Article pubs.acs.org/est
Cr(VI) Reduction and Cr(III) Immobilization by Acinetobacter sp. HK‑1 with the Assistance of a Novel Quinone/Graphene Oxide Composite Hai-Kun Zhang, Hong Lu,* Jing Wang, Ji-Ti Zhou, and Meng Sui Key Laboratory of Industrial Ecology and Environmental Engineering (China Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, # 2 Linggong Road, Dalian 116024, People’s Republic of China S Supporting Information *
ABSTRACT: Cr(VI) biotreatment has attracted a substantial amount of interest due to its cost effectiveness and environmental friendliness. However, the slow Cr(VI) bioreduction rate and the formed organo-Cr(III) in solution are bottlenecks for biotechnology application. In this study, a novel strain, Acinetobacter sp. HK-1, capable of reducing Cr(VI) and immobilizing Cr(III) was isolated. Under optimal conditions, the Cr(VI) reduction rate could reach 3.82 mg h−1 g cell−1. To improve the Cr(VI) reduction rate, two quinone/ graphene oxide composites (Q-GOs) were first prepared via a one-step covalent chemical reaction. The results showed that 2-amino-3-chloro-1,4-naphthoquinone-GO (NQ-GO) exhibited a better catalytic performance in Cr(VI) reduction compared to 2-aminoanthraquinone-GO. Specifically, in the presence of 50 mg L−1 NQ-GO, a Cr(VI) removal rate of 190 mg h−1 g cell−1, which was the highest rate obtained, was achieved. The increased Cr(VI) reduction rate is mainly the result of NQ-GO significantly increasing the Cr(VI) reduction activity of cell membrane proteins containing dominant Cr(VI) reductases. X-ray photoelectron spectroscopy analysis found that Cr(VI) was reduced to insoluble Cr(III), which was immobilized by glycolipids secreted by strain HK-1. These findings indicate that the application of strain HK-1 and NQ-GO is a promising strategy for enhancing the treatment of Cr(VI)-containing wastewater.
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INTRODUCTION Chromium is widely used in numerous industrial processes, including electroplating, ore refining, metal plating, and pigmentation.1 The improper treatment of industrial effluents containing chromium can result in the contamination of natural water sources, eventually threatening human health. Chromium predominantly exists as Cr(VI) and Cr(III) in wastewaters. Cr(VI) is highly toxic and carcinogenic, whereas Cr(III) is less toxic and generally can be precipitated out of solution using chemical methods.2 Compared with chemical methods, chromium biotreatment has received more attention due to its low cost and eco-friendliness. So far, various bacteria have been proven to be capable of reducing Cr(VI) to less toxic Cr(III) under aerobic or anaerobic conditions.3−10 However, many bacteria generally spend several days or even weeks performing the complete reduction of no more than 50 mg L−1 Cr(VI) under optimal conditions.11−13 Moreover, the formed soluble organo-Cr(III) will be discharged together with the effluents, when organic substances existed in wastewater or were secreted by bacteria. It has been reported that Cr(III) has strong biological toxicity at high concentrations.14 Therefore, it will be necessary to develop new biotechnologies that can effectively perform Cr(VI) reduction and the formed Cr(III) removal from the polluted wastewater.15 Recent studies have shown that the Cr(VI) bioreduction rate can be significantly enhanced by several quinone compounds © 2014 American Chemical Society
(QCs), including anthraquinone-2-sulfonate (AQS), anthraquinone-2,6-disulfonate (AQDS) and 2-hydroxy-1,4-naphthoquinone (Lawsone).16−18 However, in practical applications, these soluble compounds have to be continuously added into reaction systems, which can result in increases in running costs and in secondary contamination. Accordingly, various methods to immobilize QCs were developed.19,20 It has been demonstrated that reduced graphene oxide (rGO) can be prepared using many microorganisms and can mediate the bioreduction of environmental contaminations.21,22 Thus, graphene oxide (GO) was selected as a carrier, and quinone/ GO composites (Q-GOs), including anthraquinone/GO and naphthoquinone/GO composites, were prepared using a onestep covalent chemical reaction. Meanwhile, a novel strain Acinetobacter sp. HK-1 capable of reducing Cr(VI) and immobilizing Cr(III) was isolated. Accordingly, Q-GOs supplemented biosystems were established and expected to effectively remove soluble Cr. The aim of the present study is to evaluate the capability of strain HK-1 for dealing with Cr(VI)-containing aqueous solutions and to investigate the effect of Q-GOs as solid Received: Revised: Accepted: Published: 12876
August 11, October 8, October 8, October 8,
2014 2014 2014 2014
dx.doi.org/10.1021/es5039084 | Environ. Sci. Technol. 2014, 48, 12876−12885
Environmental Science & Technology
Article
donor at an initial Cr(VI) concentration of 50 mg L−1 was investigated. Next, the effects of different electron donors (glucose, formic acid, pyruvate, starch, acetate, and fructose), the optimal electron donor concentration (0−2 g L−1), pH (5− 9) and temperature (15−40 °C) on the Cr(VI) reduction rates were tested at an initial Cr(VI) concentration of 50 mg L−1. Then, the effect of initial Cr(VI) concentration (55−220 mg L−1) was also studied. Under optimal conditions, the bioreduction of 100 mg L−1 Cr(VI) was performed in the presence of 5 mg L−1 AQ-GO or NQ-GO. Moreover, the effects of different concentrations of NQ-GO (5−75 mg L−1, in the presence of 100 mg L−1 Cr(VI)) and of the initial Cr(VI) concentrations (55−220 mg L−1, in the presence of 5 mg L−1 NQ-GO) on the Cr(VI) bioreduction rate were also investigated. Control systems without cells or with heat-killed cells were also analyzed. All treatments and controls were run in triplicate. Identification of Reduced Product. To identify the location of the reduced product, strain HK-1 cells associated with the reduced product were characterized by scanning electron microscopy-energy dispersive X-ray (SEM-EDX), XPS, and FTIR. The preparation of the samples was described in the SI. Preparation of Cell Extracts and Enzyme Activity Assays. The cell extracts were prepared using the method described in the SI. The total protein content was estimated using Lowry’s method.23 The Cr(VI) reductase activity was assayed using NADH as an electron donor. The total volume of the reaction mixture was 1 mL, which contained 0.1 mL crude enzyme solution, 0.1 mg L−1 NQ-GO, 0.1 mM NADH and 1 mg L−1 Cr(VI) in a phosphate buffer (10 mM, pH = 7.5). Control assays without NQ-GO were also performed. The assay mixtures were incubated at 35 °C for 30 min in an anaerobic incubator. One unit of enzyme activity was defined as the amount of enzyme that reduced 1 nmol Cr(VI) in 1 min at 35 °C. Analytical Methods. The concentration of Cr(VI) was determined by the DPC (diphenylcarbazide) method.24 The total Cr concentration in the supernatant was determined after it was oxidized to Cr(VI) by potassium permanganate (pH = 2) at 100 °C. The Cr(VI) reduction efficiency and rate were calculated using eqs 1 and 2, respectively, as follows:
mediators on Cr(VI) bioreduction by strain HK-1. During this process, the mechanism of Cr(VI) reduction and Cr(III) immobilization by strain HK-1 with the assistance of naphthoquinone/GO composite was studied in detail.
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MATERIALS AND METHODS Materials and Chemicals. Graphite powder was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2-Aminoanthraquinone (AQ) and 2-amino-3-chloro1,4-naphthoquinone (NQ) were purchased from Tokyo Kasei Kogyo Co., Ltd. (Japan). The K2Cr2O7 used in this study was purchased from Tianjin Kaixin Chemical Co., Ltd. (China). All other reagents used in this study were of analytical grade. Isolation, Identification and Cultivation of Cr(VI)Reducing Strain. A Cr(VI)-reducing bacterium was isolated from anaerobic activated sludge (AS) from the Dalian Bio Chemical Co., Ltd. (Liaoning, China) using a dilution plate method at 30 °C. The isolated strain HK-1 was identified by scanning electron microscope (SEM) and 16S rRNA gene sequencing analysis. The anaerobic mineral salt (AMS) medium used in this study contained 1 g L−1 NH4Cl, 1.23 g L−1 K2HPO4·3H2O, 0.45 g L−1 KH2PO4, 0.17 g L−1 NaHCO3, 0.41 g L−1 MgSO4·7H2O, 0.026 g L−1 CaCl2, and a 0.5 mL trace element solution (pH = 7.5). The trace element solution contains (in ×10−3 g L−1) 140 FeCl3·6H2O, 10 MnCl2·4H2O, 10 ZnCl2, 2.5 CuCl2·2H2O, 1.0 H3BO3, 2.4 Na2MoO4·2H2O, 100 NaCl, 12 NiCl2·6H2O, 2.6 Na2SeO3·5H2O, and 2.4 CoCl2·6H2O. Preparation of Q-GOs and Their Characterization. The preparations of 2-aminoanthraquinone-GO (AQ-GO) and 2amino-3-chloro-1,4-naphthoquinone-GO (NQ-GO) were described in the Supporting Information (SI). GO was prepared from graphite powder using a modified method of Hummers.21 X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, England) and Fourier transform infrared spectroscopy (FTIR, EQUINOX55, German) were used to investigate the chemical compositional changes on the surfaces of the GO, AQ-GO and NQ-GO. The morphologies of the three materials were also analyzed using an SEM (KYKY-AMRAY-1000B, U.S.A.) and an atomic force microscope (AFM, Veeco DI 3100, U.S.A.). Cr(VI) Resistance Assays. Strain HK-1 was first aerobically grown for 12 h in Luria−Bertani (LB) medium, then 1 mL of the culture was added into the flask containing 100 mL LB medium and Cr(VI) (0−500 mg L−1). Samples were periodically taken for the analysis of cell concentration. The cell concentration was measured from optical density at 600 nm using a UV−vis spectrophotometer (V-560, JASCO, Japan). Cr(VI) Reduction Assays. After strain HK-1 was cultured for 12 h in 100 mL LB medium in a rotary incubator shaker at 150 rpm, the HK-1 cells (early stationary phase) were harvested by centrifugation (5 min, 10 000g) and washed twice with a sterile phosphate buffer solution (K2HPO4·3H2O and KH2PO4, 10 mM, pH 7.5). Then, the cell pellets were resuspended in an AMS medium and held in an anaerobic chamber for the following studies. The experimental systems utilized 135 mL serum bottles containing 50 mL deoxygenated sterile AMS medium, Cr(VI) and an electron donor. The strain HK-1 cells were added into the systems in an anaerobic incubator. After cell inoculation, samples were periodically taken with a sterile needle and a syringe for the analysis of the Cr species. Various factors affecting Cr(VI) reduction were systematically studied. First, the effect of cell concentration (0−0.5 g L−1) on the Cr(VI) reduction rate using glucose as an electron
reduction efficiency(%) =
reduction rate =
Ci − Ct mt
Ci − Ct × 100% Ci
(1)
(2)
where Ci (mg L−1) and Ct (mg L−1) are the initial and residual Cr(VI) concentrations at time zero and t, respectively; m (g cell L−1) is the dry weight of the cells; t (h) is the reaction time. A pseudo first-order model can be applied to describe the kinetics of Cr(VI) bioreduction with the assistance of NQ-GO. The first-order rate constant k (h−1) was determined according to eq 3. C t = C i e−kt
(3)
The Andrews model was applied to describe the kinetics of NQ-GO-mediated Cr(VI) reduction.25 The Andrews equation was given as in eq 4. 12877
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Figure 1. Effects of electron donors, glucose concentration, temperature, pH in 24 h (a, initial Cr(VI) concentration of 50 mg L−1), and initial Cr(VI) concentration (the relation between ln(C0/Ct) and time) (b) on Cr(VI) reduction by strain HK-1. Error bars shows mean standard deviation of three determinations.
k=
k maxC C + Ks + −1
C2 Ki
morphology of strain HK-1 is a short rod with dimensions of 3 × 0.6 μm2 (SI Figure S1). On the basis of the sequencing of the 16S rDNA gene, the homology between strain HK-1 (GenBank accession number KJ958271) and an Acinetobacter baumannii ATCC 17978 (GenBank accession number NC_009085) is 98%. Thus, it can be concluded that strain HK-1 belongs to the genus Acinetobacter. The phylogenetic tree of strain HK-1 is shown in Figure S1 (SI). Cr(VI) resistance assays showed that the aerobic growth in the LB medium had only slight delay with the increase of Cr(VI) concentration from 25 to 100 mg L−1 (SI Figure S2). When adding 150 mg L−1 Cr(VI), the strain HK-1 could recover the growth after 24 h incubation. Only when the Cr(VI) concentration was up to 500 mg L−1, was the growth of strain HK-1 inhibited severely. However, strain HK-1 can not reduce Cr(VI) under aerobic conditions. Under anaerobic conditions, strain HK-1 can reduce Cr(VI) without further proliferation. The effect of cell concentrations on the Cr(VI) reduction rate by strain HK-1 was first investigated under anaerobic conditions. In 24 h, approximately 7.4, 30.3, 56.8, 70.9, and 83.4% of the Cr(VI) were removed in
(4) −1
where C (mg L ) is the Cr(VI) concentration; kmax (h ) is the maximum specific Cr(VI) reduction rate; and Ks (mg L−1) and Ki (mg L−1) are the half-rate and inhibition constants, respectively. Glycolipids of strain HK-1 cells were extracted as described by Hošková et al.26 and separated using thin-layer chromatography (TLC) with a mixture of chloroform−methanol−glacial acetic acid (180:25:1, v/v/v). A band visualization was carried out with 2,7-dichlorofluorescein.
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RESULTS Cr(VI) Reduction by a Newly Isolated Strain. A facultative anaerobic strain capable of removing total Cr was isolated and named HK-1 for the following studies. When strain HK-1 was grown on an LB agar plate under aerobic conditions, its colony was white and circular in shape with raised centers and an irregular edge. The SEM analysis shows that the 12878
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Figure 2. FTIR spectra, XPS spectra, SEM and AFM images of GO, AQ-GO and NQ-GO. The (a) FTIR spectra of GO, AQ-GO, and NQ-GO; (b) N 1s of AQ-GO; (c) N 1s of NQ-GO; (d) C 1s of GO; (e) C 1s of AQ-GO; and (f) C 1s of NQ-GO.
the presence of 0.12, 0.23, 0.34, 0.43, and 0.47 g L−1 cells, respectively (SI Figure S3). The highest removal rate of Cr(VI) per gram of dry biomass was observed at a cell concentration of 0.34 g L−1. The Cr(VI) reduction rate is also affected by some environmental parameters, including additional electron donors, pH, and temperature. Figure 1 shows that among the
tested electron donors, glucose was proven to be the most suitable electron donor for removing Cr(VI). Moreover, when the concentration of glucose was 1.0 g L−1, the Cr(VI) removal rate reached 3.42 mg h−1 g cell−1 in 24 h. As Cr(VI) reduction rate increased slowly with further increase of glucose concentration, 1.0 g L−1 glucose was selected for the following 12879
dx.doi.org/10.1021/es5039084 | Environ. Sci. Technol. 2014, 48, 12876−12885
Environmental Science & Technology
Article
Figure 3. Effects of AQ-GO and NQ-GO on the Cr(VI) reduction rate by strain HK-1. (a) Cr(VI) reduction curves; (b) total Cr removal curves; (3) photographs of Q-GO-mediated Cr(VI) bioreduction; and (d) effect of NQ-GO concentration. Error bars shows mean standard deviation of three determinations.
absorption bands of COOH and COC at approximately 1741 and 1053 cm−1, respectively, disappeared. Simultaneously, CONH bonds at approximately 1668 cm−1 (mainly CO stretching) and 1484 cm−1 (NH deformation coupled with C N stretching) appeared (Figure 2a) in their infrared spectra. CCl bonds at approximately 700 cm−1 were also observed in the infrared spectrum of the NQ-GO. The XPS analysis showed the appearance of an N 1s peak at 394−400 eV in the spectra of the AQ-GO and NQ-GO (Figure 2b,c). Moreover, it was observed that the intensity of the CC peak increased, while the intensity of the CO peak decreased at different levels in the C 1s spectra of the two composites compared with those in the C 1s spectrum of the GO, indicating that the GO was partially reduced during the Q-GO preparation (Figures 2d−f). All these observations also suggest the successful surface modification of the GO by the AQ and NQ molecules. On the basis of the XPS analysis, the immobilization efficiencies of the two QCs are approximately 2.69 mmol AQ g−1 GO and 1.93 mmol NQ g−1 GO. Figure S4 (SI) shows that the chemical modification of GO using AQ or NQ is simple and that the reaction conditions are mild. Using SEM, it can be observed that the surface of the GO sheets is smooth. After reacting with the AQ and NQ, the surfaces of the AQ-GO and NQ-GO sheets are slightly rougher than those of the GO sheets (SI Figure S5a). The AFM analysis determined the average thickness of the GO, AQ-GO, and NQ-GO to be approximately
experiments. Figure 1 also shows that the optimal temperature for Cr(VI) bioreduction is 35 °C. When the temperature was lower or higher than 35 °C, the Cr(VI) reduction rate decreased significantly. A pH range of 6−8.5 was optimal for Cr(VI) reduction by many bacteria including Ochrobactrum and Enterobacter species.27 The optimal pH range was 7−8 for strain HK-1 to remove Cr(VI). Under optimal conditions, the effect of the initial Cr(VI) concentration on the Cr(VI) bioreduction rate was further investigated. As less than 3% Cr(VI) was removed using heatkilled cells, Cr(VI) adsorption was neglected. This reduction process follows the first-order kinetics at each initial Cr(VI) concentration tested. The first-order rate constant (k) was determined and is shown in Figure 1b. It is obvious that the Cr(VI) reduction rate constant declined significantly from 19.3 to 0.92 h−1 with increasing initial Cr(VI) concentration from 55 to 220 mg L−1. This result may be because the high concentrations of Cr(VI) adversely affect the metabolism of cells.13,28 Characterization of Q-GOs. The preparations of AQ-GO and NQ-GO via a one-step covalent chemical reaction are described in Figure S4 (SI). In this reaction, the carboxyl groups and epoxy groups of GO can react with the amino group of AQ/NQ, which was also demonstrated by the following characterization of the two composites. In the infrared spectra of the AQ-GO and NQ-GO, the characteristic 12880
dx.doi.org/10.1021/es5039084 | Environ. Sci. Technol. 2014, 48, 12876−12885
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2.76, 2.43, and 2.57 nm, respectively (SI Figure S5b). These findings indicate that the surface chemical modification only partially reduces the GO and does not remarkably alter the GO structure, which is beneficial for transferring electrons from QGOs to environmental contaminants. Effects of Q-GOs on Cr(VI) Bioreduction. As shown in Figure 3a, little Cr(VI) removal (
