Contents lists available at ScienceDirect

Chemical Geology

journal homepage: www.elsevier.com/locate/chemgeo

Biological and chemical processes of microbially mediated nitrate-reducingFe(II) oxidation by Pseudogulbenkiania sp. strain 2002

Dandan Chena,b,c,1, Tongxu Liub,1, Xiaomin Lib, Fangbai Lib,⁎, Xiaobo Luob, Yundang Wub,Ying Wangb

aGuangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, PR ChinabGuangdong Institute of Eco-Environmental Science & Technology, Guangdong Key Laboratory of Integrated Agro-Environmental Pollution Control and Management,Guangzhou 510650, PR ChinacUniversity of Chinese Academy of Sciences, Beijing 100049, PR China

A R T I C L E I N F O

Editor: Michael E. Böttcher

Keywords:ChemodenitrificationBiological processNitrate-reducing Fe(II) oxidationNitrogen isotopic fractionationMineralization

A B S T R A C T

In the microbially mediated nitrate-reducing Fe(II) oxidation system, it is recognized that chemical oxidation ofFe(II) by nitrite, which is a bioreduction intermediate of nitrate, can occur under anoxic conditions (chemo-denitrification), but it is still difficult to quantitatively evaluate the contributions of biological Fe(II) oxidationand chemodenitrification. Here, nitrate reduction coupled with Fe(II) oxidation by a suggested lithoautotrophicnitrate-reducing Fe(II)-oxidizing bacterium, Pseudogulbenkiania sp. strain 2002, was investigated in PIPES buf-fered medium without any organic cosubstrate through reaction kinetics, nitrogen isotope fractionation, andsecondary mineral characterization. Substantial Fe(II) oxidation was observed in the presence of cells and ni-trate, and nitrite (0.59 mM) was able to quickly oxidize Fe(II). Stored carbon in strain 2002 harvested duringpre-incubation can serve as carbon source for nitrate reduction. Furthermore, the N isotopic composition (δ15N)of N2O in Cell + NO3

− + Fe(II) was much more negative than those in Cell + NO3−/NO2

−, Cell + NO2− + Fe

(II), and NO2− + Fe(II), implying that Fe(II) affects N fractionation associated with the reduction of nitrate to

nitrite. Goethite was formed in Fe(II) + NO2−, while lepidocrocite was the main mineral phase in Cell + Fe(II)

+ NO3−. The morphology and cell-mineral interactions determined by electron microscopy showed that sec-

ondary minerals were formed outside of cells in Cell + NO2− + Fe(II), while cell encrustation was observed in

the periplasmic space of cells in Cell + NO3− + Fe(II). The secondary minerals present in the different treat-

ments further illustrated the co-occurrence of biological, chemical, and coupling processes in the microbiallymediated nitrate-reducing Fe(II) oxidation system. This study highlights the involvements of the biological Fe(II)oxidation and chemical Fe(II) oxidation by nitrite in microbially mediated nitrate-reducing Fe(II) oxidation.

1. Introduction

Iron is the most abundant redox-active metal in Earth's crust, andiron-bearing minerals are ubiquitous reactive constituents of aquifers,soils, and sediments (Kappler and Straub, 2005). Iron cycling in naturalsystems (aquatic sediments, soils, and subsurface environments) has amajor impact on the fate of trace metals and nutrients as well as on thedegradation of organic matter (Elliott et al., 2014; Liu et al., 2014b;Melton et al., 2014). Due to a lack of oxygen, the dominant type of Fe(II) oxidation under anoxic conditions can be microbially mediated(Weber et al., 2006a; Druschel et al., 2008). In the presence of light,phototrophic Fe(II) oxidation was dominant under anoxic conditions(Melton et al., 2014). Since microbial Fe(II) oxidation coupled to nitrate

reduction under subsurface anoxic conditions was first demonstrated(Hafenbradl et al., 1996; Straub et al., 1996; Benz et al., 1998), nitrate-reducing Fe(II) oxidation has frequently been proposed as a newpathway for biological iron cycling in the past two decades. Microbiallymediated nitrate-reducing Fe(II) oxidation should play a significant rolein coupling the redox cycles of N and Fe in neutral, anaerobic en-vironments (Melton et al., 2014; Li et al., 2015; Scholz et al., 2016;Zhou et al., 2016; Smith et al., 2017). The formation of Fe(III) (hydr)oxides could significantly affect the migration of heavy metals andradionuclides, which are strongly influenced by adsorption reactions atiron(III) oxide surfaces (Lack et al., 2002; Borch et al., 2010; Hohmannet al., 2010; Xiu et al., 2016).

Previous studies have provided evidence that nitrate-reducing Fe(II)

https://doi.org/10.1016/j.chemgeo.2017.11.004Received 25 April 2017; Received in revised form 31 October 2017; Accepted 4 November 2017

⁎ Corresponding author.

1 These authors contributed equally to this work.E-mail address: cefbli@soil.gd.cn (F. Li).

Chemical Geology 476 (2018) 59–69

Available online 09 November 20170009-2541/ © 2017 Elsevier B.V. All rights reserved.

T

oxidation is a biological process and that Fe(II) can also be oxidizedabiotically by nitrite, but no studies have directly demonstrated thecontributions of biotic and abiotic mechanisms in the microbiallymediated nitrate-reducing Fe(II) oxidation system (Klueglein andKappler, 2013). Previous studies have highlighted the biological pro-cesses of Fe(II) oxidation and nitrate/nitrite reduction, but overlookedthe chemical mechanisms (Weber et al., 2001; Senko et al., 2005; Kopfet al., 2013; Laufer et al., 2016). Recent studies have shown that theimportance of biological Fe(II) oxidation might be overestimated andthat abiotic reactions by nitrite (the intermediate of nitrate reduction)probably have a much more important influence on Fe(II) oxidation inthe Fe(II)-nitrate-microbe system (Picardal, 2012; Klueglein andKappler, 2013). Therefore, it is still difficult to distinguish the biologicaland chemical processes of microbially mediated nitrate-reducing Fe(II)oxidation (Melton et al., 2014).

Many studies have been conducted to determine the chemical andbiological mechanisms of microbially mediated nitrate-reducing Fe(II)oxidation, but it is hard to separate abiotic and biotic reactions, whichoccur simultaneously (Kopf et al., 2013; Klueglein et al., 2014; Meltonet al., 2014; Klueglein et al., 2015). Stable isotopic fractionation is anoninvasive method for investigating biological systems, as it is basedon the fact that organisms transform compounds containing lighterisotopes (e.g., 14N) at a slightly higher rate than compounds containingheavier isotopes (e.g.,15N) (Torrentó et al., 2011; Wunderlich et al.,2012; Wenk et al., 2014), and thus, it may be feasible to distinguishbiotic and abiotic nitrate reduction processes (Mandernack et al., 2009;Grabb et al., 2017). The isotopic composition (δ) of N2O is useful fortracking the origin of N2O produced by different reduction processes(Barford et al., 1999; Wrage et al., 2004; Toyoda et al., 2005; Yanget al., 2014). Isotopic composition analysis of N2O has been used todistinguish nitrification- and denitrification-mediated N2O productionin natural systems (Schmidt et al., 2004; Sutka et al., 2006; Jones et al.,2015), as well as to distinguish chemical and biological mechanisms toreduce NO2

− during the reduction of goethite and nitrate by Shewanellaputrefaciens 200 (Cooper et al., 2003; Zhang et al., 2014). Hence, Nisotopic fractionation of N2O is considered to be a useful technique todetermine the biotic and abiotic reactions that produce N2O duringmicrobially mediated nitrate-reducing Fe(II) oxidation (Melton et al.,2014).

Furthermore, while all reactions between NO3−/NO2

− and Fe(II)are thermodynamically favorable (Moraghan and Buresh, 1976; VanCleemput and Samater, 1996; Straub et al., 2001; Picardal, 2012; Zhanget al., 2012), the kinetics of the chemical reaction of NO3

− reduction byaqueous Fe2+ in the absence of catalytic ions or surfaces are relativelyslow (Ottley et al., 1997). Meanwhile, both homogeneous and hetero-geneous reactions for NO2

− reduction by Fe(II) are rapid (Tai andDempsey, 2008; Picardal, 2012). In the microbially mediated nitrate-reducing Fe(II) oxidation system, Fe(II) is oxidized via biological andchemical processes, followed by the formation of Fe(III) or Fe(III)-Fe(II)minerals (e.g., green rust) (Pantke et al., 2012; Miot et al., 2014;),which rapidly precipitate on the cell surface or in the periplasm to formcell encrustations, resulting in limited cell growth and nitrate reduction(Schädler et al., 2009; Gault et al., 2011; Klueglein et al., 2014; Schmidet al., 2014). However, it is still unknown whether the formation ofsecondary minerals is controlled by biological, chemical, or biological-chemical coupling mechanisms in the microbially mediated nitrate-re-ducing Fe(II) oxidation system (Chakraborty and Picardal, 2013a;Chakraborty and Picardal, 2013b; Klueglein and Kappler, 2013). So far,several bacteria that are capable of coupling nitrate reduction to Fe(II)oxidation have been isolated (Emerson et al., 2010; Hedrich et al.,2011). Most of them are mixotrophic and require an organic cosub-strate (e.g., acetate) for continuous Fe(II) oxidation and growth (Mueheet al., 2009). Pseudogulbenkiania sp. strain 2002 was the first anaerobic,mesophilic, neutrophilic, nitrate-dependent, and lithoautotrophic Fe(II)oxidizer described in freshwater (Weber et al., 2006b), which can al-ternatively grow heterotrophically with organic carbon as well (Weber

et al., 2009). In this study, using strain 2002 as a model bacterium, theobjectives were to (1) investigate the kinetics of the biological, che-mical and the possible enzymatic reactions between Fe(II) and NO3

−

involved in microbially mediated nitrate-reducing Fe(II) oxidation; (2)distinguish between the chemical and biological processes via nitrogenisotopic fractionation of N2O; and (3) reveal the roles of chemical andbiological processes in the formation of secondary minerals. This studywill be helpful for fundamentally and comprehensively interpreting theprocesses of microbially mediated nitrate-reducing Fe(II) oxidation.

2. Materials and methods

2.1. Bacterial cultivation and chemicals

Pseudogulbenkiania sp. strain 2002 (ATCC BAA-1479; DSM 18807),previously isolated from sediments collected from a freshwater lake inIllinois (Weber et al., 2009), was purchased from the DSMZ, Germany.Strain 2002 stock was first activated under oxic conditions in a beefextract-peptone medium (3.0 mg·L−1 beef extract, 5.0 mg·L−1 peptone,5.0 mg·L−1 NaCl) at 37 °C with shaking at 180 rpm. The activated cellsof strain 2002 were subcultured in a fresh beef extract-peptone medium(inoculum/medium ratio of 1/10) for cell preparation. At the ex-ponential phase, cells were harvested by centrifugation (6000 ×g,6 min) at 4 °C. Cells were washed three times and re-suspended insterile and anaerobic (100% N2 atmosphere) PIPES (piperazine-N, N′-bis (2-ethanesulfonic acid)) buffer (30 mM, pH 7.0) to harvest a cellsuspension with an optical density of OD600 nm = 1.9–2.1 for all nitrate-reducing Fe(II) oxidation experiments. PIPES (≥99.0%) andFeSO4·7H2O (≥99.0%) were purchased from Sigma-Aldrich Co., USA.Other chemical reagents for cell growth medium and kinetics experi-ments were of analytical grade and purchased from Guangzhou Che-mical Reagent Factory, China.

2.2. Experimental setup

PIPES buffer (30 mM) at pH 7.0 was dispensed into serum bottles,sparged with 100% N2 for at least 30 min to ensure anoxic conditions,and autoclaved. Other stock solutions were filter sterilized by passingthe solutions directly through a sterile 0.22-μm nylon filter under 100%N2 atmosphere and stocked into sterile stoppered serum bottles.Vitamin and trace mineral solutions were prepared as previously de-scribed (Weber et al., 2009). A Fe(II) stock solution (1 M) was preparedby adding FeSO4·7H2O to anoxic (100% N2 headspace) distilled deio-nized water and was then filter sterilized and sealed with a butylstopper under an anoxic atmosphere. In anaerobic experiments,NaHCO3 (10 mM) was added as a carbon source, NaNO3 (2 mM) as anelectron acceptor and Fe(II) (3.5 mM) as an electron donor. The finalcell density of strain 2002 in the anaerobic experiments was approxi-mately 6 × 107 cells mL−1. Eight batch treatments for anaerobic ex-periments were conducted (Table 1): three abiotic treatments (Fe(II)+ NO3

−, Fe(II) + NO2−, and Killed-Cell + Fe(II) + NO3

−), threebiotic treatments (Cell + NO3

−, Cell + NO2−, and Cell + Fe(II)), and

two combined treatments (Cell + Fe(II) + NO2− and Cell + Fe(II)

+ NO3−). Killed cells were obtained by autoclaving at 103.4 kPa,

121 °C for 20 mins. Standard anaerobic techniques were usedthroughout all experiments as previously described (Zhang et al.,2012). The total volume of the reaction solution was 20 mL, which wasplaced in a 110 mL serum bottle and sealed with butyl rubber stoppersand aluminum caps. All trials were conducted in triplicate and in-cubated in a BACTRON Anaerobic/Environmental Chamber II(SHELLAB, Sheldon Manufacturing Inc.) at 37 °C the in dark.

2.3. Chemical analyses

During the incubation period, triplicate bottles were used to quan-tify the total Fe(II), nitrate, nitrite, N2O, and NH4

+. The headspace gas

D. Chen et al. Chemical Geology 476 (2018) 59–69

60

of each serum bottle was sampled with a syringe and needle in a va-cuum tube for analysis of N isotopes and to determine the total con-centration of N2O (Liu et al., 2014a). The N2O concentration wasmeasured by a Techcomp GC7900 gas chromatograph using ECD de-tectors. For the quantification of total Fe(II), 1 ml of culture suspensionwas removed and dissolved in 4 ml of 40 mM sulfamic acid (pH ap-proximately 1.8) for 1.5 h on a rotary shaker at 180 rpm at 30 °C (Liet al., 2009; Klueglein and Kappler, 2013). The pH of sulfamic acid wasapproximately 1.8, which can maintain the rapid reduction of nitriteand avoid abiotic Fe(II) oxidation (Granger and Sigman, 2009). Thetotal Fe(II) concentration was determined by the 1,10-phenanthrolinemethod. For NO3

−, NO2− and NH4

+ measurements, samples were fullyexposed to O2 to rapidly oxidize Fe(II), centrifuged at 8000 ×g for5 min to remove cells and oxides, and then filtered through a 0.22 μmmembrane before analysis (Li et al., 2016). The concentrations ofNO3

−, NO2− and NH4

+ were quantified by a Continuous Flow Ana-lyzer (SAN++, Skalar). Samples were buffered at pH 8.2 and passedthrough a column containing granulated copper‑cadmium to reducenitrate to nitrite. Nitrite was quantified by diazotizing with sulfanila-mide and coupling with N-(1-naphtyl)ethylendiamine dihydrochlorideto form a highly colored azo dye, which was measured at 540 nm (Liet al., 2015). The ammonia levels in the samples were determined bythe modified Berthelot reaction and formed a green F complex that wasmeasured at 660 nm (Hou et al., 2014).

2.4. N isotope fractionation

Isotopic measurements of N2O were performed by PreconcentrationIsotope Ratio Mass Spectrometry (PreCon-IRMS) (Isoprime-100,IsoPrime). Aliquots of air were displaced from a 500 mL sample bulbusing a He carrier stream for variable periods. First, water and CO2

were removed by passing through a chemical trap filled with ascariteand Mg(ClO4)2. Second, any remaining contaminants were removed byprocessing through a gas chromatograph fitted with a thermal con-ductivity detector. The recovered N2O was then measured directly bystable isotope mass spectrometry tuned for masses of 44, 45 and 46. Therelative abundance of 15N in N2O molecules was expressed as δ=[(Rsample/Rstandard)-1] × 1000, where Rsample = 15N/14N in sample N2Oand Rstandard = 15N/14N in standard N2O. The N standard was atmo-spheric N2.

The N isotopic composition of nitrite was determined after quanti-tatively converting nitrite to N2O gas by hydroxylamine(HNO2 + NH2OH → N2O + 2H2O). Since the N atoms of the N2O wereproduced from both nitrite and hydroxylamine, the N isotopic compo-sition of nitrite was calculated with the equation 15N at.% (NO2

−)=2 × 15N at.% (N2O) −15N at.% (NH2OH), where 15N at.% (NH2OH)was about 0.365 at.%.

For the method of determining the N isotopic composition of nitrate,nitrate was completely reduced to nitrite by spongy cadmium and thegenerated nitrite was analyzed for N isotopic composition as described

above.

2.5. Secondary mineral characterization

Biogenic and abiogenic mineral samples were collected from theincubations by filtration inside an anoxic N2/H2 atmosphere glovebox.Samples were filtered onto 0.22 μm filters (VCTP Millipore Isopore),washed twice with deoxygenated DDI water, and dried in the glovebox.The dry minerals were then characterized. The morphology was in-vestigated by scanning electron microscopy (SEM, Merlin, Zeiss) andtransmission electron microscopy (TEM, Tecnai, FEI). FourierTransform Infrared Spectroscopy (FTIR) measurements were carriedout using KBr disks on a Bruker VERTEX 70 FTIR spectrometer. Forstructural analysis of minerals, an X-ray diffractometer (XRD, D8ADVANCE, Bruker) using Cu Kα radiation was utilized with a diffrac-tion angle range of 2θ= 20–80°. The scan speed was 0.2° per minute,and the step size was 0.01°. MDI Jade 7 software was used for theidentification of mineral phases. This software utilizes the InternationalCenter for Diffraction Date Powder Diffraction File Database (ICDDPDF-2, Sets 1–46, 1996) as a reference source (Habibi, 2014).

3. Results

3.1. Kinetics of nitrate-reducing Fe(II) oxidation

The Cell + Fe(II) + NO3− results showed that Fe(II) was com-

pletely oxidized within 60 h (Fig. 1a). Nitrate was fully reduced by cellsonly (Cell + NO3

−) within 8 h suggesting that cell-stored carbon wasused as electron donor (Fig. 1b). However, in the presence of Fe(II)(Cell + Fe(II) + NO3

−), nitrate was reduced quickly in the first threehours and only approximately 44% of nitrate was reduced at the end ofthe incubation. The first-step intermediate of NO3

− reduction, NO2−,

increased quickly to its highest concentration (2.05 mM) within 8 h inthe Cell + NO3

− experiment (Fig. 1c) and then slowly decreased to1.6 mM in the following 86 h. Meanwhile, only 30% of nitrate wasreduced to nitrite in the Cell + Fe(II) + NO3

− experiment. The con-centrations of N2O in the Cell + Fe(II) + NO3

− experiment (Fig. 1d)slowly increased to 55.3 μM at the end of the incubation, which wasmuch higher than that for Cell + NO3

−. The above kinetics suggestedthat Fe(II) oxidation and nitrate reduction in the presence of strain2002 occur simultaneously.

To illustrate the roles of NO3−, Fe(II), and cells for Fe(II) oxidation

and nitrate reduction in Cell + Fe(II) + NO3−, the control treatments

were also investigated. No Fe(II) oxidation was observed in the Cell+ Fe(II), Fe(II) + NO3

−, and Killed-cell + Fe(II) + NO3− treatments

(Fig. 1a), and no nitrate was reduced in the Fe(II) + NO3− and Killed-

cell + Fe(II) + NO3− treatments (Fig. 1b). Furthermore, no NH4

+ wasobserved in any treatment. The above kinetics of Fe(II) oxidation andnitrate reduction revealed that Fe(II) was neither microbially oxidizedby cells alone nor chemically oxidized by NO3

− and that the killed-cellscould not stimulate the reaction between Fe(II) and NO3

−. Hence, cellsof strain 2002 may mediate the nitrate-reducing Fe(II) oxidation pro-cesses.

Although strain 2002 was suggested to be an autotrophic nitrate-reducing Fe(II)-oxidizing bacterium (Weber et al., 2006b), we need toinvestigate the effects of stored carbon on the observed reactions. Tofurther confirm whether strain 2002 can grow with Fe(II) and nitrateunder autotrophic conditions, the experiments with Cell + Fe(II)+ NO3

− and Cell + NO3− were conducted in the presence/absence of

NaHCO3. Results in Fig. S1a showed that the kinetics of Fe2+ decay inCell + Fe(II) + NO3

− with NaHCO3 was very close to that withoutNaHCO3, suggesting that no significant influence on Fe(II) oxidationwas induced by the presence of NaHCO3. The NO3

− reduction andNO2

−/N2O production were further examined. For Cell + Fe(II)+ NO3

−, results in Fig. S1b and S1c showed that the nitrate reductionand nitrite production in with/without NaHCO3 were almost the same,

Table 1The batch treatments with the pseudo-first-order rate constants (k) of Fe(II) oxidation andNO3

−/NO2− reduction.

Treatments Fe(II) oxidation NO3−/NO2

− reduction

k (h−1) R2 k (h−1) R2

Fe(II) + NO3− 0 0 0 0

Fe(II) + NO2− 0.092 ± 0.007 0.978 0.038 ± 0.004 0.941

Killed-cell + Fe(II) + NO3− 0 0 0 0

Killed-cell + Fe(II) + NO2− 0.035 ± 0.003 0.955 0.047 ± 0.005 0.949

Cell + Fe(II) 0 0 / /Cell + NO3

− / / 0.436 ± 0.041 0.957Cell + NO2

− / / 0.006 ± 0.000 0.889Cell + Fe(II) + NO2

− 0.043 ± 0.005 0.949 0.037 ± 0.003 0.975Cell + Fe(II) + NO3

− 0.101 ± 0.030 0.779 0.127 ± 0.010 0.982

D. Chen et al. Chemical Geology 476 (2018) 59–69

61

but the results in Fig. S1d showed that N2O production withoutNaHCO3 was obviously higher than that with NaHCO3. To further re-veal the impact of NaHCO3 on nitrate reduction and NO2

−/N2O pro-duction, the kinetics of NO3

− and nitrite/N2O in Cell + NO3− were

further examined. Results in Fig. S1b showed that the residual nitratewithout NaHCO3 was lower than that with NaHCO3, indicating that theNaHCO3 may inhibit nitrate reduction. The nitrite production withoutNaHCO3 was higher than that with NaHCO3 before the cross point at35 h, and became lower, which may be attributed to the further re-duction of nitrite to other nitrogen products (i.e., N2O). The productionof N2O without NaHCO3 was remarkably higher than that withNaHCO3, suggesting that the presence of NaHCO3 could substantiallyinhibit the reduction of nitrite to N2O.

The reduction of NO3− was observed in the absence of NaHCO3,

probably because the strain was growing with the potential carbonstorage during pre-incubation. The NO3

− reduction was inhibited bythe presence of NaHCO3, because the NaHCO3 may involve in the NO3

−

reduction under autotrophic conditions, resulting in the low enzymaticreduction of NO3

−. To further clarify the role of stored carbon, strain2002 was cultured in Cell + Fe(II)-NTA + NO3

− in order to deplete thecells from stored carbon, then subcultured in Cell + NO3

− and Cell+ NO3

− + Fe(II)-NTA, respectively. Nitrate reduction or nitrite pro-duction was hardly observed in Cell + NO3

− (Fig. S2a), and no cellgrowth was observed in the second generation of Cell + NO3

− + Fe(II)-NTA (Fig. S2b). These results suggested that the stored carbonharvested from organic rich pre-incubation played a key role in nitratereduction and Fe(II) oxidation.

3.2. Kinetics of nitrite-reducing Fe(II) oxidation

Since it was reported that the redox reaction between the nitratereduction intermediate (nitrite) and Fe(II) can occur spontaneously(Picardal, 2012), the kinetics of nitrite reduction and formation of N2O(Fig. 2) must be affected by the presence of Fe(II). To clearly reveal thereaction between nitrite and Fe(II) in the presence of cells, the kineticsof Fe(II) oxidation and nitrite reduction for the treatments (Cell + Fe(II) + NO2

−, Cell + NO2−, Fe(II) + NO2

−, and Killed-cell + Fe(II)+ NO2

−) were examined. The results presented in Fig. 2a show that inCell + Fe(II) + NO2

−, Fe(II) + NO2−, and Killed-cell + Fe(II)

+ NO2−, Fe(II) was completely oxidized within 60 h. The pseudo-first-

order reaction rate constants (k) of Fe(II) oxidation were 0.043 h−1

(R2 = 0.949) for Cell + Fe(II) + NO2−, 0.092 h−1 (R2 = 0.978) for Fe

(II) + NO2−, and 0.035 h−1 (R2 = 0.955) for Killed-cell + Fe(II)

+ NO2−, which were lower than that for Cell + Fe(II) + NO3

−, whichwas 0.101 h−1 (R2 = 0.779) (Fig.S3 and Table 1). NO2

− reduction wasobserved in all the treatments (Fig. 2b). The pseudo-first-order reactionrate constants (k) of nitrite reduction were 0.037 h−1 (R2 = 0.975) forCell + Fe(II) + NO2

−, 0.006 h−1 (R2 = 0.889) for Cell + NO2−,

0.038 h−1 (R2 = 0.941) for Fe(II) + NO2−, and 0.047 h−1

(R2 = 0.949) for Killed-cell + Fe(II) + NO2−, (Table 1). No NH4

+ wasobserved in any treatment. The production of N2O in the above fourtreatments is shown in Fig. 2c. It is noted that the N2O concentrationsfor the treatments Cell + Fe(II) + NO2

−, Fe(II) + NO2−, and Killed-

cell + Fe(II) + NO2− at the end of the incubation were as high as

0.25 mM, which were much higher than those of Cell + NO3−

(0.015 mM) and Cell + NO2− (0.030 mM).

The above results suggested that nitrite can be reduced by both ofcells and Fe(II), so there is competition between nitrite reduction by thebacteria and nitrite reduction by Fe(II). The chemical reaction betweenFe(II) and NO2

− (Fe(II) + NO2−) was favorable for nitrite reduction

and N2O production, while the presence of strain 2002 slightly slowednitrite reduction and N2O production (Cell + Fe(II) + NO2

− and Cell+ NO2

−). In addition, the nitrite reduction and N2O production inKilled-cell + Fe(II) + NO2

− was slightly lower than that in Fe(II)+ NO2

−, suggesting that the presence of biomass just had minor in-fluence on the nitrite reduction and N2O production, which might beattributed to the lower mobility of Fe(II) due to the adsorption on cellsurfaces.

Furthermore, the additional experiments were conducted for mea-suring the cell growth during the incubations with Fe(II) and nitrate/nitrite. Since it is difficult to enumerate cells in the presence of ironminerals, cell protein concentrations of Cell + Fe(II) + NO3

−, Cell+ NO3

−, Cell + Fe(II) + NO2−, and Cell + NO2

− were measured toevaluate the relative changes (Zhao et al., 2013). Results in Fig. S4showed that no obvious change was observed in the cell protein con-centrations in Cell + Fe(II) + NO3

−, while the concentrations in Cell+ NO3

− increased from 20 to 30 μg mL−1. Similarly, no significantchanges were observed for both Cell + Fe(II) + NO2

− and Cell

(a)

(c)

(b)

(d)

Fig. 1. Fe(II) oxidation (a), NO3− reduction (b),

NO2− formation (c) and N2O formation (d) in

treatments with nitrate and Fe(II). Initial con-centrations: 3.5 mM Fe(II), 2 mM NO3

−,6 × 107 strain 2002 cells mL−1, and 10 mMNaHCO3 in a 30 mM PIPES buffer medium atpH = 7.0. Error bars represent the standarddeviation of the mean (n = 3).

D. Chen et al. Chemical Geology 476 (2018) 59–69

62

+ NO2−. From these results, nitrate reduction may be coupled to the

cell growth, while Fe(II) oxidation might not be coupled to cell growth.

3.3. N isotopic fractionation of N2O

The initial δ15N of NO3− and NO2

− added to the incubations were−0.2 ± 0.1‰ and −7.25 ± 0.25‰, respectively (Fig. 3a). At thebeginning of the incubation, the initial δ15N-N2O of the treatment Cell+ Fe(II) + NO3

− (−33.8 ± 0.68‰) was extremely more negativethan that in the other four treatments (around −13‰). The δ15N-N2Oof all the treatments increased during the incubation period, but theincreasing extents were different in all the treatments. At the end of theincubation, the δ15N-N2O values of NO3

−/NO2− reduction by strain

2002 (Cell + NO3− and Cell + NO2

−) increased substantially to7.52 ± 1.36‰ and 1.50 ± 2.02‰, respectively. The δ15N-N2O ofCell + Fe(II) + NO3

− increased from −33.8 ± 0.68‰ to−28.34 ± 0.64‰. The δ15N-N2O for the treatments (Fe(II) + NO2

−

and Cell + Fe(II) + NO2−) slightly increased to −9.83 ± 0.17‰ and

−8.01 ± 0.95‰. Results in Fig. 3b showed that the maximum in-creasing extent was 20.58 ± 0.69‰ for Cell + NO3

−, while theminimum value was 2.45 ± 0.09‰ for Fe(II) + NO2

−. It was notedthat δ15N-N2O values increased less in treatments Fe(II) + NO2

− andCell + Fe(II) + NO2

− with relatively high concentrations N2O (about0.3 mM) than in treatments Cell + NO3

− and Cell + NO2− with very

little N2O (< 15 μM). The statistical evaluation in Fig. 3b showed thatsignificant differences were observed in the extents to which δ15N-N2Ovalues increased among all the treatments except the two treatments(Cell + Fe(II) + NO3

− and Cell + Fe(II) + NO2−). Results suggested

that the addition of Fe(II) in Cell + NO3− substantially reduced the

δ15N-N2O, but that Fe(II) had very little effect in the Cell + NO2−

treatment.

3.4. Mineralization of iron

Fe(II) oxidation to Fe(III) can result in the formation of secondaryminerals. However, different oxidation processes have different kineticsof Fe(III) formation that eventually influence the types of secondaryminerals. The morphology of the solid samples obtained by SEMshowed that the minerals from the treatment of Fe(II) + NO2

− wereneedle-like on the first day (Fig. 4a) and did not change remarkably

(a)

(b)

(c)

Fig. 2. Fe(II) oxidation (a), NO2− reduction (b) and N2O formation (c) in the treatments

with nitrite and Fe(II). Initial concentrations: 4.7 mM Fe(II), 2.4 mM NO2−, 6 × 107

strain 2002 cells mL−1, and 10 mM NaHCO3 in a 30 mM PIPES buffer medium atpH = 7.0. Error bars represent the standard deviation of the mean (n = 3).

(a)

(b)

Fig. 3. (a) Time-course changes of δ15N values of N2O produced by chemical and biotictreatments. The symbols “★” and “☆” represent the δ15N values of NO3

− and NO2−

added to the incubations (−0.2 ± 0.1‰ and −7.25 ± 0.25‰); (b) The average in-creasing values of δ15N-N2O (from 8 h to 92 h) in different treatments. Dates aremeans ± standard errors (n = 3). Letters a, b c, and d indicate significant differencesamong treatments with different P level at the p < 0.05 level according to One-WayANOVA.

D. Chen et al. Chemical Geology 476 (2018) 59–69

63

over the entire incubation period (Fig. 4b and c). In the presence ofstrain 2002 (Cell + Fe(II) + NO2

−), the shape of the product was stillneedle-like, but the sizes were smaller than those in Fe(II) + NO2

−

(Fig. 4d). With increasing incubation time, no obvious change in themorphology was observed in Cell + Fe(II) + NO2

− (Fig. 4e and f). Inthe combined treatments (Cell + Fe(II) + NO3

−), scale-like mineralsattached to the cell surfaces were observed on the 1st day (Fig. 4g). Asthe incubation time elapsed, the shapes of the minerals changed toneedle-like structures (Fig. 4h and i).

The TEM image of Fe(II) + NO2− (Fig. 5a) showed that the clear

needle-shaped mineral was well dispersed. In the image of Cell + Fe(II) + NO2

− (Fig. 5b), similar to that of Fe(II) + NO2−, the minerals

were also needle-like shapes. In addition, the edges of the aggregationarea also contacted cells to some extent, reflecting a potential Fe(II)oxidation reaction on the cell surface. For Cell + Fe(II) + NO3

−, theimage (Fig. 5c) clearly shows cell encrustation, as cells of strain 2002were mostly enclosed by a capsule of Fe(III) minerals (Fig. S5) either indirect contact with or in close proximity to the cell surface, implyingthat the interfacial reaction between the cell surface and minerals ac-counted for the cell encrustation. According to the literature (Zhaoet al., 2013; Klueglein et al., 2014), Fe(II) oxidation occurs in theperiplasmic space of strain 2002 cells during Cell + NO3

− + Fe(II)treatment, resulting in the cell encrustation and further inhibition of thenitrate-reducing Fe(II) oxidation.

XRD analysis of the minerals in the Fe(II) + NO− 2 and Cell + Fe(II) + NO2

− treatments showed that the main Fe(III) mineral was

goethite (Fig. 6a and b), while lepidocrocite (~70%) and goethite(~25%) were semi-quantified from the Scherrer Equation in the Cell+ Fe(II) + NO3

− treatment (Fig. 6c). Green rust was observed pre-viously (Pantke et al., 2012), but it was not observed here. The results(Fig. 7) showed that the broad band observed from 3369 to 3438 cm−1

was assigned to the OeH stretching vibration of water and an OH groupof Fe(III) oxyhydroxides (Rong et al., 2010). The band at 2925 cm−1

was due to an asymmetric eCH2 stretching vibration from bacteria,which was only observed in strain 2002 samples (Habibi, 2014). Twobands at 1630 cm−1 and 1780 cm−1 were also assigned to the waterbending vibration (Liao et al., 2007). The band at 475 cm−1 was as-signed to the FeeO vibration, and the absorption bands located at 885and 790 cm−1 were ascribed to the Fe-OH of goethite (Rong et al.,2010). Two bands at 1150 and 1025 cm−1 were attributed to lepido-crocite (Hug and Leupin, 2003; Antony et al., 2004;). In addition, for allof the patterns of XRD and FTIR at the 1st, 5th, and 12th days, nosignificant change was observed, implying that the secondary mineralswere mainly formed at the beginning and the phase transformation ofthe secondary minerals might be a very slow process.

4. Discussion

Microbially mediated nitrate-reducing Fe(II) oxidation was pre-viously considered to be nitrate-dependent Fe(II) oxidation, whichseemed to occur via a biological process (Straub et al., 1996; Weberet al., 2006b). However, due to the involvement of very fast Fe(II)

Fig. 4. SEM images of minerals produced by the abiotic treatment Fe(II) + NO2− (a, b and c) biotic treatments Cell + Fe(II) + NO2

− (d, e and f) and Cell + Fe(II) + NO3− (g, h and i)

for 1, 5, and 12 days.

D. Chen et al. Chemical Geology 476 (2018) 59–69

64

oxidation by an intermediate of nitrate bioreduction (nitrite), it is hardto define Fe(II) oxidation as either a biological or chemical process(Picardal, 2012; Klueglein and Kappler, 2013). Although some aspectsof microbially mediated nitrate-reducing Fe(II) oxidation have beenassessed, including the characterization of new bacteria, kinetics, mi-crobial community, mineralization and so on (Chakraborty andPicardal, 2013a; Chakraborty and Picardal, 2013b; Klueglein et al.,2014), it is still difficult to distinguish biological and chemical pro-cesses. In this study, several techniques (kinetics, N isotopic fractiona-tion, and identification of minerals formed) were used on detailedsystems, including biotic, abiotic, and the combined treatments, andthus, the chemical mechanism, biological mechanism, and their cou-pling mechanism were described more clearly.

The Gibbs free energies of the reactions (Rxns. (1) to (4)) betweenFe(II) and nitrate were calculated as described below, which showedthat the ΔGr

0 of all of the reactions were negative, indicating that thechemical reactions might be thermodynamically feasible. However,nitrate cannot be directly reduced by Fe(II) without a catalyst (Picardal,2012), which was further confirmed by the kinetics of Fe(II) and nitratein the (Fe(II) + NO3

− and Killed-cell + Fe(II) + NO3−) treatments

(Fig. 1).

+ + → + +

= −

− + − +γ ΔG12

NO Fe 32

H O ‐FeOOH 12

NO 2H

7.24kJ mol

ro

32

2 2

‐1 (1)

+ + → + +

= −

− + − +α ΔG12

NO Fe 32

H O ‐FeOOH 12

NO 2H

16.94kJ mol

ro

32

2 2

‐1 (2)

+ + → + +

= −

− + +γ g ΔG14

NO Fe 118

H O ‐FeOOH 18

N O( ) 74

H

34.93kJ mol

ro

32

2 2

‐1 (3)

Fig. 5. TEM images of the minerals produced by the abiotic treatment (a) Fe(II) + NO2−

as well as the biotic treatments (b) Cell + Fe(II) + NO2− and (c) Cell + Fe(II) + NO3

−

for 12 days.

(a)

(b)

(c)

Fig. 6. X-ray diffraction patterns of the abiotic treatment Fe(II) + NO2− (a) and biotic

treatments Cell + Fe(II) + NO2− (b) and Cell + Fe(II) + NO3

− (c) for 1, 5, and 12 days.G indicates goethite, L indicates lepidocrocite.

D. Chen et al. Chemical Geology 476 (2018) 59–69

65

+ + → + +

= −

− + +α g ΔG14

NO Fe 118

H O ‐FeOOH 18

N O( ) 74

H

44.63 kJ mol

ro

32

2 2

‐1 (4)

Despite the low feasibility of direct Fe(II) oxidation by nitrate, strain2002 may play a role of a catalyst which can obviously facilitate thereaction between Fe(II) and nitrate. Given the negative values of all ofthese reactions (Rxns. (1) to (4)), strain 2002 might gain energy fromthe redox reactions between Fe(II) and nitrate. However, two possiblepathways of nitrate reduction coupled with Fe(II) oxidation can beproposed: (i) Fe(II) oxidation by biogenic nitrite in which nitrate is firstreduced to nitrite by strain 2002, followed by Fe(II) oxidation by nitrite;

(ii) biological metabolism of Fe(II) and nitrate in which Fe(II), as anelectron donor of strain 2002, is enzymatically oxidized via the electrontransfer chain, with nitrate as an electron acceptor.

4.1. The pathway of Fe(II) oxidation by biogenic nitrite

Chemical oxidation of Fe(II) by nitrite was discovered several dec-ades ago (Moraghan and Buresh, 1976), and very fast reaction rateswere observed from the kinetics of the treatment (Fe(II) + NO2

−).Many bacteria can reduce nitrate to nitrite and other intermediates(Emerson et al., 2010; Carlson et al., 2013), which was proven by theresults of nitrate reduction by strain 2002 in this study, and reactionsbetween Fe(II) and nitrite or other intermediates may play more im-portant roles in the microbially mediated nitrate-reducing Fe(II) oxi-dation system, which has been previously largely overlooked untilstudied by Klueglein and Kappler (2013). As a result of Fe(II) oxidation,Fe(III) minerals or Fe(II)/Fe(III) minerals are produced. From XRD andFTIR, lepidocrocite and goethite were identified. N2O was detected as aproduct of nitrite reduction, so the chemical reactions between Fe(II)and nitrite can be written as Rxns. (5) and (6)

+ + → + +

= −

− + +γ ΔG12

NO Fe 54

H O ‐FeOOH 14

N O(g) 32

H

60.14 kJ mol

ro

22

2 2

‐1 (5)

+ + → + +

= −

− + +α ΔG12

NO Fe 54

H O ‐FeOOH 14

N O(g) 32

H

69.84 kJ mol

ro

22

2 2

‐1 (6)

Therefore, the importance of the well-confirmed chemical reactionbetween Fe(II) and nitrite is highlighted by all of the processes involvedin microbially mediated NO3

−-reducing Fe(II) oxidation. Since a sig-nificant amount of nitrite was generated by Cell + Fe(II) + NO3

−,chemical Fe(II) oxidation by biogenic nitrite may contribute to theoverall Fe(II) oxidation. Hence, the measured nitrite did not reflect thetotal amount of nitrite formed but the remaining nitrite after some ofthe nitrite has reacted with Fe(II).

4.2. The pathway of biological reactions between Fe(II) and nitrate

While the enzymes required to oxidize Fe(II) might play a major rolein biological Fe(II) oxidation, no enzymes involved in Fe(II) oxidationby NO3

−-reducing Fe(II) oxidation bacteria have been identified to date(Laufer et al., 2016). Nevertheless, cytochrome c of strain 2002 wasconfirmed to be related to Fe(II) oxidation (Ilbert and Bonnefoy, 2013).The reduction potentials of the possible Fe(III)/Fe(II) redox pairs rangefrom −0.314 V to +0.014 V, indicating that electrons can readily bedonated to the more electron positive type b, c, or a cytochrome com-ponents of an electron transport chain to reduce nitrate (Weber et al.,2006a). From the kinetics of Fe(II), Fe(II) oxidation in the treatment ofFe(II) + NO2

− was very close to that of the treatment of Cell + Fe(II)+ NO2

−, so both of these treatments can represent pure chemicaloxidation of Fe(II). In addition, cells alone in the absence of nitrateshowed no ability to oxidize Fe(II). However, the Fe(II) oxidation rateof Cell + Fe(II) + NO3

− was higher than that in the chemical treat-ment, implying that besides chemical Fe(II) oxidation, biological Fe(II)oxidation by strain 2002 might be stimulated by the presence of nitratevia biological oxidation pathway. Hence, competition between che-mical Fe(II) oxidation and biological Fe(II) oxidation occurred in theCell + Fe(II) + NO3

− system. The enhancement of Fe(II) oxidation inCell + Fe(II) + NO3

− compared with that in Fe(II) + NO2− (Table 1)

indicated that the rate of Fe(II) oxidation in biological oxidationpathway might be higher than that in chemical oxidation pathway.

The kinetic results in Fig. 1d and 2c suggested that N2O in Cell + Fe(II) + NO3

− was produced from two pathways: direct biological re-duction by strain 2002 and chemical reduction of nitrite by Fe(II).Regarding to the N isotopic fractionation of N2O, the initial δ15N-N2O of

(a)

(b)

(c)

Fig. 7. Fourier transform the infrared spectra of abiotic treatment Fe(II) + NO2− (a), and

biotic treatments Cell + Fe(II) + NO2− (b) and Cell + Fe(II) + NO3

− (c) for 1, 5, and12 days.

D. Chen et al. Chemical Geology 476 (2018) 59–69

66

all the treatments were more negative than the starting δ15N of NO3−

(−0.2 ± 0.1‰) and NO2−(−7.25 ± 0.25‰), which were well

matched with the existing studies on chemodenitrification and bio-de-nitrification (Bryan et al., 1983; Barford et al., 1999; Granger et al.,2008; Jones et al., 2015; Grabb et al., 2017). The large difference ofδ15N between NO3

−/NO2− and N2O can be attributed to a heavy pool

of the intermediate product (NO) in which the 14N could be pre-ferentially reacted to N2O, resulting in the more negative δ15N in N2O(Jones et al., 2015).

The δ15N-N2O of Cell + NO3− and Cell + NO2

− treatments clearlyincreased over time, which was probably caused by the further reduc-tion of N2O to N2 with 14N being preferentially reacted (Barford et al.,1999; Weber et al., 2009; Zhao et al., 2013). The increase of the δ15N-N2O in treatment Cell + Fe(II) + NO2

−(4.71 ± 0.21‰) was sig-nificantly higher than that in treatment Fe(II) + NO2

−

(2.45 ± 0.09‰), probably because of greater N isotope fractionationassociated with biological NO2

− reduction in incubations with cellsthan without. The δ15N-N2O in the treatment Cell + Fe(II) + NO3

−

also increased (from −33.8 ± 0.68‰ to −28.34 ± 0.64‰), but theextent was smaller than the Cell + NO3

− and Cell + NO2− treatments

and bigger than that in Fe(II) + NO2−, indicated that NO2

− might bereduced by both biological and chemical processes in Cell + Fe(II)+ NO3

− system.The very large difference of the δ15N-N2O was observed in Cell + Fe

(II) + NO3− and Cell + NO3

− treatments. Since nitrate was completelyreduced to nitrite in Cell + NO3

− treatment, the isotopic compositionof NO2

− was rapidly (< 8 h) reset to that of nitrate at the start of theexperiment. However, the kinetics results showed that only a portion ofNO3

− (about 30%) was converted to NO2− in Cell + Fe(II) + NO3

−

treatment, as a result, the isotopic composition of NO2− likely had a

more negative δ15N because of the fractionation during nitrate reduc-tion. The NO2

− with negative δ15N was further reduced to N2O, re-sulting in extremely negative values of δ15N-N2O in the Cell + Fe(II)+ NO3

− treatment. Hence, the δ15N-N2O produced from NO2− in the

Cell + NO3− treatment started at a more positive value than that in the

Cell + Fe(II) + NO3− treatment, and the very negative δ15N-N2O in

the Cell + Fe(II) + NO3− treatment reflects the sum of the isotope

fractionations associated with the reduction of NO3− to NO2

− plus thatof the reduction of NO2

− to N2O.Since the N mass balance and the N isotopes of other nitrogen

species (i.e., NO3−, NO2

−, and N2) over time were not determined inthis study, the above interpretation on various treatments cannot bedirectly based on the N isotope ratios of produced N2O. Therefore, thedetailed analysis of nitrogen species and the nitrogen isotope fractio-nations would be essential for clearly explaining the underlying reac-tion mechanisms in the microbially mediated nitrate-reducing Fe(II)oxidation processes. Furthermore, it was well recognized that N2O inCell + Fe(II) + NO3

− was produced from two pathways: direct biolo-gical reduction by strain 2002 and chemical reduction of nitrite by Fe(II). Since the enzymatic reaction between Fe(II) and nitrate was ver-ified (He et al., 2017; Liu et al., 2017), it would be very interesting toinvestigate the effect on the N isotope fractionation in the future stu-dies.

Mineralization occurred in the treatments with Fe(II) after Fe(II)oxidation reactions. The secondary minerals in the chemical treatment(Fe(II) + NO2

−) were different from those in the Cell + Fe(II) + NO3−

treatment. It was shown that the observed Fe(III) mineral diversity maybe the result of different mechanisms of Fe(II) oxidation, different cell-Fe(III) interactions, and different geochemical solution conditions(Larese-Casanova et al., 2010). The geochemical solution conditions(pH, medium composition, concentrations of co-substrates and in-cubation conditions) were the same in the different treatments used inthis study; thus, they could not influence the mineralogy of the Fe(III)phases. In this study, the Fe(II) oxidation rates were obviously differentfor the different Fe(II) oxidation mechanisms, which may play a keyrole in determining the mineralogy of the secondary minerals (Kappler

et al., 2005). With different Fe(II) oxidation rates (Table 1), the sec-ondary minerals produced by the chemical and combined experimentswere different. Both produced goethite, but lepidocrocite was onlyobserved in the combined treatment (Cell + Fe(II) + NO3

−). It wasalso found that the crystal size of the secondary minerals in the che-mical treatment (Fe(II) + NO2

−) was larger than that in the Cell + Fe(II) + NO3

− treatment, implying that the chemical reaction was fa-vorable for crystal growth and the presence of strain 2002 might slowcrystal growth. This result was also supported by the fact that lepido-crocite with low crystallinity was only observed in the Cell + Fe(II)+ NO3

− treatment. It was reported that the surface reactivity of thecell surface under environmental pH conditions conferred a net nega-tive charge to the cell wall, which led to the binding of soluble Fe(III)and Fe(II), and eventually to the precipitation of iron (oxyhydr)oxides(Fortin and Langley, 2005). Furthermore, it was suggested that biolo-gical Fe(II) oxidation leading to cell encrustation took place on the cellsurface or in the periplasmic space of cells (Carlson et al., 2012;Klueglein et al., 2014). In this study, morphological observationsshowed that cells were encrusted by Fe(III) minerals in the Cell + Fe(II) + NO3

− treatment, resulting in incomplete NO3− reduction, as

shown in Fig. 1b, which was consistent with other reports (Weber et al.,2009; Klueglein et al., 2014; Miot et al., 2015), because encrustationmay inhibit nitrate reduction physically by blocking its transport intocells. The cells and minerals weakly contacted each other in the Cell+ Fe(II) + NO2

− treatment, while whole cells of strain 2002 werecovered by Fe(III) minerals in the Cell + Fe(II) + NO3

− treatment,implying that biological oxidation pathway mainly occurred on the cellsurfaces but that chemical oxidation pathway occurred outside of thecells.

5. Conclusions

In this study, biological and chemical processes were investigatedthrough reaction kinetics, nitrogen isotope fractionation and secondaryminerals characterization. The chemical reaction between Fe(II) andnitrite definitely occurred via microbially mediated NO3

− reducing Fe(II) oxidation. Enhancement of Fe(II) oxidation was observed in theCell + Fe(II) + NO3

− treatment, suggesting that the rate of Fe(II)oxidation via microbially mediated processes might be higher thanthose via chemical processes. The extremely negative δ15N-N2O valuein the Cell + Fe(II) + NO3

− treatment may be caused by the in-complete reduction of nitrate due to the inhibition by newly-formedminerals on cell surfaces. The mineralization results further revealedthat Fe(II) oxidation via microbially mediated processes mainly oc-curred on the cell surfaces, while Fe(II) oxidation via chemical pro-cesses occurred outside of cells. The observations in this study clearlydistinguish the differences between the biological and chemical pro-cesses involved in the process of microbially mediated NO3

−-reducingFe(II) oxidation.

Acknowledgments

This work was funded by the National Natural Science Foundationof China (41571130052 and 41522105), the Guangdong NaturalScience Funds for Distinguished Young Scholars (2014A030306041),the Excellent Talent Fund of Guangdong Academy of Sciences(2017GDASCX-0408), and the SPICC program (2016GDASPT-0105).We are grateful for the help for the δ15N test from the Public ServiceTechnology Center, Institute of Subtropical Agriculture, ChineseAcademy of Sciences. We are also grateful for the very careful andhelpful comments from the anonymous reviewers.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemgeo.2017.11.004.

D. Chen et al. Chemical Geology 476 (2018) 59–69

67

References

Antony, H., Peulon, S., Legrand, L., Chausse, A., 2004. Electrochemical synthesis of le-pidocrocite thin films on gold substrate - EQCM, IRRAS, SEM and XRD study.Electrochim. Acta 50 (4), 1015–1021.

Barford, C.C., Montoya, J.P., Altabet, M.A., Mitchell, R., 1999. Steady-state nitrogenisotope effects of N2 and N2O production in Paracoccus denitrificans. Appl. Environ.Microbiol. 65 (3), 989–994.

Benz, M., Brune, A., Schink, B., 1998. Anaerobic and aerobic oxidation of ferrous iron atneutral pH by chemoheterotrophic nitrate-reducing bacteria. Arch. Microbiol. 169,159–165.

Borch, T., Kretzschmar, R., Kappler, A., Cappellen, P.V., Ginder-vogel, M., Voegelin, A.,Campbell, K., 2010. Biogeochemical redox processes and their impact on con-taminant dynamics. Environ. Sci. Technol. 44 (1), 15–23.

Bryan, B.A., Shearer, G., Skeeters, J.L., Kohl, D.H., 1983. Variable expression of the ni-trogen isotope effect associated with denitrification of nitrite. J. Biol. Chem. 258,8613–8617.

Carlson, H.K., Clark, I.C., Melnyk, R.A., Coates, J.D., 2012. Toward a mechanistic un-derstanding of anaerobic nitrate-dependent iron oxidation: balancing electron uptakeand detoxification. Front. Microbiol. 3, 57–62.

Carlson, H.K., Clark, L.C., Blazewicz, S.K., Lavarone, A.T., Coates, J.D., 2013. Fe(II)oxidation is an innate capability of nitrate-reducing bacteria that involves abiotic andbiotic reactions. J. Bacteriol. 195 (14), 3260–3268.

Chakraborty, A., Picardal, F., 2013a. Induction of nitrate-dependent Fe(II) oxidation by Fe(II) in Dechloromonas sp strain UWNR4 and Acidovorax sp strain 2AN. Appl.Microbiol. Biotechnol. 79 (2), 748–752.

Chakraborty, A., Picardal, F., 2013b. Neutrophilic, nitrate-dependent, Fe(II) oxidation bya Dechloromonas species. World J. Microbiol. Biotechnol. 29 (4), 617–623.

Cooper, D.C., Picardal, F.W., Schimmelmann, A., Coby, A.J., 2003. Chemical and biolo-gical interactions during nitrate and goethite reduction by Shewanella putrefaciens200. Appl. Environ. Microbiol. 69 (6), 3517–3525.

Druschel, G.K., Emerson, D., Sutka, R., Suchecki, P., Luther, G.W., 2008. Low-oxygen andchemical kinetic constraints on the geochemical niche of neutrophilic iron(II) oxi-dizing microorganisms. Geochim. Cosmochim. Acta 72 (14), 3358–3370.

Elliott, A.V.C., Plach, J.M., Droppo, I.G., Warren, L.A., 2014. Collaborative microbial Fe-redox cycling by pelagic floc bacteria across wide ranging oxygenated aquatic sys-tems. Chem. Geol. 366, 90–102.

Emerson, D., Fleming, E.J., McBeth, J.M., 2010. Iron-oxidizing bacteria: an environ-mental and genomic perspective. Annu. Rev. Microbiol. 64, 561–583.

Fortin, D., Langley, S., 2005. Formation and occurrence of biogenic iron-rich minerals.Earth-Sci. Rev. 72 (1–2), 1–19.

Gault, A.G., Ibrahim, A., Langley, S., Renaud, R., Takahashi, Y., Boothman, C., Lloyd, J.R.,Clark, L.D., Ferris, F.G., Fortin, D., 2011. Microbial and geochemical features suggestiron redox cycling within bacteriogenic iron oxide-rich sediments. Chem. Geol. 281(1–2), 41–51.

Grabb, K.C., Buchwald, C., Hansel, C.M., Wankel, S.D., 2017. A dual nitrite isotopic in-vestigation of chemodenitrification by mineral-associated Fe(II) and its production ofnitrous oxide. Geochim. Cosmochim. Acta 196, 388–402.

Granger, J., Sigman, D.M., 2009. Removal of nitrite with sulfamic acid for nitrate N and Oisotope analysis with the denitrifier method. Rapid Commun. Mass Spectrom. 23,3753–3762.

Granger, J., Sigman, D.M., Lehmann, M.F., Tortell, P.D., 2008. Nitrogen and oxygenisotope fractionation during dissimilatory nitrate reduction by denitrifying bacteria.Limnol. Oceanogr. 53 (6), 2533–2545.

Habibi, N., 2014. Preparation of biocompatible magnetite-carboxymethyl cellulose na-nocomposite: characterization of nanocomposite by FTIR, XRD, FESEM and TEM.Spectrochim. Acta Part A 131, 55–58.

Hafenbradl, D., Keller, M., Dirmeier, R., Rachel, R., Roßnagel, P., Burggraf, S., Huber, H.,Stetter, K.O., 1996. Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilicarchaeum that oxidizes Fe2+ at neutral pH under anoxic conditions. Arch. Microbiol.166, 308–314.

He, S., Barco, R.A., Emerson, D., Roden, E.E., 2017. Comparative genomic analysis ofneutrophilic iron(II) oxidizer genomes for candidate genes in extracellular electrontransfer. Front. Microbiol. 8, 1584–1601.

Hedrich, S., Schlomann, M., Johnson, D.B., 2011. The iron-oxidizing proteobacteria.Microbiology 157 (6), 1551–1564.

Hohmann, C., Winkler, E., Morin, G., Kappler, A., 2010. Anaerobic Fe(II)-oxidizing bac-teria show as resistance and immobilize as during Fe(III) mineral precipitation.Environ. Sci. Technol. 44, 94–101.

Hou, L., Zheng, Y., Liu, M., Gong, J., Zhang, X., Yin, G., You, L., 2014. Anaerobic am-monium oxidation (anammox) bacterial diversity, abundance, and activity in marshsediments of the Yangtze Estuary. J. Geophys. Res. 118, 1237–1246.

Hug, S.J., Leupin, O., 2003. Iron-catalyzed oxidation of arsenic(III) by oxygen and byhydrogen peroxide: pH-dependent formation of oxidants in the Fenton reaction.Environ. Sci. Technol. 37 (12), 2734–2742.

Ilbert, M., Bonnefoy, V., 2013. Insight into the evolution of the iron oxidation pathways.Biochim. Biophys. Acta 1827 (2), 161–175.

Jones, L.C., Peters, B., Pacheco, J.S.L., Casciotti, K.L., Fendorf, S., 2015. Stable isotopesand iron oxide mineral products as markers of chemodenitrification. Environ. Sci.Technol. 49 (6), 3444–3452.

Kappler, A., Straub, K.L., 2005. Geomicrobiological cycling of iron. Rev. Mineral.Geochem. 59 (1), 85–108.

Kappler, A., Schink, B., Newman, D.K., 2005. Fe(III) mineral formation and cell en-crustation by the nitrate-dependent Fe(II)-oxidizer strain BoFeN1. Geobiology 3 (4),235–245.

Klueglein, N., Kappler, A., 2013. Abiotic oxidation of Fe(II) by reactive nitrogen species incultures of the nitrate-reducing Fe(II) oxidizer Acidovorax sp. BoFeN1 - questioningthe existence of enzymatic Fe(II) oxidation. Geobiology 11 (2), 180–190.

Klueglein, N., Zeitvogel, F., Stierhof, Y.D., Floetenmeyer, M., Konhauser, K., Kappler, A.,2014. Potential role of nitrite for abiotic Fe(II) oxidation and cell encrustation duringnitrate reduction by denitrifying bacteria. Appl. Microbiol. Biotechnol. 80 (3),1051–1061.

Klueglein, N., Picardal, F., Zedda, M., Zwiener, C., Kappler, A., 2015. Oxidation of Fe(II)-EDTA by nitrite and by two nitrate-reducing Fe(II)-oxidizing Acidovorax strains.Geobiology 13 (2), 198–207.

Kopf, S.H., Henny, C., Newman, D.K., 2013. Ligand-enhanced abiotic iron oxidation andthe effects of chemical versus biological iron cycling in anoxic environments.Environ. Sci. Technol. 47 (6), 2602–2611.

Lack, J.G., Chaudhuri, S.K., Kelly, S.D., Kemner, K.M., O'Connor, S.M., Coates, J.D., 2002.Immobilization of radionuclides and heavy metals through anaerobic bio-oxidation ofFe (II). Appl. Environ. Microbiol. 68 (6), 2704–2710.

Larese-Casanova, P., Haderlein, S.B., Kappler, A., 2010. Biomineralization of lepidocro-cite and goethite by nitrate-reducing Fe(II)-oxidizing bacteria: effect of pH, bi-carbonate, phosphate, and humic acids. Geochim. Cosmochim. Acta 74 (13),3721–3734.

Laufer, K., Røy, H., Jørgensen, B.B., Kappler, A., 2016. Evidence for the existence ofautotrophic nitrate-reducing Fe(II)-oxidizing bacteria in marine coastal sediment.Appl. Microbiol. Biotechnol. 82 (20), 6120–6131.

Li, X., Zhou, S., Li, F., Wu, C., Zhuang, L., Xu, W., Liu, L., 2009. Fe(III) oxide reduction andcarbon tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strainL17. J. Appl. Microbiol. 106, 130–139.

Li, X., Hou, L., Liu, M., Zheng, Y., Yin, G., Lin, X., Cheng, L., Li, Y., Hu, X., 2015. Evidenceof nitrogen loss from anaerobic ammonium oxidation coupled with ferric iron re-duction in an intertidal wetland. Environ. Sci. Technol. 49, 11560–11568.

Li, X., Zhang, W., Liu, T., Chen, L., Chen, P., Li, F., 2016. Changes in the composition anddiversity of microbial communities during anaerobic nitrate reduction and Fe(II)oxidation at circumneutral pH in paddy soil. Soil Biol. Biochem. 94, 70–79.

Liao, S., Wang, J., Zhu, D., Ren, L., Lu, J., Geng, M., Langdon, A., 2007. Structure andMn2+ adsorption properties of boron-doped goethite. Appl. Clay Sci. 38 (1–2),43–50.

Liu, T., Li, X., Li, F., Zhang, W., 2014a. Fe(III) oxides accelerate microbial nitrate re-duction and electricity generation by Klebsiella pneumoniae L17. J. Colloid InterfaceSci. 423, 25–32.

Liu, T., Zhang, W., Li, X., Li, F., Shen, W., 2014b. Kinetics of competitive reduction ofnitrate and iron oxides by Aeromonas hydrophila HS01. Soil Sci. Soc. Am. J. 78 (6),1903–1912.

Liu, T., Wang, Y., Li, X., Li, F., 2017. Redox dynamics and equilibria of c-type cyto-chromes in the presence of Fe(II) under anoxic conditions: insights into enzymaticiron oxidation. Chem. Geol. 468, 97–104.

Mandernack, K.W., Mills, C.T., Johnson, C.A., Rahn, T., Kinney, C., 2009. The δ15N andδ18O values of N2O produced during the co-oxidation of ammonia by methanotrophicbacteria. Chem. Geol. 267 (1–2), 96–107.

Melton, E.D., Swanner, E.D., Behrens, S., Schmidt, C., Kappler, A., 2014. The interplay ofmicrobially mediated and abiotic reactions in the biogeochemical Fe cycle. Nat. Rev.Microbiol. 12 (12), 797–808.

Miot, J., Li, J., Benzerara, K., Sougrati, M.T., Ona-Nguema, G., Bernard, S., Jumas, J.C.,Guyot, F., 2014. Formation of single domain magnetite by green rust oxidationpromoted by microbial anaerobic nitrate-dependent iron oxidation. Geochim.Cosmochim. Acta 139, 327–343.

Miot, J., Remusat, L., Duprat, E., Gonzalez, A., Pont, S., Poinsot, M., 2015. Fe biomi-neralization mirrors individual metabolic activity in a nitrate-dependent Fe(II)-oxi-dizer. Front. Microbiol. 6, 879–891.

Moraghan, J.T., Buresh, R.J., 1976. Chemical reduction of nitrite and nitrous oxide byferrous iron. Soil Sci. Soc. Am. J. 41, 47–50.

Muehe, E.M., Gerhardt, S., Schink, B., Kappler, A., 2009. Ecophysiology and the energeticbenefit of mixotrophic Fe(II) oxidation by various strains of nitrate-reducing bacteria.FEMS Microbiol. Ecol. 70 (3), 335–343.

Ottley, C.J., Davison, W., Edmunds, W.M., 1997. Chemical catalysis of nitrate reductionby iron(II). Geochim. Cosmochim. Acta 61 (9), 1819–1828.

Pantke, C., Obst, M., Benzerara, K., Morin, G., Onanguema, G., Dippon, U., Kappler, A.,2012. Green rust formation during Fe(II) oxidation by the nitrate-reducingAcidovorax sp. strain BoFeN1. Environ. Sci. Technol. 46, 1439–1446.

Picardal, F., 2012. Abiotic and microbial interactions during anaerobic transformations ofFe(II) and NO- x. Front. Microbiol. 3 (3), 112–118.

Rong, X., Chen, W., Huang, Q., Cai, P., Liang, W., 2010. Pseudomonas putida adhesion togoethite: studied by equilibrium adsorption, SEM, FTIR and ITC. Colloids Surf., B 80(1), 79–85.

Schädler, S., Burkhardt, C., Hegler, F., Straub, K.L., Miot, J., Benzerara, K., Kapper, A.,2009. Formation of cell-iron-mineral aggregates by phototrophic and nitrate-redu-cing anaerobic Fe(II)-oxidizing bacteria. Geomicrobiol J. 26 (2), 93–103.

Schmid, G., Zeitvogel, F., Hao, L., Ingino, P., Floetenmeyer, M., Stierhof, Y.D., Schroeppel,B., Burkhardt, C.J., Kappler, A., Obst, M., 2014. 3-D analysis of bacterial cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovoraxsp. strain BoFeN1 using complementary microscopy tomography approaches.Geobiology 12, 340–361.

Schmidt, H.L., Werner, R.A., Yoshida, N., Well, R., 2004. Is the isotopic composition ofnitrous oxide an indicator for its origin from nitrification or denitrification? A the-oretical approach from referred data and microbiological and enzyme kinetic aspects.Rapid Commun. Mass Spectrom. 18 (18), 2036–2040.

Scholz, F., Löscher, C.R., Fiskal, A., Sommer, S., Hensen, C., Lomnitz, U., Wuttig, K.,Göttlicher, J., Kossel, E., Strininger, R., Canfield, D.E., 2016. Nitrate-dependent iron

D. Chen et al. Chemical Geology 476 (2018) 59–69

68

oxidation limits iron transport in anoxic ocean regions. Earth Planet. Sci. Lett. 454,272–281.

Senko, J.M., Dewers, T.A., Krumholz, L.R., 2005. Effect of oxidation rate and Fe(II) stateon microbial nitrate-dependent Fe(III) mineral formation. Appl. Environ. Microbiol.71 (11), 7172–7177.

Smith, R.L., Kent, D.B., Repert, D.A., Böhlke, J.K., 2017. Anoxic nitrate reduction coupledwith iron oxidation and attenuation of dissolved arsenic and phosphate in a sand andgravel aquifer. Geochim. Cosmochim. Acta 196, 102–120.

Straub, K.L., Benz, M., Schink, B., Widdel, F., 1996. Anaerobic, nitrate-dependent mi-crobial oxidation of ferrous iron. Appl. Environ. Microbiol. 62 (4), 1458–1460.

Straub, K.L., Benz, M., Schink, B., 2001. Iron metabolism in anoxic environment at nearneutral pH. FEMS Microbiol. Ecol. 34 (3), 181–186.

Sutka, R.L., Ostrom, N.E., Ostrom, P.H., Breznak, J.A., Gandhi, H., Pitt, A.J., Li, F., 2006.Distinguishing nitrous oxide production from nitrification and denitrification on thebasis of isotopomer abundances. Appl. Environ. Microbiol. 72 (1), 638–644.

Tai, Y.L., Dempsey, B.A., 2008. Nitrite reduction with hydrous ferric oxide and Fe(II):stoichiometry, rate, and mechanism. Water Res. 43 (2), 546–552.

Torrentó, C., Urmeneta, J., Otero, N., Soler, A., Viñas, M., Cama, J., 2011. Enhanceddenitrification in groundwater and sediments from a nitrate-contaminated aquiferafter addition of pyrite. Chem. Geol. 287 (1–2), 90–101.

Toyoda, S., Mutobe, H., Yamagishi, H., Yoshida, N., Tanji, Y., 2005. Fractionation of N2Oisotopomers during production by denitrifier. Soil Biol. Biochem. 37 (8), 1535–1545.

Van Cleemput, O., Samater, A.H., 1996. Nitrite in soils: accumulation and role in theformation of gaseous N compounds. Fert. Res. 45, 81–89.

Weber, K.A., Picardal, F.W., Roden, E.E., 2001. Microbially catalyzed nitrate-dependentoxidation of biogenic solid-phase Fe(II) compounds. Environ. Sci. Technol. 35 (8),1644–1650.

Weber, K.A., Achenbach, L.A., Coates, J.D., 2006a. Microorganisms pumping iron:anaerobic microbial iron oxidation and reduction. Nat. Rev. Microbiol. 4 (10),752–764.

Weber, K.A., Pollock, J., Cole, K.A., O'Connor, S.M., Achenbach, L.A., Coates, J.D., 2006b.Anaerobic nitrate-dependent iron(II) bio-oxidation by a novel lithoautotrophic

betaproteobacterium, strain 2002. Appl. Environ. Microbiol. 72 (1), 686–694.Weber, K.A., Hedrick, D.B., Peacock, A.D., Thrash, J.C., White, D.C., Achenbach, L.A.,

Coates, J.D., 2009. Physiological and taxonomic description of the novel autotrophic,metal oxidizing bacterium, Pseudogulbenkiania sp strain 2002. Appl. Microbiol.Biotechnol. 83 (3), 555–565.

Wenk, C.B., Zopfi, J., Blees, J., Veronso, M., Niemann, H., Lehmann, M.F., 2014.Community N and O isotope fractionation by sulfide-dependent denitrification andanammox in a stratified lacustrine water column. Geochim. Cosmochim. Acta 125,551–563.

Wrage, N., Lauf, J., Prado, A.D., Pinto, M., Pietrzak, S., Yamulki, S., Oenema, O., Gebauer,G., 2004. Distinguishing sources of N2O in European grasslands by stable isotopeanalysis. Rapid Commun. Mass Spectrom. 18 (11), 1201–1207.

Wunderlich, A., Meckenstock, R., Einsiedl, F., 2012. Effect of different carbon substrateson nitrate stable isotope fractionation during microbial denitrification. Environ. Sci.Technol. 46 (9), 4861–4868.

Xiu, W., Guo, H., Shen, J., Liu, S., Ding, S., Hou, W., Ma, J., Dong, H., 2016. Stimulation ofFe(II) oxidation, biogenic lepidocrocite formation and arsenic immobilization byPseudogulbenkiania sp. strain 2002. Environ. Sci. Technol. 50 (12), 6449–6458.

Yang, H., Hasand, G., Ostrom, N.E., Hegg, E.L., 2014. Isotopic fractionation by a fungalP450 nitric oxide reductase during the production of N2O. Environ. Sci. Technol. 48(18), 10707–10715.

Zhang, W., Li, X., Liu, T., Li, F., 2012. Enhanced nitrate reduction and current generationby Bacillus sp. in the presence of iron oxides. J. Soils Sediments 12 (3), 354–365.

Zhang, W., Li, X., Liu, T., Li, F., Shen, W., 2014. Competitive reduction of nitrate and ironoxides by Shewanella putrefaciens 200 under anoxic conditions. Colloids Surf. APhysicochem. Eng. Asp. 445 (6), 97–104.

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

Zhou, J., Wang, H., Yang, K., Ji, B., Chen, D., Zhang, H., Sun, Y., Tian, J., 2016.Autotrophic denitrification by nitrate-dependent Fe(II) oxidation in a continuous up-flow biofilter. Bioprocess Biosyst. Eng. 39 (2), 1–8.

D. Chen et al. Chemical Geology 476 (2018) 59–69

69