Biogenic iron mineralization of polyferric sulfate by dissimilatory iron reducing bacteria: Effects of medium composition and electric field stimulation

Science of the Total Environment 684 (2019) 466–475

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Biogenic iron mineralization of polyferric sulfate by dissimilatory iron reducing bacteria: Effects of medium composition and electric field stimulation Qin Wang, Ziliang Wei, Xiaoyun Yi ⁎, Jie Tang, Chunhua Feng ⁎, Zhi Dang The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Fe(II) is produced from bioreduction of PFS in the presence of DIRB. • Biogenic Fe(II) catalyzes biomineralization of PFS to various secondary products. • Green rust and vivianite are the major minerals in the phosphate-buffered medium. • Magnetite is the primary product in the PIPES system. • Electric field stimulates Fe(II) production and induces more crystalline minerals.

a r t i c l e

i n f o

Article history: Received 4 March 2019 Received in revised form 3 May 2019 Accepted 21 May 2019 Available online 22 May 2019 Editor: Zhen (Jason) He Keywords: Biomineralization Dissimilatory iron reduction Electrical stimulation Bioelectrochemical system Sediment remediation Coagulant

a b s t r a c t Polyferric sulfate (PFS) is a coagulant widely used for removing contaminants from the aqueous phase; however, PFS destabilizes and recrystallizes in the solid phase in the presence of dissimilatory iron reducing bacteria (DIRB), which has a profound influence on the cycle of Fe and the fate of the associated pollutants. Our objective is to investigate the combined effects of medium composition and electric field stimulation on the biomineralization of PFS. Batch experiments were conducted with PFS and the DIRB Shewanella oneidensis MR-1 under anoxic conditions to examine the microbial reduction of PFS to Fe(II) and its subsequent biotransformation. The high concentration of phosphorous in phosphate buffer solution (PBS) is responsible for slower and less extensive Fe(II) generation compared to the lower concentration of phosphorous in a medium of 1,4piperazinediethanesulfonic acid (PIPES). The PBS system induces the formation of green rust (SO2− 4 ) and vivianite as the major minerals; in contrast, magnetite is the predominant end product in the PIPES system. The application of an anodic potential of 0.2 V significantly stimulates Fe(II) release from PFS, leading to precipitation and transformation of more crystalline minerals in increased quantities. The results demonstrate that Fe (II) catalyzes biomineralization of PFS to a variety of secondary products; this electron transfer process is highly dependent on the rate and magnitude of PFS reduction and the surface reaction with the host compound and adsorbed ions. © 2019 Published by Elsevier B.V.

⁎ Corresponding authors. E-mail addresses: [email protected] (X. Yi), [email protected] (C. Feng).

https://doi.org/10.1016/j.scitotenv.2019.05.322 0048-9697/© 2019 Published by Elsevier B.V.

Q. Wang et al. / Science of the Total Environment 684 (2019) 466–475

1. Introduction Polyferric sulfate (PFS; Fe at valence state of +3) is a common coagulant in water and wastewater treatment for destabilizing/adsorbing colloids and dissolved contaminants and generating large floc aggregates that are separable from the aqueous phase. Owing to their ability to rapidly remove pollutants in water, PFS coagulants are frequently used to manage accidental discharge of contaminants into surface water (Duan and Gregory, 2003; Song et al., 2006). Nearly 300 tons per year of synthetic PFS is poured into rivers and precipitated into the underlying sediment every year in China for emergency control of such contaminants (Fu and Wang, 2011). The dissolution of PFS flocs and the corresponding biogeochemical cycles of Fe in anoxic sediments remain of great concern because they play a critical role in the fate of associated contaminants, nutrients, and organic matter (Li et al., 2016). PFS flocs are amorphous and should be easily subject to a phase transformation, forming a crystalline Fe mineral, a process driven by dissimilatory iron reducing bacteria (DIRB), which is ubiquitous in the sediment. This is evidenced from many previous investigations showing that biogenic iron (Fe(II)) as a consequence of dissimilatory iron reduction in oxygen-depleted environments can thermodynamically induce transformation of iron minerals from less crystalline to more crystalline forms (Behrends and Van Cappellen, 2007; Borch et al., 2010; Xiao et al., 2018). However, despite its environmental significance, very little information is available on the Fe(II)-aided mineralization of PFS. A variety of biomineralization products result from the DIRBcatalyzed conversion of poorly crystalline Fe(III) compounds; the nature and distribution of these products are substantially influenced by the rate of Fe(II) formation, the presence of sorbed ions, and the medium composition (Zachara et al., 2002; Fredrickson et al., 1998). Among these, the Fe(II) generation rate has proved to be a primary factor governing secondary Fe mineralization, which is closely linked to microbial bioavailability (Roden and Urrutia, 2002). In recent years, studies have shown that microbes can actively interact with electrodes (either as electron acceptors or electron donors) (Wang et al., 2011; Chun et al., 2013), and our previous work has demonstrated that the application of a low electric field can stimulate the growth and enrichment of DIRB in Fe-containing sediment (Liu et al., 2017). This process generally utilizes the organic matter available in the sediment as the fuel source for electricity generation and pollutant removal; it is thus of great interest for bioremediation. It is expected that the increase in DIRB population caused by electric field stimulation can benefit reduction of Fe(III) to Fe(II), which should in turn affect biomineralization of Fe solids. The medium composition is also relevant with regard to which secondary products are formed due to the effects of adsorbed/ coprecipitated ions. For example, in a 1,4-piperazinediethanesulfonic acid (PIPES)-buffered medium, magnetite was the primary mineral product formed from DIRB-facilitated hydrous ferric oxide (HFO) reduction in an anoxic solution (Fredrickson et al., 1998; Li et al., 2012). Ferrous carbonate (siderite) in association with ferrous phosphate (vivianite), however, were identified as the major products of bioreduction of HFO by Shewanella putrefaciens CN32 (Liu et al., 2001; Glasauer et al., 2003; Salas et al., 2010) in a HCO− 3 -buffered medium. In addition, some previous studies indicate that the presence of PO3− 4 in the solution retards Fe(III) reduction because of its strong affinity for the surface of Fe(III) mineral (Zachara et al., 2002; Bocher et al., 2004; Refait et al., 2007); other studies suggest that PO3− 4 can improve Fe(III) reduction by complexation with the produced Fe(II) (Fredrickson et al., 1998; O'Loughlin et al., 2013). It has been previously reported that in the absence of phosphate, magnetite was the primary secondary mineral formed during bioreduction of lepidocrocite and akaganeite by Shewanella putrefaciens CN32; on the contrary, carbonate green rust was the principle secondary mineral in the presence of phosphate (O'Loughlin et al., 2010; O'Loughlin et al., 2013; O'Loughlin et al., 2015).

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The purpose of this study was therefore to investigate the biomineralization of PFS by DIRB in an anoxic solution, focusing on the combined effects of medium composition and electric field stimulation on the form and extent of secondary products. Fe(II) production rates and the concomitant secondary mineral formation were evaluated under a variety of conditions. Of particular interest was the mechanism governing electric field-stimulated Fe(II) production and the role of biogenic Fe (II) in promoting the crystallization of iron oxides. To achieve this, a three-electrode bioelectrochemical reactor was constructed with a poised potential of 0.2 V on the working electrode. The PFS in the reactor underwent bioreduction by Shewanella oneidensis MR-1 (a model dissimilatory iron reducing microorganism) in either phosphate- or PIPES-buffered solution. The crystallographic and morphologic features of the mineral products were analyzed based on physicochemical characterizations. 2. Materials and methods 2.1. Preparation of cell suspension and PFS flocs for biotic experiments Shewanella oneidensis MR-1 was selected as the model DIRB for the biotic experiments and purchased from ATCC (700550). The strain was stored in an agar (Luria-Bertani) slope tube at 4 °C and then activated (30 °C, 150 rpm, 18 h) in LB culture medium (5 g L−1 beef extract, 10 g L−1 tryptone, and 5 g L−1 NaCl). A 1 mL aliquot of the activated seed strain was used to inoculate another 50 mL of the sterile LB medium, which was then incubated aerobically (30 °C, 150 rpm, 18 h). The cells were harvested via centrifugation (6500 rpm, 10 min, 15 °C) and washed twice with the corresponding mineral basal medium to remove residual LB. Two medium formulas were adopted for the abiotic experiments, namely phosphate buffer solution (PBS) and the piperazine-1,4bisethanesulfonic acid (PIPES) buffer solution. The PBS medium (pH 6.8) consists of 5.88 g L−1 NaCl, 0.10 g L−1 KCl, 0.25 g L−1 NH4Cl, 22.2 g L−1 Na2HPO4·12H2O, 5.92 g L−1 NaH2PO4·2H2O, 10 mL of vitamin solution, and 10 mL of mineral solution (Wan et al., 2018). The PIPES medium (pH 6.8) contains 5.88 g L−1 NaCl, 0.25 g L−1 NH4Cl, 0.10 g L−1 K2HPO4, 0.30 g L−1 KH2PO4·3H2O, 3.02 g L−1 PIPES, 10 mL of vitamin solution, and 10 mL of mineral solution (Wan et al., 2018). After harvesting and rinsing, the wet biomass was transferred to the sterile medium to obtain cell suspensions with a concentration of 1 g L−1. The PFS flocs were prepared by mixing with 5.0 M and 1.0 M NaOH adjusted to pH of 6.8 at a stirring speed of 200 rpm. After 4 h of gravity sedimentation, the flocs were obtained and the upper transparent liquid supernatant was discarded. The general formula of PFS flocs is [Fe2(OH)n(SO4)3−n/2]m (n N 2). 2.2. Configuration of the bioreactor and operation of biotic experiments Batch biotic experiments were performed in a two-chamber microbial reactor, each chamber of which has a volume of 220 mL. A cation exchange membrane (Zhejiang Qianqiu Group Co., Ltd. China) separated the chambers. Carbon felt (5.0 cm × 5.0 cm × 0.5 cm; Sanye Co., Ltd., Beijing, China) was used as the anode and the cathode. The felt was pretreated in a hot H2O2 (10%, 90 °C) solution for 2 h (Wang and Feng, 2017), followed by thorough rinsing with deionized (DI) water (90 °C); this procedure was repeated two times before drying at 60 °C. To run the electrolysis test, the anode and the cathode were connected to an external circuit via twisted-pair titanium wire (0.6 mm in diameter). A saturated calomel electrode (SCE) inserted into the anode chamber was used as the reference electrode. All potentials reported throughout this study are in reference to the SCE. Chronoamperometry (CA) experiments were performed at a controlled temperature of 30 °C with a poised potential of 0.2 V, which was controlled by a CHI 1000C potentiostat. The selection of 0.2 V to stimulate the growth MR-1 is owing to the fact that this potential value has been widely used in the bioelectrochemical systems to promote the enrichment of

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electrochemical active bacteria with high activity towards electricity generation (Nakamura et al., 2009; Feng et al., 2013; Min et al., 2017). To enrich the electrochemically active biofilms on the anode, the anode chamber was injected with the PBS medium containing cell suspension (1 g L−1) and lactate (25 mM). Three repeatable cycles were run to gain stable bio-current production; for each cycle, the chamber was supplemented with additional fresh medium containing 25 mM lactate. Before each replacement, the anaerobic condition was maintained by purging with O2-free N2 for 20 min. The electric field-stimulated microbial reduction of PFS was then performed by replacing the old medium with fresh medium as well as 40 mL of PFS flocs. Two treatments were undertaken: applied potential of 0.2 V and applied potential of −0.2 V. For comparison, one open-circuit control experiment was conducted with the enriched biofilms and another open-circuit control experiment was conducted without the biofilms, analogous to the traditional anaerobic test performed with PBS medium containing PFS flocs (4 g L −1), cell suspension (1 g L−1), and lactate (25 mM). Solid and aqueous samples were taken at particular time intervals for chemical and physicochemical analyses. For all tests, duplicate experiments were conducted to confirm their reproducibility. For the data presented with the error bar, it is the average value of the results from samples drawn from duplicate reactors; otherwise, it refers to the result from a representative reactor.

2.3. Abiotic Fe(II)-induced PFS transformation The abiotic PFS transformation experiments were conducted in a 100 mL headspace bottle. The PBS or PIPES medium was added to the bottle. After purging with O2-free N2 for 20 min, the solution was amended with 40 mL of PFS flocs and different volumes of 1 M FeSO4 solution to final Fe(II) concentrations of 0, 20, 50, 100, 300, and 500 mg L−1. All bottles were sealed with butyl rubber stoppers with aluminum seals and then placed in a constant-temperature oscillator (150 rpm, 30 °C). The solid phases after operation times of 6 and 13 days were analyzed via physicochemical characterizations.

2.4. Analytical methods The total Fe(II) concentration of the sample (including aqueous and solid phases) was determined via 0.5 M HCl extraction (150 rpm, 30 °C, 2 h). Following extraction, the desorbed solution sample was filtered through 0.22 μm sterile filters prior to aqueous analysis. The Fe(II) concentration and the dissolved Fe2+ concentration in the aqueous phase were quantified via ultraviolet-visible spectrophotometry (Shimadzu UV-2550, Japan) at λ = 510 nm according to the o-phenanthroline photometry method (Sarradin et al., 2005). The non-turnover cyclic voltammetry (CV) characterizations were performed to investigate the electron transfer capability of MR-1. These tests were run in the threeelectrode system with anode as the working electrode, cathode as the counter electrode, and a sterilized SCE placed into the anode chamber as the reference electrode. Solid samples were characterized via X-ray diffraction (XRD) and scanning electron microscopy (SEM) with regard to their structure, composition, and morphology. The samples were first centrifuged at 5000 rpm, then washed three times with DI water, then freeze-dried for 24 h to obtain fine powder. The morphology of the resulting solid products was examined by SEM (Merlin, Zeiss Co., Berlin, Germany). The distributions of major elements in the mineral surfaces were determined via energy dispersive spectroscopy analysis (EDS). The crystalline structure of the mineral phase was determined via XRD (Empyrean, Malvern Panalytical, Netherlands). Spectra were elucidated using Jade 6.5 software with reference to the Joint Committee on Powder Diffraction Standards (JCPDS).

3. Results and discussion 3.1. Effects of buffer composition and electric field stimulation on biogenic Fe(II) formation The rate and extent of Fe(II) production resulting from DIRB-driven reduction of poorly crystalline iron minerals plays a pivotal role in the nature of the secondary biomineralization products formed because the biogenic Fe(II) catalyzes the transformation via the electron transfer pathway in the surface (Roden and Urrutia, 2002). Fig. 1 shows the time course of biogenic Fe(II) concentration as a consequence of microbial reduction of PFS, which is impacted by both the presence of an electric field and the type of buffer (PBS or PIPES). Insignificant production of Fe(II) was observed in the abiotic control. Because of its amorphous and thermodynamically unstable characteristics, PFS was substantially reduced without lag time, causing the release of large amounts of Fe (II) for all treatments. Three phrases can be distinguished from the concentration curves of total Fe(II) plotted against incubation time (Fig. 1a and b): rapid Fe(II) production with dramatically increasing concentration (Phase I), slow Fe(II) production with gently increasing concentration (Phase II), and a decline in Fe(II) concentration (Phase III). Phase I is correlated to an initial quick dissolution of PFS, generating aqueous Fe (II). Phase II can be attributed to two causes: the depletion of efficient electron donors (i.e., lactate) and adsorption of Fe(II) on PFS surfaces, which might retard microbial reduction of PFS. Phase III results from the transformation of Fe(II) into crystalline biomineralization products, from which some Fe species (e.g., magnetite) can hardly be extracted with 0.5 M HCl (Fredrickson et al., 1998). It was noticeable from Phase I that compared to the PIPES-buffered conditions, the PBS condition resulted in smaller quantities of Fe(II) for all treatments. The relatively slower reaction rate was due to the adsorption of PO3− 4 to the surface of PFS, which acted as an inhibitory barrier to further dissolution of PFS (Zachara et al., 2002; Bocher et al., 2004; Refait et al., 2007). Indeed, the release of biogenic Fe(II) is dependent on the concentration of phosphorous in the solution. Fig. S1 shows the time course of biogenic Fe(II) concentration as a function of the phosphorous content, clearly suggesting its significant effect on both the rate and extent of bioreduction of PFS. At lower phosphorous concentrations (from 1 to 20 mM), the increasing content resulted in an increase in the Fe(II) production rate, with a maximum Fe(II) concentration of 468.1 mM achieved at day 10 in the treatment with 20 mM phosphorous. This phenomenon can be attributable to the complexation and/or adsorption of PO3− with the produced Fe(II) 4 (Fredrickson et al., 1998; O'Loughlin et al., 2013), thus thermodynamically driving the bioreduction. In contrast, at higher phosphorous concentrations (from 20 to 100 mM), the increasing content inhibited bioreduction of PFS, as a peak Fe(II) concentration of 214.8 mM was obtained in the treatment with 100 mM phosphorous. This observation can be explained by the strong affinity of Fe(III) surface by PO3− 4 , which obscures the reaction sites (Zachara et al., 2002). Under both buffer conditions, the application of an electric field promoted Fe(II) production (Phase I and Phase II), with a more pronounced effect observed for the 0.2 V potential. The application of a poised potential of −0.2 V slightly enhanced Fe(II) generation in comparison to no electric field with and without preformed electroactive biofilms. A remarkable observation in Phase III is that electric field stimulation at 0.2 V resulted in a more rapid and significant decrease in Fe(II) concentration; in contrast, its concentration stabilized at a high level for the open-circuit control experiment. The treatment order with regard to Fe(II) concentration at the end of incubation was Treatment #1 (0.2 V) b Treatment #2 (−0.2 V) b Control #2 b Control #1. These findings imply that electric field stimulation alters the rate and extent of microbial PFS reduction and the subsequent formation of secondary mineralization solids; a poised potential of 0.2 V facilitates bioreduction and gives rise to the formation of stable Fe-bearing products that are resistant to being released via HCl extraction.

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Fig. 1. HCl-extractable Fe(II) and aqueous Fe2+ concentrations in (a, c) PBS and (b, d) PIPES-buffered suspensions inoculated with Shewanella oneidensis MR-1 in the presence of PFS. Treatment #1: application of an anodic potential of 0.2 V; Treatment #2: application of an anodic potential of −0.2 V; Abiotic control: treatment at 0.2 V in the absence of bacteria; Control #1: open-circuit operation in the system without pre-enriched biofilms; Control #2: open-circuit operation in the system with pre-enriched biofilms.

Fig. 1c and d show that the concentration of aqueous Fe2+ for all treatments first increases, reaches a peak amount, and subsequently declines, reflecting generation, then adsorption, followed by transformation associated with the solids. Some studies (Dong et al., 2000;

Kukkadapu et al., 2004) of microbial reduction of ferrihydrites by DIRB have reported that the aqueous Fe2+ concentrations were far below the total Fe(II) concentrations, suggesting that most of the biogenic Fe (II) was rapidly bound and/or precipitated. However, the aqueous Fe2

Fig. 2. Biocurrent generation in the PBS and PIPES-buffered suspensions inoculated with Shewanella oneidensis MR-1 in the (a) absence and (b) presence of PFS. For Phase a, four reactors were run for bacteria enrichment at a poised potential of 0.2 V, with two for PBS and two for PIPES. For Phase b, four reactors were operated to investigate biogenic iron mineralization of PFS at different potentials, with two for PBS respectively at 0.2 V and −0.2 V, and two for PIPES respectively at 0.2 V and −0.2 V.

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+

concentrations resulting from microbial reduction of PFS were quite close to the concentrations of total Fe(II), particularly in the early incubation period. This suggests that the interaction between Fe2+ and PFS surfaces might not be strong. Fig. 1c and d also show that the application of 0.2 V potential enabled more rapid rates of generation and disappearance of aqueous Fe2+ in comparison to those obtained under other conditions. The results are in accordance with those associated with Fe(II) evolution, indicative of electric field-promoted PFS reduction and biotransformation. 3.2. Enhanced biocurrent generation with added PFS Biocurrent generation in two buffer solutions in the absence and presence of PFS was examined. Fig. 2a shows that after three consecutive cycles, biocurrent production upon a poised potential was successfully achieved. In comparison to the abiotic control, which exhibited no anodic current, the application of 0.2 V potential stimulates the growth of MR-1 and enables a maximum current density in the range from 2.5 to 3.0 × 10−2 A m−2 for the PBS and PIPES systems. This result indicates the successful enrichment of electrochemically active bacteria biofilms at the anode, in accordance with our previous findings (Feng et al., 2013; Yu et al., 2016). Fig. 2b shows that the addition of PFS noticeably magnifies the production of biocurrent at a poised potential of 0.2 V. This promoting effect is analogous to the addition of iron oxides, which enhances extracellular electron transfer from the microbes to the anode and thus bioelectricity generation (Peng et al., 2013; Liu et al., 2017; Grobbler et al., 2018). The promoted electron transfer is expected to accelerate PFS reduction, resulting in elevated rates of Fe(II) production as compared to the open-circuit treatments. In addition, a higher peak current was observed in the PBS buffer in comparison to the PIPES buffer, likely due to the promotion of MR-1 activity in the phosphorus-rich environment (Torres et al., 2008). The poised potential of −0.2 V, despite being lower than 0.2 V, is higher than the experimentally observed redox potentials for bioelectrochemical lactate oxidation under similar conditions (−0.55 to −0.6 V) (Yu et al., 2016). As such, the application of −0.2 V potential also prompted biocurrent generation in both the PBS and PIPESbuffered medium, although the levels of currents were lower than those achieved at 0.2 V. CV tests were conducted to measure relative activity of MR-1 in the treatments with and without electric field stimulation (a poised potential of 0.2 V) by comparing catalytic currents and waves. As shown in Fig. S2a, the 0.2 V-enriched electrode exhibited a clear redox wave and much higher currents compared to the non-enriched electrode. The wave with a midpoint potential of −0.19 V is in agreement with the reported value of active c-type cytochromes secreted from Shewanella oneidensis (Nakamura et al., 2009; Feng et al., 2010; Min et al., 2017). This indicates an enhanced activity of MR-1 as a result of electric field stimulation. When the PFS was added to the reactor, the peak current was significantly enhanced (Fig. S2b), suggesting the long-distance electron transfer conduit in the colloidal network (Nakamura et al., 2009). The effect of electric filed stimulation on the extent of current was also pronounced in the PFS-amended systems. These results demonstrate that the inoculation with a poised potential of 0.2 V can substantially promote the extracellular electron transfer capability of MR-1. 3.3. Physicochemical characterizations of secondary biomineralization products XRD tests were performed to identify the crystalline phases of the secondary biomineralization products formed after incubation. Fig. 3 shows and compares the XRD patterns of these products obtained under different conditions in the PBS (Fig. 3a) or PIPES (Fig. 3b) buffer solution. The effect of incubation time on the biomineralization solids

was also evaluated. From Day 6 to Day 10, and to Day 20, the intensities of most of the XRD peaks in Fig. 3a and b were augmented, suggesting the formation of increasing quantities of crystalline compounds. The distinct discrepancy between Fig. 3a and b is that more peaks appear when more phosphate is available in the solution. The excess peaks observed in the XRD patterns in relation to the PBS medium are ascribed to hydroxysulfate green rust GR (SO2− 4 ; JCPDS No. 41-0014) and vivianite (JCPDS No. 26-1137). This can be explained by the strong surface complexation of phosphate with Fe(II), which impedes microbial reduction of Fe(III) and prevents the solid-state transformation of Fe(III) (Zachara et al., 2002). The layered Fe(II–III) GR, represented by the general for3+ mula Fe2+ 4 Fe2 (OH)12SO4·3H2O, is metastable and can be transformed to vivianite (in the phosphate-laden solution) and magnetite (Hansen, 1999). Similar observation of vivianite formation was reported in a recent study (Wang et al., 2018) concerning dissimilatory iron reduction of Fe(III) in the wastewater containing phosphate. In the PIPES medium, no peaks ascribable to GR are displayed and the proportion of XRD peaks assigned to magnetite (JCPDS No. 19-0629) increase substantially. Additionally, a close observation of Fig. 3a and b indicates that the magnitudes of all the peaks are sensitive to the presence of an electric field. Stimulation with an anodic potential of 0.2 V significantly boosted peak intensity, particularly for those peaks related to magnetite. This is in good accordance with the chemical analysis results, which show that the Fe(II) concentration was the lowest at the end of incubation for Treatment #1 (0.2 V) compared to the other systems (Fig. 3). The SEM morphology characterization (Fig. 4) further supports the variations in the biomineralization products as a consequence of the presence of an applied anodic potential and the type of buffer. The relevant SEM-EDS experiments were also conducted to predict the resultant minerals (Table 1) based on inference from the relative EDS elemental percentages. The initial PFS exhibits an amorphous aggregate morphological structure, while the microbial reduction of PFS by MR-1 yields mineral products with diverse morphology. Vivianites, featuring lathshaped crystals, were seen in all samples, and the relevant EDS spectra clearly verify the presence of a substantial amount of elemental phosphorous. Platelets with typical irregular edges were found in the PBS buffer system, indicating the presence of GR. The EDS observation confirms the inclusion of elemental sulfur, implying the formation of GR (SO2− 4 ). A substantial amount of magnetite, appearing as aggregates of individual nanometer-sized crystals, was also seen in the PIPESbuffered system. The confirmation of the presence of magnetite was also evident from the EDS result, which displayed predominantly iron and oxygen atoms (trace quantities of phosphorous were also seen, possibly due to adsorption). Moreover, it is evident from the SEM images that the solid phase resulting from the 0.2 V simulation contains a larger proportion of GR and vivianite in the PBS medium and a larger proportion of magnetite in the PIPES medium. The SEM images (Fig. S3) of the solids obtained under the open-circuit condition and the associated SEM-EDS results (Table S1) also suggest the formation of secondary minerals like GR and vivianite in PBS and magnetite in PIPES, but they appear in a less crystalline structure. These observations are in good agreement with the XRD results. 3.4. Pathways of mineral biotransformation Based on the above data, the major pathways for the biotransformation of PFS under Fe(III)-reducing conditions are proposed in Fig. 5. The biotransformation of PFS is a complex process that is highly dependent on the composition of the medium and on electric-field stimulation. As illustrated in Fig. 5a, these variables strongly affect the abundance of the resulting secondary Fe minerals such as GR, vivianite, and magnetite. Analogous to biotransformation of low-crystalline Fe(III) minerals such as ferrihydrite (Hansel et al., 2003; Dippon et al., 2015) and hydrous ferric oxide (HFO) by DIRB (Zachara et al., 2002), the microbial reduction of PFS coupling the

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Fig. 3. XRD patterns of solid products formed under different operational conditions in (a) the PBS and (b) the PIPES-buffered systems at different incubation times (V = vivianite; M = magnetite; GR = green rust (SO2− 4 )).

oxidation of lactate proceeds according to the following reaction (Eq. (1)): h

Fe2 ðOHÞn ðSO4 Þ3−n=2 −

i m

þ CH3 CHOHCOO− þ H2 O→CO2 þ Fe2þ

of GR (SO2− 4 ) as a result of biogenic Fe(II) catalysis can be represented by Eq. (2): 2Fe3þ þ 4Fe2þ þ 12OH− þ SO4 2− þ 3H2 O→Fe4 2þ Fe2 3þ ðOHÞ12 SO4  3H2 O

ð1Þ

ð2Þ

The reaction results in the formation of biogenic Fe(II), which is key to yielding secondary Fe(II)–Fe(III) products, because Fe(II) adsorbed on the solid surface can catalyze the recrystallization of poorly crystalline Fe(III) compounds to thermodynamically more stable minerals via surface reaction (surface complexation, electron transfer; (Mao et al., 2016). In the PBS medium, microbial reduction of PFS and the associated Fe(II)-induced mineralogical evolution appear to yield substantial amounts of GR and vivianite and smaller amounts of magnetite. In contrast, in the PIPES-buffered medium, magnetite becomes increasingly prevalent and vivianite is present in low proportions of the solid phase. The production

Previous studies of Fe(II)-induced biotransformation of ferrihydrite have also observed that the presence of high-concentration phosphate is essential for GR formation (Refait et al., 2007). This effect is attributed to the strong surface complexation between phosphate and ferrihydrite, which might impede structural ordering (Bocher et al., 2004; Feng et al., 2015). In the phosphate-rich system, GR can act as a precursor for vivianite formation (Hansen, 1999). This is because the adsorbed phosphate slows F(III) reduction and meanwhile replaces sulfate in the GR. Continuous reduction and substitution eventually leads to the production of vivianite (Eq. (3); Schoepfer et al., 2019). It is also possible that vivianite precipitation results from complexation between phosphate

þ OH þ SO4

2−

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high concentrations of phosphate (Hansen, 1999). Relatively lower quantities of magnetite were generated in the PBS medium, which might be originating from GR, a metastable intermediate that can be conversed to vivianite (Eq. (6)). The PIPES-buffered system, which contains lower concentrations of phosphate (added as a nutrient for bacterial growth), showed an elevated rate of Fe(III) reduction. The released Fe(II) immediately reacted with phosphate, resulting in precipitation of vivianite (Eq. (7)), facilitating continued Fe(III) reduction (Wilfert et al., 2016). Magnetite was found to be the dominant end product in the PIPES-buffered medium as a result of biogenic Fe(II)-catalyzed PFS transformation (Eq. (8)). This observation is in good accordance with the bioreduction of HFO by DIRB reported previously (Fredrickson et al., 1998): 3Fe4 2þ Fe2 3þ ðOHÞ12 SO4  3H2 O þ 8H2 PO4 − þ 2Hþ þ 5H2 O→ ð3Þ 4Fe3 ðPO4 Þ2  8H2 O þ 6FeðOHÞ3 þ 3SO4 2−

Fig. 4. SEM images of (a) the initial PFS, and the solid products produced from (b) the PBS and (c) the PIPES-buffered systems with applied potential of 0.2 V. Numbers refer to EDS spot analysis, and the corresponding EDS elemental percentages can be found in Table 1.

and Fe(III) and subsequent Fe(III) reduction (Eqs. (4) and (5)), which may be another pathway for vivianite formation in the presence of Table 1 Elemental percentages of Fe, P, O, and S at each analysis spot at Day 20, as analyzed by SEM-EDS. Spectrum values are related to numbers in Fig. 4. Sample

Spectrum Fe at. %

P at.%

O at. %

PFS

1

30.18

0

2

29.99

0

1 2 3 4 5 6 7 1 2 3 4 5

26.32 27.89 26.78 26.15 16.19 16.08 40.18 40.64 40.57 40.02 14.33 12.98

0.67 0.53 0.89 0.95 8.55 9.09 0.52 0.56 0.68 0.17 8.71 8.87

66.71 3.11 [Fe2(OH)n(SO4) 3-n/2]m 66.85 3.16 [Fe2(OH)n(SO4) 3-n/2]m 68.58 4.43 GR (SO2− 4 ) 67.23 4.35 GR (SO2− 4 ) 66.32 4.90 GR (SO2− 4 ) 68.12 4.78 GR (SO2− 4 ) 75.25 0 Vivianite 74.84 0 Vivianite 59.30 0 Magnetite 58.81 0 Magnetite 58.75 0 Magnetite 59.81 0 Magnetite 76.96 0 Vivianite 78.15 0 Vivianite

PBS Treatment 1# (0.2 V)

PIPES Treatment 1# (0.2 V)

S at.%

Mineralogy

Fe3þ þ PO3− 4 →FePO4

ð4Þ

12FePO4 þ CH3 CHOHCOO− þ 11OH− →4Fe3 ðPO4 Þ2 þ 4PO3− 4 þ 3CO2 þ 8H2 O

ð5Þ

Fe4 2þ Fe2 3þ ðOHÞ12 SO4  3H2 O→Fe3 O4 þ 3Fe2þ þ 4OH− þ SO4 2− þ 7H2 O

ð6Þ

3Fe2þ þ 2PO3− 4 →Fe3 ðPO4 Þ2

ð7Þ

2Fe3þ þ Fe2þ þ 4H2 O→Fe3 O4 þ 8Hþ

ð8Þ

Fe2þ →Fe3þ þ e−

ð9Þ

To confirm Fe(II)-catalyzed transformation of PFS to different secondary crystalline products, control experiments were undertaken under abiotic conditions in which FeSO4 was used as the precursor and mixed with PFS. The concentrations of FeSO4 were varied from 0 to 500 mg L−1, mirroring the amounts of Fe(II) released from microbial reduction of PFS. Fig. 6 shows the XRD patterns of the resultant solid phases, which show clear evidence of the formation of crystalline compounds as a consequence of the surface reaction between Fe(II) and PFS. The crystalline peaks appearing in the XRD patterns are ascribed to magnetite and vivianite. In both the PBS and PIPES media, the intensities of these peaks, which are related to the quantities of the newly formed species, were sensitive to the reaction time and the concentration of FeSO4. Longer reaction time was linked to increasingly abundant crystals. Increasing the amount of Fe(II) from 0 to 50 mg L−1 promoted the formation of magnetite according to Eq. (8); this result is consistent with previous studies establishing the relationship between the concentration of Fe(II) produced and the transformation rate of ferrihydrite (Kukkadapu et al., 2004; Han et al., 2018). Interestingly, further increases in the amount of Fe(II) from 100 to 500 mg L−1 resulted in declines in the intensities of the peaks. Almost no peaks were visible at Fe(II) concentration of 500 mg L−1. This implies that redundant Fe(II) on the PFS surface may block structural ordering. Furthermore, in contradiction to the observations in the PBS medium under biotic conditions, no appreciable peaks ascribed to GR were displayed under abiotic conditions. The formation of GR in the presence of bacteria is possibly due to the slower microbial reduction of PFS and the corresponding lower rate of Fe(II) dissolution caused by phosphate inhibition. Fe(II) is present in insufficient amounts to compete against phosphate for the PFS surface sites; thus phosphate complexation favors GR formation and inhibits PFS transformation to magnetite (Bocher et al., 2004). In contrast, this inhibition effect might be attenuated when a certain amount of Fe(II) is immediately added, as the reaction between Fe(II) and PFS is thus less influenced by the presence of phosphate, leading to the formation of magnetite and vivianite.

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Fig. 5. (a) Proposed pathway of biomineralization of PFS to different secondary products in the PBS and PIPES-buffered systems. (b) Biogenic Fe(II)-mediated electron transfer from the bacteria to the electrode. (c) Summary of the effects of medium composition and electric field stimulation on types of secondary products formed and their relative quantities.

Fig. 6. XRD patterns of solid products resulting from the abiotic experiments in (a) the PBS and (b) the PIPES-buffered systems at different operation times. In the abiotic experiments, FeSO4 with various concentrations was directly mixed with PFS (M = magnetite; V = vivianite).

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The application of an electric field during the microbial reduction of PFS, despite not affecting the type of secondary Fe minerals produced, has a strong impact on the timing of their formation and on their abundance. The secondary product content was substantially increased in the system with an anodic potential of 0.2 V. This effect is attributed to the enhanced electron transfer ability from the microbes to the solid phase and then to the electrode (Fig. 5b), as demonstrated previously (Feng et al., 2013). On one hand, the higher electron transfer rate is linked to greater Fe(II) production, which plays a vital role in increasing the amount of secondary minerals (Klein et al., 2014). On the other hand, electron transfers from Fe(II) to the electrode result in its oxidation to Fe(III) (Eq. (9)); this appears to kinetically promote bioreduction via removal of the passivating byproducts as shown in Eq. (9) It should be noted that reducing excess Fe(II) via this pathway is also conducive to production of magnetite, as evidenced by the abiotic results (Fig. 6). To summarize, the relative abundances of different secondary biomineralization products in response to different medium composition and electric field stimulation are illustrated in Fig. 5c. The application of 0.2 V potential in the PIPES-buffered medium is responsible for the formation of the dominant end product, magnetite; in contrast, the same treatment in the PBS medium allows the generation of GR (SO2− 4 ) and vivianite. 4. Conclusions The stability of PFS, usually accommodated with heavy metals and organic pollutants, in bottom sediments remains a critical environmental issue as its decomposition/recrystallization plays an important role in the cycling of Fe and the fate of contaminants. Despite its environmental significance, very little information is available on the Fe (II)-aided mineralization of PFS. Herein, we make the attempt to show that PFS is subjected to bioreduction by DIRB, followed by subsequent transformation to secondary solid products. We demonstrate that the rate and extent of PFS reduction are strongly dependent on the medium composition and the electric field stimulation, which in turn determine which secondary products are formed and in what quantities. A slower rate of reduction was noted in the PBS medium in comparison to the PIPES-buffered medium owing to the surface complexation between phosphate and the PFS surface, which retards PFS dissolution. As such, the phosphorous richness of the PBS medium is essential for formation of abundant GR and vivianite. In contrast, the PIPES-buffered medium enables a quicker rate and higher degree of PFS reduction and induces the transformation of amorphous PFS to magnetite as the predominant end mineral. Furthermore, the application of an electric field (0.2 V) can substantially increase microbial reduction of PFS, as evidenced by the higher amounts of Fe(II) evolution in the early stage in both buffer solutions. The elevated rate of Fe(II) release from PFS then results in the formation of more crystalline solids in appreciable quantities. Given the recent research interest in using electrochemical methods for bioremediation of anoxic sediments, the current study can hopefully provide information on electric field-stimulated biotransformation of poorly crystalline Fe-bearing compounds (i.e., PFS, ferrihydrite, and HFO), an important process impacting the fate of contaminants. Future studies should therefore be conducted with heavy metal-entrapped PFS with the aim of understanding how the electric field influences the interaction between the bacteria, PFS, and heavy metals, and accordingly, the availability and mobility of heavy metals. Acknowledgements We gratefully acknowledge financial support from the National Natural Science Foundation of China (nos. 41673090, 21876052, and 21577041), and the Natural Science Foundation of Guangdong Province, China (no. 2016A030311023).

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