Insoluble Fe-Humic Acid Complex as a Solid-Phase Electron

(1) PCP is still classified as a priority pollutant(2) and is listed in the drinking water ... in 250 mL bottles, using 0.5 g of sample and 50 mL of e...
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Insoluble Fe-Humic Acid Complex as a Solid-Phase Electron Mediator for Microbial Reductive Dechlorination Chunfang Zhang,‡,§ Dongdong Zhang,† Zhiling Li,‡,∥ Tetsuji Akatsuka,‡ Suyin Yang,‡ Daisuke Suzuki,‡ and Arata Katayama*,‡ †

Graduate School of Engineering, Nagoya University, Chikusa, Nagoya, Aichi 464-8603, Japan EcoTopia Science Institute, Nagoya University, Chikusa, Nagoya, Aichi 464-8603, Japan

‡

S Supporting Information *

ABSTRACT: We report that the insoluble Fe-HA complex, which was synthesized with both commercial Aldrich humic acid (HA) and natural HA, functions as a solid-phase electron mediator (EM) for the anaerobic microbial dechlorination of pentachlorophenol. Spectroscopic characterizations and sequential Fe extraction demonstrated that the Fe-HA complex was predominated with Na4P2O7-labile Fe (represented as the organically bound Fe fraction) and poorly ordered Fe fraction (the fraction left in the residue after the sequential extraction), which were associated with different possible binding processes with carboxylate and phenolic groups. The change in the electron-mediating activity caused by Fe extraction indicated that the electron-mediating function of the Fe-HA complex is attributable to the Na4P2O7-labile Fe fraction. The Fe-HA complex also accelerated the microbial reduction of Fe(III) oxide, which suggested the presence of multiple electron-mediating functions in the complex. The electron shuttle assay showed that the Fe-HA complex had an electron-accepting capacity of 0.82 mequiv g−1 dry Fe-HA complex. The presence of redox-active moieties in the Fe-HA complex was verified by cyclic voltammetry analysis of the sample after electrical reduction, with a redox potential estimated at 0.02 V (vs a standard hydrogen electrode).

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INTRODUCTION Pentachlorophenol (PCP) is a highly recalcitrant chlorinated organic pollutant found extensively in soils, sediments, and aquatic environments. It was widely used as an antiseptic, herbicide, and wood preservative and was banned because of its high toxicity.1 PCP is still classified as a priority pollutant2 and is listed in the drinking water standards of the United States and the World Health Organization.3 Recently, bioremediation through reductive dechlorination coupled with anaerobic aromatic ring oxidation has been proposed as a cost-effective strategy for the cleanup of PCP under anaerobic conditions. In our laboratory, we have achieved complete mineralization of PCP by a combination of microbial reductive PCP dechlorination and phenol degradation.4−6 However, PCP dechlorination activity requires the presence of soil or humin in the system.6−8 This phenomenon is not exceptional, because many dehalogenating cultures require the presence of soil or sediment to maintain their activities.9−13 As the most widely spread natural complexing ligands, humic substances (HSs) tend to chelate the metals present in the environment. In our previous study, we found that humins, the fraction of HSs that is not water-soluble under any pH conditions, contained considerably high concentrations of iron.8 This iron-rich, insoluble humin showed a stable electron-mediating ability for the microbial reductive dechlorination of PCP, whereas the humic acid (HA) obtained from the © 2014 American Chemical Society

same soil source was unstable in mediating the activity in dissolved form. Drawing inspiration from this fact, an insoluble iron humate (Fe-HA) complex was prepared by the complexation of HAs with ferrous sulfate to activate and stabilize the electron-mediating groups harbored in HAs. Although the complex formation of HSs with iron (Fe), which is an essential micronutrient and one of the most abundant metals in soils and sediments, has been widely studied and the insoluble Fe-HA complex has already been reported to serve as a plant Fe source14 or as the sorbent for basic dyes,15 its effect as an electron mediator (EM) for the anaerobic microbial reaction has not been explored so far. Here, we demonstrate the function of the solid-phase Fe-HA complex as an EM in microbial reductive dechlorination of PCP, with physicochemical, electrochemical, and spectrophotometric characterization.

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MATERIALS AND METHODS Preparation of the Fe-HA Complex. Commercial Aldrich humic acid sodium salt (AHA, Aldrich Chemical Co., Milwaukee, USA) was used in this study. For the preparation of the Fe-HA complex, AHA was dissolved in ultrapure water at a concentration of 1.5 g L−1 and centrifuged at 15,000g for 15 Received: September 10, 2013 Accepted: April 23, 2014 Published: April 23, 2014 6318

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1 mL of trace element SL-10 solution;16 1 mL of Se/W solution;16 and 15 mM MOPS buffer (pH 7.2). PCP and its metabolites were analyzed using a QP2010 gas chromatography−mass spectrometry system (Shimadzu, Kyoto, Japan) equipped with a DB-5MS column (J&W Scientific, Folsom, CA), as described previously.17 The influence of the Fe-HA complex and control samples on the microbial PCP-to-phenol dechlorination activity was examined via an incubation experiment, which was carried out in the same way as humin culture maintenance with 10 mM formate as a sole electron donor. An excess amount of formate was provided as electron donor compared to the electrons required guaranteeing the reducing power enough for PCP dechlorination. The Fe-HA complex and Control-Fe samples were added at a concentration of 2.5 g L−1 and autoclaved with the medium. For Control-HA, 10 mL of the sample was added into the bottle containing 10 mL of medium and autoclavesterilized. The influence on the dechlorination was judged after at least two generations of incubation. Sorption Experiment. PCP or phenol was added at concentrations of 5, 10, or 20 μM to 60 mL serum bottles containing 20 mL of mineral medium amended with/without Fe-HA complex (2.5 g L−1) and incubated for 5 days under the same conditions as those used for the culture incubation experiments. The Fe-HA complex was removed by centrifugation, and the concentration of PCP or phenol in the supernatant was analyzed. Microbial Fe(III) Reduction Assay. The influence of the Fe-HA complex on the microbial reduction of Fe(III) oxide was carried out by adding Fe-HA complex or the Fe-HA complex subjected to various treatments (2.5 g L−1) to 30 mL of NaHCO3-buffered (30 mM, pH 6.8) fresh medium amended with 20 mM lactate and 10 mM synthesized Fe(III) oxide and inoculated with about 109 cell mL−1 of washed Shewanella putrefaciens CN-32 grown in a Tryptic soy broth (Becton Dickinson, Sparks, USA). All the experiments were conducted in duplicate while providing four controls: two abiotic controls, a control using inoculum but without the Fe-HA complex, and a control with both inoculum and the Fe-HA complex but without the addition of Fe(III) oxide. The Fe(III) oxide was synthesized as previously described.18 The medium was rendered anoxic by bubbling with N2/CO2 (80%/20%). Electron Accepting Capacity Measurement. Both microbial and chemical reduction assays were carried out to calculate the electron-accepting capacity (EAC) of the samples in an anaerobic chamber following the protocol described previously.19,20 The microbial reduction assays were carried out by adding Fe-HA complex or the Fe-HA complex subjected to various treatments (2.5 g L−1) or Control-HA (1.5 g L−1) to 30 mL of NaHCO3-buffered fresh medium amended with 20 mM lactate and inoculated with about 109 cell mL−1 of washed S. putrefaciens CN-32. The chemical reduction assays were performed by adding the samples in 10 mM PIPES buffer (pH 6.8) with a concentration of 2.5 g L−1 (1.5 g L−1 for Control-HA) and incubated for 5 days with shaking (150 rpm) under a 100% H2 atmosphere (nominal 0.78 mM in the aqueous solution) in the presence of five Pd-coated aluminum oxide pellets. To quantify the amount of electrons transferred to the FeHA complex during microbial or chemical reduction, 1 mL of sample suspension was reacted with 2 mL of 10 mM ferric iron complexed with nitrilotriacetic acid [Fe(III)-NTA] for 1 h. This reaction mixture was then filtered (0.22 μm), and 0.5 mL of

min to remove the insoluble particles. To this purified AHA solution, 5 mM of Fe(SO4)·7H2O was added during rapid agitation of the mixture. The pH of the solution was then neutralized to 7.0. The suspensions, held in polypropylene containers, were maintained for 1 week at 30 °C, keeping the pH constant. The precipitated compounds, i.e., the Fe-HA complex, were collected by centrifugation (15,000g, 20 min) and freeze-dried. Two control samples were provided, following the exact procedures described above, but with the addition of either AHA or Fe(SO4)·7H2O, and they were designated as Control-HA (liquid solution) and Control-Fe (freeze-dried powder), respectively. HAs extracted from paddy soils in Kamajima (KM) and Yatomi (YA), Aichi prefecture, Japan, designated as KMHA and YAHA, were also provided for the complex formation with ferrous sulfate following the same procedures described above, except that KMHA or YAHA was dissolved in 0.01 M Na4P2O7 instead of ultrapure water. The formed complex was designated as Fe-KMHA and Fe-YAHA, respectively. The HAs were extracted on the basis of the conventional alkaline methods following procedures described previously.8 The elemental composition of carbon, hydrogen, and nitrogen in the samples was determined on a Yanaco MT-5 CHN-corder (Yanaco New Science, Kyoto, Japan). The ash content was determined by weight. Iron Fraction Responsible for the Electron-Mediating Function. A three-step sequential extraction was performed on the Fe-HA complex using (i) MgCl2 (1 M, pH 7), (ii) sodium acetate (1 M, pH 5, adjusted by acetic acid), and (iii) Na4P2O7 (0.1 M, pH 10) to obtain information on the (i) exchangeable Fe fraction, (ii) acid-soluble Fe fraction, and (iii) Na4P2O7extractable Fe represented as an organically bound Fe fraction. The extraction was performed at room temperature, in 250 mL bottles, using 0.5 g of sample and 50 mL of extractant for 24 h. After each treatment, the extract was separated by centrifugation (15,000g, 10 min). This extraction process was repeated twice. Fe content was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES, PerkinElmer, Yokohama, Japan) in the supernatant. The total Fe content and the poorly available Fe fraction (represented by Fe content in the residue fraction after the extraction with Na4P2O7) of the Fe-HA complex was analyzed by ICP-AES after digesting with perchloric acid and nitric acid. In addition, NaOH (0.1 M) was used as a milder extractant to remove the organically bound Fe fraction, while H2O2 oxidation (30% of H2O2, 12 h at room temperature) and heat treatment (300 °C, 5 h) were applied to destroy the organic fraction in the Fe-HA complex. The Fe-HA complex after each treatment was rinsed with distilled water until neutralized and was referred to as MgCl2-, NaOAc-, Na4P2O7-, NaOH-, H2O2-, or heat-treated Fe-HA complex, respectively. Microbial PCP-Phenol Dechlorination Experiment. An anaerobic PCP-to-phenol dechlorinating humin culture8 was used as an inoculum source in this study. This humin culture was maintained through serial transfer with 5% by volume of the inoculum, 10 mM formate, and 20 μM PCP to serum bottles containing 20 mL of mineral medium and 5 g L−1 humin, with 10−25 days of incubation time needed for dechlorination. The information on the microbial community composition was provided in a previous study.8 The cultures were incubated statically at 30 °C in the dark in an incubator. The mineral medium consisted of (per liter) 1.0 g of NH4Cl; 0.05 g of CaCl2·2H2O; 0.1 g of MgCl2·6H2O; 0.4 g of K2HPO4; 6319

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Figure 1. Influence of the Fe-HA complex on PCP dechlorination in the enrichment culture and on the concentration of PCP and phenol in solution. (a) Dechlorination of PCP (Δ) with the production of a final metabolite of phenol (○) in the culture with 2.5 g L−1 Fe-HA complex and 5% inoculum; (b) no dechlorination of PCP under the control conditions with 2.5 g L−1 Fe-HA and without inoculum (○) and with inoculum but without Fe-HA (Δ); (c) no dechlorination of PCP in the cultures with AHA (□), Control-Fe (◇), or Control-HA samples (△); (d) sorption of PCP and phenol: comparison of the concentrations of PCP (Δ) and phenol (○) between the media supplemented with and without the Fe-HA complex (2.5 g L−1) after equilibrium. Arrows indicate the transfer of 5% of the microcosm to the fresh medium spiked with PCP. The experiments were carried out at least three times, and the representative results are shown. Data show the mean values of duplicate parallel cultures, and vertical bars show the difference in duplicate samples.

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filtrate was added to 5 mL of ferrozine (1 g L−1) solution in 50 mM HEPES buffer (pH adjusted to 7.0 with NaOH); the absorbance at 562 nm was measured. Meanwhile, 1 mL of sample suspension was reacted with ferrozine and its absorbance at 562 nm was measured as a background value to exclude the influence from the Fe(II) released from the FeHA complex during the incubation. The difference between the Fe(II) concentration of the final (after reduction) and initial (before reduction) samples was defined as the sample EAC (in mequiv g−1 dry Fe-HA complex). Electrochemical Measurement. Electrochemical experiments were performed using a potentiostat (HSV-110; Hokuto Denko, Osaka, Japan) equipped with a bioelectrochemical cell that consisted of a graphite working electrode (5 mm × 15 cm; Tokai Carbon, Tokyo, Japan), a twisted platinum counter electrode (0.8 mm × 1 m; Nilaco, Tokyo, Japan), and a Ag/ AgCl (saturated KCl) reference electrode (HX-R8 Hokuto Denko, Osaka, Japan). Cyclic voltammetry measurements were performed with a scan rate of 10 mV s−1, a sample concentration of 2.5 g L−1, and a potential range from −0.8 to 0.6 V (vs Ag/AgCl) with a mineral medium as the electrolyte. A set of the samples was electrically reduced using a mineral medium as electrolyte at an applied potential of −500 mV [vs a standard hydrogen electrode (SHE)] for 2.5 h. Fourier-Transform Infrared (FTIR) Spectroscopy. FTIR spectra were determined using a KBr pellet technique in the 400−4000 cm−1 range by using a JASCO FT/IR-6100 spectrometer (JASCO, Tokyo, Japan), with a resolution of 4 cm−1 and eight accumulations. The spectra were corrected against pure KBr and ambient air.

RESULTS Physicochemical Characterization of the Fe-HA Complex. Purified AHA contained 52.8% carbon, 3.9% hydrogen, 1.0% nitrogen, and 0.8% ash, while the Fe-HA complex had a carbon content of 23.2%, hydrogen content of 2.8%, nitrogen content of 0.4%, and ash content of 33.6%. ICPAES analysis showed that the Fe-HA complex had a total Fe content of 149.6 ± 13.8 g kg−1. The results from the sequential Fe extraction revealed that the Fe-HA complex contained 4.6% MgCl2-labile Fe (represented as the exchangeable Fe fraction), 9.6% NaOAc-labile Fe (represented as the acid-soluble Fe fraction), 52.7% Na4P2O7-labile Fe (represented as the organically bound Fe fraction), and 33.1% poorly available Fe (the fraction left in the residue after the sequential extraction). An average yield of 0.58 g of insoluble Fe-HA complex (as freeze-dried powder) was obtained by the complexation of 1.5 g of purified AHA with 1.39 g of ferrous sulfate in aqueous solution, which corresponded to a recovery rate of 20.0%. Influence of Fe-HA Complex on Microbial Dechlorination of PCP. We have reported previously that humins extracted as a solid fraction from the soil and sediment exhibit a very stable ability in sustaining both PCP dechlorination and tetrabromobisphenol A (TBBPA) debromination activities in two anaerobic cultures, respectively, whereas HA extracted from the same soil was unstable in mediating the activity in dissolved form since the dehalogenation activity was lost after subculture.8,13 In this study, the insoluble Fe-HA complex displayed stable PCP-to-phenol dechlorination activity (Figure 1a), while the intact AHA showed negative activity (Figure 1c). The PCP dechlorination activity was not observed without 6320

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Figure 2. Influence of the Fe extraction of the Fe-HA complex on microbial reductive dechlorination of PCP. Reactions in the enrichment cultures with (a) MgCl2-treated Fe-HA complex, (b) NaOAc-treated Fe-HA complex, (c) NaOH-treated Fe-HA complex, and (d) Na2P4O7-, H2O2-, and heat-treated Fe-HA complex samples. Symbols: Δ = PCP; ○ = phenol. Arrows indicate the transfer of 5% of the microcosm to fresh medium spiked with PCP. Data show mean values of duplicate parallel cultures, and vertical bars show the difference in duplicate samples.

treated sample and to 5.8% in the Na4P2O7-treated sample, while the carbon content of the Fe-HA complex increased from 21.3% to 55.6% in the NaOH-treated sample and to 59.1% in the Na4P2O7-treated sample. Meanwhile, the Fe content of the Fe-HA complex decreased from 149.6 ± 13.8 to 35.4 ± 0.01 g kg−1 in the NaOH-treated sample and to 8.7 ± 0.04 g kg−1 in the Na4P2O7-treated sample. Influence of the Fe-HA Complex on Microbial Reduction of Fe(III) Oxide. The Fe(II) concentration increased with time; however, only a minor amount of Fe(II) was detected in the bottles without the inoculation of the cells regardless of the presence of the Fe-HA complex (Figure 3). Meanwhile, the addition of the Fe-HA complex accelerated the microbial reduction of Fe(III) oxide compared with the nonamended culture. As high as 5.1 mM of Fe(II) was detected in the cultures supplemented with the Fe-HA complex, whereas the control sample without the addition of Fe(III) oxide produced ca. 1.7 mM of Fe(II), which is

inoculation or without the presence of the Fe-HA complex, suggesting that PCP dechlorination was due to microbial activity and that the Fe-HA complex was required for dechlorination to take place (Figure 1b). No PCP dechlorination was observed in incubation experiments in the presence of the Control-Fe or the Control-HA sample as a substitute for the Fe-HA complex (Figure 1c). The complex prepared using natural HAs, i.e., Fe-KMHA and Fe-YAHA, also showed positive activity for mediating the microbial reductive dechlorination of PCP (Supporting Information Figure S1). A sorption experiment showed that, although the PCP concentration was decreased by 15% by the sorption to the FeHA complex, metabolite production was not observed in the system where phenol was not absorbed to Fe-HA at all (Figure 1d), suggesting only a slight sorption of PCP to the Fe-HA complex and no chemical reaction between the two compounds. Electron-Mediating Fraction in the Fe-HA Complex. The solid-phase Fe-HA complex displayed a stable electronmediating property, and the MgCl2- or NaOAc-treated Fe-HA complex did not influence the PCP dechlorination activity (Figure 2a,b). The NaOH treatment for removal of Fe represented as an organic-bonded Fe fraction did not result in the loss of mediating ability in PCP dechlorination (Figure 2c). However, the activity became unstable after the sample was treated with Na4P2O7 solution as the dechlorination activity only occurred in the first generation, or no dechlorination was observed at all (Figure 2d), indicating that the functional group is associated with the Na4P2O7-labile Fe fraction, representing organically bound Fe fraction. In addition, the H2O2- and heattreated Fe-HA complex showed negative PCP dechlorination activity (Figure 2d), indicating that the removal of organic fraction in the Fe-HA complex would result in the loss of mediating ability in PCP dechlorination. The ash content of the Fe-HA complex (33.6%) decreased to 13.7% in the NaOH-

Figure 3. Influence of Fe-HA complex on microbial reduction of Fe(III) oxide in the culture of S. putrefaciens CN-32 with 20 mM of lactate and 10 mM of Fe(III) oxide. 6321

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attributable to the Fe reduced from the Fe-HA complex. Therefore, ca. 3.4 mM of Fe(II) was produced from the accelerated microbial reduction of Fe(III) oxide by the Fe-HA complex, which is 1.9-fold higher than that recovered from the nonamended culture (1.8 mM) at the end of the incubation. The MgCl2-, NaOAc-, and NaOH-treated Fe-HA complex samples enhanced the microbial reduction of Fe(III) oxide, while the H2O2-, Na4P2O7-, and heat-treated Fe-HA complex samples showed little enhancement for the microbial reduction of Fe(III) oxides (Supporting Information Figure S2). Electron Accepting Capacity of Fe-HA Complex. The EAC of the Fe-HA complex was evaluated through a microbial (S. putrefaciens CN-32) or chemical (H2/Pd) reduction assay (Figure 4). For the microbial reduction experiment, a large

Fe-HA complex in 2 days (Figure 4a). For the Control-HA, a much lower amount of electron equivalents was produced, with an EAC value of 0.13 mequiv g−1 dry Control-HA (Figure 4a). The EAC values for the MgCl2-, NaOAc-, NaOH-, H2O2-, Na4P2O7-, and heat-treated samples were 0.42, 0.37, 0.34, 0.02, 0.03, and 0.01 mequiv g−1, respectively (Supporting Information Figure S3a). Exposure of the samples to H2 in the presence of Pd catalyst readily reduced the Fe-HA complex, and an EAC value of 0.36 mequiv g−1 Fe-HA complex was obtained after 2 days of incubation (Figure 4b). The Control-HA exposed to the H2/Pd system showed similar trends in the production of electron equivalents to that of the microbial reduction assay, with an EAC value of 0.10 mequiv g−1 dry Control-HA (Figure 4b). Only a small amount of electron equivalent was recovered in the Control-Fe sample (Figure 4). The EAC values for the MgCl2-, NaOAc-, NaOH-, H2O2-, Na4P2O7-, and heat-treated samples were 0.18, 0.18, 0.14, 0.01, 0.01, and 0.01 mequiv g−1, respectively (Supporting Information Figure S3b). Spectroscopic Properties. The FTIR spectrum of the FeHA complex (Figure 5a) showed a clear difference as compared with that of AHA and Control-Fe (Figure 5b). AHA gave the typical spectral bands of HSs, while no peak was observed in a broad range from 1100 to 4000 cm−1 for Control-Fe. When AHA interacted with ferrous sulfate, an obvious change was observed at a wavenumber from 1750 to 1200 cm−1, with the appearance of clear new bands at 1735, 1703, and 1233 cm−1 and several small ones in the region from 1500 to 1370, 1605 to 1500, and 1735 to 1605 cm−1 (Figure 5a). The peaks at 1703, 1585, and 1370 cm−1 were also observed for the Fe-HA complex subjected to MgCl2, NaOAc, and NaOH treatments (Supporting Information Figure S4). Meanwhile, the peaks in the region from 1705 to 1500 cm−1 were diminished or vanished for the H2O2-, Na4P2O7-, and heat-treated Fe-HA complex, which exhibited unstable mediating activity in the microbial PCP dechlorination. Also, the peak at 1233 cm−1 disappeared in the Na4P2O7- and heat-treated Fe-HA complex (Supporting Information Figure S4). The Fe-HA complex showed an electron spin resonance (ESR) signal at g = 2.0042 whereas the Control-Fe sample showed little ESR signal (Supporting Information Figure S5a). The Fe-HA complex adjusted at pH 11 produced a remarkable

Figure 4. Electron accepting reaction of the Fe-HA complex in microbial and chemical reduction. (a) Reduction by S. putrefaciens strain CN-32 cells. (b) Abiotic reduction by H2/Pd.

quantity of electron equivalents was recovered in the Fe-HA complex samples, and the EAC value reached 0.82 mequiv g−1

Figure 5. FTIR spectra of the (a) Fe-HA complex and Na4P2O7-treated Fe-HA complex and (b) Aldrich HA and Control-Fe samples. 6322

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of the Na4P2O7-treated Fe-HA complex displayed CVs with irregular shape, regardless of the electrical reduction, as no redox couple was observed (Figure 6c).

increase in the radical signal compared with that of the Fe-HA complex at pH 3 (Supporting Information Figure S5b). The increase in the ESR signal at high pH is typical of semiquinonetype radicals; however, we cannot exclude the contribution of ESR signals from other origins such as metal-organic complexes, thiols, or nitrogen functional groups. Voltammetric Behavior of the Fe-HA Complex. HSs and their metal complexes generally produce CVs with little or no useful structure because of the lack of electrode activity,21 as evidenced by the irregular CV shape of the Fe-HA complex (Figure 6a) and the Control-HA (Figure 6b). However, when

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DISCUSSION In this study, we have demonstrated that the synthetic Fe-HA complex functions as a stable solid-phase EM for anaerobic respiration. The Fe-HA complex prepared through the complexation of AHA with ferrous sulfate displayed positive mediating activity in the PCP-to-phenol dechlorination process (Figure 1a). It also accelerated the microbial reduction of Fe(III) oxide (Figure 3), which was consistent with the finding observed by Roden et al.20 Meanwhile, the electron-mediating ability was not observed in the intact AHA (Figure 1c), suggesting that the function of Fe-HA complex as an EM was produced by complexation. These findings indicated that FeHA plays an important role as a solid-form electron mediator for the anaerobic microbial reaction in the natural environment, especially regarding the fact that Fe is one of the most abundant metals in soils and sediments, where HSs are ubiquitous. The results from the sequential Fe extraction revealed that the Na4P2O7-labile Fe fraction (represented as the organically bound Fe fraction) was predominant in the Fe-HA complex, followed by poorly ordered Fe fraction (the fraction left in the residue after the sequential extraction), with MgCl2-labile and NaOAc-labile Fe fractions accounting for 14.2%. This was interpreted as a result of the interactions between HA and the hydrolytic Fe fraction, which restrains the formation of crystalline Fe oxides but favors the formation of amorphous Fe oxide such as ferrihydrite.22 This feature of the Fe-HA complex makes it a stable solid-phase EM against the change of the ambient environment, as evidenced by the positive activity after treatments with 1 M MgCl2, 1 M sodium acetate, and 0.1 M NaOH (Figure 2a−c). Meanwhile, the Na4P2O7-treated FeHA complex exhibited unstable electron-mediating ability as the PCP dechlorination activity only occurred in the first generation, or no dechlorination was observed at all (Figure 2d). In addition, the H2O2- and heat-treated Fe-HA complex showed the functional loss in the PCP dechlorination (Figure 2d), indicating that the removal of organic fraction in the FeHA complex would result in the loss of mediating ability in PCP dechlorination. Therefore, the electron-mediating function of the Fe-HA complex would be attributable to the Na4P2O7labile organically bound Fe fraction. Microbial Fe(III) oxide reduction assay, EAC analysis, and cyclic voltammetry analysis further supported the association of Na4P2O7-labile organically bound Fe fraction as functional group. The microbial Fe(III) oxide reduction assay showed that the MgCl2-, NaOAc-, and NaOH-treated Fe-HA complexes enhanced the reduction of Fe(III) oxide, though with varied accelerating rate. However, the H2O2-, heat-, or Na4P2O7treated Fe-HA complex showed little enhancement (Supporting Information Figure S2). Also, the MgCl2-, NaOAc-, and NaOH-treated samples exhibited high EAC ranging from 0.14 to 0.42 mequiv g−1, while the H2O2-, heat-, or Na4P2O7-treated Fe-HA complex showed little EAC (Supporting Information Figure S3). These results corresponded with those of electron mediating functions for the microbial PCP dechlorination (Figure 2) and iron reduction (Figure 3). Moreover, the CV analysis showed that the same redox couple was observed for each MgCl2-, NaOAc-, and NaOH-treated Fe-HA complex samples, while the shape of the redox couple was diminished in the H2O2- and heat-treated samples (Supporting Information

Figure 6. Cyclic voltammograms of the samples. Cyclic voltammograms of (a) Fe-HA complex and mineral medium before and after the electrical reduction at −500 mV (vs SHE), (b) Control-Fe and Control-HA samples, and (c) Na4P2O7-treated Fe-HA complex before and after the electrical reduction at −500 mV (vs SHE). Mineral medium was used as electrolyte and background for the analysis.

we assessed the voltammetric behavior of the Fe-HA complex after electrically reducing the samples at an applied potential of −500 mV for 2.5 h, a CV with well-defined peaks was obtained (Figure 6a). On the basis of the correction to the peak potentials, the redox potential of the Fe-HA complex was estimated at 0.02 V vs SHE. This redox couple was also observed for each MgCl2-, NaOAc-, and NaOH-treated Fe-HA complex sample which retain active PCP dechlorination ability. However, very small redox peaks were observed in the H2O2and heat-treated Fe-HA complex which lost the electron mediating ability for the PCP dechlorination (Supporting Information Figure S6). Meanwhile, the voltammetry analysis 6323

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mentally benign are urgently desired. Recently, there are some new reports that documented the role of solid-phase HSs on the enhanced Fe oxide reduction,20 as well as the immobilized HSs as EMs for the biotransformation of contaminants.28−31 In this study, the finding that the synthetic insoluble Fe-HA complex functions as a solid-form EM for the microbial reductive PCP dechlorination supports a prospective application for in situ bioremediation (e.g., wastewater treatment system), especially regarding the fact that Fe is one of the most abundant metals in natural environments, where HSs are also ubiquitous. In any case, the findings that the insoluble Fe-HA complex in its solid form is redox-active have important implications for both engineering scale application and geobiochemical basic study.

Figure S6). No redox couple was observed for the Na4P2O7treated sample (Supporting Information Figure S6). The FTIR spectrum of the Fe-HA complex showed clear differences in the region from 1735 to 1370 cm−1, which was influenced mainly by dissociated COO− groups and C−O stretching of phenol groups (Figure 5a). The obvious presence of dissociated carboxylic and phenolic groups was evidenced by the appearance of new peaks at 1735 and 1700 cm−1, which was likely involved in the complexation with Fe.23 Meanwhile, the FTIR analysis of the Fe-HA complex subjected to various treatments suggested the functional moieties were associated with the groups in the region from 1750 to 1500 cm−1. To verify this, HA was methylated with methyl iodide, which results in the formation of methyl esters of carboxyl groups and methyl ethers of phenolic OH groups (Supporting Information Figure S8). The Fe-HA-methylated complex prepared using this methylated HA showed negative activity in mediating the microbial reductive PCP dechlorination (Supporting Information Figure S9). ESR analysis suggested the presence of the quinone-type structure. Together, these results suggested that the chemical structure of the functional groups in the Fe-HA complex was attributable to the carboxylic and phenolic carbon plus quinone-type structure complexed with Fe. In addition, the electron shuttle assay showed that the Fe-HA complex contained microbially and chemically reducible redox-active moieties (Figure 4). The extent of microbial reduction was over 2.2-times higher than chemical reduction, which suggested that the functional groups were more readily reduced by microbial reduction, in contrast to dissolved HSs, where microbial and chemical reduction resulted in the same extent of reduction.24 The presence of redox-active moieties in the Fe-HA complex is shown by the CV of the sample after electrical reduction (Figure 6a), with a redox potential estimated at 0.02 V (vs SHE). The production of well-defined cyclic voltammetry peaks of the electrically reduced Fe-HA complex suggested that the redox-active moieties harbored in the Fe-HA sample were refractory to be reduced or oxidized, which made them insusceptible to the conventional scans of the cyclic voltammetry analysis, but they can be reduced gradually at a poised potential of −500 mV (vs SHE). However, the accumulation of a small fraction of Fe(II) was observed after complete PCP dechlorination (Supporting Information Figure S7) or when the Fe-HA complex was incubated together with the S. putrefaciens CN-32 cells. Nevertheless, there was no relevant report on the dissolution of Fe from the Fe-associated EMs during the redox reactions, and although the dissolution of Fe has been observed in the cathode of Fe-containing polymer used for fuel cells (Fe-nitrogen-carbon structure), the dissolution mechanism in relation with the functional potential remains unknown.25 Therefore, further study would be required to explore this phenomenon by studying the interaction mechanism of Fe with HA and the structure of the functional groups, as well as the change of the functional groups during the redox reactions. The effect of HSs as EMs in promoting anaerobic bioremediation processes by transferring electrons has been studied extensively during the past two decades with a focus on dissolved HSs.26,27 However, dissolved EMs would be easily flushed away in the remediation sites, especially in the wastewater treatment systems, resulting in the demands of continuous dose. Besides, some of the soluble EMs themselves are toxic (e.g., methyl viologen). Therefore, solid-form EMs that are of natural origin, chemically stable, and environ-

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ASSOCIATED CONTENT

S Supporting Information *

Method description for microbial Fe(III) oxide reduction experiment and electron spin resonance analysis; reduction of the Fe-HA complex during the dechlorination of PCP; preparation of the Fe-HA-methylated complex. Results for the dechlorination activity of the Fe-HA complex prepared using (a) KMHA and (b) YAHA (Figure S1); influence of pretreatment of Fe-HA complex on microbial meduction of Fe(III) oxide (Figure S2); microbial and chemical reduction of Fe-HA complex subjected to various treatments (Figure S3); Fourier-transform infrared spectra of the samples (Figure S4); ESR spectra of the Fe-HA complex and control sample (Figure S5); cyclic voltammetry of Fe-HA complex samples subjected to various treatments (Figure S6); production of Fe(II) during the dechlorination of PCP (Figure S7); FTIR spectra of the methylated samples (Figure S8) and the dechlorination activity of the Fe-HA-methylated complex (Figure S9). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81 52 789 5856; fax: +81 52 789 5857; e-mail: [email protected]. Present Addresses

§ C.Z.: Ocean College, Zhejiang University, Hangzhou, Zhejiang 310058, China. ∥ Z.L.: State Key Laboratory of Urban Water Resources and Environment, Harbin Institute of Technology, Harbin 150090 China.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research (23310055, 23658272), by a university grant for “Design of cascade utilization system for unused biological resources in the Tokai area” from the Ministry of Education, Culture, Sports, Science and Technology in Japan, and by the Sekisui Chemical Grant Program for Research on Manufacturing Based on Innovations Inspired by Nature.

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REFERENCES

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