4d Transmittance
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Earth and Space Chemistry
3d 2d 1d
3d 2d 1d 0d
0d
1200
664
1000
800
600
400
1200
1000
Wavenumber (cm-1)
Wavenumber (cm-1)
665 666
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ACS Earth and Space Chemistry
Figure 4.
667 (a)
(b)
No cell
H
WT
6d
Relative Intensity
M
Relative Intensity
4d 3d 2d
M
M
H
G
M
G
H
M
G
H
H
M
H
M
6d M
M
4d M
M
3d
G H
H
HH
2d
H
H H
1d
G H
1d
0d
0d 20
30
40
50
60
20
70
30
40
G G
H
H
H
H H
G
6d
H
G
H G
H
G
H
H
H
H H
H
H
4d 3d
H
2d
70
60
H
H
ΔmtrC
M G
H
G
H
H H G
H
H
H
H
H
H
H
H
H
H M H H
3d
H
2d 1d
1d
0d
0d 20
40
50
60
20
70
30
40
2 Theta ( )
a (e)
H
Relative Intensity
M
(f)
∆mtrD M
H
M
M G
6d
H M
G
M
G G
M
H
H
H
H
G H
M
M
4d M
M
3d
H
2d
H
1d
60
H
M
G
50
70
2 Theta (o)
o
∆mtrF
G
M
H
M
M
6d
H
Relative Intensity
669
30
M M
G
M
G
H
G H G H
H
M
H
M
M
4d
M
3d
H
2d
H
1d 0d
0d 20
30
40
50
60
70
20
30
40
2 Theta (o)
670 (g)
(h)
G
∆omcA G
G
M
H
6d
H
G
M
G G
H
H M
M
M
4d
H H
H G H
60
3d 2d
H
G H
H
H
M
M
Relative Intensity
M
50
G
H
G
H
H
G
H
G
H
ΔcymA
H
H
H
H
H
H
H
H
671
30
H H
6d
HH
4d
H
3d
H
2d
H
1d
1d 0d
0d 20
70
2 Theta (o)
H
G
6d 4d
H
G H
H
G
G
ΔmtrA
H
H
Relative Intensity
(d)
H
Relative Intensity
(c)
50
2 Theta (o)
2 Theta (o)
668
Relative Intensity
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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40
50
60
70
20
30
40
50
2 Theta (o)
2 Theta (o)
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70
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Figure 5.
672 12000
WT ∆mtrA ∆mtrC ∆mtrD ∆mtrF ∆omcA ∆cymA
(a)
Relative Intensity
9000
6000
Goethite
3000
0 0
1
2
3
4
5
6
Time (day)
673 30000
WT ∆mtrA ∆mtrC ∆mtrD ∆mtrF ∆omcA ∆cymA
(b)
Relative Intensity
25000
20000
Hematite
15000
10000
5000
0 0
1
2
3
4
5
6
Time (day)
674 (c)
Magnetite
WT ∆mtrA ∆mtrC ∆mtrD ∆mtrF ∆omcA ∆cymA
12000
Relative Intensity
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Earth and Space Chemistry
9000
6000
3000
0
1
675 676
2
3
4
Time (day)
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6
ACS Earth and Space Chemistry
677 678 679
Table of Contents Fe(II) production
Secondary mineral formation
Fe2+
Goethite
Ferrihydrite
Hematite
eMagnetite
MtrCAB
680 681 682
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Fe2+
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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