Supporting Information

Electron Transfer Between Sorbed Fe(II) and Structural Fe(III) in Smectites and its Effects

on Nitrate-Dependent Iron Oxidation by Pseudogulbenkiania sp. strain 2002

Li Zhang1, Hailiang Dong1,2*, Ravi K. Kukkadapu3*, Qusheng Jin4, and Libor Kovarik3

1Department of Geology and Environmental Earth Science

Miami University, Oxford, OH 45056, USA

2State Key Laboratory of Biogeology and Environmental Geology

China University of Geosciences, Beijing 100083, China

3EMSL, Pacific Northwest National Laboratory, Richland, WA 99352, USA

4Department of Earth Sciences, University of Oregon, Eugene, OR 97403

*Corresponding authors: Hailiang Dong

Tel: 513 529 2517; Fax: 513 529 1542; Email: dongh@miamioh.edu

Ravi Kukkadapu: 509-371-6384; Email: Ravi.Kukkadapu@pnnl.gov

Revised for Geochimica et Cosmochimica Acta

August 10, 2019

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S1 Measurements of Fe(II) and total Fe concentrations

To prevent any underestimation of Fe(II) concentration due to its rapid reaction with

nitrite under acidic conditions that is required for a typical Fe(II) assay (Zhao et al., 2013), nitrite

was separated from Fe(II)-bearing clay minerals by three washes with sterile and anoxic buffers

followed by centrifugation at 10,000 × g for 10 min. (Zhao et al., 2017). Fe(II) concentration was

measured colorimetrically with 1,10 phenanthroline (Amonette and Templeton, 1998) after clay

digestion with a combination of HF and H2SO4. Total Fe concentration was determined by first

reducing Fe(III) to Fe(II) with hydroxylamine hydrochloride and subsequently measured with the

same method. Fe(III) concentration was determined by the difference between total Fe and

Fe(II). To monitor any Fe(II) desorption from clay surface, time course change of aqueous Fe2+

concentration was measured on the supernatant following centrifugation (10,000 × g for 10 min).

The result showed that there was essentially no Fe(II) desorption under the conditions used in

this study.

S2 Biogeochemical Reaction Modeling

Model validation and sensitivity analysis

We validate the model of microbial iron oxidation using independent laboratory

observations – the observations that have not been applied in our model development.

Specifically, Weber and colleagues monitored the metabolism of strain 2002 in a medium

containing Fe2+ and nitrate (Weber et al., 2001). We apply the model by using the chemical

conditions of their experiments, and simulate the progress of Fe2+ oxidation, nitrate reduction,

and the growth of strain 2002. As shown in Fig. S5, by using a rate constant kFe of 5.0±0.7×106

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kg·gcdw-1·s1, the model describes well the progress of Fe2+, nitrate consumption and the growth of

strain 2002 observed in the laboratory experiments.

We also analyze the sensitivity of the model to microbial parameters. Following the

framework of metabolic control analysis (Fell, 1992), we calculate the sensitivity of microbial

iron reduction rate r to model parameter p according to

We compute the sensitivity by simulating the growth of strain 2002 in batch reactors that contain

3 mM Fe(II) and 6 mM nitrate at pH 6, the same conditions used in our experiments. According

to the modeling results, the rate is most sensitive to the rate constant kFe (a sensitivity of

0.94), but relatively insensitive to the growth yield Y, the specific maintenance rate D, or the

half-saturation constant KA for nitrate (a sensitivity of 0.01, 0.001, and 0, respectively).

Based on these results, we conclude that the progress of microbial iron oxidation observed in our

experiments can be applied to estimate the rate constant kFe of microbial iron oxidation.

Thermodynamic database

We added into the thermodynamic dataset the entries for ferrous iron sorbed onto the two

different sites, the basal plane and the edge site, of nontronite and montmorillonite, and saved the

dataset as thermo.com.V8.R6+.S2002.tdat. Specifically, the entry for ferrous iron sorbed on the

basal plan of montmorillonite is:

Montmor-6B type= Smectite formula= Fe.107Na.33Mg.33Al1.67Si4O10(OH)2.214 mole vol.= 0.0000 cc mole wt.= 376.6322 g 7 species in reaction -6.214 H+ .330 Mg++ .330 Na+

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1.670 Al+++ 4.214 H2O 4.000 SiO2(aq) .107 Fe++ 3.5459 2.4844 .4327 -1.7940 -4.1631 -6.1636 -7.9650 -9.7369

The entry for ferrous iron sorbed on the edge site is:

Montmor-8E type= Smectite formula= Fe.319Na.33Mg.33Al1.67Si4O10(OH)2.638 mole vol.= 0.0000 cc mole wt.= 395.6828 g 7 species in reaction -6.638 H+ .330 Mg++ .330 Na+ 1.670 Al+++ 4.638 H2O 4.000 SiO2(aq) .319 Fe++ 3.5459 2.4844 .4327 -1.7940 -4.1631 -6.1636 -7.9650 -9.7369

The entry for ferrous iron sorbed on the basal plan of nontronite is:

Nontronite-6B type= Smectite formula= Ca.165Fe2.152Al.33Si3.67H2.304O12.304 mole vol.= 131.1000 cc mole wt.= 437.9521 g 7 species in reaction -7.624 H+ .165 Ca++ .330 Al+++ 2.000 Fe+++ 3.670 SiO2(aq) 4.964 H2O 0.152 Fe++ -11.3915 -11.5822 -12.6234 -13.9486 -15.4751 -16.8671 -18.2346 -19.7093

The entry for ferrous iron sorbed on the edge site is:

Nontronite-8E type= Smectite formula= Ca.165Fe2.267Al.33Si3.67H2.534O12.534 mole vol.= 131.1000 cc mole wt.= 448.2861 g 7 species in reaction -7.854 H+ .165 Ca++ .330 Al+++ 2.000 Fe+++ 3.670 SiO2(aq) 5.194 H2O 0.267 Fe++ -11.3915 -11.5822 -12.6234 -13.9486 -15.4751 -16.8671 -18.2346 -19.7093

Input Scripts

The input script for simulating microbial oxidation of dissolved ferrous and reduction of

nitrate to nitrogen gas (N2) at pH 6:

data = thermo.com.V8.R6+.S2002.tdat

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time start = 0 day, end = 6 daytemperature = 25 Cdecouple ALLH2O = 1 free kgNa+ = 50 mmol/kgCl- = 50 mmol/kgbalance on Cl-Fe++ = 3 mmol/kgswap Goethite for Fe+++Goethite = .1 free mmol/kgNO3- = 6.14 mmol/kgNO2- = .01 mmol/kgpH = 6swap Acetate for Acetic_acid(aq)Acetate = .5 mmol/kgN2(aq) = .001 mmol/kgHCO3- = 1 mmol/kgkinetic microbe-1 rxn = "Fe++ + 1.5 H2O + .5 NO3- -> Goethite + 2 H+ + .5 NO2-" biomass = 100 rate_law = file "FeOx.bas" rate_con = 1.5e-8 KD = 2.0e-3 KA = 2.3e-6 mpower(NO3-) = 1 mpowerA(NO3-) = 1 growth_yield = 200 decay_con =1e-7

kinetic microbe-2 rxn = "Acetate + .6 H+ + 1.6 NO3- -> 2 HCO3- + .8 N2(aq) + .8 H2O" biomass = 50 rate_con = 1e-9 KA = 2.3e-6 mpower(NO3-) = 1 mpowerA(NO3-) = 1 ATP_energy = 1 ATP_number = -45 growth_yield = 11000 decay_con = 1e-7fix pHprecip = off

The rate law file "FeOx.bas":Fe_II = totmolal("Fe++")Threshold = KDFK = Fe_II*molality("NO3-")/(molality("NO3-")+KA)rate = Wmass*rate_con*biomass*FK*(1-QoverK)IF (Fe_II < Threshold) THEN rate = 0rprime0 = dndt0/(biomass0 * Wmass)rpave = (1.0-Theta)*rprime0 + Theta*rprimebiom = biomass0*exp((growth_yield*rpave-decay_con)*Deltat)IF biom < 0.0 THEN biom = 0.0setgwbvar("biomass", biom)end_block:return rprime * biomass * Wmass

Note that we use KD in the input file represents the threshold of ferrous iron oxidation. To

simulate the experiments at pH 8, we set pH at 8 and KD at 4.0e-4.

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To simulate microbial oxidation of ferrous iron sorbed onto the basal plan of

montmorillonite, we swapped Montmor-6B for aqueous ferrous iron and added the

corresponding aqueous components for the mineral:

pH = 6swap Montmor-6B for Fe++Montmor-6B = 28 free mmol/kgMg++ = .05 mmol/kgAl+++ = .05 mmol/kgSiO2(aq) = .05 mmol/kgprecip off

We also replace the entry for microbial reaction:

kinetic microbe-1 rxn = "Montmor-6B + .0535 NO3- -> .0535 H2O + Montmor-Na\ + .107 Goethite + .0535 NO2-"\ rate_law = file "Montmor-6B_OX.bas" biomass = 100 rate_con = 2.1e-8 KA = 2.3e-6\ KD = 2.03e-2 mpower(NO3-) = 1 mpowerA(NO3-) = 1 growth_yield = 39.4\ decay_con =1e-7

The rate law "Montmor-6B_OX.bas" contains the following entries:Fe_II = mass("Montmor-6B")*0.107Threshold = KD*0.107FK = Fe_II*molality("NO3-")/(molality("NO3-")+KA)rate = Wmass*rate_con*biomass*FK*(1-QoverK)IF (Fe_II < Threshold) THEN rate = 0rprime0 = dndt0/(biomass0 * Wmass)rpave = (1.0-Theta)*rprime0 + Theta*rprimebiom = biomass0*exp((growth_yield*rpave-decay_con)*Deltat)IF biom < 0.0 THEN biom = 0.0setgwbvar("biomass", biom)end_block:return rprime * biomass * Wmass

Note that the 0.107 mol ferrous iron is sobred onto the basal plane of 1 mol of montmorillonite

(see Table S1).

Likewise, we used the following entries to simulate microbial oxidation of ferrous iron

sorbed on the edge sites of montmorillonite:

pH = 8swap Montmor-8E for Fe++

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Montmor-8E = 9.5 free mmol/kgMg++ = .05 mmol/kgAl+++ = .05 mmol/kgSiO2(aq) = .05 mmol/kgprecip off

The entry for microbial reaction is

kinetic microbe-1 rxn = "Montmor-8E + .1595 NO3- -> .1595 H2O + Montmor-Na + .319 Goethite + .1595 NO2-" biomass = 100 rate_law = file "Montmor-8E_OX.bas.bas" rate_con = 3.1e-8 KA = 1e-5 KD = 2.0e-3 mpower(NO3-) = 1 mpowerA(NO3-) = 1 growth_yield = 63.8 decay_con = 1e-7

The rate law "Montmor-8E_OX.bas" contains the following entries:

Fe_II = mass("Montmor-8E")*0.319Threshold = KD*0.319FK = Fe_II*molality("NO3-")/(molality("NO3-")+KA)rate = Wmass*rate_con*biomass*FK*(1-QoverK)IF (Fe_II < Threshold) THEN rate = 0rprime0 = dndt0/(biomass0 * Wmass)rpave = (1.0-Theta)*rprime0 + Theta*rprimebiom = biomass0*exp((growth_yield*rpave-decay_con)*Deltat)IF biom < 0.0 THEN biom = 0.0setgwbvar("biomass", biom)end_block:return rprime * biomass * Wmass

To simulate microbial oxidation of ferrous iron sorbed onto the basal plan of nontronite,

we used the following entries:

pH = 6swap Nontronite-6B for Fe++Nontronite-6B = 21 free mmol/kgMg++ = .05 mmol/kgAl+++ = .05 mmol/kgSiO2(aq) = .05 mmol/kgkinetic microbe-1 rxn = " Nontronite-6B + .076 NO3- -> .076 H2O + \ Nontronite-Ca + .152 Goethite + .076 NO2-" \ rate_law = file "Nontronite-6B_OX.bas" biomass = 100 rate_con = 1.1e-8\ KA = 2.3e-6 KD = 9.92e-2 mpower(NO3-)=1 mpowerA(NO3-) = 1\ growth_yield = 30.4 decay_con = 1e-7

The rate law "Nontronite-6B_OX.bas" contains the following entries:Fe_II = mass("Nontronite-6B")

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Threshold = KD*0.152FK = Fe_II*molality("NO3-")/(molality("NO3-")+KA)rate = Wmass*rate_con*biomass*FK*(1-QoverK)IF (Fe_II < Threshold) THEN rate = 0rprime0 = dndt0/(biomass0 * Wmass)rpave = (1.0-Theta)*rprime0 + Theta*rprimebiom = biomass0*exp((growth_yield*rpave-decay_con)*Deltat)IF biom < 0.0 THEN biom = 0.0setgwbvar("biomass", biom)end_block:return rprime * biomass * Wmass

To simulate microbial oxidation of ferrous iron sorbed onto the edge sites of nontronite,

we used the following entries:

pH = 8swap Nontronite-8E for Fe++Nontronite-8E = 11.25 free mmol/kgMg++ = .05 mmol/kgAl+++ = .05 mmol/kgSiO2(aq) = .05 mmol/kgprecip offkinetic microbe-1 rxn = " Nontronite-8E + .1335 NO3- -> .1335 H2O + \ Nontronite-Ca + .267 Goethite + .1335 NO2-" \ rate_law = file "Nontronite-8E_OX.bas" biomass = 100 rate_con = 3.0e-8 \ KA = 2.3e-6 KD = 1.38e-2 mpower(NO3-)=1 mpowerA(NO3-) = 1\ growth_yield = 53.4 decay_con = 1e-7

The rate law "Nontronite-8E_OX.bas" contains the following entries:Fe_II = mass("Nontronite-8E")Threshold = KD*0.267FK = Fe_II*molality("NO3-")/(molality("NO3-")+KA)rate = Wmass*rate_con*biomass*FK*(1-QoverK)IF (Fe_II < Threshold) THEN rate = 0rprime0 = dndt0/(biomass0 * Wmass)rpave = (1.0-Theta)*rprime0 + Theta*rprimebiom = biomass0*exp((growth_yield*rpave-decay_con)*Deltat)IF biom < 0.0 THEN biom = 0.0setgwbvar("biomass", biom)end_block:return rprime * biomass * Wmass

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S3 Electron balance calculation

In order to determine the extent of interfacial electron transfer (IET) and help identify

various Fe(II) species as a result of IET, electron balance calculations were carried out using

published data.(Schaefer et al., 2011; Latta et al., 2017) The basic principle is that upon sorption

of Fe(II) to clay minerals, there is electron transfer between sorbed Fe(II) and structural Fe(III).

These two previous studies performed sorption of Mössbauer-invisible 56Fe(II) to NAu-2 at pH

7.5(Schaefer et al., 2011) and to montmorillonite at pH 4-7.5(Latta et al., 2017). The extent of

reduction of structural Fe(III) (of natural isotopic abundance) in these clay minerals was

monitored over time and a stoichiometric ratio between the amount of sorbed Fe(II) oxidation

and the amount of structural Fe(III) reduction was established. Although it was not explicitly

stated, this electron transfer process should be driven by a gradient in electron density. In other

words, if the clay minerals are fully reduced, there should not be any IET. When the NAu-2 or

SWy-2 structure has a certain amount of initial structural Fe(II), the amount of IET should be

proportionally decreased. No published data are available to perform electron balance calculation

for NAu-2 at pH 6. In the following paragraphs, we describe electron balance calculation for the

other three cases (NAu-2 pH 8, SWy-2 pH 6 & 8).

Schaefer et al. reported that at a similar pH (7.5) reduction of structural Fe(III) in NAu-2

by sorbed Fe(II) is a 1:1 stoichiometric ratio when sorbed Fe(II)/Total Fe in clay is < 15%.

(Schaefer et al., 2011) Our sorbed Fe(II)/total Fe ratio in NAu-2 is 12.6% (e.g.,

(3.51-0.583)%/(0.583+22.6)%, Table 1, here total Fe in NAu-2 is before sorption), thus, after

IET, the Fe(II)/total Fe ratio in the NAu-2 structure should be ~12.6% (or 2.93% Fe(II) by clay

weight or 2.93/(3.51+22.0) = 11.5% by Fe(II)/total Fe ratio, Table S3, here total Fe in NAu-2 is

after sorption to make it comparable to Mössbauer-fitted ratio). Considering that an initial Fe(II)

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present in the pristine NAu-2 (0.583% by weight), 2.35% structural Fe(II) (or 9.21% Fe(II)/total

Fe ratio) should be newly produced, and the other 0.580% (or 2.27% Fe(II)/total Fe ratio) should

remain sorbed. Thus the structural Fe(II)/total Fe ratio in the NAu-2 structure (11.5%) is fairly

consistent with our Mössbauer fitted ratio of 9.8% for structural Fe(II) (Table S2). Residual

sorbed Fe(II) (2.27%) should be associated with newly generated goethite and magnetite.

For SWy-2 at pH 6, the ratio of sorbed Fe(II)/total Fe in SWy-2 is

(1.63-0.202)/(0.202+2.88) = 46.4%, which, according to the stoichiometric reaction between

sorbed Fe(II) and structural Fe(III) in SWy-2(Latta et al., 2017) should produce ~46.4%

Fe(II)/total Fe in the SWy-2 structure (or 1.43 % Fe(II) by clay weight). Considering there is

0.202% initial Fe(II) in the original SWy-2 (Table 1), 1.23% structural Fe(II) should be newly

generated through reduction of structural Fe(III) by sorbed Fe(II) through IET. Therefore, 1.23%

sorbed Fe(II) should have been consumed to reduce structural Fe(III), which, together with the

initial amount of Fe(II) in the SWy-2 structure (0.202% wt.), should produce ~32.1% Fe(II) [e.g.,

1.43/(1.63+2.83) = 32.1% Fe(II)/total Fe ratio], which is close to our Mössbauer fitted value of

38.4% (150 K) or 41.7% (12 K). Correspondingly the amount of sextet production should be

~32.1%, however, for this sample, the pristine SWy-2 also has some sextet to begin with,

therefore the Mössbauer fitted value of sextet area is greater than 32.1%. A small fraction of

original sorbed-Fe(II) is not oxidized and should remain sorbed (onto goethite or the

nanoparticles).

For SWy-2 at pH 8, using the same method, we estimate that approximately 70%

structural Fe(II) can be achieved from 151% sorbed Fe/total Fe ratio (e.g.,

(4.86-0.202)/(0.202+2.88) = 151%), which amounts to 2.16% structural Fe(II) by weight or

28.4% in terms of Fe(II)/total Fe ratio (Table S3). Therefore, the amount of sorbed Fe(II) that is

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not oxidized should be 4.86-2.16 = 2.70% by weight or 35.5% in terms of Fe(II)/total Fe ratio).

Considering there is an initial amount of Fe(II) in SWy-2 (0.202 %), the amount of new sextet

formation from the oxidation of a fraction of sorbed Fe(II) should be 2.16-0.202 = 1.96% (or

1.96/7.61 = 25.8% in terms of Fe(II)/total Fe ratio), which is higher than the Mossbauer-derived

fitting value of 16.8% (Table S2), suggesting that some newly formed Fe(III) may not be present

in sextet, but instead as a doublet. The residual amount of Fe(II) (2.70% by weight or 35.5% in

terms of Fe(II)/total Fe ratio) occurs as the inner doublet (the mixed Fe(II)-Fe(III) nanoparticles)

and is magnetically ordered at 12 K (Table S2).

In summary, 80.2% of the Fe(II) sorbed to NAu-2 at pH 8 (2.35/2.93 = 80.2% in Table

S3) is oxidized to goethite and a mixed-valence magnetite phase. However, the trend of

oxidation of sorbed-Fe(II) is opposite for SWy-2: 86.0% (1.23/1.43 = 86.0%) of the Fe(II)

sorbed to SWy-2 is oxidized to goethite and nanoparticles at pH 6, but only 42% (1.96/4.66 =

42.1%) at pH 8 is to oxidized goethite and the remaining Fe(II) occurred as highly reactive

Fe(II)-Fe(III) nanoparticles (either sorbed or structural).

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Table S1 Summary of studies on Fe2+ sorption capacity onto various clay minerals

Clay Mineral pH Method to measure Fe2+

Fe2+ Sorption capacity (mmol/g)

Reference

NAu-2 6.0 1,10-phen 0.25 This study8.0 0.52

SWy-2 6.0 0.268.0 0.84

NAu-2 6.0 Ferrozine ~0.22 (Jaisi et al., 2008)(Jaisi et al., 2008)

8.0 ~0.22

SYn-1 (0% wt. Fe(III)) 4.0 0.0477 (Neumann et al., 2013)(Neumann et al., 2013)

7.5 0.312NAu-1 4.0 0.241

6.0 0.2826.5 0.3277.5 0.920

NAu-1 6.0 1,10-phen 0.364 (calculated average from time course)

(Neumann et al., 2015)(Neumann et al., 2015)

7.5 0.907NAu-2 6.0 0.637

7.5 0.955SWa-1 (15.4% wt. Fe(III))

6.9 0.0222

MAu-1(2.1% wt. Fe(III)) 6 ICP-OES ~0.15 (Tsarev et al., 2016)(Tsarev et al., 2016)

8 ~1NAu-1(25.2% wt. Fe(III)) 6 ~0.15

8 ~1NAu-2 (26.5% wt. Fe(III))

6 ~0.15

8 ~1

SWy-2 4.0 1,10-phen 0.142 (Latta et al., 2017)(Latta et al., 2017)

6.0 0.3637.0 0.5847.5 0.954

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Table S2 Mössbauer spectrum parameters

Sample Temp Site CS1 QS2 σQS3 ε4 HFD5 σHFD

6 %7 χ2 8 Chemical9

(t final) (mm/sec)

(mm/sec)

(mm/sec)

(mm/sec)

(Tesla) (Tesla) (%)

NAu-2 12 K Fe(III)-1 0.48 0.35 0.3 __ __ __ 65.9 0.83 97.4Fe(III)-1' 0.53 1.24 0.06 __ __ __ 29.3Fe(III)-tet 0.42 0.5 0.09 __ __ __ 5.1

NAu-2 Fe(II) 12 K Fe(II)-1 1.25 2.68 0.125 __ __ __ 8.6 0.98 8.2pH6 Fe(III)-1 0.51 0.36 0.19 __ __ __ 68.5 91.8

Fe(III)-1' 0.59 1.03 0.35 __ __ __ 28.5

NAu-2 Fe(II) 12 K Fe(II)-1 1.27 2.68 0.07 __ __ __ 8.5 1.3 6.6bacteria Fe(III)-1 0.5 0.33 0.17 __ __ __ 65.5pH6 Fe(III)-1' 0.48 1.03 0.17 __ __ __ 20.8 93.4

lepidocrocite 0.39 __ __ 0.18 43.9 3 5.2

NAu-2 Fe(II) 12 K Fe(II)Fe(III)

1.22 2.86 0.1 __ __ __ 9.8 0.95 13.8pH8 0.47 0.46 0.29 __ __ __ 55.2 86.2

Ferrihydrite-like Fe

oxides 0.48 __ __ -0.11 39.31 21.3 35.0

NAu-2 Fe(II) 12 K Fe(II)-1 1.22 2.8 0.0008 __ __ __ 5.4 0.96 4.6bacteria Fe(III)-1 0.48 0.47 0.3 __ __ __ 57.5 95.4pH8 Ferrihydrite-like Fe

oxides 0.51 __ __ -0.14 40.5 21.7 37.1

SWy-2 Fe(II) 150 K Fe(II)-1 1.27 3.03 0.18 __ __ __ 38.4 0.82 36.6pH6 Fe(III) 0.32 0.33 0.18 __ __ __ 18.5 63.4

Goethite + clay-Fe(III) 0.52 __ __ -0.05 44.7 3.08 43.4

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12 K Fe(II)-1 1.28 3.05 0.11 __ __ __ 41.7 0.93 36.6Fe(III) 0.35 0.43 0.26 __ __ __ 14.9 63.4Goethite + clay-Fe(III) 0.48 __ __ -0.11 49.5 0.75 43.4

SWy-2 Fe(II) 150 K Fe(II)-1 1.25 3.05 0.18 __ __ __ 26.2 1.7 25.8bacteria Fe(III) 0.48 0.71 0.31 __ __ __ 44.6 74.2pH6 Goethite + clay-Fe(III) 0.49 __ __ -0.07 43.2 6.92 29.2

12 K Fe(II)-1 1.28 3.08 0.13 __ __ __ 24.2 1.8 25.8Fe(III) 0.49 0.69 0.39 __ __ __ 22.7 74.2Goethite + clay-Fe(III)-1 0.5 __ __ -0.09 49 1.5 24.3Goethite + clay-Fe(III)-2 0.42 __ __ 0.02 47.4 7.1 28.8

SWy-2 Fe(II) 150 K Fe(II)-1 1.27 2.85 0.22 __ __ __ 38.9 0.8 63.9pH8 Fe(II)-2 1.35 2.07 0.12 __ __ __ 33

Fe(III) 0.55 0.55 0.41 __ __ __ 14.4 36.1Goethite + clay-Fe(III) -0.09 __ __ -0.09 45.7 3 13.7

__ __ __12 K Fe(II)-1 1.29 2.83 0.1 42.2 0.85 63.9

Fe(II)-2-magnetic 1.35 __ __ 1.04 18.1 0.01 28Fe(III) 0.44 0.6 0.2 __ __ __ 13 36.1Goethite + clay-Fe(III) 0.41 __ __ 0.007 49.2 2 16.8

SWy-2 Fe(II) 150 K Fe(II)-1 1.22 2.96 0.3 __ __ __ 10.1 1.2 13.3bacteria Fe(III) 0.49 0.59 0.29 __ __ __ 35.5 86.7pH8 Goethite + clay-Fe(III) 0.46 __ __ 0.14 46.3 2.3 54.4

12 K Fe(II) -1 1.33 3.1 0.3 __ __ __ 11.1 0.9 13.3Fe(III) 0.52 0.58 0.29 __ __ __ 30.1 86.7Goethite + clay-Fe(III) 0.48 __ __ -0.12 49.6 1.3 58.2

1center shift; 2quadrupole splitting (QS); 3QS standard deviation;4 quadrupole splitting parameter; 5magnetic hyperfine field (HFD);6HFD standard deviation; 7relative contribution (assuming identical recoilless fractions for all the species);8goodness of fit; 9Chemical method can only determine total Fe(II) and Fe(III) contents, not contents of individual Fe species.

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Table S3 Summary of electron balance calculations following Fe2+ sorption

Clay pH [Fe(II)initially sorbed]

(wt.% in clay or

mmol/g)

[Fe(II)Initially sorbed]/Total Fein clay1

(%)

[Structural Fe(II)]/Total Fe

in clay after IET2

(%)

Structural Fe(II) after IET

Initial clay structural

Fe(II) 7

(wt.% in clay or

mmol/g)

Newly produced clay structural Fe(II)

from IET

Residual fraction of the sorbed Fe(II)

after IET

5(wt.% in clay or mmol/g)

6(% of total Fe)

8(wt.% in clay or mmol/g)

9(% in total Fe)

10(wt.% in clay or mmol/g)

11(% in total Fe)

NAu-2 8.0 2.93 (0.525)

12.6 ~12.63 2.93 (0.525)

11.5 0.583 (0.104)

2.35 (0.421)

9.23 0.580 (0.104)

2.27

SWy-2 6.0 1.43 (0.256)

46.4 ~46.44 1.43 (0.256)

32.1 0.202 (0.036)

1.23 (0.220)

27.6 0.200 (0.036)

4.48

8.0 4.66

(0.835)

151 ~704 2.16

(0.387)

28.4 0.202

(0.036)

1.96

(0.351)

25.8 2.70

(0.484)

35.5

Fe(II) in clay is expressed in both wt% and mmol/g (in parenthesis). Fe(II) relative to total Fe is in % only. 1Calculted using data in Table 1 as sorbed Fe(II) divided by total Fe in pre-sorption clay;2 This column of data is obtained based on references on stoichiometric electron transfer between sorbed Fe(II) and structural Fe(III).3 based on data in Schaefer et al., for pH 7.5 (their Fig. 4)(Schaefer et al., 2011); 4Latta et al. (their Fig. 8)(Latta et al., 2017); 5Calculated by multiplying [structural Fe(II)]/Total Fe in clay (column 5) by Total Fe in pre-sorption clay (Table 1, last column); 6Calculated by dividing structural Fe(II) by total Fe in clay (with Fe2+ sorption); 7Initial Fe(II) in each clay (Table 1); 8Calculated by the difference between the total Fe(II) and initial amount of Fe(II); 9Calculated by dividing Fe(II)8 by total Fe in clay (with Fe2+ sorption); 10Calculated by the difference between the total amount of sorbed Fe(II) and the amount used to reduce structural Fe(III)8; 11Calculated by dividing Fe(II)10 by total Fe in clay (with Fe2+ sorption).

S15

332

333

334335336337338339340

Fig. S1. Nitrate reduction during the growth of strain 2002 in the presence of either acetate or

aqueous Fe2+ (10 mM) as electron donor.

S16

341

342

343

344

Fig. S2. Nitrate reduction by strain 2002 cells at pH 6 & 8. Electron donor is inferred to be from

stored carbon within cells.

S17

345

346

347

348

-20 0 20 40 60 80 100 120 140 1600.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Nitr

ite/m

M

Time/h

NAu-2 pH 8 (HEPES) SWy-2 pH 8 (HEPES) NAu-2 pH 6.1 (PIPES) SWy-2 pH 6.1 (PIPES)

Fig. S3. Negligible amounts of abiotic nitrite reduction by clay-associated Fe(II) at pH 6 and pH

8.

S18

349

350

351

352

Fig. S4 Time-course change of aqueous Fe2+ concentration in the supernatant of bio-oxidation

experiments with clay-associated Fe(II). Total Fe(II) concentration is shown in Fig. 1A, D, and

G.

S19

353

354

355

356

357

358

0 2 4 6Time (d)

0.0

0.2

0.4

0.6

Bio

mas

s (m

gkg

1)

B

0

5

10

Fe2+

(mM

)

1

2

3

4

Nitr

ate

(mM

)

A

Fe2+

Nitrate

Fig. S5. Variations in the concentrations of Fe(II) and nitrate (A), and biomass (B) during the

growth of strain 2002 in laboratory batch reactors. Data points are from a previous study (Weber

et al., 2006) and lines are the modeling results.

S20

359

360

361

362

363

364

Fig. S6 TEM images for SWy-2 at pH 8 after Fe(II) sorption (A) and after bio-oxidation (B): A)

before oxidation, amorphous nanoparticles are located on the edge of clay; B) after bio-

oxidation, goethite are still present with a bundle-shaped morphology, but the nanoparticles are

absent.

S21

365

366

367

368

369

370

Fig. S7 Room temperature (RT, A) and 150 K (B-E) Mössbauer spectra of pristine, Fe(II)-

sorbed, and Fe(II)-sorbed, bio-oxidized NAu-2 samples at pH 6 and pH 8.

S22

371

372

373

Fig. S8 150-K Mössbauer spectra of pristine (A), Fe(II)-sorbed (B), and Fe(II)-sorbed, bio-

oxidized SWy-2 (C) samples at pH 6. At pH 8, Mössbauer spectra for the latter two samples are

shown (D and E, respectively).

S23

374

375

376

377

378

References

Amonette J. E. and Templeton J. C. (1998) Improvements to the quantitative assay of nonrefractory minerals for Fe (II) and total Fe using 1, 10-phenanthroline. Clays Clay Miner. 46, 51–62.

Fell D. A. (1992) Metabolic control analysis: a survey of its theoretical and experimental development. Biochem. J. 286, 313–330.

Jaisi D. P., Liu C., Dong H., Blake R. E. and Fein J. B. (2008) Fe2+ sorption onto nontronite (NAu-2). Geochim. Cosmochim. Acta 72, 5361–5371.

Latta D. E., Neumann A., Premaratne W. A. P. J. and Scherer M. M. (2017) Fe(II)–Fe(III) Electron Transfer in a Clay Mineral with Low Fe Content. ACS Earth Space Chem. 1, 197–208.

Neumann A., Olson T. L. and Scherer M. M. (2013) Spectroscopic Evidence for Fe(II)–Fe(III) Electron Transfer at Clay Mineral Edge and Basal Sites. Environ. Sci. Technol. 47, 6969–6977.

Neumann A., Wu L., Li W., Beard B. L., Johnson C. M., Rosso K. M., Frierdich A. J. and Scherer M. M. (2015) Atom Exchange between Aqueous Fe(II) and Structural Fe in Clay Minerals. Environ. Sci. Technol. 49, 2786–2795.

Schaefer M. V., Gorski C. A. and Scherer M. M. (2011) Spectroscopic Evidence for Interfacial Fe(II)−Fe(III) Electron Transfer in a Clay Mineral. Environ. Sci. Technol. 45, 540–545.

Tsarev S., Waite T. D. and Collins R. N. (2016) Uranium Reduction by Fe(II) in the Presence of Montmorillonite and Nontronite. Environ. Sci. Technol. 50, 8223–8230.

Weber K. A., Picardal F. W. and Roden E. E. (2001) Microbially Catalyzed Nitrate-Dependent Oxidation of Biogenic Solid-Phase Fe(II) Compounds. Environ. Sci. Technol. 35, 1644–1650.

Weber K. A., Pollock J., Cole K. A., O’Connor S. M., Achenbach L. A. and Coates J. D. (2006) Anaerobic Nitrate-Dependent Iron(II) Bio-Oxidation by a Novel Lithoautotrophic Betaproteobacterium, Strain 2002. Appl. Environ. Microbiol. 72, 686–694.

Zhao L., Dong H., Edelmann R. E., Zeng Q. and Agrawal A. (2017) Coupling of Fe(II) oxidation in illite with nitrate reduction and its role in clay mineral transformation. Geochim. Cosmochim. Acta 200, 353–366.

Zhao L., Dong H., Kukkadapu R., Agrawal A., Liu D., Zhang J. and Edelmann R. E. (2013) Biological oxidation of Fe(II) in reduced nontronite coupled with nitrate reduction by Pseudogulbenkiania sp. Strain 2002. Geochim. Cosmochim. Acta 119, 231–247.

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392393394

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399400401

402403404

405406407

408409410

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