Bioremediation of Cr(VI) and Immobilization as Cr(III) - ACS Publications

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Environ. Sci. Technol. 2010, 44, 6357–6363

Bioremediation of Cr(VI) and Immobilization as Cr(III) by Ochrobactrum anthropi YANGJIAN CHENG,† FENBO YAN,† FENG HUANG,† WANGSHENG CHU,‡ DANMEI PAN,† ZHI CHEN,† JINSHENG ZHENG,† MEIJUAN YU,‡ Z H A N G L I N , * ,† A N D Z I Y U W U * ,‡ State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fujian 350002, China, National Synchrotron Radiation Facility, USTC 230026 Hefei, China, and Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China

Received January 19, 2010. Revised manuscript received June 10, 2010. Accepted June 28, 2010.

Bioremediation of Cr(VI) through reduction relies on the notion that the produced Cr(III) may be precipitated or efficiently immobilized. However, recent reports suggest that soluble organo-Cr(III) complexes are present in various chromatereducing bacterial systems. This work was designed to explore the factors that affect the immobilization of Cr(III) in the Ochrobactrum anthropi system. X-ray absorption fine structure analysis on the cell debris clearly verified that coordination of Cr(III) occurs on the surfaces via the chelating coordination with carboxyl- and amido-functional groups. However, competitive coordination experiments of Cr(III) revealed that the small molecules such as amino acids and their derivatives or multicarboxyl compounds hold stronger coordination ability with Cr(III) than with cell debris. We speculate that it is the preferential coordination of Cr(III) to the soluble organic molecules in the bacterial culture medium that inhibits effective immobilization of Cr(III) on the cells. On the basis of this understanding, a strategy with two-step control of the medium was proposed, and this achieved successful immobilization of Cr(VI) as Cr(III) by O. anthropi and Planococcus citreus in 5-50 L pilot-scale experiments.

Introduction Cr(VI) is highly toxic, mutagenic, and carcinogenic. It is introduced into environment as the byproduct of industries. Low cost and recyclable strategies for treating Cr(VI)containing wastes are in great demand all over the world, especially in China. It was estimated that the traditional chromate salt plants in China have already generated over 2500 kilotons of Cr(VI)-containing wastes by the end of 2006 (1). Currently, technologies including chemical reduction and ion-exchange are mainly used for removing chromate from industrial wastewater (2-4). The chemical reduction method * Corresponding authors e-mail: [email protected] (Z.L.); phone and fax: (+086)591-83705474 (Z.L).; e-mail: [email protected] (Z.W.); phone: (+086)551-3602077 (Z.W). † State Key Laboratory of Structural Chemistry. ‡ National Synchrotron Radiation Facility and Institute of High Energy Physics. 10.1021/es100198v

 2010 American Chemical Society

Published on Web 07/07/2010

is more suitable for disposing high concentration Cr(VI)containing wastewater, while ion-exchange is restricted by the high cost ion-exchange resins. Recently, the use of a biological method to remediate chromate contaminated wastes has been regarded as a promising, safe, and costeffective technology, especially when dealing with Cr(VI)containing wastewater at low-to-mid concentrations (10-200 mg/L). Various microbes were found to have the ability to reduce highly toxic Cr(VI) to less toxic Cr(III) (2, 5, 6). Some earlier researchers believed that such activity can ‘fix’ Cr(VI) into an insoluble form such as Cr(OH)3 that cannot easily be dispersed (7). However, recent investigations revealed that the microbial reduction of Cr(VI) may form soluble organoCr(III) complexes (8-11). In oxidative environments, the mobile organo-Cr(III) complexes can be reoxidized into Cr(VI) (12, 13). This is a relevant obstacle to implement industrial applications of chromate-reducing bacteria. Hence, how to immobilize the reduced Cr(III) in microbial systems becomes a challenge and at the same time a necessary research subject. Here, we present a study of the immobilization mechanism of Cr(III) by bacteria. A chromate reducing strain, Ochrobactrum anthropi, isolated from a Cr(VI)-contaminated site of China, was selected as the representative research object. In our previous work, we found this strain could tolerate as high as 500 mg/L Cr(VI) (14). Microscopic investigations show that Cr(III) ‘fixed’ by O. anthropi cells were mostly accumulated on the surfaces, addressing the critical role of the chemical interaction between cell surface and Cr(III) (15). Fourier transform-infrared spectroscopy (FTIR) analysis implied that the -COOH and -NH2 in the living bacteria may play an important role during the Cr(III) immobilization process (15). However, it was also found that the immobilization efficiency of Cr(III) was different in the Tris-HCl buffer and Luria-Bertani medium (LB medium) (15). Hitherto, the relationship between the immobilization mechanism and immobilization efficiency of Cr(III) is still unclear. More in-depth investigations are needed. We designed experiments to explore the immobilization mechanism of Cr(III) at a molecular level. To achieve this goal, we disrupted cells and separated them into two parts, cell debris (sediment) and cytoplasm (supernatant) (16), for testing their corresponding roles during Cr(VI) reduction and Cr(III) immobilization processes. The Cr(III) competitive coordination effect of a series representative of small molecules that coexisted in the solution was studied individually. On the basis of the understanding of the immobilization mechanism of Cr(III), we propose a strategy for treating Cr (VI) with O. anthropi cells. We expect that the present results may be common to many other Cr(VI)reducing bacterial systems and hope this finding could provide important information for helping the engineering of the bioremediation program of Cr(VI) in the future.

Material and Methods Chemicals. All chemicals were ACS reagent grade and purchased from Sigma-Aldrich Shanghai Trading Co. Ltd. (Shanghai, China). The stock solutions of Cr(VI) and Cr(III) were prepared by dissolving potassium dichromate and chromium chloride in deionized-distilled water at the concentration of 10000 mg/L, respectively. Bacteria. Strain CTS-325, isolated from a chromatecontaminated site in Changsha, China (28.264° N, 112.957° E) was identified as O. anthropi on the basis of its biochemical properties and 16S rDNA sequence homology analysis. The VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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16S rDNA sequence could be found in GenBank (accession No. EF493855). Preparation of Cell Debris. The routine cultivation of the O. anthropi was performed at 37 °C with shaking at a speed of 180 rpm in LB medium for 24 h. Cells were collected by centrifugation at 8000 g for 10 min at 4 °C. The cell pellet was then washed with Tris-HCl buffer (pH 7.4) twice and a repeated freezing and thawing cycle with liquid nitrogen three times. Then the cell pellet was resuspended in 25 mL of a Tris-HCl buffer that contained 0.5 mM phenylmethylsulfonyl fluoride (PMSF) as protease inhibitor. Cells were then disrupted by sonication for five 15 s pulses at 40 W, with 30 s intervals under ice cooling treatment. (Cell lysate produced from sonication treatment was spread onto an agar plate for testing the viability. After being cultured at 37 °C for 24 h, no colony could be observed on the agar plate, indicating that the sonication treatment is an effective method to disrupt the cells). Cell debris and cytoplasm were separated by centrifugation at 20000 g for 20 min at 4 °C, and then resuspended in 25 mL of a Tris-HCl buffer (16). The protein concentrations of the cytoplasm and cell debris were measured by the Bradford method, with bovine serum albumin as the standard (17). Before the protein assay, the cell debris was treated with 0.66 M NaOH at 30 °C for 24 h (18). Chromium analysis. The concentration of Cr(VI) was detected by the 1,5-diphenylcarbazide method at 540 nm with a Perkin-Elmer Lamda 35 UV/vis spectrometer (19). For the experiments starting from Cr(VI), before each measurement experiment, the cell suspension was centrifuged at 20000 g for 20 min (4 °C); thus Cr(VI) and soluble Cr(III) remained in the supernatant. The concentration of Cr(VI) can be detected directly with an UV/vis spectrometer method (19). Total Cr in the supernatant was determined via oxidizing all the soluble chromium forms into Cr(VI) by the addition of excess potassium permanganate and then was detected as Cr(VI). Measurement experiments were conducted in triplicate. Soluble Cr(III) could be acquired by deducting the remained Cr(VI) from the total Cr in the supernatant. Cr(III) in the sediment could be acquired by deducting the total Cr in the supernatant from the initial concentration of Cr(VI). For the experiments that directly started from Cr(III), the same centrifugation treatments were conducted prior to the chromium assay. Cr(III) remaining in the supernatant was analyzed via conversing Cr(III) into Cr(VI) first with the addition of excess potassium permanganate and then measured as above. Active Component for Cr(VI) Reduction. Cell debris, cytoplasm, intact cells, and LB medium were used for the reduction experiment of Cr(VI). The initial concentration of Cr(VI) was 200 mg/L. The corresponding protein concentration of 0.1 g/mL cell debris (wet weight) was about 2.5 mg/ mL. The protein concentration of the cytoplasm was around 1.5 mg/mL. Cell debris was treated with 5 mM HgCl2, 10 mM AgNO3, and 0.5% sodium dodecyl sulfate (SDS) at room temperature and heat denaturalization (60 °C/95 °C) for 10 min. The treated and untreated (control) cell debris were used for Cr(VI) reduction experiments. The reducing ability of the cell debris after DNase treatment was also conducted as a control experiment. XAFS Analysis. Cr(VI)-cell debris and Cr(III)-cell debris samples were obtained by interacting the cell debris with 4 mM Cr(VI) at pH 7.4 and 4 mM CrCl3 · 6H2O at pH 4.2 for 24 h, respectively. After centrifugal-washing by deionized-distilled water and freeze drying, the samples were analyzed by X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy. The spectra were collected at the Cr K-edge (5989 eV) at the U7C beamline of National Synchrotron Radiation Laboratory (NSRL) in Hefei, a storage ring working at 800 MeV with a maximum 6358

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current of 300 mA. The absolute energy positions were calibrated using a Cr metal foil. A Si (111) double crystal monochromator was utilized. CrCl3 · 6H2O, Cr(NO3)3 · 9H2O, Cr(OH)3, K3Cr2(µ-OH)(D-Glucose) · 4H2O (20), Na[Cr(Lcysteine)2] · 2H2O (Cr-Cys) (21), Cr(Glycine)3 · H2O (Cr-Gly) (22), Cr(Aspartic acid)3 · 4H2O (Cr-Asp) (23), Cr2(SO4)3, K2Cr2O7, and Cr(CH3COO)3 (Cr(Ac)3) were recorded as model compounds. The Cr(III)-cell debris, Cr(VI)-cell debris, and Cr(Ac)3 samples were measured in the fluorescence mode, using a ion chamber to record I0 and a Lytle detector to record If, which were flowed with N2 and Ar2, respectively. Solid models were coated on scotch tape and measured in the transmission mode, while both I0 and I1 were recorded by two ion chambers, both flowed with N2. The analysis of the experimental XAFS spectra was performed by WinXAS3.1 (24). Fitting of the First Shell of the Fourier Transform (FT) of EXAFS. The Feff8 program (24, 25) was used to generate phase and amplitude parameters on the basis of the crystal structure of the Cr-Gly compound. To explore Cr coordination numbers in the cell debris, we fitted the first shell of the FT of the Cr(III)-cell debris sample with a model where Cr coordinates six oxygen or nitrogen (O/N) atoms. The range of K space and R space used for the fitting is 2.5-11 Å-1 and 1-2 Å, respectively. Thus, the EXAFS data can give five independent data points, according to the standard formula Nind ∼(2∆k∆R)/π (26), where ∆k and ∆R are the K and R intervals in which data have been analyzed, respectively. During the fitting process, we fitted the isolated EXAFS data of the Cr-N/O pair of the reference sample Cr-Gly first. Then we fixed the coordinator number and varied S02, distance, ∆E, and σ2. At last, we fitted the isolated EXAFS data of the Cr(III)-cell debris sample using the same S02 with that of the reference sample Cr-Gly. Modification of the Functional Groups. The esterification (E) of the carboxyl groups on the cell debris was performed with addition of acidic methanol. Briefly, 1 g of wet cell debris was suspended in 10 mL of methanol and 5.4 mL of concentrated HCl (the final concentration of HCl is 0.1 M) with continuous agitation for 24 h (27). The sample was dialyzed against a Tris-HCl buffer (pH 7.4) in dialysis bags at room temperature to eliminate unreacted methanol and then was lyophilized. The acetylation (A) of amino groups on the cell debris was carried out by suspending cell debris with 1:10 (v/v) acetic anhydride/alcohol solution for 1 h (27). The samples were then dialyzed and lyophilized as mentioned above. The double modification was performed with first esterification then acetylation (E-A) or first acetylation then esterification (A-E) as specifically described above. Two control experiments were designed as follows: control 1 was treated the same as the E process except for the lack of methanol in the reaction solution; control 2 was treated the same as the A process except for the lack of acetic anhydride in the reaction solution. The modified samples and controls were further interacted with 4 mM CrCl3 · 6H2O at pH 4.2 for 24 h. The concentration of the Cr(III) remaining in the suspensions was determined as mentioned above. Competitive Coordination Experiment. An equal amount of cell debris was suspended in solution containing different types of small organic molecules. The initial concentration of Cr(VI) was 200 mg/L. No additional carbon source was added because the preliminary experiment revealed that the cell debris contains enough reducing agents to convert Cr(VI) into Cr(III). After incubating at room temperature for 24 h, the samples were centrifuged at 20000 g for 20 min. The concentration of the reduced Cr(III) that remained in the supernatant was analyzed as mentioned above. Pilot-Scale Treatments. With two types of chromatereducing bacteria (O. anthropi and Planococcus citreus) as biomass, 5 or 50 L pilot-scale experiments were performed for treating three types of Cr(VI)-containing wastewater,

FIGURE 1. Enzymatic reduction of Cr(VI) by O. anthropi. (a) The reducing capability of cell debris, cytoplasm, intact cells, and LB medium to Cr(VI). (b) The reducing capability of cell debris to Cr(VI) after heating denaturalization and protein denaturant treatments (25 °C) for 10 min. Error bars represent standard deviation (s.d.).

TABLE 1. Reduction Ratio and Immobilization Ratio of Cr at Different Times interaction time (h)

reduction ratio (RR)a (%)

immobilization ratio (IR) (%)

IR/RR (%)

3 6 12 24

42.6 82.6 95.8 99.6

27.3 59.9 87.2 98.7

64.1 72.5 91.0 99.1

a We counted all the reduced Cr as Cr(III) because the Cr(V) intermediate is unstable. The initial concentrations of Cr(VI) and cell debris were used as 100 mg/L and 0.1 g/mL at wet weight, respectively.

including chlorate plant wastewater, plating Cr-contaminated wastewater, and lixivium from the chromium slag. Cells were cultured from organic wastes and filtered out first, then the cells were plunged into wastewater for 24 h, with 1% saccharose as the carbon source. After that, the immobilized efficiency was detected via measuring total Cr in the supernatant.

Results and Discussion Enzyme-Mediated Reduction of Cr(VI) by Cell Debris. The bacteria were disrupted and separated into cell debris and cytoplasm. The reducing ability of these components to Cr(VI) was analyzed, respectively. The preliminary data related to the experimental design, including the amount of biomass, concentration of Cr(VI), and effect of contact time and pH can be found in Tables S1-S2 and Figure S1 of the Supporting Information. As shown in Figure 1a, when the cell debris and intact cell were introduced into 200 mg/L Cr(VI), the concentration of Cr(VI) decreased to 0 mg/L within 24 h in both situations. On the contrary, no significant change of the Cr(VI) concentration was observed when cell cytoplasm and LB medium were introduced. This revealed that the cytoplasm and LB medium could not reduce Cr(VI) directly. Cell debris was further treated with heat denaturalization and protein denaturants, aiming to find out whether the reduction process was enzyme-mediated or not. As shown in Figure 1b, the reduction of Cr(VI) can be halted by heat denaturalization. However, this also conceives an explanation that heat treatment destroyed some unidentified heat-labile reductants in the cell debris, which were responsible for the reduction of Cr(VI). To exclude the possible alternatives, we treated cell debris with other protein denaturants at room temperature (28). It showed that both heavy metals (Hg2+ or Ag+) and surfactants (SDS) may significantly affect or even halt the effective reduction of Cr(VI). A control experiment of DNase treating cell debris was also conducted. It showed that the reducing ability could not be caused by the DNA molecule. The results indicate that the reduction of Cr(VI) by cell debris of O. anthropi is enzyme-mediated.

We found during this process that Cr(VI) reduction and Cr(III) immobilization happened asynchronously. As shown in Table 1, 42.6% of Cr(VI) was reduced after interacting with cell debris for 3 h, while only 64.1% of the reduced Cr(III) was immobilized. From 3 to 24 h, the Cr(III) that binded to the cell debris increased from 64.1% to 99.1%, which indicated that Cr(VI) reduction was a relatively faster step than that of Cr(III) immobilization. Status of Cr on Cell Debris. After incubating cell debris with Cr(VI) for 24 h, the Cr(VI)-cell debris sample was obtained and analyzed by XANES and EXAFS spectroscopy (Figure 2a,b). The corresponding spectroscopy of seven Cr(III) model compounds and the Cr(III)-cell debris sample were collected as well. As shown in Figure 2a, for the Cr(VI)-cell debris sample, the typical feature of Cr(VI) at 5980 eV disappeared and a small peak in accordance with Cr(III) presented at 5982 eV, indicating the original Cr(VI) had been totally changed into Cr(III) after interacting with cell debris for 24 h. The coordinating status of the reduced Cr in cell debris was further studied by comparing the FT of the EXAFS spectra of the Cr(VI)-cell debris sample with the model compounds (29-31). We found the FT of the Cr(VI)-cell debris sample has three coordination shells with radial distances of 1.5, 2.5, and ∼3.3 Å, which matched that of Cr-Gly for both the distances of the three coordination shells and their relative amplitudes (Figure 2b). But the FT of the Cr(VI)-cell debris is different from that of the Cr-Asp, indicating that the Cr in the cell debris has a low possibility to coordinate with six O atoms. (Asp has an extra carboxyl functional group comparing with Gly). It is difficult to distinguish the FT of both of the Cr(VI)-cell debris and Cr-Cys, while chemically the mercapto groups were normally passivated in peptides or proteins. Thus, the possibility of Cr to coordinate via thiol group in cell debris is negligible. Combining with our previous FT-IR analysis (15), we concluded that the reduced Cr(III) could bind to the cell debris via the chelating coordination with the carboxyl and amido functional groups, which is similar to the coordination situation of Cr-Gly (Figure 2c) (22). VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. (a) Comparison of XANES spectra of the Cr(VI)-cell debris with model compounds. (b) Comparison of the FT of the Cr(VI)-cell debris with above model compounds. (c) Scheme of the binding situation of Cr-Gly (22). (d) Fitting of the first shell of the FT of Cr(III)-cell debris on the basis of a model where Cr is coordinated with six O/N atoms. (e) Comparison of the immobilization efficiency of the cell debris after passivated by single and double modifications. Error bar represents the standard deviation (s.d.), which was calculated with three times independent experimental data.

TABLE 2. FT Fitting Results for the Cr(III)-Cell Debris Sample and Cr-Gly sample

coordination type

coordination number

R(Å)

Debye-Waller factor σ2(Å2)

∆E

R-factor

Cr(III)-cell debris Cr-Gly

N/O N/O

6.1 ( 1.1a 6.0 ( 0.6

1.98 ( 0.02 2.00 ( 0.01

0.0038 ( 0.0013 0.0031 ( 0.0008

4.87 4.25

0.9% 0.2%

a

The number that follows the ( sign represents a computing error in this table.

Moreover, it is worthy to mention that the EXAFS spectroscopy of the Cr(VI)-cell debris shows the same characteristic as that of the Cr(III)-cell debris sample, indicating that the mixing of inorganic Cr(III) with cell debris could lead to the same organo-Cr(III) complexes during Cr(VI) reduction. Thus the coordination number of Cr(III) in the cell debris could be obtained by fitting the first shell of the FT of the Cr-cell debris sample with the model compound Cr-Gly (Table 2 and Figure 2d) (22, 32). The good fitting results further confirmed the similarity of the coordination of Cr in cell debris with that in the Cr-Gly compound. Selective passivation experiments were performed to confirm the hypothesis that carboxyl and amido functional groups were responsible for the immobilization of Cr(III) on the cell debris. Because the mixing of inorganic Cr(III) with cell debris could lead to the same organo-Cr(III) complexes during Cr(VI) reduction, in this specific experimental section, CrCl3 · 6H2O instead of K2Cr2O7 was used directly for binding capability test. Obviously, if carboxyl or amido was respon6360

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sible for the binding of Cr(III) individually, at least one of the binding capabilities should decrease no more than 50% when either carboxyl or amido was inactivated. However, as shown in Figure 2e, the binding capability of the cell debris to Cr(III) was significantly decreased over 75% when the amido group was acetylated, while the cell debris almost could not immobilize Cr(III) when the carboxyl group was esterificated. The same phenomenon was observed when the cell debris was double modified (E-A or A-E). Thus, we conclude that the chelating activity of the carboxyl and amido functional groups are responsible for the binding of Cr(III) to the cell debris. A t test was further used to analyze the significance of difference of these four kinds of passivation treatments; the details of the t test statistical results are shown in Table S3 of the Supporting Information. Competitive Coordination Experiment of Cr(III). The above results clearly demonstrated that the immobilization of Cr(III) on the cell surface occurred via the chelating coordination with carboxyl and amido functional groups.

TABLE 3. Competitive Coordination between Cell debris and Typical Small Molecules to Reduced Cr(III) small molecules introduced into the Cr(VI)-cell debris system

soluble Cr(III)a (%)

pure water LB mediumb D-glucose (multihydroxyl) D-sucrose (multihydroxyl) glycine (H-CH(NH2)COOH) amber acid (HOOC-(CH2)2COOH) asparagine (H2NOC-CH2CH(NH2)COOH) aspartic acid (HOOC-CH2CH(NH2)COOH) cysteine (HS-CH2CH(NH2)COOH) serine (HO-CH2CH(NH2)COOH) malate (HOOC-CH2CH(OH)COOH) GSH (Gly-Cys-Glu) ammonium hydroxide(HCl, pH7.4) c (NH3 · H2O · HCl) ethylene Diamine (HCl, pH7.4) c (NH2-CH2CH2 -NH2 · HCl)

3.5 ( 2.01 61.2 ( 3.10 2.9 ( 1.81 3.1 ( 2.29 35.9 ( 2.35 62.2 ( 3.21 40.2 ( 2.78 82.8 ( 3.47 97.6 ( 2.84 58.8 ( 3.08 60.4 ( 2.94 76.8 ( 3.17 3.4 ( 2.42 6.3 ( 2.56

a Cr(VI) was reduced completely to Cr(III) after incubation with cell debris for 24 h. The number that follows the ( sign is the s.d., which was calculated with three times independent experimental data. b LB medium (pH 7.2) is composed of tryptone (10 g/L), yeast extract (5 g/L), and NaCl (10 g/L). The concentration of the other small molecules introduced into the Cr-cell debris system is 4 mM. c Because of the lack of selection, we used these two small molecules to demonstrate the coordination capability of Cr(III) to small molecules containing single- and multi-amido functional groups.

By this way, we observed that the cell debris suspended in pure water can immobilize about 96.5% of Cr(III) in the solution. However, once the culture medium was introduced, the reduced Cr(III) could not be completely immobilized on the cell surfaces (Figure S2a of the Supporting Information). Consequently, we speculate that the low immobilization efficiency of Cr(III) on cells is due to one or several types of small organic molecules that exist in the bacterial culture medium. These small organic molecules may have a stronger affinity for chelating coordination toward Cr(III) to form a soluble Cr(III)complex than that of functional groups on the bacterial surface, thus a part of Cr(III) remained in the supernatant. It is known that the possible coordinating functional groups to Cr(III) include hydroxyl, carboxyl, amido, and mercapto groups. To identify the key ligands, we introduced several types of small molecules with the above functional groups into the Cr(VI)-cell debris system. The criteria adopted to select these typical small organic molecules were as follows: It must be a common small molecule present in the organism, in the bacterial culture medium, or in the metabolite of the organism. These may include (i) sugars, (ii) basic amino acids and their derivatives, (iii) small organic acids containing multicarboxyl functional groups, and (iv) some small organic molecules containing multiamido functional groups. As shown in Table 3, when there were no organic molecules in the solution, the reduced Cr(III) could be almost immobilized on the cell debris. Moreover, the addition of sugars (D-glucose or D-sucrose) containing multihydroxyl functional groups or molecules containing multiamido functional groups did not affect the solubility of Cr(III). On the contrary, the addition of typical amino acids and their derivatives or small organic molecules containing multicarboxyl groups resulted in a dissociative behavior of Cr(III), so that 36-98% of Cr(III) was detected in the supernatant. Thus, the basic amino acids and their derivatives or small organic acids containing multicarboxyl functional groups represents the most active chelating coordination structures for Cr(III). Strategy for Immobilizing Cr(III) and Pilot-Scale Tests. As mentioned above, the industrial application of chromatereducing bacteria is subject to the reality that reduced Cr(III) could not be effectively precipitated under most conditions. The effect can be explained as follows: The general strategy for the Cr(VI) treatment by microbes is to reduce Cr(VI) by harnessing a specific metabolic pathway in bacteria. Theo-

retically, the more nutrition is supplied, the more bacteria proliferate. Therefore, the more Cr(VI) can be reduced, and also the more functional groups immobilize Cr(III). However, because the bacteria cannot consume all the amino acids during growth, the reduced Cr(III) cannot be fully immobilized by the bacteria on the basis of the competitive coordination effect caused by amino acids or other soluble small organic molecules (Figure 3a). For the O. anthropi-Cr(VI) system, a full immobilization of Cr(III) can hardly be achieved (Figure S2a of the Supporting Information). Here we propose a practical strategy to achieve the effective immobilization of Cr(III) by bacteria as follows (Figure 3b): (i) We first cultured O. anthropi in a solution containing carbon and nitrogen sources. Organic waste can be utilized as a nutrition source for growing O. anthropi, which guarantees a low cost for culturing bacteria (Figure S2b of the Supporting Information). (ii) Filtration was performed to separate the harvested bacteria from the residual soluble amino acids and other small organic molecules. (iii) O. anthropi, Cr(VI)-containing wastewater, and an appropriate amount of pure carbon sources (about 1% sugars) were mixed to facilitate effective reduction of Cr(VI) and immobilization as Cr(III). (iv) The Cr(III)-containing bacteria could be filtered out, and the Cr-contaminated wastewater could be purified. The above strategy with a two-step control of the bacterial culture medium was tested in 5-50 L pilot-scale experiments for treating with three types of typical Cr(VI)containing wastewater. The process is shown in Figure S3 of the Supporting Information. It was revealed that O. anthropi could be applicable to treat chromium-containing wastewater in a wide scope of situations. Because the maximum immobilization capability of O. anthropi is 23.2 mg/g cell at dry weight, when the concentration of the original Cr(VI) was less than 200 mg/L, pH was from 3.6-10. The bacterial amount was appropriate (≈0.1 g/mL at wet weight), and no matter what kind of Cr-contaminated wastewater was used, successful immobilization of Cr was obtained within 24 h (Table S4 of the Supporting Information). After treatment, the three types of Cr-contaminated wastewater all achieved the wastewater release standard of China [Cr(VI) e 0.5 mg/L, total Cr e 1.5 mg/L]. The Cr(III) collected by bacteria can further be recycled (Figure S4 of the Supporting Information). VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. (a) Scheme of treating Cr(VI)-containing wastewater via the growth of chromate-reducing bacteria in the wastewater directly. Reduced Cr(III) cannot be fully immobilized by the bacteria because the bacterial culture medium contains some favorite coordinating ligands to Cr(III) such as amino acids and their derivatives. (b) Scheme of the treatment of Cr(VI)-containing wastewater via the two-step control of the bacterial culture medium.

In order to ascertain the universality of the strategy with control of the medium, other bacterial classes should be used. Because the cell surface of Gram-positive and Gram-negative bacteria is obviously different, a Gram-positive Cr(VI)reducing strain (P. citreus) that is isolated from the same pollution area was used for testing the feasibility of the strategy. As shown in Table S4 of the Supporting Information, in a 5 L pilot-scale experiment for treating chlorate plant wastewater, successful immobilization of the Cr(III) was achieved, indicating that the present understanding related to the factors that affect the immobilization of Cr(III) may be common to other chromate-reducing bacterial systems. However, it is worthy to mention that cautions should be paid to chromate-reducing bacterial systems that can produce extracellular secretion or biomacromolecules during growth. Active functional groups for coordinating with Cr(III) could possibly exist in these components, which might increase the complexity of the systems. More indepth studies should still be conducted when trying to apply the strategy to other bacterial systems.

Acknowledgments Financial support for this study was provided by the National Basic Research Program of China (973 Program) (2007CB815601, 2010CB933501), National Natural Science Foundation of China (40902097, 20803082, 40772034), the Knowledge Innovation Project of the Chinese Academy of Sciences (KJCX2.YW.W01), the Outstanding Youth Fund (10125523, 50625205) and the Opening Project of Key Laboratory of Solid Waste Treatment and Resource Recycle (09ZXGK05), Ministry of Education.

Supporting Information Available Additional figures and tables summarizing the methods and results including (i) preliminary data related to the experimental design, (ii) reduction and immobilization efficiency of Cr in different culture media, (iii) t test analysis of selective passivation experiments, and (iv) pilot-scale treatment of three types of Cr(VI)-containing wastewater. This material 6362

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is available free of charge via the Internet at http:// pubs.acs.org.

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