Dependence of Secondary Mineral Formation on Fe(II) Production

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 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

1 2

Dependence of Secondary Mineral Formation on Fe(II) Production from

3

Ferrihydrite Reduction by Shewanella oneidensis MR-1

4

Rui Han,a,b Tongxu Liu,a,* Fangbai Li,a Xiaomin Li,a,c Dandan Chena, and Yundang Wua

5 6 7

a

Guangdong Institute of Eco-environmental Science & Technology, Guangdong Key Laboratory of Integrated Agro-environmental Pollution Control and Management, Guangzhou 510650, China

8 9 10

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,

11 12

South China Normal University, Guangzhou 510006, China

13 14 15

*

16

E-mail address: [email protected] (T.X.Liu)

Corresponding author. Tel.: +86 20 37021396.

17 18


19

(Re-submitted March, 2018)

20 21

1

ACS Paragon Plus Environment

ACS Earth and Space Chemistry 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

22

ABSTRACT: Although dissimilatory iron reduction and secondary mineral formation by

23

Shewanella oneidensis MR-1 have been widely recognized, questions remain about the effects

24

of biogenic Fe(II) on the rate and extent of secondary mineral formation and the importance

25

of biogenic Fe(II)-induced crystallization processes. In this study, we investigated the effects

26

of different mutants of MR-1 on the bioreduction and mineralization of ferrihydrite. The

27

results indicate that while the reduction rates of ferrihydrite by ∆mtrD, ∆mtrF, and ∆omcA are

28

similar to that of the wild type (WT), the capacity to reduce ferrihydrite decreased

29

dramatically in the mutants ∆cymA and ∆mtrA. The order for Fe(III) reduction by MR-1 WT

30

and mutants was ranked as follows: WT ≈ ∆mtrD ≈ ∆mtrF >∆omcA > ∆mtrC > ∆cymA >

31

∆mtrA. Secondary minerals of ferrihydrite were characterized using X-ray diffraction, Fourier

32

transform infrared spectra, and scanning electron microscopy. The results show that goethite

33

and hematite were the main secondary minerals formed during the first two days in all

34

treatments, and then magnetite appeared in the WT, ∆mtrD, ∆mtrF and ∆omcA treatments,

35

while magnetite began to appear from the sixth day onwards in the ∆mtrC treatment.

36

However, no magnetite was observed during the six days in the ∆mtrA and ∆cymA incubation

37

treatments. The plausible electron transfer pathways of bioreduction and phase transformation

38

were also verified using thermodynamic calculations of elementary reactions. This study

39

clarified the importance of Fe(II) production in secondary mineral formation processes and

40

highlighted the significance of biogenic Fe(II)-catalyzed crystallization. This information may,

41

in turn, help us to better understand natural microbe−mineral interaction processes.

42 43

KEYWORDS: Biotransformation; ferrihydrite; mutant; Shewanella oneidensis MR-1;

44

extracellular electron transfer.

45

2


Page 2 of 34

Page 3 of 34 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


46

1. INTRODUCTION

47

Iron is the most abundant redox-active metal in the Earth’s crust, and iron-bearing minerals

48

are ubiquitous reactive constituents of aquifers, soils, and sediments. Dissimilatory Fe(III)

49

reduction is widely observed in natural systems (aquatic sediments, soils, and subsurface

50

environments) and has major impacts on iron geochemistry, the fate of trace metals and

51

nutrients, and the degradation of organic matter.1 Dissimilatory iron-reducing bacteria (DIRB)

52

can reduce Fe(III) to Fe(II) under anoxic conditions relying on hydrogen or the oxidation of

53

organic matter to obtain energy for maintenance and cell growth.2,3 A wide phylogenetic

54

diversity of microorganisms, including archaea and bacteria, are capable of dissimilatory

55

Fe(III) reduction. Of all the identified strains of DIRB, Geobacter and Shewanella are by far

56

the most extensively investigated.4

57

Iron exists predominantly in an insoluble solid phase (e.g., ferrihydrite, goethite, and

58

hematite) in the environment under circumneutral pH conditions.4 Shewanella oneidensis

59

MR-1 (MR-1) has two main pathways of extracellular electron transfer (EET): (i) to directly

60

transfer electrons from outer membrane (OM)-bound cytochromes and porin-cytochrome

61

complexes toward iron oxides, or along organic appendages named “nanowires,” which are

62

extensions of the outer membrane and periplasm produced by MR-1 5; (ii) to use endogenous

63

organic molecules (e.g., flavin or riboflavin mononucleotides) as electron shuttles that diffuse

64

in the medium and mediate EET; thus, DIRB must pump electrons from the internal

65

cytoplasmic membrane to iron oxides located outside the cell.6 To overcome this physical

66

barrier, MR-1 developed a pathway (i.e., Mtr) that requires multihaem c-type cytochromes

67

(c-Cyts) for transferring electrons from the inner membrane via the periplasm and OM to

68

external minerals.7,8 Electron transport proteins of the Mtr pathway include multihaem c-Cyts

69

(CymA, MtrA, MtrC, OmcA, and a porin-like OM protein MtrB).7,8 The roles of these

70

proteins in the electron transfer process have been documented as follows: CymA is an inner 3



71

membrane-anchored quinol dehydrogenase that oxidizes quinol in the inner membrane and

72

transfers the released electrons to the periplasmic MtrA; MtrA delivers electrons to the

73

OM-anchored MtrC and OmcA, which can directly reduce solid metal (hydr)oxides.9-12 For

74

most Shewanella members, the EET process is generally performed by an MtrCAB complex.

75

Biochemical characterization of this complex indicates that MtrB functions as a

76

trans-membrane sheath into which both MtrC and MtrA are partially inserted. 8,13,14 MtrF,

77

MtrD, and MtrE are the homologues of MtrC, MtrA, and MtrB, respectively. MtrF, MtrD, and

78

MtrE are thought to form an OM-spanning complex, namely MtrFDE, which has the same

79

overall structure as MtrCAB but only makes a minor contribution to the process of iron

80

reduction.15 Despite progress in understanding the key proteins in the Mtr pathway via

81

mutants and purified proteins,10,15-18 there is a critical need to quantify the roles of these

82

proteins in EET to iron minerals owing to their implications for biogeochemical redox

83

processes. Therefore, it is necessary to further investigate the effects of key proteins and their

84

homologues on the kinetics of iron reduction by wild type (WT) and mutants of MR-1.

85

Poorly crystalline hydrous ferric oxides (e.g., ferrihydrite), which commonly exist in soils

86

and sediments, are thermodynamically unstable and, with time, transform to more crystalline

87

Fe(III)-oxides, e.g., goethite and/or hematite,19 resulting in a loss of ability to scavenge other

88

trace metals from solution.20 Ferrihydrite is considered the most (bio)available Fe(III)

89

(hydr)oxide for DIRB, and electron transfer proteins from Shewanella play an important role

90

in the extracellular reduction of ferrihydrite, followed by biomineralization. Previous studies

91

have focused on the factors influencing secondary mineralization, such as iron reduction rates,

92

electron donor/acceptor concentrations, cell density, pH, and redox potential.19,21,22

93

Although the roles of some OM c-Cyts (MtrC and OmcA) have been documented with regard

94

to biomineralization of ferrihydrite by MR-1,23 questions still remain about the relationship

95

between the rate of iron bioreduction and the extent of secondary mineral formation, and the 4


Page 4 of 34

Page 5 of 34 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


96

importance of Fe(II)-compounds as catalysts in these chemical processes.24

97

In this study, MR-1 was selected because it is a DIRB that is well known for its abilities to

98

use a variety of iron oxides and oxyhydroxides as terminal electron acceptors.25 The

99

objectives of this study were to examine the rates of Fe(III) reduction and the concomitant

100

secondary mineral formation by wild type and mutants of MR-1, with implications for

101

quantifying the effects of biogenic Fe(II) production on the rates and extents of secondary

102

mineral formation in the dissimilatory iron reduction processes of ferrihydrite.

103 104

2. MATERIALS AND METHODS

105

2.1. Materials. MR-1 wild type (WT) that had been isolated from anoxic sediments from

106

Lake Oneida, NY26 was purchased from MCCC (Marine Culture Collection of China, China).

107

All mutant-deletion strains ∆mtrA, ∆mtrC, ∆mtrD, ∆mtrF, ∆omcA, and ∆cymA were provided

108

by Professor Gao Haichun from Zhejiang University, as documented in previous studies.

109

8,15,27,28

110

98%). Piperazine-N–N-bis-2-ethanesulfonic acid (PIPES) was purchased from J&K Chemical

111

(Beijing, China). Lactate was purchased from Sinopharm Chemical Reagent Co., Ltd.

112

(Shanghai, China). Other chemicals were purchased from Guangzhou Chemical Reagent

113

Factory (Guangzhou, China). All solutions were prepared using Milli-Q deoxygenated

114

ultrapure water (18 MΩ cm, EASYpure II RF/UV, USA).

All chemicals were of analytical reagent grade and the highest available purity (≥

115

2.2. Preparation of ferrihydrite. Ferrihydrite was synthesized by neutralizing 0.4 mM

116

FeCl3·6H2O with 1 mM NaOH in a high-density polyethylene container.29 Firstly, aqueous

117

solutions of 0.4 mol L-1 FeCl3·6H2O and 1 mol L-1 NaOH were prepared. Then the FeCl3

118

solution was stirred intensely and evenly while the NaOH solution was trickled steadily into it.

119

As the circumneutral pH approached, the NaOH solution was added more slowly, and

120

a thick red slurry of ferrihydrite formed. At pH 7.0, the slurry was allowed to ripen for 2–6 h, 5



121

with a slight decrease in pH (usually < 1 pH unit). The slurry was adjusted back to pH 7.0 and

122

centrifuged at 3 600 g for 20 min at 4 °C. After decanting the supernatant, the solid was

123

re-suspended, washed with Milli-Q water, and centrifuged again. This procedure was repeated

124

seven times to reduce the residual chloride content to less than 1 mM. The slurry was then

125

kept in Milli-Q water prior to use.30

126

2.3. Biotransformation of ferrihydrite by wild type and mutants of MR-1. MR-1 WT

127

and various mutant cells (∆mtrA, ∆mtrC, ∆mtrD, ∆mtrF, ∆omcA, and ∆cymA) were grown

128

aerobically in a Luria-Bertani medium (10 g L-1 NaCl, 5 g L-1 yeast extract, 10 g L-1 tryptone)

129

at 30 °C with shaking at 180 rpm. When approaching the exponential phase, cells were

130

harvested by centrifuging at 7 000 g at 4 °C for 10 min. The pellets were then washed three

131

times and re-suspended in 30 mM PIPES buffer at pH 7.0 ± 0.2. The Fe(III) reduction

132

medium contained 30 mM PIPES (pH 7.0 ± 0.2) as a buffer, 50 mM ferrihydrite as the sole

133

electron acceptor, and 20 mM lactate as the sole electron donor. The bacterial concentration in

134

each treatment was OD600nm = 1.0, and the number of bacterial cells (wild and mutants) for an

135

OD600nm = 1.0 were measured using the DAPI staining method.31 Growth curves of the

136

bacterial cells (wild and mutants) were plotted using the method outlined in Supporting

137

Information, and the total protein contents of wild type and mutants at OD600nm = 1.0 were

138

measured. An experimental group without bacteria was used as a control. The pH was also

139

maintained at approximately 7.0 ± 0.2 using 30 mM PIPES. The medium was dispensed into

140

100-mL serum bottles purged with O2-free N2 for at least 30 min, capped with butyl rubber

141

closures, and crimp sealed. All anaerobic media were sterilized by autoclaving at 121 °C for

142

20 min and then cooled to room temperature in a Bactron Anaerobic/Environmental Chamber

143

II (Shellab, Sheldon Manufacturing Inc., Cornelius, OR, USA) before use. Inoculation and

144

sampling were conducted using sterile syringes and needles. All experiments were incubated

145

in an anaerobic chamber in the dark at 30 °C. Three reactor bottles per treatment were taken 6


Page 6 of 34

Page 7 of 34 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


146 147

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)

148

from the suspensions using 0.5 M HCl for 1.5 h32 and assaying the extract using the

149

1,10-phenanthroline colorimetric method at 510 nm on a UV−Vis spectrophotometer

150

(TU-1810PC, Beijing Purkinje General Instruments, China). Standard curves were made

151

using ammonium ferrous sulfate dissolved in 0.5 M hydrochloric acid. The mineral samples

152

were collected from the incubations by filtration inside the anaerobic chamber. Samples were

153

filtered onto 0.22 µm filters (VCTP Millipore Isopore), washed twice with deoxygenated

154

double deionized (DDI) water, and dried in the anaerobic chamber. The dry minerals were

155

then characterized. The X-ray diffraction (XRD) patterns of powder samples before and after

156

reactions were recorded on a Bruker Advance Diffractometer (Bruker Co., USA) at room

157

temperature, operating at 40 kV and 40 mA, using Cu Kα radiation (λ = 0.15418 nm). Data

158

were acquired in the range of 15° to 70°, using a step of 0.04°. The phases were identified by

159

comparing diffraction patterns with those on standard powder XRD cards compiled by the

160

Joint Committee on Powder Diffraction Standards (JCPDS).33 Fourier transform infrared

161

spectroscopy (FTIR) was recorded using an FTIR spectrometer (Perkin−Elmer, model Vector

162

33, Bruker Co., USA) at room temperature. The morphology was investigated by scanning

163

electron microscopy, and the samples were dispersed onto carbon-coated Cu grids in the

164

anaerobic chamber. SEM images were obtained using field-emission scanning electron

165

microscopy (LEO1530VP, Zeiss, Germany).

166 167

3. RESULTS AND DISCUSSION

168

3.1. Effect of mutants on bioreduction of ferrihydrite. The kinetics of Fe(II) generation

169

during ferrihydrite reduction by WT and mutants are shown in Figure 1a. While no Fe(II) was

170

generated in the ferrihydrite control without MR-1, the presence of MR-1 WT and mutants 7



171

obviously stimulated Fe(II) generation, indicating that the reduction of ferrihydrite to Fe(II)

172

occurred via a biological reaction. Fe(II) generation by WT increased quickly to ~ 4 mM in

173

the first two days and then decreased gradually to ~2.8 mM at the end of incubation (day 6).

174

The Fe(II) generation by ∆mtrD and ∆mtrF was very close to that of WT. Fe(II) generation by

175

∆omcA also increased at the beginning and then decreased, but the Fe(II) concentration was

176

lower than that generated by WT, ∆mtrD, or ∆mtrF. Fe(II) generation by ∆mtrC, ∆cymA, and

177

∆mtrA increased over time during the six days of incubation. To clearly illustrate Fe(II)

178

generation rates of all the treatments, the zero-order reduction rates of Fe(II) generation

179

during the increasing stage of the kinetic curves were calculated (Figure 1b). Because the

180

concentration of Fe(II) in these systems decreases after two days due to the reaction between

181

Fe(II) and ferrihydrite, production rates of Fe(II) in the WT and mutants without MtrD or

182

MtrF were calculated using the data of the first two days. Compared to the Fe(II) production

183

rate for WT (2.05 mM d-1), a slight decrease was observed for ∆mtrD (1.80 mM d-1) and

184

∆mtrF (1.75 mM d-1); the rates for other mutants decreased substantially, by 64.9%, 77.1%,

185

88.3%, and 93.7% for ∆omcA, ∆mtrC, ∆cymA, and ∆mtrA, respectively. Therefore, the order

186

for the capacity of reducing ferrihydrite by MR-1 WT and mutants was ranked as follows: WT

187

≈ ∆mtrD ≈ ∆mtrF > ∆omcA > ∆mtrC > ∆cymA > ∆mtrA, indicating their roles in controlling

188

the electron transfer pathway. The initial cell number at OD600 nm = 1.0 added in all treatment

189

groups (Figure 1c) was not identical for each strain of MR-1. WT had an initial cell number of

190

2.35×109 which is 2.35 times and 4.7 times more than those of the ∆mtrA and ∆cymA

191

treatments, respectively. The total protein contents of WT and mutants at OD600nm = 1.0

192

(Figure S2) were consistent with the cell numbers in Figure 1c, thus the initial cell numbers

193

were different. To clearly differentiate the effects of gene deletion and quantitative variation

194

of cells used in Fe(III) oxide reduction, the cell numbers in Figure 1c were used to calibrate

195

the rates in Figure 1b. The calibrated rates in Figure S3 for various treatments with WT and 8


Page 8 of 34

Page 9 of 34 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


196

mutants are consistent with the original rates for Fe(II) production in Figure 1b. The above

197

results indicated that the knock outs of different electron transfer proteins influenced the cell

198

numbers of bacteria, and also affected the reduction rate of ferrihydrite and the secondary

199

mineral formation.

200

3.2. Effect of mutants on secondary mineral formation. The mineralogy of ferrihydrite

201

changed along with ferrous release during the bioreduction processes, which was indicated by

202

visible color changes of the incubated suspension (inserts of Figure 2). After six days of

203

incubation, the color of the ferrihydrite suspension was dark red after reacting with ∆cymA,

204

∆mtrA, and ∆mtrC, while those with WT, ∆mtrD, ∆mtrF and ∆omcA had turned black. The

205

different colors of the reaction suspensions might be attributed to different secondary

206

minerals.34

207

Morphology. The samples were examined using SEM techniques to identify the changes in

208

surface morphology of the solid phases during ferrihydrite reduction by WT and mutants. The

209

ferrihydrite image showed a smooth flat surface (Figure 2a), which might be attributed to an

210

aggregation of the tiny ferrihydrite particles after the drying processes. After incubation with

211

MR-1 WT and mutants, large particles formed with a wide range of particle sizes between 20

212

nm and 200 nm (Figure 2b-h). It was apparent that the particle sizes of minerals with WT,

213

∆mtrD, ∆mtrF, and ∆omcA were much smaller than those with ∆mtrC, ∆cymA and ∆mtrA.

214

The difference in particle sizes was similar to that of the iron reduction rates. Since the

215

particle sizes during microbial mineralization might be determined by the mineral

216

components, crystallization conditions, and the location relatively to the cells, the single SEM

217

images did not provide crucial information for the secondary minerals except the surface

218

morphology of the overall mixtures with cells and minerals. Therefore, specific differences of

219

the secondary minerals were further characterized by FTIR and XRD as discussed below.

220

Crystal structures. The solid samples of ferrihydrite before and after reduction by MR-1 9



221

WT and mutants were further examined by FTIR. For the control treatment with ferrihydrite

222

alone, the FTIR patterns of ferrihydrite remained unchanged, as no bioreduction or

223

biomineralization occurred during the six-day incubation. The major peaks at 590 cm-1, 1040

224

cm-1, 1120 cm-1, and 1175 cm-1 (Figure 3a) represent the characteristic peaks of ferrihydrite.34

225

After reduction of ferrihydrite by MR-1 WT and mutants, the peaks for ferrihydrite changed

226

by differing amounts (Figure 3b-h). Specifically, while the peaks for ferrihydrite at 1120 cm-1

227

and 1175 cm-1 for WT, ∆mtrD, and ∆mtrF nearly disappeared after six days of incubation, the

228

intensity of these peaks for the other mutants were only slightly lower. Two sharp peaks at

229

797 cm-1 and 887 cm-1 appeared in all the treatments with MR-1 and were ascribed to the

230

Fe–OH bond of goethite.35 It can also be seen that the peak at 590 cm-1 shifted to 564 cm-1 for

231

the treatments with WT, ∆mtrD, ∆mtrF, and ∆omcA, which may be attributed to the Fe–O

232

bond of magnetite.36 These results indicate that ferrihydrite was gradually reduced and

233

transformed by WT and mutants over time, followed by the formation of goethite and

234

magnetite.

235

Microbial reduction of ferrihydrite was followed by recrystallization processes and

236

formation of more crystalline iron (hydr)oxides.34 The XRD patterns of minerals in the

237

control treatment of ferrihydrite showed no obvious diffraction peaks owing to its amorphous

238

structure that remained unchanged over the six-day incubation (Figure 4a). For MR-1 WT or

239

mutants, new diffraction peaks appeared, and their relative intensities changed over time

240

(Figure 4b-h). After comparing those peaks with JCPDS cards, the newly formed crystal

241

structures were found to include goethite (No. 29-0713), hematite (No. 33-0664), and

242

magnetite (No. 19-0629). Goethite (2θ = 33.2°) and hematite (2θ = 35.7°, 54.1°, 62.4°, 64.0°)

243

were the main forms of secondary minerals in the first two days for all the treatments with

244

MR-1. For WT, ∆mtrD, ∆mtrF and ∆omcA, magnetite (2θ = 33.2°, 43.2°, 57.1°, 62.5°) began

245

to appear on the third day, followed by a gradual disappearance of hematite, and then 10


Page 10 of 34

Page 11 of 34 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


246

magnetite became the main form on the sixth day. For ∆omcA and ∆mtrC, magnetite began to

247

appear on the fourth day and the sixth day, respectively. For ∆mtrA and ∆cymA, only hematite

248

and goethite were observed, with no magnetite observed during the six-day incubation.

249

Equal amounts of ferrihydrite were used as the starting materials and all the samples were

250

characterized at the same time. Thus, the relative contents of all the minerals can be compared

251

according to the diffraction peaks and the kinetics of each mineral can be estimated from the

252

changes of the peak intensities over time. The relative peak areas of goethite (2θ = 33.2o),

253

magnetite (2θ = 62.5o) and hematite (2θ = 35.7o) were plotted as a function of incubation time

254

(Figure 5). For goethite, the peak areas of all treatments with MR-1 WT and mutants

255

increased with incubation time (Figure 5a). During the first day, the order of goethite

256

formation amounts was similar to that for Fe(II) production in Figure 1b. The peak areas for

257

∆mtrA, ∆mtrC and ∆cymA on the sixth day were slightly higher than those of WT, ∆mtrD,

258

∆mtrF and ∆omcA. The changing trend for hematite was opposite to that for goethite with all

259

the treatments. The peak intensities of hematite (2θ = 35.7°) in the treatments of WT, ∆mtrD,

260

∆mtrF and ∆omcA on the sixth day were slightly higher than those of ∆mtrA, ∆mtrC and

261

∆cymA (Figure 5b). Meanwhile, the peak of magnetite (2θ = 57.1°) appeared and increased as

262

the reaction proceeded, indicating that the minerals were transformed into magnetite from the

263

third day of reaction. Since goethite are just intermediate mineral products, the order of

264

formation of goethite in the following days was not well matched with that of Fe(II)

265

production. For magnetite (Figure 5c), diffraction peaks began to appear on the second day

266

for WT, ∆mtrD, and ∆mtrF; the third day for ∆omcA; and the fourth day for ∆mtrC. No

267

magnetite peaks were observed for ∆mtrA and ∆cymA over the six days of incubation. Hence,

268

the order of formation rates of magnetite as the final mineral product was consistent with

269

Fe(II) production.

270

3.3. Thermodynamics of bioreduction and biomineralization. The above experimental 11



271

results clearly indicate that Fe(II) generation can occur via bioreduction processes and iron

272

(oxyhdr)oxides formation via re-crystallization. These bioreduction processes were identified

273

as enzymatic reactions between outer membrane c-type cytochromes (OM c-Cyts) and iron

274

(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

276

biomineralization processes, the thermodynamics of all possible reactions were calculated

277

according to the reported values of various species and minerals involved in the

278

biotransformation processes (Table 1).

279

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

282

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

285

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,

287

∆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.

290

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

292

bioreduction system, more negative ∆G 0r values may reflect greater difficulty in iron

293

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


Page 12 of 34

Page 13 of 34 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


295

non-redox reactions among them may directly induce phase transformation. In addition, the

296

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).

298

These reactions are written according to the mass balance law as Rxns. 5 – 10 in Table 3. It

299

has been reported that ferrihydrite is thermodynamically metastable and transforms into the

300

more-crystalline products goethite and hematite via a long-term aging process.42 Therefore, it

301

is essential to clarify whether the aging processes of iron (oxyhydr)oxides plays a role in

302

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

304

(am-FeOOH and α-FeOOH) in Table 4 were lower than zero, indicating that the reactions of

305

am-FeOOH → α-FeOOH and am-FeOOH→ α-Fe2O3 may spontaneously occur, though they

306

may be very slow.42 The reaction α-FeOOH→ α-Fe2O3 (Rxn. 7), and all three reactions

307

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

309

hematite only occurred under heating at about 250 oC.43,44

310

Recently, the Fe(II)-catalyzed phase transformation of iron (oxyhydr)oxides to more

311

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

314

(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

316

“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



320

reaction can be written as Rxns. 11 – 13. The first step (Rxn. 11) is surface complexation with

321

the formation of the reactive intermediate (≡Fe(III)OFe(II)+). The second step (Rxn. 12) is the

322

internal electron transfer to another Fe(III) atom in the structure, forming ≡Fe(II)OFe(III)+. In

323

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.

327

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

343

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


Page 14 of 34

Page 15 of 34 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


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.

353

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

356

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



370

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

373

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

375

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,

379

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

16


Page 16 of 34

Page 17 of 34 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

395


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



420

of experiment method, Figures S1-S3 with illustrations. This material may be found in the

421

online version of this article.

422 423

REFERENCES

424

(1) Lovley, D. Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes. in: The Prokaryotes: Volume 2:

425

Ecophysiology and Biochemistry, (Eds.) Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H.,

426

Stackebrandt, E. Springer New York, New York, 2006; pp 635–658.

427

(2) Bose, S.; Hochella, M.F.; Gorby, Y.A.; Kennedy, D.W.; McCready, D.E.; Madden, A.S.; Lower, B.H.

428

Bioreduction of hematite nanoparticles by the dissimilatory iron reducing bacterium Shewanella

429

oneidensis MR-1. Geochim. Cosmochim. Acta

2009, 73, 962–976. doi. 10.1016/j.gca.2008.11.031

430

(3) Scala, D.J.; Hacherl, E.L.; Cowan, R.; Young, L.Y.; Kosson, D.S. Characterization of Fe(III)-reducing

431

enrichment cultures and isolation of Fe(III)-reducing bacteria from the Savannah River site, South

432

Carolina. Res. Microbiol. 2006, 157, 772–783. doi:10.1016/j.resmic.2006.04.001.

433 434

(4) Esther, J.; Sukla, L.B.; Pradhan, N.; Panda, S. Fe (III) reduction strategies of Dissimilatory Iron Reducing Bacteria. Korean J. Chem. Eng. 2015, 32, 1–14. doi:10.1007/s11814-014-0286-x.

435

(5) Miot, j. and Etique, M. Formation and transformation of iron-bearing minerals by iron(II)-oxidizing and

436

iron(III)-reducing bacteria, in: Iron Oxides from Nature to Applications, WILEY-VCH, Germany,

437

2016; pp 53–98.

438

(6) Lower, B.; Shi, L.; Yongsunthon, R.; Droubay, T.C.; Mccready, D.E.; Lower, S.K. Specific bonds between an

439

iron oxide surface and outer membrane cytochromes MtrC and OmcA from Shewanella oneidensis

440

MR-1. J. Bacteriol. 2007, 189, 4944–4952. doi:10.1128/JB.01518-06.

441

(7) Shi, L.; Squier, T.C.; Zachara, J.M.; Fredrickson, J.K. Respiration of metal (hydr)oxides by Shewanella and

442

Geobacter: a key role for multihaem c-type cytochromes. Mol. Microbiol. 2007, 65, 12–20.

443

doi:10.1111/j.1365-2958.2007.05783.x.

444

(8) Shi, L.; Rosso, K.M.; Zachara, J.M.; Fredrickson, J.K. Mtr extracellular electron-transfer pathways in

445

Fe(III)-reducing or Fe(II)-oxidizing bacteria: a genomic perspective. Biochem. Soc. Trans. 2012, 40,

446

1261–1267. doi:10.1042/bst20120098.

447

(9) Myers, C.R. and Myers, J.M. Cloning and sequence of cymA, a gene encoding a tetraheme cytochrome c

448

required for reduction of iron (III), fumarate, and nitrate by Shewanella putrefaciens MR-1. J. Bacteriol. 18


Page 18 of 34

Page 19 of 34 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

449


1997, 179, 1143–1152. doi:10.1128/jb.179.4.1143-1152.1997.

450

(10) Myers, C.R. and Myers, J.M. MtrB is required for proper incorporation of the cytochromes OmcA and

451

OmcB into the outer membrane of Shewanella putrefaciens MR-1. Appl. Environ. Microbiol. 2002, 68,

452

5585–5594. doi:10.1128/AEM.68.11.5585-5594.2002.

453

(11) Pitts, K.E.; Dobbin P.S.; Reyes-Ramirez F.; Thomson A.J.; Richardson D.J.; Seward H.E. Characterization

454

of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA: expression in Escherichia coli confers

455

the ability to reduce soluble Fe(III) chelates. J. Biol. Chem.

456

10.1074/jbc.M302582200.

2003 ， 278, 27758–27765. doi:

457

(12) Shi, L.; Richardson, D.J.; Wang, Z.; Kerisit, S.N.; Rosso, K.M.; Zchara, J.M. The roles of outer membrane

458

cytochromes of Shewanella and Geobacter in extracellular electron transfer. Env. Microbiol. 2009, 1,

459

220–227. doi:10.1111/j.1758-2229.2009.00035.x.

460

(13) Hartshorne, R.S.; Reardon, C.L.; Ross, D.; Nuester, J.; Clarke, T.A.; Gates, A.J.; Mills, P.C.; Fredrickson,

461

J.K.; Zachara, J.M.; Shi, L.; Beliaev, A.S.; Marshall, M.J.; Tien, M.; Brantley, S.; Butt, J.N.; Richardson,

462

D.J. Characterization of an electron conduit between bacteria and the extracellular environment. P. Nati.

463

Acad. Sci. USA 2009, 106, 22169–22174. doi: 10.1073/pnas.0900086106

464

(14) Ross, D.; Ruebush, S.S.; Brantley, S.L.; Hartshorne, R.S.; Clarke, T.; Richardson, D.J. Characterization of

465

protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1. Appl. Environ.

466

Microbiol. 2007, 73, 5797–5808. doi:10.1128/aem.00146-07.

467 468

(15) Dan, C.; and Gralnick, J. A. Modularity of the mtr respiratory pathway of shewanella oneidensis strain

MR-1. Mol. Microbiol. 2010,77(4), 995–1008.

469

(16) Marritt, S.J.; Lowe, T.G.; Bye, J.; McMillan, D.G.; Shi, L.; Fredrickson, J.; Zachara, J.; Richardson, D.J.;

470

Cheesman, M.R.; Jeuken, L.J.; Butt, J.N. A functional description of CymA, an electron-transfer hub

471

supporting

472

465–474. doi:10.1042/BJ20120197.

anaerobic

respiratory

flexibility

in

Shewanella.

Biochem.

J.

2012,

444,

473

(17) Ross, D.E.; Brantley, S.L.; Tien, M. Kinetic characterization of OmcA and MtrC, terminal reductases

474

involved in respiratory electron transfer for dissimilatory iron reduction in Shewanella oneidensis MR-1.

475

Appl. Environ. Microbiol. 2009, 75, 5218–5226. doi:10.1128/AEM.00544-09.

476

(18) Schuetz, B.; Schicklberger, M.; Kuermann, J.; Spormann, A.M.; Gescher, J. Periplasmic electron transfer

477

via the c-type cytochromes MtrA and FccA of Shewanella oneidensis MR-1. Appl. Environ. Microb.

478

2009, 75, 7789–7796. doi:10.1128/aem.01834-09. 19



Page 20 of 34

479

(19) Zachara, J.M.; Kukkadapu, R.K.; Fredrickson, J.K.; Gorby, Y.A.; Smith, S.C. Biomineralization of poorly

480

crystalline Fe(III) oxides by dissimilatory metal reducing bacteria (DMRB). Geomicrobiol. J. 2002, 19,

481

179–207. doi:10.1080/01490450252864271.

482 483

(20) Ford, R.G.; Bertsch, P.M.; Farley, K.J. Changes in transition and heavy metal partitioning during hydrous iron oxide aging. Environ. Sci. Technol. 1997, 31, 2028–2033. doi: 10.1021/es960824+.

484

(21) Hansel, C.M.; Benner, S.G.; Neiss, J.; Dohnalkova, A.; Kukkadapu, R.K.; Fendorf, S. Secondary

485

mineralization mathways induced by dissimilatory iron reduction of ferrihydrite under advective flow.

486

Geochim.Cosmochim. Acta 2003, 67, 2977–2992. doi: 10.1016/s0016-7037(03)00276-x.

487

(22) Zegeye, A.; Ruby, C.; Jorand, F. Kinetic and thermodynamic analysis during dissimilatory γ-FeOOH

488

reduction: formation of green rust 1 and magnetite. Geomicrobiology 2007, 24, 51–64.

489

doi:10.1080/01490450601134325.

490

(23) Reardon, C.L.; Dohnalkova, A.C.; Nachimuthu, P.; Kennedy, D.W.; Saffarini, D.A.; Arey, B.W.; Shi, L.;

491

Wang, Z.; Moore, D.; McLean, J.S.; Moyles,D.; Marshall, M.J.; Zachara, J.M.; Fredrickson, J.K.;

492

Beliaev, A.S. Role of outer-membrane cytochromes MtrC and OmcA in the biomineralization of

493

ferrihydrite

494

10.1111/j.1472-4669.2009.00226.x.

495 496 497 498 499 500 501 502

by

Shewanella

oneidensis

MR-1.

Geobiology

2010,

8,

56–68. doi:

(24) Fredrickson, J.K.; Zachara, J.M. Electron transfer at the microbe-mineral interface: A grand challenge in biogeochemistry. Geobiology 2008, 6, 245–253. doi: 10.1111/j.1472-4669.2008.00146.x. (25) Lovley, D.R.; Holmes, D.E.; Nevin, K.P. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol. Rev. 1991, 55, 259–287. doi:10.1007/springerreference_3664. (26) Nies, D.H. and Silver, S. Metal ion uptake by a plasmid-free metal-sensitive Alcaligenes eutrophus strain. J. Bacteriol. 1989, 171, 4073–4075. doi:10.1128/jb.171.7.4073-4075. (27) Dan, C. and Gralnick, J.A. Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR–1. Mol. Microbiol. 2010, 77, 995–1008. doi: 10.1111/j.1365-2958.2010.07266.x.

503

(28) Gao, H.; Barua, S.; Liang, Y.; Wu, L.; Dong, Y.; Reed, S.; Chen, J.; Culley, D.; Kennedy, D.; Yang, Y.; He,

504

Z.; Nealson, K.H.; Fredrickson, J.K.; Tiedje, J.M.; Romine, M.; Zhou, J. Impacts of Shewanella

505

oneidensisc-type cytochromes on aerobic and anaerobic respiration. Microb.Biotechnol. 2010, 3,

506

455–466. doi:10.1111/j.1751-7915.2010.00181.x.

507 508

(29) Mccormick, M.L. and Peter, A. Carbon tetrachloride transformation on the surface of nanoscale biogenic magnetite particles. Environ. Sci. Technol. 2004, 38, 1045–1053. doi:10.1021/es030487m. 20


Page 21 of 34 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


509

(30) Li, X.M.; Zhou, S.G.; Li, F.B.; Wu, C.Y.; Zhuang, L.; Xu, W.; Liu, L. Fe(III)oxide reduction and carbon

510

tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strain L17. J. Appl. Microbiol.

511

2009, 106, 130–139. doi: 10.1111/j.1365-2672.2008.03985.x

512

(31) Glavin D.P.; Cleaves H. J.; Schubert M.; Aubrey A.; Bada J.L. New Method for Estimating

513

Bacterial Cell Abundances in Natural Samples by Use of Sublimation. Appl. Environ. Microbiol.

514

2004, 5923–5928.

515 516 517 518

(32) Fredrickson, J.K. and Gorby, Y.A. Environmental processes mediated by iron-reducing bacteria. Curr. Opin. Biotechnol. 1996, 7, 287–294. doi:10.1016/s0958-1669(96)80032-2. (33) Schwertmann, U. and Cornell, R.M. Iron oxides in the laboratory : preparation and characterization, First ed. Wiley-VCH, Weinheim. 1991.

519

(34) Li, X.; Liu, T.; Li, F.; Zhang, W.; Zhou, S.; LI, Y. Reduction of structural Fe(III) in oxyhydroxides by

520

Shewanella decolorationis S12 and characterization of the surface properties of iron minerals. J. Soils

521

Sediment. 2012, 12, 217–227. doi: 10.1007/s11368-011-0433-5.

522

(35) Rong, X.; Chen, W.; Huang, Q.; Peng, C.; Wei, L. Pseudomonas putida adhesion to goethite: studied by

523

equilibrium adsorption, SEM, FTIR and ITC. Colloids Surf. B Biointerfaces 2010，80, 79–85.

524

(36) Yang, K.; Peng, H.; Wen, Y.; Ning, L. Re-examination of characteristic FTIR spectrum of secondary layer in

525

bilayer

oleic

acid-coated

Fe3O4

526

doi:10.1016/j.apsusc.2009.11.079.

nanoparticles.

Appl.

Surf.

Sci.

2010,

256:

3093–3097.

527

(37) Mitsunobu, S.; Muramatsu, C.; Watanabe, K.; Sakata, M. Behavior of antimony(V) during the

528

transformation of ferrihydrite and its environmental implications. Environ. Sci. Technol. 2013, 47,

529

9660−9667. doi:10.1021/es4010398.

530

(38) Boland, D.D.; Collins, R.N.; Miller, C.J.; Glover, C.J.; Waite, T.D. Effect of solution and solid-phase

531

conditions on the Fe (II)-accelerated transformation of ferrihydrite to lepidocrocite and goethite.Environ.

532

Sci. Technol. 2014, 48, 5477–5485. doi: 10.1021/es4043275.

533

(39) Field, S.J., Dobbin, P.S., Cheesman, M.R., Watmough, N.J., Thomson, A.J. and Richardson, D. J.

534

Purification and magneto-optical spectroscopic characterization of cytoplasmic membrane and outer

535

membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400. J. Biol. Chem.

536

2000, 275, 8515–8522. doi: 10.1074/jbc.275.12.8515.

537

(40) Beliaev, A.S.; Saffarini, D.A.; Mclaughlin, J.L.; Hunnicutt, D. MtrC, An outer membrane decahaem c

538

cytochrome required for metal reduction in Shewanella putrefaciens MR-1. Mol. Microbiol. 2001, 39, 21



539

Page 22 of 34

722–730. Doi: 10.1046/j.1365-2958.2001.02257.x.

540

(41) Bretschger, O.; Obraztsova, A.; Sturm, C.A.; Chang, I.S.; Gorby, Y.A.; Reed, S.B.; Culley, D.E.; Reardon,

541

C.L.; Barua, S.; Romine, M.F.; Zhou, J.; Beliaev, A.S.; Bouhenni, R.; Saffarini, D.; Mansfeld, F.; Kim,

542

B.H.; Fredrickson, J.K.; Nealson, K.H. 2007. Current production and metal oxide reduction by

543

Shewanella oneidensis MR-1 wild type and mutants. Appl. Environ. Microbiol. 2007, 73,

544

7003–7012. doi: 10.1128/jb.00925-09.

545 546 547 548

(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.

549

(44) Gualtieri A .F, Venturelli P. In situ study of the goethite-hematite phase transformation by real time

550

synchrotron powder diffraction. Am. Mineral., 1999, 84:895-904. doi:10.2138/am-1999-5-625.

551

(45) Shi, L.; Chen, B.; Wang, Z.; Elias, D.A.; Mayer, M.U.; Gorby, Y.A.; Ni, S.; Lower, B.H.; Kennedy, D.W.;

552

Wunschel, D.S.; Mottaz, H.M.; Marshall, M.J., Hill, E.A.; Beliaev, A.S.; Zachara, J.M.; Fredrickson,

553

J.K.; Squier, T.C. Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two

554

outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1. J. Bacteriol. 2006, 188,

555

4705–4714. doi:10.1128/JB.01966-05.

556

(46) Myers, C.R. and Myers, J.M. Cell surface exposure of the outer membrane cytochromes of Shewanella

557

oneidensis

MR-1.

Lett.

Appl.

558

doi:10.1046/j.1472-765X.2003.01389.x.

Environ.

Microbiol.

2003,

37,

254–258.

559

(47) Myers, C.R. and Myers J.M. The outer membrane cytochromes of Shewanella oneidensis MR-1 are

560

lipoproteins. Lett. Appl. Microbiol. 2004, 39, 466–470. doi: 10.1111/j.1472-765X.2004.01611.x.

561

(48) Cutting, R.S.; Coker, V.S.; Fellowes, J.W.; Lloyd, J.R.; Vaughan, D.J. Mineralogical and morphological

562

constraints on the reduction of Fe(III) minerals by Geobacter sulfurreducens. Geochim.Cosmochimi.

563

Acta 2009, 73, 4004–4022. doi: 10.1016/j.gca.2009.04.009.

564

(49) Breuer M., Zarzycki P.; Shi L.; Clarke T.A.; Edwards M.J.; Butt J.N.; Richardson D.J.; Fredrickson

565

J.K.;Zachara J.M.; Blumberger J.; Rosso K.M. Molecular structure and free energy landscape for

566

electron transport in the decahaem cytochrome MtrF. Biochem. Soc. Trans. 2012, 40, 1198–1203. doi:

567

10.1042/bst20120139.

568

(50) Breuer, M.; Rosso, K.M.; Blumberger, J.; Butt, J.N. Multi-haem cytochromes in Shewanella oneidensis 22


Page 23 of 34 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


569

MR-1: Structures, functions and opportunities. J. R. Soc. Interface 2015, 12, 1117. doi:

570

10.1098/rsif.2014.1117.

571

(51) Liu, T.; Li, X.; Li, F.; Zhang, W.; Chen, M.; Zhou, S. Reduction of iron oxides by Klebsiella pneumoniae

572

L17: Kinetics and surface properties. Colloids Surf. A Physicochem. Eng. Asp. 2011, 379, 143–150.

573

doi:10.1016/j.colsurfa.2010.11.061.

574

(52) Fredrickson, J.K.; Zachara, J.M.; Kukkadapu, R.K.; Gorby, Y.A.; Smith, S.C.; Brown, C.F.

575

Biotransformation of Ni-substituted hydrous ferric oxide by an Fe(III)-reducing bacterium. Environ. Sci.

576

Technol. 2001, 35, 703–712. doi:10.1021/es001500v.

577 578

(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.

579

(54) Wagman, D.D.; Evans, W.H.; Parker, V.B.; Schumm, R.H.; Halow, I.; Bailey, S.M.; Churney, K.L.; Nuttal,

580

R.L. The NBS tables of chemical thermodynamic properties: selected values for inorganic and C1 and

581

C2 organic substances in SI units. J. Phys. Chem. 1982, 11, 1-392. doi:10.1063/1.555845.

582

(55) Hiemstra T. Formation, stability, and solubility of metal oxide nanoparticles: Surface entropy, enthalpy, and

583

free

energy

of

ferrihydrite.

584

10.1016/j.gca.2015.02.032.

Geochim.

Cosmochim.

Acta

2015,

158,

179–198.

doi:

585

(56) Diakonov, I.; Khodakovsky, I.; Schott, J.; Sergeeva, E. Thermodynamic properties of iron oxides and

586

hydroxides. I. Surface and bulk thermodynamic properties of goethite (α-FeOOH) up to 500 K. Eur. J.

587

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

23



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

24


37 37 37 38 39 40 41

Page 25 of 34 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


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



Page 26 of 34

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

26


Page 27 of 34 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


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

27



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


Page 28 of 34

Page 29 of 34

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


0

653 29



654 655

Figure 2.

656 657 658

30


Page 30 of 34

Page 31 of 34

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


3d 2d 1d

3d 2d 1d 0d

0d

1200

664

1000

800

600

400

1200

1000

Wavenumber (cm-1)

Wavenumber (cm-1)

665 666

31


400


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

Page 32 of 34

40

50

60

70

20

30

40

50

2 Theta (o)

2 Theta (o)

32


60

70

Page 33 of 34

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


(b)

Relative Intensity

25000

20000

Hematite

15000

10000

5000

0 0

1

2

3

4

5

6

Time (day)

674 (c)

Magnetite


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


9000

6000

3000

0

1

675 676

2

3

4

Time (day)

33


5

6


677 678 679

Table of Contents Fe(II) production

Secondary mineral formation

Fe2+

Goethite

Ferrihydrite

Hematite

eMagnetite

MtrCAB

680 681 682

34


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

Page 34 of 34

Dependence of Secondary Mineral Formation on Fe(II) Production

Recommend Documents