es3049459 1..10 - es3049459.pdf

Ligand-Enhanced Abiotic Iron Oxidation and the E

ff

ects of Chemical

versus Biological Iron Cycling in Anoxic Environments

Sebastian H. Kopf,

†

Cynthia Henny,

and Dianne K. Newman

†

‡

∥

†

Division of Geologial and Planetary Sciences and

‡

Division of Biology, California Institute of Technology, Pasadena, California,

United States

Research Center for Limnology, LIPI, Cibinong, Indonesia

∥

Howard Hughes Medical Institute, Pasadena, California, United States

*

Supporting Information

ABSTRACT:

This study introduces a newly isolated, genet-

ically tractable bacterium (

Pseudogulbenkiania

sp. strain MAI-1)

and explores the extent to which its nitrate-dependent iron-

oxidation activity is directly biologically catalyzed. Speci

cally,

we focused on the role of iron chelating ligands in promoting

chemical oxidation of Fe(II) by nitrite under anoxic conditions.

Strong organic ligands such as nitrilotriacetate and citrate can

substantially enhance chemical oxidation of Fe(II) by nitrite at

circumneutral pH. We show that strain MAI-1 exhibits

unambiguous biological Fe(II) oxidation despite a signi

cant

contribution (

∼

−

35%) from ligand-enhanced chemical

oxidation. Our work with the model denitrifying strain

Paracoccus denitri

cans

further shows that ligand-enhanced

chemical oxidation of Fe(II) by microbially produced nitrite can be an important general side e

ect of biological denitri

cation.

Our assessment of reaction rates derived from literature reports of anaerobic Fe(II) oxidation, both chemical and biological,

highlights the potential competition and likely co-occurrence of chemical Fe(II) oxidation (mediated by microbial production of

nitrite) and truly biological Fe(II) oxidation.

■

INTRODUCTION

Fe(II)/Fe(III) is an important redox couple in natural

environments.

In anoxic systems, iron oxidation can be

mediated by several biological agents, such as anoxygenic

phototrophs

2,3

and nitrate-dependent chemotrophs.

4,5

While

the enzymatic machinery for Fe(II) oxidation has been

identi

ed and characterized for two anoxygenic photo-

trophs,

3,6,7

comparable catalysts have not yet been identi

for nitrate-dependent chemotrophs. Toward this end, we

isolated a fast growing Fe(II) oxidizing, nitrate-dependent

chemotroph from the iron-rich tropical Lake Matano,

with the

intention of developing it into a model genetic system.

However, work with the isolate highlighted a second, often

overlooked aspect of Fe(II) oxidation in anoxic environments:

direct chemical interaction with nitrite (a form of chemo-

denitri

cation

). Being able to distinguish the mechanisms and

turnover rates of direct biological versus abiotic components of

anaerobic Fe(II) oxidation is necessary to gain a complete

understanding of the biogeochemical coupling of the N and Fe

redox cycles. Here, we expand our understanding of chemo-

denitri

cation by experimental elucidation of how organic

ligands promote abiotic Fe(II) oxidation by nitrite, and discuss

its relevance to assessing the potential co-occurrence of

chemical and biological Fe(II) oxidation.

The isolation and characterization of an increasing number of

microorganisms capable of nitrate-dependent anaerobic Fe(II)

oxidation in recent years

4,5,10

−

has revealed the potential for

chemotrophic recycling of Fe(II) in anoxic systems. However,

deconvolving the chemical and biological aspects of this process

remains challenging in many environmental settings

17,18

and

even laboratory studies.

19,20

The complication arises whenever

denitrifying organisms reduce nitrate in iron-rich anoxic

systems, where the metabolic intermediate nitrite can oxidize

Fe(II).

−

This was recently highlighted in a review by

Picardal,

which underscored that while biologically induced

(through the production of nitrite during biological denitri

cation), Fe(II) oxidation can be abiotically catalyzed and

proceed by chemodenitri

cation. Because Fe(II) oxidation may

also be directly catalyzed by (potentially the same) denitrifying

organisms, two competing pathways exist whose precise

mechanisms and relative importance in nature are poorly

understood. While the physiology of nitrate-dependent Fe(II)-

oxidizing bacteria has been the subject of a growing number of

studies,

16,24,28

−

the chemical aspect of anaerobic Fe(II)

Received:

March 22, 2012

Revised:

February 6, 2013

Accepted:

February 12, 2013

Published:

February 12, 2013

Article

pubs.acs.org/est

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Environ. Sci. Technol.

2013, 47, 2602

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oxidation by nitrite has received less attention,

27,31

despite its

relevance to constraining the extent of its microbial counter-

part.

Rapid oxidation of Fe(II) by nitrite in strongly acidic

conditions was described as early as 1936,

with high reaction

rates linked to the generation and subsequent degradation of

nitrous acid (p

= 3.4). At circumneutral pH, nitrite is stable

and anaerobic Fe(II) oxidation requires a catalyst or suitable

Fe(II)-containing mineral to proceed at appreciable rates.

Acceleration of this process has been reported with a number of

speci

c Fe(II) mineral phases and catalysts, such as Cu

iron

oxides and hydroxides,

31,34

−

green rust,

25,38

as well as

siderite

and vivianite,

and even microbial surfaces,

providing possible reaction mechanisms for Fe(II)-oxidizing

chemodenitri

cation. The same is true for nitrate, which is

generally less reactive toward Fe(II) than nitrite at circum-

neutral pH,

but can similarly bene

t from metal and mineral

catalysis.

41,42

However, metals and surfaces are not the only

agents for chemical catalysis. While the kinetic e

ects of ligands

(including EDTA, NTA, and citrate) on iron redox processes in

oxic environments have been explored before

−

and often

lead to acceleration of Fe(II) oxidation, much less is known

about their e

ects in the absence of molecular oxygen. Several

studies have investigated the e

ect of ligands on iron redox

processes in acidic conditions and solvents,

47,48

but with the

notable exception of studies on microbial Fe(II) oxidation in

the presence of EDTA,

13,49

little is known about the impact of

ligands at circumneutral pH.

Here, we investigate the e

ect of several Fe(II)-chelating

ligands on iron-oxidizing chemodenitri

cation to (1) assess true

biological Fe(II) oxidation in the newly isolated

-proteobacte-

rium

Pseudogulbenkiania

sp. strain MAI-1 and (2) elucidate the

role ligands could play more generally in abiotic Fe(II)

oxidation in laboratory and environmental settings. We use

Paracoccus denitri

cans

as a model strain to show how Fe(II)

oxidation can appear to be directly biologically catalyzed when,

in fact, much of this activity may only be indirectly biologically

mediated. We describe the kinetics and potential reaction

mechanism of the chemical oxidation of Fe(II) by nitrite

observed in these experiments and discuss their relevance for

the interpretation of laboratory and environmental studies. We

place our

ndings in the context of chemical and biological

oxidation rates reported in the literature to evaluate their

relative importance in anaerobic Fe(II) oxidation.

■

MATERIALS AND METHODS

Media.

All reagent solutions were autoclaved or

lter-

sterilized prior to use. The basal medium for all experiments

was a freshwater medium containing 500 mg/L MgSO

300 mg/L NH

Cl, 100 mg/L CaCl

O, and 5.4 mg/L

. For microbial cultures, the medium was

amended with a 1000

vitamin mix (

nal concentrations in

the medium: 40

g/L 4-aminobenzoic acid, 10

g/L D-biotin,

100

g/L nicotinic acid, 50

g/L Ca pantothenate, 100

g/L

pyridoxamine

2HCl, 100

g/L thiamine

2Cl) and a 1000

trace element solution (

nal concentrations in the medium: 1.1

mg/L FeSO

O, 42

g/L ZnCl

,50

g/L MnCl

190

g/L CoCl

O, 2

g/L CuCl

O, 24

g/L

NiCl

O, 18

g/L Na

MoO

O, 300

g/L H

For aerobic cultures, the medium was bu

ered to pH 7.2 with

20 mM phosphate. For anoxic experiments, the medium was

pH bu

ered with 22 mM NaHCO

and adjusted to pH 7 with

1 M HCl under an oxygen free atmosphere containing 15%

. Phosphate addition was minimal (but not microbially

growth inhibiting) to avoid

precipitation of vivianite

(Fe

(PO

)

O) at high Fe(II) concentrations. The

nal

ionic strength was

∼

0.04 M. Anoxic solutions were prepared

using O

-free deionized water and stored anoxically for at least

three days prior to use. Reactant solutions containing nitrite

were always prepared fresh from an anoxic stock solution kept

at pH 11 to avoid degradation through self-decomposition. All

glassware and plastics were autoclaved and stored anoxically for

at least three days prior to use.

Bacterial Strains.

Paracoccus denitri

cans

strain ATCC

19367 was obtained from the United States Department of

Agriculture culture collection and was grown routinely in

anoxic freshwater medium under denitrifying conditions with

succinate as the growth substrate.

Pseudogulbenkiania

sp. strain

MAI-1 is a newly isolated

-proteobacterium that was routinely

growninanoxicfreshwatermediumunderdenitrifying

conditions with acetate as the growth substrate.

Isolation.

Cultures of anaerobic Fe(II) oxidizing chemo-

trophs were enriched by inoculating freshwater medium

supplemented with 10 mM FeCl

,10mMNa

NTA, 2 mM

Na acetate, and 5 mM NaNO

with samples from a microbial

mat in the litterol zone of iron-rich tropical Lake Matano,

Sulawesi Island, Indonesia.

Enrichments were incubated at 30

C in the dark. After a few days, some enrichments developed

the characteristic dark green color of Fe(III)-NTA, indicating

Fe(II) oxidation. Cultures exhibiting fast Fe(II) oxidation were

transferred successively to fresh Fe(II)-containing medium.

After four transfers, serial dilutions of enrichments were plated

on YP agar pates (0.3% yeast extract, 0.3% Difco Bacto

Peptone, 1.2% agarose) and incubated aerobically at 30

Cin

the dark to identify strains potentially suitable for genetic

manipulation. Colonies were picked and subcultured in the

Fe(II) enrichment medium. Fast Fe(II) oxidizers were plated

again, and the purity was assessed by phase-contrast

microscopy. The 1497-bp 16S rRNA gene sequence of strain

MAI-1 was deposited in the GenBank database under the

accession number HQ714499. The pure strain was deposited

with the American Type Culture Collection under the ATCC

number BAA-2177.

Analytical Techniques.

The concentration of Fe(II) was

determined colorimetrically at 562 nm using the ferrozine [3-

(2-pyridyl)-5,6 bis(4-phenylsulfonic acid)-1,2,4-triazine, mono-

sodium salt] assay

without prior acidi

cation of analyte.

Sample acidi

cation in the presence of nitrite led to

underestimation of Fe(II) concentrations

and was therefore

avoided (see Supporting Information Figure S4). The assay was

calibrated using ferrous ammonium sulfate hexahydrate of

known concentration. Nitrite was determined colorimetrically

at 520 nm using sulfanilamide and N-1-napthylethylenediamine

dihydrochloride.

The chelator EDTA is incompatible with

this assay,

but none of the ligands used in this study interfere

with nitrite determination (Supporting Information Figure S5).

The assay was calibrated using a commercial nitrite standard

(Fluka Analytical TraceCERT). Samples for Fe(II) and nitrite

determination in microbial cultures were obtained with a sterile

disposable syringe

ushed for 30 s with 20%CO

/80%N

. The

evolution of N

O in abiotic reactions was assessed qualitatively

by gas-chromatography using a Hewlett-Packard 5890 Series II

Plus Gas Chromatograph equipped with a Thermal Con-

ductivity Detector. Samples were injected onto a HP-MOLSIV

column (30m, 0.32 mm inner diameter (ID), 12

lm) and

eluted with helium at a

ow rate of 10 mL/min using a

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temperature gradient from 35 to 240

C (4 min at 35

C, 35

C/min up to 140

C, 25

C/min up to 240

C). Formation of

the nitrosyliron-NTA complex (Fe(II)-NTA-NO) was assessed

qualitatively by monitoring its characteristic absorption peaks

(440 nm and 600 nm)

54,55

spectroscopically. Growth of

microbial cultures was followed by optical density at 600 nm

(OD

600

) in cultures without iron and at 700 nm (OD

700

)in

cultures with iron. This wavelength was used to decrease

distortion by Fe(III)-NTA, which absorbs strongly at 600 nm.

700

underestimates optical density as compared to OD

600

Experimental Procedure.

Kinetic Fe(II) oxidation experi-

ments were conducted inside an anaerobic chamber (Coy

Laboratory Products, Inc.) equipped with palladium catalysts

for O

removal. The chamber contained

∼

3%H

/15% CO

82% N

and experiments were performed at 25

C using a

digital heat block. Samples were taken at varying time points

and analyzed immediately for Fe(II) and nitrite concentrations

using a BioTek Synergy 4 Microplate Reader housed inside the

chamber. Oxidation experiments were conducted in sterile basal

freshwater medium containing 2 mM Fe(II) and 2 mM NO

−

and were amended alternatively with 2 mM nitrilotriacetate

(NTA), 300 mg/L Pahokee Peat Humic Acid (PPHA,

International Humic Substances Society), 0.1, 0.5, or 2 mM

citrate, 300 mg/L PPHA + 2 mM citrate. PPHA was selected as

the humic acid of choice due to its high solubility and low

capacity for storing redox equivalents that could rereduce

Fe(III) and interfere with the experiment.

Control experi-

ments included incubations of Fe(II) with or without NTA in

the absence of nitrite or in the presence of 2 mM nitrate. pH

was measured at the beginning and conclusion of each

experiment.

Pseudogulbenkiania

sp. strain MAI was grown in triplicate at

C in the dark in freshwater medium amended with 0.5 mM

acetate, 4 mM Fe(II), and 8 mM NTA, and a headspace of

∼

3%H

/15% CO

/82% N

. Cultures were sampled regularly

for nitrite accumulation and Fe(II) oxidation.

Paracoccus denitri

cans

was grown in triplicate at 30

C in the

dark in freshwater medium amended with 10 mM succinate and

20 mM nitrate and sampled regularly for nitrite accumulation.

Upon reaching a nitrite concentration of

∼

5 mM, 5 mL of each

culture was withdrawn and processed anaerobically as follows:

each withdrawn sample was divided in four. Two aliquots were

left unchanged while the other two were

lter sterilized using a

0.2

m syringe

lter. All aliquots were spiked with

∼

5mM

Fe(II) and one of each set (one un

ltered

P. denitri

cans

and

one

lter-sterilized aliquot) was further amended with 10 mM

citrate (all from 1 M stock solutions to avoid sample dilution).

No citrate was present in cultures prior to spiking. Aliquots

were incubated at 25

C for 4 h and sampled at regular intervals

as described in the kinetic Fe(II) oxidation experiments. The

remaining cultures were reincubated at 30

C for continued

monitoring of growth and nitrite accumulation.

Computation.

Nonlinear least-squares model

ts and

parameter estimates for kinetic data were computed using the

statistical model analysis functionality provided by Wolfram

Mathematica

(v. 8.0). Fe(II) speciation in solution was

estimated using the Visual MINTEQ equilibrium speciation

model (v. 3.0) with stability constants provided by King

(Fe(II)-carbonate complexes) and the MINTEQ database

(all other Fe(II) species) and precomputed humic substance

properties based on the NICA-Donnan model.

Chemical

oxidation of Fe(II) with nitrite produced by MAI-1 was

modeled using Euler

’

s method to calculate stepwise solutions of

eq 5. Nitrite concentrations at each time step were calculated

by linear interpolation between closest measurement time

points. Chemical oxidation with concomitant biological NO

consumption was modeled by assuming complete NO removal

and subsequent lack of Fe(II)-NTA-NO complex formation.

■

RESULTS

The enrichment of fast growing anaerobic Fe(II) oxidizing

chemotrophs lead to the successful isolation of

Pseudogulben-

kiania

sp. strain MAI-1, a novel

-proteobacterium closely

related to the lithoautotrophic Fe(II) oxidizer

Pseudogulbenkia-

nia

sp. 2002

16,28

(96.9% 16S rRNA gene sequence similarity,

97.3% to the type strain

Pseudogulbenkiania sub

ava

BP-5

MAI-1 has several key characteristics necessary for routine

genetic manipulation: the strain forms colonies on plates

(aerobically within 24 h), grows rapidly both aerobically and

anaerobically (overnight at 30

C), is sensitive to antibiotics,

and cryopreserves well. Most importantly, it displays the

desired phenotype: rapid nitrate dependent Fe(II) oxidation

(10 mM in less than 24 h, Supporting Information Figure S1)

in the presence of a chelator, nitrilotriacetate (NTA), that

prevents the formation of mineral precipitates (which could

obscure cells in automated assays) but does not serve as a

growth substrate for the organism (Supporting Information

Figure S2). When

rst isolated, MAI-1 appeared to be an ideal

candidate for elucidating the genes required for nitrate

dependent Fe(II) oxidation. However, although Fe(II)-NTA

is highly stable in abiotic controls in the presence of nitrate

(Figure 1; Supporting Information Figure S1), adding Fe(II)-

NTA to

lter-sterilized spent MAI-1 growth medium that had

Figure 1.

Ligands a

ect the abiotic oxidation of Fe(II) by NO

−

. Error

bars omitted for clarity (relative standard deviation of Fe(II) and

−

quantitation from all seven experiments estimated at 3% and

2%, respectively).

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accumulated substantial amounts of nitrite lead to rapid Fe(II)

oxidation with concomitant nitrite reduction (Supporting

Information Figure S3). The strain

’

s ability to use a wide

range of chelators as a carbon substrate (e.g., citrate, humic

acids, DTPA) and its inability to grow and oxidize free Fe

(Supporting Information Figure S1) precluded avoiding NTA.

Additionally, MAI-1 cannot use alternate electron acceptors

(e.g., DMSO, TMAO, fumarate), requiring the use of nitrate

(and consequentially risking the production of nitrite) for

anaerobic culturing.

To quantitatively assess the e

ect of Fe(II) chelation on

chemical oxidation by nitrite at circumneutral pH, we

conducted kinetic experiments with NTA as well as two

environmentally relevant Fe(II)-chelating ligands (citrate, CIT,

and Pahokee Peat Humic Acid, PPHA). Attempts to investigate

the e

ect of Fe(II) chelation with the siderophore desferox-

amine (DFO) and the organic pollutant ethylenediaminete-

traacetate (EDTA) proved unsuccessful because of interference

with the ferrozine assay and the nitrite assay, respectively

(Supporting Information Figure S5). They were not pursued

further. Figure 1 shows the oxidation of Fe(II) and

concomitant reduction of NO

−

over the course of

∼

100 h

(4.2 days) for each condition. Nitrite-free controls without any

oxidant or amended with nitrate show little Fe(II) oxidation (a

maximum of 2% without oxidant, 5% with nitrate; see Table 1)

over the course of the experiment. This provided con

dence

that O

contamination is not a signi

cant source of error in our

experimental setup and suggested that nitrate is relatively

unreactive toward Fe(II) even in the presence of ligands (see

abiotic control in Supporting Information Figure S1). Nitrite in

the absence of iron shows high stability, con

rming the

expected absence of nitrite self-decomposition that occurs at

acidic pH.

In the absence of any chelating moieties, less than

9% of Fe(II) is oxidized by nitrite within the

rst 22 h. Similar

control experiments in previous reports have yielded Fe(II)

oxidation rates at

∼

8% Fe(II) within 10 h,

∼

9% within 20 h,

and

∼

1% within 24 h.

Complexation by both citrate and

NTA, however, leads to rapid depletion of Fe(II) and nitrite,

indicating that these organic ligands can accelerate Fe(II)

oxidation by nitrite (Figure 1).

Equipped with an estimate for the extent of chemical Fe(II)

oxidation by nitrite in the presence of NTA, we grew MAI-1 in

the presence of Fe(II)

−

NTA while closely monitoring the

accumulation of nitrite (Figure 2) to model the maximal abiotic

Fe(II) oxidation resulting from an abiotic reaction with nitrite.

Given the strong e

ect of citrate on the chemical oxidation of

Fe(II) by nitrite, we also tested the hypothesis that abiotic

Fe(II) oxidation could be mediated by the biological

production of nitrite during denitri

cation in general. For this

purpose,

P. denitri

cans

, a model denitrifying microorganism,

was grown anaerobically on succinate and nitrate, such that

substantial quantities of nitrite accumulated during early

exponential growth (Supporting Information Figure S6).

After accumulation of

∼

5 mM nitrite,

lter sterilized culture

medium as well as active cultures of

P. denitri

cans

were

amended with

∼

5 mM Fe(II) with or without 10 mM citrate.

Figure 3 illustrates the resulting oxidation of Fe(II) over the

course of 4 h. Moderate oxidation occurred in the absence of

chelation both with

P. denitri

cans

cultures as well as in spent

medium (up to 21% and 12%, respectively). Higher oxidation

rates for cultures are likely a consequence of continued

denitri

cation by

P. denitri

cans

, increasing the measured pool

of nitrite by up to 13%. However, the most striking feature is

the rapid depletion of Fe(II) and nitrite (up to 76% Fe(II),

38% NO

−

) observed with the addition of 10 mM citrate,

regardless of the presence of

P. denitri

cans

(Table 2, Figure 3).

■

DISCUSSION

Reaction Mechanism and Kinetics.

Understanding the

kinetics of Fe(II) oxidation in the presence of ligands provides

the tools for predicting the potential e

ects of ligand-enhanced

Fe(II) oxidation in microbial systems. The total consumption

of Fe(II) and nitrite (Table 1) suggests that Fe(II) oxidation by

nitrite proceeds with 2:1 Fe(II)/NO

−

stoichiometry regardless

Table 1. Summary of Kinetic Fe(II) Oxidation Experiments by Nitrite

reactant changes within

∼

100 h

Fe(II) oxidation

−

reduction

Δ

Fe(II)

Δ

−

Δ

Fe(II)/

Δ

−

model

app

(LCI;UCI

)

model

app

(LCI;UCI

)

[

M] (%

)[

M] (%

)(

)

[10

−

]

[10

−

]

Controls

2mMNO

−

only

−

3 (0%)

2 mM Fe(II) only

−

3 (0%)

+2 mM NTA

−

35 (2%)

+2 mM NO

−

91 (5%)

+2 mM NTA + 2 mM NO

−

64 (3%)

Kinetically Unresolved

2 mM Fe(II) + 2 mM NO

−

963 (48%)

−

478 (24%)

2.0

0.3

+0.1 mM citrate

−

933 (50%)

−

480 (24%)

1.9

0.2

+300 mg/L PPHA

−

592 (30%)

−

303 (16%)

2.0

0.4

Second-Order Kinetics

+0.5 mM citrate

−

1281 (66%)

−

686 (34%)

1.9

0.2

0.9995

0.98 (0.92;1.04)

0.9995

1.04 (0.88;1.19)

+2 mM citrate

−

1883 (96%)

−

945 (47%)

2.0

0.1

0.9979

4.67 (4.18;5.17)

0.9992

4.31 (3.57;5.06)

+2 mM citrate +300 mg/L PPHA

−

1773 (90%)

−

931 (48%)

1.9

0.1

0.9963

3.31 (2.85;3.78)

0.9997

3.59 (3.24;3.93)

+2 mM NTA

−

1119 (55%)

−

1065 (54%)

1.1

0.1

0.9987

6.66 (5.19;8.13)

0.9993

6.11 (5.15;7.07)

The rate constant

app

is reported for reactions that are described well by second-order kinetics. The experiments were conducted at 25

C, pH 6.9

to 7.1. The p-values for the model parameter

app

are <0.001 for all conditions.

is the adjusted regression coe

ffi

cient for the least-squares

Percentage change of [Fe(II)] and [NO

−

] relative to starting concentrations.

Derived by error propagation from measurement errors (relative

standard deviation of Fe(II) and NO

−

quantitation during experiments estimated at 3% and 2% respectively).

Lower (LCI) and upper (UCI) 95%

con

dence interval of parameter derived from model

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of complexation (no ligand, PPHA, citrate), with the notable

exception of NTA, which appears to deplete Fe(II) and NO

−

in a 1:1 ratio. The 2:1 stoichiometry is in agreement with

literature reports that the predominant product of nitrite

reduction at pH regimes between 6 and 8 is N

22,33,36,37,62

according to the following representative net reaction:

++→++

+−++

4Fe

2NO

4Fe

N O 3H

(1)

where Fe

can be unbound Fe

or a ligand-bound Fe(II)-L

species, and Fe

can be ligand-bound Fe(III)-L or contained

within an (oxy)hydroxide mineral (e.g., FeOOH). This net

reaction likely comprises a number of elementary reaction

steps; we consider the following three to contextualize our

observations:

++→++

+−++

(slow)

NO H

(2)

+→ −

(fast) Fe

(Fe(II) NO)

(3)

−+→++

(fast)

(Fe(II) NO)

(4)

Equations 3

and 4

proceed rapidly at circumneutral pH,

with eq 2 being the rate limiting step (

≈

). Accordingly,

the reaction consumes 2 Fe(II) for every NO

−

, except in the

case of NTA. Both citrate and NTA complexes with ferrous

iron can bind nitric oxide such that the following reactions can

occur in competition with eq 3:

−+→ −−

−

(Fe(II) CIT) NO

(Fe(II) CIT NO)

5aq

(5)

−+→−−

−

(Fe(II) NTA) NO

(Fe(II) NTA NO)

([6])

Figure 2.

Fe(II) oxidation by

Pseudogulbenkiania

sp. strain MAI-1

during anaerobic growth with nitrate. Nitrite accumulation during

growth depicted in top panel, concomitant Fe(II) oxidation in middle

panel, modeled abiotic Fe(II) oxidation in bottom panel (see Materials

and Methods for details on computation). Solid and dashed lines

indicate Fe(II) oxidation without/with biological NO consumption,

respectively. Dotted line indicates Fe(II) oxidation with 6

higher rate

constant and NO consumption. Model range for three biological

replicates shaded in gray. Vertical line indicates time point addressed

in text. Experiment conducted in biological triplicates (solid markers)

and with abiotic control (empty circles,

○

). All data are shown.

Figure 3.

Fe(II) oxidation in

P. denitri

cans

cultures and

lter-

sterilized spent medium. Fe(II) concentrations shown as solid lines,

−

concentrations as dashed lines. Samples are drawn from

triplicate cultures (Supporting Information Figure S6) after accumu-

lation of

∼

5mMNO

−

and spiked with Fe(II)

citrate at 0 h. All

data are shown.

Table 2. Summary of Kinetic Fe(II) Oxidation Experiments

by Nitrite in

P. denitri

fi

cans

Cultures and Spent Medium

reactant changes

within

∼

4 h

Fe(II) oxidation

−

reduction

Δ

e(II)

Δ

−

model

app

(LCI;UCI

) model

app

(LCI;UCI

)

[mM]

)

[mM]

)

[10

−

]

[10

−

]

P. denitri

cans

−

3.7

(76%)

−

1.9

(36%)

0.9991 12 (11;14) 0.9991 11 (8;15)

−

3.3

(73%)

−

1.8

(36%)

0.9984 11 (9;13)

0.9996 10 (8;12)

−

3.2

(69%)

−

1.8

(38%)

0.9977 10 (7;13)

0.9985 11 (6;17)

Filter Sterilized

−

3.6

(73%)

−

1.8

(33%)

0.9990 11 (9;12)

0.9981 10 (6;15)

−

3.2

(71%)

−

1.7

(34%)

0.9985 11 (9;13)

0.9995 10 (8;12)

−

3.2

(65%)

−

1.7

(37%)

0.9983 9 (7;11)

0.9988 12 (7;17)

The experiment was conducted at 25

C. P-values for the model

parameter

are <0.01.

is the adjusted regression coe

ffi

cient for the

least-squares

Percentage change of [Fe(II)] and [NO

−

] relative

to starting concentrations.

Lower (LCI) and upper (UCI) 95%

con

dence interval of parameter derived from model

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However, Fe(II)-NTA forms a considerably stronger complex

with NO (

≈

2.1

−

=10

6.26

)

54,65,66

than

Fe(II)-citrate (

≈

4.4

−

=10

2.83

)

or Fe

alone (

≈

6.2

−

=10

2.65

potentially

preventing eq 4 from proceeding. For example, if 100

Fe(II) reacted with 100

MNO

−

to form NO in the presence

of 2 mM NTA, more than 99.98% of the produced NO would

form the highly stable Fe(II)-NTA-NO complex. The 1:1

stoichiometry of Fe(II) oxidation by nitrite observed in the

presence of NTA is likely a consequence of this stable Fe(II)

−

NTA

−

NO complex formation. As expected, we con

rmed

evolution of N

O during Fe(II) oxidation by nitrite by gas

chromatography in the presence of citrate, but no N

O formed

in the presence of NTA (Supporting Information Figure S8);

the formation of the Fe(II)

−

NTA

−

NO complex could be

observed instead (Supporting Information Figure S9).

Based on the rate-limiting, Fe(II) and NO

−

dependent

rst

reaction step (eq 2), a plausible scheme for the overall reaction

kinetics is a second-order rate expression with overall rate

constant

app

in analogy with oxidation of Fe(II) and Mn(II) by

57,67

=−

−

dFe(II)/d

2 [Fe(II)][NO ]

app

(7)

=−

−−

dNO /d

[Fe(II)][NO

]

2app 2

(8)

where Fe(II) comprises the total pool of ferrous iron (free Fe

as well as all complexed Fe(II)). Given the equimolarity of

initial total Fe(II) and NO

−

in our experimental setup, we

integrate eqs 7 and 8 to yield the following decay equations

(see the Supporting Information for details):

−+

Fe(II)( )

Fe(II)

12e

Fe(II)

0app

(9)

−+

−

NO ( )

NO e

12e

app

(10)

Least-squares

ts of eqs 9 and 10 to our experimental results

for Fe(II) and NO

−

depletion provide two separate estimates

of the overall rate constant

app

for each condition (Tables 1

and 2). Reactions without a ligand and with low citrate or

PPHA are better described by a linear least-squares

(apparent zero-order kinetics) and are therefore considered

kinetically unresolved (no

app

determined). Elementary

reaction steps and kinetic constraints for these conditions

cannot be deduced from our observations, and it remains

unclear why the reactions appear to be zero-order. Oxidation in

these conditions likely proceeds as a consequence of ferric

(oxy)hydroxide precipitation (observed visually) and subse-

quent heterogeneous autocatalysis as reported by Tai and

Dempsey (2009).

Apparent zero-order kinetics could re

ect

the complex balance between the generation of catalytic

mineral surfaces and depletion of dissolved Fe(II) and nitrite.

At higher concentrations of citrate and NTA, the reactions

remained homogeneous and are in agreement with a second-

order kinetic interpretation of our data (Tables 1 and 2 and

Supporting Information Figure S7). Rate constants derived

from Fe(II) oxidation and nitrite reduction agree well within

their 95% con

dence intervals, lending further credence to the

model. The pH remained close to 7.0 in all conditions, with an

average change of 0.1 by the end of the experiment (Supporting

Information Table S1), suggesting that the presence of the

ligands, rather than

uctuations in pH are responsible for the

observed di

erences in reaction kinetics. The reaction

progression observed in the presence of PPHA suggests that

chelation of Fe(II) by the humic acid moieties (10% of the

initial Fe(II) pool is organically complexed) has little to no

ect on the kinetics of iron oxidation (see Figure 1, PPHA and

CIT + PPHA). Rather than accelerating Fe(II) oxidation,

PPHA appears to have a slight retarding e

ect. In contrast to

experiments without a ligand, PPHA is likely to impede iron

oxide formation and autocatalysis as a result of its high a

ffi

nity

for Fe(III). In combination with citrate, PPHA leads to

diminished formation of the Fe(II)-citrate complex (Support-

ing Information Table S2), which appears to reduce the overall

reaction rate (Table 1).

Additional information for predicting the contribution of

chemical Fe(II) oxidation, especially in well-de

ned laboratory

systems, can be gained from identifying the reactive species. In

analogy to Fe(II) and Mn(II) oxidation by O

, the overall rate

constant

app

observed in our experiments can likely be

explained in terms of the weighted sum of the oxidation rates of

individual Fe(II) species

57,67

app

∑

where

is the

fraction of each Fe(II) species in solution and

the species-

speci

c second-order rate constant for oxidation by nitrite. A

comparison of

app

with the extent of Fe(II) complexation for

each experimental condition (Figure 4; Supporting Information

Table S2) suggests that the Fe(II)-L complex is involved in

accelerating Fe(II) oxidation, although the e

ect is ligand-

speci

c (no e

ect for PPHA, variable magnitude for citrate and

NTA). The observed reaction rates at low species fractions of

Fe(II)-L (<20%) suggest the existence of other Fe(II) species

with appreciable nitrite-dependent oxidation rates. We

speculate that the carbonate species Fe(II)

−

and

Fe(II)

−

(CO

)

−

(Supporting Information Table S2) could

provide such reactive species in analogy to their role in Fe(II)

oxidation by molecular oxygen.

However, the precise

mechanism and species-speci

c reaction rates

for the

Figure 4.

Rate constants increase with increasing degree of Fe(II)

complexation. Second-order rate constants for oxidation experiments

in the presence of citrate (black symbols) and NTA (gray symbols) are

plotted against the degree of Fe(II) complexation by citrate/NTA.

Rate constants derived from [Fe(II)] depicted as circles (

○

constants derived from [NO

−

] as squares (

□

). Error bars indicate

95% con

dence intervals (Tables 1 and 2). Details on speciation can

be found in Supporting Information Table S1. Larger con

dence

intervals for data reported in Table 2 are a consequence of reduced

temporal resolution and greater deviation from the assumption that

initial Fe(II) and NO

−

concentrations are equimolar.

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observed oxidation of Fe(II) by nitrite are beyond the scope of

this report and await further study. Due to the uncertainty

surrounding the reactive species involved, we recommend

caution in applying the rate constants derived in Tables 1 and 2

to aqueous environments with widely di

ering Fe(II) complex-

ation, pH, or ionic strength.

Biological Fe(II) Oxidation by

Pseudobulkeniania

sp.

Strain MAI-1.

Using the kinetic rate constants derived for the

oxidation of Fe(II) by nitrite in the presence of NTA with the

nitrite accumulation measured in culture of MAI-1 (Figure 2),

we modeled the purely abiotic Fe(II) oxidation that would

result from the interaction of Fe(II) with the accumulated

nitrite (Figure 2, bottom), assuming the presence of cell

Table 3. Maximal Rates of Fe(II) Oxidation Reported for Various Anaerobic Processes at Circumneutral pH (25

−

°

C, Except

Where Otherwise Indicated)

experimental conditions

max. rates

Fe(II)

nitrite

nitrate

Δ

Fe(II)

[

M/h]

reference

Chemical (Abiotic)

+30 mg/L lepidocrocite (

-FeOOH)

7.5 autotitration

0.2 mM

−

36, Figure 5

+30 mg/L lepidocrocite (

-FeOOH)

8.5 autotitration

0.2 mM

−

36, Figure 5

Fe(II) as siderite (10 g/L

∼

80 mM)

MES/PIPES/

HEPES

10 g/L

4.6 mM

−

265

39, Figure 5

Fe(II) as siderite (10 g/L

∼

80 mM)

6.5 MES/PIPES/

HEPES

10 g/L

4.6 mM

−

169

39, Figure 5

Fe(II) as siderite (10 g/L

∼

80 mM)

7.9 MES/PIPES/

HEPES

10 g/L

4.6 mM

−

140

39, Figure 5

+2.5 mM Fe(II) as HFO, 64

M average solid-bound

Fe(II)

6.8 PIPES

0.38 mM 0.38 mM

−

158

37, Table 1,

+17.5 mM Fe(III) as HFO, 188

M average solid-

bound Fe(II)

6.8 PIPES

0.34 mM 0.32 mM

−

301

37, Table 1,

F(II) as green rust

8.25 autotitration

10.81 mM

14.2 mM

−

139

42, Table 1

+2 mM NTA

bicarbonate

2 mM

−

192

this study,

Table 1

+2 mM CIT

bicarbonate

2 mM

−

134

this study,

Table 1

+10 mM CIT,

P. denitri

cans

spent medium

bicarbonate

5 mM

−

1695

this study,

Table 2

+10 mM CIT,

P. denitri

cans

culture

bicarbonate

5 mM

−

1910

this study,

Table 2

Mixed (Chemical + Biological)

D. frappieri

strain G, Fe(II) complexed by

10 mM NTA

∼

bicarbonate

4.8 mM

1.4 mM

2.5 mM

−

294

20, Figure 5

D. frappieri

strain G, Fe(II) as smectite

∼

bicarbonate

3 mM

1.4 mM

5 mM

−

175

20, Figure 6

Pseudogulbenkiania

sp. MAI-1, Fe(II)-NTA

bicarbonate

4 mM

5 mM

10 mM

−

360

this study,

Figure 2

Chemotrophic

enrichment culture, +1 mM acetate

bicarbonate

10 mM

3 mM

−

106

4, Figure 1

enrichment culture containing

Sideroxydans

species

6.8 bicarbonate

10 mM

4 mM

−

156

29, Figure 1a

Pseudogulbenkiania

strain 2002

6.8 bicarbonate

10 mM

2.2 mM

−

16, Figure 4

strain HidR2, +1 mM acetate

6.7 bicarbonate

6 mM

<30

M5mM

−

14, Figure 2

Ferroglobus placidus

, 85C

bicarbonate

2 mM

up to

550

0.64 mM

−

173

5, Figure 4

cell suspension of

D. suillum

, grown on acetate +

nitrate

6.8 bicarbonate

10 mM

−

4700

12, Figure 3a

Paracoccus ferrooxidans

, +25 mM EDTA,

+1 mM ethanol

bicarbonate

25 mM

5 mM

−

1600

13, Figure 3a

Acidovorax

sp. strain BoFeN1, +2 mM acetate

6.8 bicarbonate

2.5 mM

<1 mM

5 mM

−

15, Figure 2

Acidovorax

sp. strain BoFeN1, +5 mM acetate

bicarbonate

10 mM

0 mM

10 mM

−

240

30, Figure 1a

Acidovorax

sp. strain 2AN, +1.6 mM acetate

6.85 bicarbonate

8.3 mM

up to 1 mM 5 mM

−

158

24, Figure 2a

Acidovorax

sp. strain 2AN, + 4 mM EDTA,

+1.2 mM ethanol

PIPES

4 mM

5 mM

−

970

49, Figure 3c

Dechloromonas

sp. UWNR4, + 4 mM EDTA,

+1.2 mM ethanol

PIPES

4 mM

5 mM

−

950

49, Figure 3d

lake sediment slurry

∼

bicarbonate

1.4 mM

0.01 mM

1 mM

−

69, Figure 3

Phototrophic

Rhodopseudomonas palustris

strain TIE-1, + 0.2 mM

citrate

bicarbonate

4.5 mM

−

3, Figure 2

Rhodobacter capsulatus

strain SB1003,

+0.2 mM citrate

bicarbonate

0.1 mM

−

3, Figure 4

Rhodobacter capsulatus

strain SB1003, +1 mg/L HA

bicarbonate

0.1 mM

−

70, Figure 4

Rhodobacter capsulatus

strain SB1003, +0.2 mM NTA

bicarbonate

0.1 mM

−

112

70, Figure 4

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surfaces

to have negligible e

ects on purely chemical

oxidation. Even if we conservatively assume the upper 95%

con

dence interval for the rate constant (8.13 M

−

; see

Table 1) and that produced NO is biologically consumed (thus

leaving more Fe(II) free to react by preventing formation of the

highly stable Fe(II)-NTA-NO complex), abiotic oxidation

would maximally account for

∼

30%/35% (solid vs dashed

curve) of the observed Fe(II) oxidation after 28 h (time point

indicated by vertical line in Figure 2). In fact, a 6

higher rate

constant (combined with biological consumption of any

produced NO) would be required to attribute observed Fe(II)

oxidation to purely chemical processes (Figure 2, dotted

model). Based on the kinetic quanti

cation of chemical

oxidation of Fe(II), it thus becomes evident that

Pseudogulben-

kiania

sp. MAI-1 can directly oxidize Fe(II), establishing the

organism as a novel neutrophilic nitrate-dependent chemotroph

with unambiguous biological Fe(II)-oxidizing activity. The

potential to easily genetically manipulate this strain makes it a

good candidate for elucidating the machinery involved in

biological Fe(II) oxidation. Whether the biological component

of Fe(II) oxidation in MAI-1 occurs via a dedicated enzyme

system or via nonspeci

creactionswithredoxactive

components of the cell, such as periplasmic thiols or

components of the electron transport chain,

25,26

is a question

that could be addressed in the future.

Chemical vs Biological Fe(II) Oxidation in Laboratory

and Environmental Studies.

Given the aforementioned

ffi

culty in discriminating between chemical and biological

contributions to anaerobic Fe(II) oxidation in many systems, it

can be informative to compare Fe(II) oxidation rates observed

in a variety of environmental and laboratory settings. Table 3

provides an overview of the maximal Fe(II) oxidation rates

reported in a number of publications on chemical and biological

Fe(II) oxidation in nitrite/nitrate rich anoxic environments at

circumneutral pH. Several observations are particularly note-

worthy:

(i) The majority of observed maximal rates of chemical and

biological Fe(II) oxidation fall within a similar range of

values (

∼

−

100

M/h), highlighting the likely

competition and co-occurrence of chemical and bio-

logical processes involved in the coupled biogeochemical

cycling of iron and nitrogen. Moreover, because nitrite is

produced and often accumulates during the microbial

denitri

cation process, they are intrinsically coupled.

This biologically induced chemical oxidation of iron (via

the microbial production of nitrite) in organic rich

environments such as soils and wetlands is likely to

contribute signi

cantly to the cycling of iron and

immobilization of metal contaminants and organic

pollutants on iron (oxy)hydroxides. High oxidation

rates reported for environmental samples with mixed

contributions from biological and chemical catalysis

illustrate the interplay of these processes and call for

caution in interpreting an observed e

ect to stem from

solely one or the other mechanism.

(ii) In the case of mineral accelerated Fe(II) oxidation, the

presence of amorphous hydrous ferric oxide (HFO/

ferrihydrite)

9,31,37

and green rust

appears to cause the

most signi

cant acceleration of Fe(II) oxidation (see

Table S3 for additional detail on rate constants derived

for mineral catalysis). This e

ect is likely to be highly

relevant in natural settings where poorly crystalline iron

oxides are ubiquitous. However, it is also important to

consider this e

ect in laboratory studies where iron

oxides precipitate over the course of an experiment and

can provide catalytic surfaces for chemodenitri

cation as

suggested previously.

−

(iii) In the case of ligand-enhanced Fe(II) oxidation by

nitrite, the absence of a major e

ect of the humic acid

representative PPHA and low environmental abundance

of the anthropogenic ligand NTA (maximal levels of 10

−

100 nM in aqueous systems),

suggests that citrate

(detected in soil solutions in appreciable quantities,

∼

100

M range)

is likely to be the only ligand investigated in

this study that could be relevant in natural systems. In

laboratory studies of iron oxidizing microorganisms in

the presence of citrate or NTA, the ligands

’

ect on

oxidation kinetics is a crucial aspect of Fe(II) depletion

that cannot be disregarded. This is particularly clear from

the experiment reported in Figure 3 that con

rms ligand-

enhanced chemical oxidation of Fe(II) by nitrite can be

an important side e

ect of microbial denitri

cation.

Here, chemical Fe(II) oxidation could be mistaken for

direct biological catalysis by

P. denitri

cans

; while direct

catalysis may indeed be at play, it would simply be

challenging to unambiguously identify without appro-

priate controls. In conclusion, this study serves as a

reminder of the complex interplay between direct and

indirect biological e

ects involving metal transforma-

tions. In the case of denitrifying microorganisms, the

extent to which these di

erent processes catalyze Fe(II)

oxidation likely depends on the precise culturing

conditions and must be evaluated on a case-by-case basis.

■

ASSOCIATED CONTENT

*

Supporting Information

Derivation of reaction equations and additional tables and

gures as described in the text. This material is available free of

charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

Phone: 626-395-3543. Fax: 626-395-4135. E-mail: dkn@

caltech.edu.

Notes

The authors declare no competing

nancial interest.

■

ACKNOWLEDGMENTS

We thank Jim Morgan for many insightful conversations and

inspiring S.H.K. to pursue this project, Sean Crowe, CarriAyne

Jones, Arne Sturm, Sulung Nomosatryo, David Fowle, and Don

Can

eld for sample acquisition and

eldwork in Indonesia,

Nathan Dalleska and the Caltech Environmental Analysis

Center for instrumentation that bene

ted this project, Andreas

Kappler, Nicole Klu

̈

glein, and Jay Labinger for helpful

discussions, members of the Newman Lab and the anonymous

reviewers for constructive criticism that improved the manu-

script. This work was supported by grants to D.K.N from the

Dreyfus Foundation and the Howard Hughes Medical Institute

(HHMI). D.K.N. is an HHMI Investigator. S.H.K. is an HHMI

International Student Research Fellow.

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2013, 47, 2602

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■

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