Microscopic Investigations of the Cr(VI) Uptake Mechanism of Living

Aug 8, 2008 - State Key Laboratory of Structural Chemistry, Fujian Institute of Research ... This work also proved that the control of Cr immobilizati...
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Langmuir 2008, 24, 9630-9635

Microscopic Investigations of the Cr(VI) Uptake Mechanism of Living Ochrobactrum anthropi Bin Li,† Danmei Pan,† Jinsheng Zheng,†,‡ Yangjian Cheng,† Xiaoyan Ma,† Feng Huang,† and Zhang Lin*,† State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China, and Department of Chemistry and the Key Laboratory of Analytical Sciences of the Ministry of Education, College of Chemistry and Chemical Engineering, Xiamen UniVersity, Xiamen, Fujian 361005, China ReceiVed February 12, 2008 A basic understanding related to the immobilization of chromium by bacteria is essential for chromate pollutant remediation in the environment. In this work, we studied the Cr(VI) uptake mechanism of living Ochrobactrum anthropi and the influence of a bacterial culture medium on the Cr-immobilization process. It was found that the Cr-immobilization ratio of bacteria in Tris-HCl buffer is higher than in LB medium. X-ray photoelectron spectroscopy (XPS) and electron paramagnetic resonance (EPR) analysis revealed that the chromium accumulated on bacteria were mostly in Cr(III) states. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) observations showed that noticeable Cr(III) precipitates were accumulated on bacterial surfaces. AFM roughness analysis revealed that the surface roughness of bacteria increased greatly when the bacteria-Cr(VI) interaction was in Tris-HCl buffer rather than in LB solution. Transmission electron microscopy (TEM) thin section analysis coupled with energydispersive X-ray spectroscopy showed that Cr(III) is also distributed in bacterial inner portions. A chromiumimmobilization mechanism considering the participation of both bacterial inner portions and bacterial surfaces of living Ochrobactrum anthropi was proposed, whereas the bacterial surface was the dominant part of the immobilization of Cr(III). This work also proved that the control of Cr immobilization by living Ochrobactrum anthropi could be achieved via adjusting the bacterial culture medium.

1. Introduction Chromium has been a common contaminant because of the wide use of chromate in industry. Chromium occurs in the environment primarily in the Cr(VI) and Cr(III) states. Cr(VI) compounds are considered to be highly toxic, mutagenic, and carcinogenic,1,2 whereas Cr(III) compounds are less toxic than Cr(VI) compounds.3 In recent years, increased attention has been focused on the effects and related mechanisms occuring during Cr(VI) uptake by microbes. Both Cr(VI) biosorption and reduction by bacteria were taken into account. Biosorption is commonly known as a passive sorption and/or complexation process.4 Some researchers used various bacterial strains to investigate Cr(VI) biosorption processes.5-9 However, many bacteria have the ability to reduce highly toxic Cr(VI) to less toxic Cr(III).10-15 After a biotransformation process, the reduced Cr(III) could precipi* Corresponding author. Tel/Fax: +86 591 83705445. E-mail: zlin@ fjirsm.ac.cn. † Chinese Academy of Sciences. ‡ Xiamen University. (1) Pellerin, C.; Booker, S. M. EnViron. Health Perspect. 2000, 108, A402-A407. (2) Srivastava, S.; Thakur, I. S. Biodegradation 2007, 18, 637–646. (3) Ishibashi, Y.; Cervantes, C.; Silver, S. Appl. EnViron. Microbiol. 1990, 56, 2268–2270. (4) Park, D.; Yun, Y. S.; Kim, J. Y.; Park, J. M. Chem. Eng. J. 2008, 136, 173–179. (5) Srinath, T.; Verma, T.; Ramteke, P. W.; Garg, S. K. Chemosphere 2002, 48, 427–435. (6) Bai, R. S.; Abraham, T. E. J. Sci. Ind. Res. 1998, 57, 821–824. (7) Kiran, B.; Kaushik, A.; Kaushik, C. P. J. Hazard. Mater. 2007, 141, 662– 667. (8) Rapoport, A. I.; Muter, O. A. Process Biochem. 1995, 30, 145–149. (9) Zhou, M.; Liu, Y. G.; Zeng, G. M.; Li, X.; Xu, W. H.; Fan, T. World J. Microbiol. Biotechnol. 2007, 23, 43–48. (10) Klonowska, A.; Clark, M. E.; Thieman, S. B.; Giles, B. J.; Wall, J. D.; Fields, M. W. Appl. Microbiol. Biotechnol. 2008, 78, 1007–1016. (11) Srivastava, S.; Thakur, I. Biodegradation 2007, 18, 637–646. (12) Sarangi, A.; Krishnan, C. Bioresour. Technol. 2008, 99, 4130–7.

tate,16-18 whereas recent investigations revealed that significant amounts of reduced Cr(III) could still remain in the supernatant in a soluble form.15,19-21 Thus, investigations of the interaction of Cr(VI)-reducing bacteria with Cr and the factors that affect the immobilization of Cr(III) on bacteria are needed. Ochrobactrum sp. bacteria are capable of degrading organic pollutants such as organophosphorus pesticides, phenol, and dimethylformamide or removing heavy metals including chromium.22-25 Ozdemir and co-workers used the dead biomass of Ochrobactrum anthropi to study the Cr(VI) biosorption process.26 Francisco and co-workers found that Cr(VI) could partially be reduced by Ochrobactrum anthropi in an NB medium.27 The Ochrobactrum anthropi strain used in this work was isolated (13) Lee, S. E.; Lee, J. U.; Chon, H. T.; Lee, J. S. EnViron. Geochem. Health 2008, 30, 141–145. (14) Opperman, D. J.; van Heerden, E. FEMS Microbiol.y Lett. 2008, 280, 210–218. (15) Puzon, G. J.; Roberts, A. G.; Kramer, D. M.; Xun, L. Y. EnViron. Sci. Technol. 2005, 39, 2811–2817. (16) Cervantes, C.; Silver, S. Plasmid 1992, 27, 65–71. (17) Garbisu, C.; Alkorta, I.; Llama, M. J.; Serra, J. L. Biodegradation 1998, 9, 133–141. (18) McLean, J. S.; Beveridge, T. J.; Phipps, D. EnViron. Microbiol. 2000, 2, 611–619. (19) Campos, J.; Martinez-Pacheco, M.; Cervantes, C. Antonie Van Leeuwenhoek 1995, 68, 203–208. (20) McLean, J.; Beveridge, T. J. Appl. EnViron. Microbiol. 2001, 67, 1076– 1084. (21) Shen, H.; Wang, Y. T. Appl. EnViron. Microbiol. 1993, 59, 3771–3777. (22) Branco, R.; Alpoim, M. C.; Morais, P. V. Can. J. Microbiol. 2004, 50, 697–703. (23) El-Sayed, W. S.; Ibrahim, M. K.; Abu-Shady, M.; El-Beih, F.; Ohmura, N.; Saiki, H.; Ando, A. J. Biosci. Bioeng. 2003, 96, 310–312. (24) Veeranagouda, Y.; Paul, P. V. E.; Gorla, P.; Siddavattam, D.; Karegoudar, T. B. Appl. Microbiol. Biotechnol. 2006, 71, 369–375. (25) Zhang, R. F.; Cui, Z. L.; Jiang, J. D.; He, J.; Gu, X. Y.; Li, S. P. Can. J. Microbiol. 2005, 51, 337–343. (26) Ozdemir, G.; Ozturk, T.; Ceyhan, N.; Isler, R.; Cosar, T. Bioresour. Technol. 2003, 90, 71–74.

10.1021/la801851h CCC: $40.75  2008 American Chemical Society Published on Web 08/08/2008

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Figure 3. XPS results of Cr-loaded bacteria in Tris-HCl buffer (Tris) or in LB medium (LB). The standard Cr(III) compound Cr2O3 had Cr 2p1/2 at 586.0 eV and Cr 2p3/2 at 576.2 eV, and the standard Cr(VI) compound K2Cr2O7 had Cr 2p1/2 at 589.0 eV and Cr 2p3/2 at 579.2 eV.

Figure 1. Cr(VI) reduction and total Cr uptake by living Ochrobactrum anthropi suspended in different media with a final cell concentration of 0.1 g mL-1: (a) in Tris-HCl buffer and (b) in LB medium. The electron donor for the Cr(VI) reduction process might be the organic molecules in LB medium or NADH, NADPH, and electrons of endogenous reserve inside the bacteria when Tris-HCl buffer is used.

situations of Cr(VI) by living Ochrobactrum anthropi in two typical media (an LB medium and Tris-HCl buffer). The binding sites and the valence states of chromium were analyzed. We found that Cr(VI) uptake happened mostly on the bacterial surfaces and was closely related to the bacterial culture medium. We hope that the understanding of the mechanism during the Cr(VI) uptake process can provide much fundamental information for help in constructing feasible strategies for chromate bioremediation in the environment.

2. Materials and Methods

Figure 2. EPR spectrum from a solid sample of Ochrobactrum anthropi incubated with 400 mg L-1 Cr(VI) in Tris-HCl buffer for 24 h. The EPR spectrum of a bacterial sample from LB medium was the same as a bacterial sample from Tris-HCl buffer.

from a chromate-contaminated site. Previously, we found that this strain showed a strong ability to reduce Cr(VI) to Cr(III) in an LB medium, whereas the reduced Cr(III) could not be effectively adsorbed by living bacteria (Supporting Information, Figure S1).28 By using cell debris of Ochrobactrum anthropi as an active component of Cr(VI) reduction, we further recognized that some small organic components in solution such as soluble polypeptides might affect the fate of reduced chromium.29 To study the relationship between the chromium accumulation states of Ochrobactrum anthropi and the culture media, we focused on the microscopic investigations of the bioaccumulation (27) Francisco, R.; Alpoim, M. C.; Morais, P. V. J. Appl. Microbiol. 2002, 92, 837–843. (28) Li, B.; Cheng, Y. J.; Ma, X. Y.; Wang, Y. H.; Zheng, J. S.; Chu, W. S.; Wu, Z. Y.; Lin, Z. Geol. J. China UniV. 2007, 13, 651–656. (29) Cheng, Y. J.; Yan, F. B.; Huang, F.; Li, B.; Zheng, J. S.; Yu, M. J.; Wu, Z. Y.; Lin, Z. Study on the immobilization of Cr(III) in Ochrobactrum anthropi. Submitted to EnViron. Sci. Technol.

2.1. Organism and Growth Conditions. The strain was isolated from a chromate-contaminated site in China and identified as Ochrobactrum anthropi. Bacteria were cultured in LB medium (NaCl 10 g L-1, tryptone 10 g L-1, yeast extract 5 g L-1, pH 7.2) aerobically at 37.0 °C with shaking at a speed of 160 rpm. 2.2. Interaction of Bacteria with Cr(VI). After being cultivated for 24 h, cells were collected by centrifugation at 8000g for 10 min and washed three times with 50 mmol pH 7.2 Tris-HCl buffer. Then the intact cells were resuspended in Tris-HCl buffer or LB medium for further use. The final cell concentration was 0.1 g mL-1 (wet weight). The stock solution of Cr(VI) was prepared by dissolving potassium dichromate (Aldrich, analytical grade) in deionized-distilled water. The interaction of bacteria with Cr(VI) experiments were conducted with 400 mg L-1 Cr(VI) at 37 °C and repeated three times. 2.3. Chromium Analysis. At certain time intervals, samples were centrifuged at 12 000g for 3 min and separated into supernatant and cell residual. The total chromium concentration in the supernatant was determined by atomic absorption spectroscopy (Perkin-Elmer PE-306). Experiments were conducted in triplicate. The Cr(VI) concentration was analyzed by the 1,5-diphenylcarbazide method at 540 nm with a Perkin-Elmer Lamda 35 UV/vis spectrometer.30 The total Cr immobilization ratio (%) is (1 - (total chromium in supernatant/initial Cr(VI) concentration)) × 100%. 2.4. XPS Analysis. To investigate the valence states of chromium adsorbed on the surfaces of bacteria, XPS analysis was carried out. After 24 h of reaction, bacterial samples were washed three times to remove the impurities in the supernatant. Samples were dried for 6 h at 60 °C for further analysis. Cr2O3 was taken as the Cr(III) model compound and K2Cr2O7 was taken as the Cr(VI) model compound. XPS was performed using an ESCALAB MK II spectrometer (VG Scientific Co.). 2.5. EPR Analysis. Bacterial samples for EPR detection were the same as forXPS. The EPR measurements were conducted with (30) Urone, P. F. Anal. Chem. 1955, 27, 1354–1355.

9632 Langmuir, Vol. 24, No. 17, 2008 a Bruker ER-420 spectrometer with 100 kHz modulation at 9.4 GHz. Experiments were performed in a 1-mm-diameter quartz tube at room temperature (300 K). 2.6. SEM-EDS. The SEM procedure was as follows: briefly, samples were washed twice and fixed with 2% glutaraldehyde for 1.5 h. Samples were then postfixed with 1% osmium tetroxide for 30 min and dehydrated in an acetone series (35, 50, 70, 80, 95, and 100%) for 3 min each. After dehydration by 100% acetone, samples were dried in air. SEM investigations were performed using a LED1530 scanning electron microscope coupled with EDS (Oxford). 2.7. AFM. Samples were first centrifuged at 3000g for 2 min. Theacquiredcellpelletsweregentlywashedtwicewithdeionized-distilled water and resuspended in it. A 5 µL portion of the cell suspension was dropped on a freshly cleaved mica substrate and then immediately dried under N2 gas for 3 min. All AFM experiments were carried out by a Veeco Multimode NS3A-02 Nanoscope III atomic force microscope. AFM imaging was done in tapping mode. A Si tip from Veeco was used. All experiments were done within 1 h. 2.8. TEM-EDS. The process for the thin-section TEM sample preparation was the same as for SEM from the harvest to dehydration steps. Cells were then embedded in SPI-PON 812 resin. Ultrathin sections were cut using an ultramicrotome (Leica EM UC6) and stained with uranyl acetate and lead citrate. All the TEM measurements were carried out by JEOL-2010 transmission electron microscope coupled with an EDS (Oxford) system operated at 200 kV.

3. Results 3.1. Immobilization Ratio of Chromium by Ochrobactrum anthropi in Different Media. The bacteria can reduce Cr(VI) with time. As shown in Figure 1, after reaction for 24 h, the Cr(VI) concentration in the solution could decrease to approximately zero in both Tris-HCl buffer and LB medium, but the total Cr immobilization ratio of the bacteria was different with the medium. After incubating in 400 mg L-1 Cr(VI) for 24 h, up to 95% of the chromium was immobilized by bacteria in Tris-HCl buffer (Figure 1a) whereas only 70% of the chromium was immobilized by bacteria in LB medium (Figure 1b). As we know, the chemical components of the Tris-HCl buffer contain tri(hydroxymethyl)aminomethane and hydrochloric acid, which do not favor Cr(III) coordination. On the contrary, when LB medium was used, organic components such as amino acids and organic acids could compete with bacteria for Cr(III) coordination, thus soluble chromium complexes could be formed, and the immobilization ratio of chromium decreased correspondingly.29 Similar observations were reported in previous work. For example, it was found that though Escherichia coli could reduce Cr(VI) completely in LB medium the majority of the Cr(III) products remained soluble.15 3.2. Valence States of Chromium Binding to Bacteria. To detect the possible valence states of chromium in the whole cell after Cr accumulation, solid-state EPR analysis was conducted. As shown in Figure 2, a broad signal (about 500 G) centered at a g factor of 1.96 was observed, which could be attributed to the Cr(III) paramagnetic signal on the basis of a series of references.31-33 No other paramagnetic Cr species such as Cr(V) were found when the bacteria interacted with Cr(VI) for 24 h. EPR results showed that Cr(III) was binding to the bacteria. The depth resolution of XPS is on the nanometer scale,34 which is active for detecting the signal on the bacterial surface to a thickness of several nanometers.35 To determine the valence (31) Charlet, L.; Karthein, R. Aquat. Sci. 1990, 52, 93–102. (32) Lopez-Navarrete, E.; Caballero, A.; Orera, V. M.; Lazaro, F. J.; Ocana, M. Acta Mater. 2003, 51, 2371–2381. (33) Nakajima, A.; Baba, Y. Water Res. 2004, 38, 2859–2864. (34) Tougaard, S. Microsci. Microanal. 2005, 11, 676–677. (35) Ojeda, J. J.; Romero-Gonzalez, M. E.; Bachmann, R. T.; Edyvean, R. G. J.; Banwart, S. A. Langmuir 2008, 24, 4032–4040.

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states of chromium on bacterial surfaces after the bioaccumulation process, XPS was employed. Figure 3 showed the XPS results of bacteria treated with 400 mg L-1 Cr(VI) for 24 h both in Tris-HCl buffer and LB medium. It revealed that Cr-loaded bacteria in both solutions gave two distinct peaks: 585.0-588.0 eV (Cr 2p1/2) and 576.0-578.0 eV (Cr 2p3/2). Compared with two standard model compounds, Cr2O3 and K2Cr2O7, the spectrum of bacteria samples indicated that chromium binding to the bacterial surfaces was mostly for Cr(III) states. 3.3. Distribution of Chromium on Bacterial Surfaces. SEM observation of the bacterial surfaces is shown in Figure 4. Figure 4a,h revealed that bacterial cells without chromate treatment were plump and had smooth surfaces, indicating that the components at bacterial surfaces were uniformly distributed both in Tris-HCl buffer and LB medium. When the bacteria were chromate treated in Tris-HCl buffer, obvious changes were observed. As shown in Figure 4d, uneven features of bacterial surfaces were found, indicating that the bacterial surfaces apparently interfered during the Cr(VI) uptake process. A close image with detailed information revealed that precipitates were densely aggregated over the whole bacterial surfaces (Figure 4e). The chromium signal including these precipitates were exhibited correspondingly by the EDS spectrum (Figure 4f). The changes in the surface situation for the bacterial sample in LB medium were not as apparent as in Tris-HCl buffer. As shown in Figure 4k,l, although some irregular shapes occurred, the amounts of precipitates on the bacterial surfaces were not so much. In comparison with SEM, the sample-preparation process of AFM is simpler. The bacteria can be imaged by AFM without pretreatment, thus possible artifacts can be reduced.36,37 AFM results are shown in Figure 5. Similar to the above SEM observation, before treatment with Cr(VI), the bacteria had smooth surfaces both in Tris-HCl buffer and in LB medium. After incubation with 400 mg L-1 Cr(VI) for 24 h, the morphologies of the bacterial cells changed obviously. The bacterial surfaces changed more markedly in Tris-HCl buffer than in LB medium. AFM roughness analysis can provide bacterial surface characteristics and present numerical-data-related surface changes. To quantify the differences in bacterial surfaces in Tris-HCl buffer and in LB medium, the root-mean-square roughness (Rq) within the Ochrobactrum anthropi cell surface was analyzed. A 300 nm × 300 nm box area was chosen as the analytic area on the basis of previous experience on surface roughness analysis.38 As shown in Table 1, before chromate treatment, the Rq value was almost the same in both LB medium and Tris-HCl solution, 7.8 ( 3.0 and 7.2 ( 2.7 nm, respectively. After chromate treatment, the Rq values increased to 10.3 ( 2.2 nm in LB medium and increased more obviously to 16.5 ( 8.0 nm in Tris-HCl buffer, indicating that the bacterial surfaces roughness is proportional to the immobilization ratio of chromium. 3.4. State of Chromium Inside the Bacteria. To investigate the distribution of chromium inside the bacteria, conventional thin-section TEM of bacteria together with EDS was analyzed. As demonstrated in Figure 6a, if Cr(VI) was not introduced, the bacterial cells cut by transverse section were round, and the cell wall structure could be seen clearly. After chromate treatment, the bacterial cell wall became rough, indicating that the bacterial surface was greatly affected during Cr(VI) reduction and (36) Cricenti, A.; Generosi, R.; Girasole, M.; Scarselli, M. A.; Perfetti, P.; Bach, S.; Colizzi, V. J. Vacuum Sci. Technol., A 1999, 17, 1141–1144. (37) Sharma, S.; Sen, P.; Mukhopadhyay, S. N.; Guha, S. K. Colloids Surf., B 2003, 32, 43–50. (38) Wang, J.; He, S. Y.; Xu, L. N.; Gu, N. Chin. Sci. Bull. 2007, 52, 2919– 2924.

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Figure 4. SEM images of bacteria treated with different concentrations of Cr(VI) for 24 h. (a-f) In Tris-HCl buffer. (a, b) 0 mg L-1, control; (d, e) 400 mg L-1; (c) EDS spectrum of bacteria treated without Cr(VI); (f) EDS spectrum of bacteria treated with Cr(VI). (g-l) In LB medium. (g, h) 0 mg L-1, control; (j, k) 400 mg L-1; (i) EDS spectrum of bacteria treated without Cr(VI); (l) EDS spectrum of bacteria treated with Cr(VI). Scale bar: (a, d, h, k): 1 µm; (b, e, h, l): 0.5 µm.

chromium (Figure 6d) and the amount of chromium inside the bacteria was so small that the intracellular chromium was distributed evenly and no obvious precipitates were formed. On the contrary, as shown in Figure 6e,f, considerable intercellular Cr precipitates were formed. These precipitates were amorphous and did not produce regular diffraction patterns during selectedarea electron diffraction analysis. The above observation revealed that chromium accumulated by living Ochrobactrum anthropi was mostly outside the bacterial cell wall.

4. Discussion

Figure 5. AFM images of Ochrobactrum anthropi in different media for 24 h: (a, b) in Tris-HCl buffer; (c, d) in Tris-HCl buffer with 400 mg L-1 Cr(VI); (e, f) in LB medium; and (g, h) in LB medium with 400 mg L-1 Cr(VI). Scale bar: 1 µm. Table 1. Surface Roughness Analysis of Ochrobactrum anthropi in Different Media for 24 h sample in in in in

LB medium LB medium with 400 mg L-1 Cr(VI) Tris-HCl buffer Tris-HCl buffer with 400 mg L-1 Cr(VI)

Rq (nm) 7.8 ( 3.0 10.3 ( 2.2 7.2 ( 2.7 16.5 ( 8.0

immobilization processes (Figure 6c). EDS analysis showed that after bioaccumulation the inner parts of bacteria contained

4.1. Possible Cr(VI) Uptake Mechanism of Living Ochrobactrum anthropi. As we know, Cr(VI) could not only have been adsorbed by biomass directly but also could have been reduced to less toxic Cr(III) and then immobilized. The key points for understanding the Cr(VI) uptake mechanism are (i) the immobilization sites of chromium and (ii) the valence state of chromium after bioimmobilization. It was found that different biomass has a different mechanism. For example, Cheung and co-workers reported that when Bacillus megaterium TKW3 was incubated with Cr(VI) the aggregation of chromium deposited along the intracellular membrane region.39 Similary, Srivastava and co-workers found a circular electron-dense inclusion within the cell cytoplasm.11 The valence states were not determined. Daulton et al. utilized TEM coupled with the electron energy(39) Cheung, K. H.; Lai, H. Y.; Gu, J. D. J. Microbiol. Biotechnol. 2006, 16, 855–862.

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Figure 6. Conventional TEM images of bacterial thin sections of bacteria in different media for 24 h: (a) in pure Tris-HCl buffer; (c) in Tris-HCl buffer with 400 mg L-1 Cr(VI); (b, d) EDS analysis of bacterial inner parts in panels a and c, respectively; (e) TEM results of precipitates (shown by arrows) generated near bacteria; and (f) EDS spectrum of these precipitates (black circular portion). Pb peaks were TEM specimen preparation artifacts from lead citrate used to stain the cells.

loss spectroscopy technique and found that precipitates that encrust Shewanella oneidensis were predominantly Cr(III) solids.40 A similar observation had also found that the valence state of Cr(VI) actually changed to Cr(III) during the Craccumulation process of various biomass, whereas the microscopic observation of the binding sites of Cr(III) was not provided.41,42 In this work, a systematic survey of the binding sites and the valence states of chromium in living Ochrobactrum anthropi was conducted. AFM surface roughness analysis, SEM, TEM, and AFM observations revealed that, during the Cr(VI) uptake process, not only the bacterial surfaces but also the intracellular portions participated whereas the surfaces were the main sites for Cr immobilization. The immobilized chromium was mostly in the Cr(III) state by XPS and EPR analysis. Combined with these results, we proposed a possible mechanism of the Cr(VI) uptake process of living Ochrobactrum anthropi. As shown in Figure 7, (1) corresponded to the surface enzymatic reduction process of bacteria, and during this process, Cr(VI) can be reduced efficiently to Cr(III).29 The reduced chromium was mostly coordinated with the functional groups on the bacterial surface or partially released into the supernatant. Process (2) showed a possible pathway by which chromium accumulated in the intracellular parts. Cr(VI) in the form of CrO42- could readily traverse cell membranes via the sulfate transport system.43,44 The electron donors for the reduction of intracellular Cr(VI) (40) Daulton, T. L.; Little, B. J.; Lowe, K.; Jones-Meehan, J. J. Microbiol. Methods 2002, 50, 39–54. (41) Park, D.; Lim, S.-R.; Yun, Y.-S.; Park, J. M. Chemosphere 2007, 70, 298–305. (42) Park, D.; Yun, Y. S.; Jo, J. H.; Park, J. M. Water Res. 2005, 39, 533–540. (43) Cervantes, C.; Campos-Garcia, J.; Devars, S.; Gutierrez-Corona, F.; LozaTavera, H.; Torres-Guzman, J. C.; Moreno-Sanchez, R. FEMS Microbiol. ReV. 2001, 25, 335–347. (44) Codd, R.; Dillon, C. T.; Levina, A.; Lay, P. A. Coord. Chem. ReV. 2001, 216, 537–582.

Figure 7. Cr(VI) uptake mechanism by living Ochrobactrum anthropi.

could be NADH, NADPH, and electrons of endogenous reserve inside the bacteria.45,46 4.2. Influence of Bacterial Culture Medium on the CrImmobilization Efficiency. The study of the immobilization of Cr(III) by bacteria is an important topic. In this study, both total Cr immobilization data and microscopic observation showed that in Tris-HCl buffer solution the bacterial surfaces of Ochrobactrum anthropi could accumulate more chromium than in LB medium. As shown by our separate work, small organic molecules in the solution can compete with the cell debris for Cr(III) coordination.29 In this work, we proved for the first time that the Cr-immobilization efficiency of living bacteria was closely related to the culture medium. We also notice that not only the culture medium but also the relative amount of bacterial culture medium to the number of cells can affect the Cr-immobilization efficiency. If bacteria were suspended in LB medium with a cell density of 0.01 g mL-1, then the Cr-immobilization ratio was arround 0%.28 By increasing the cell density to 0.1 g mL-1 in LB medium, though the relative amount of LB medium to the cell decreased by only a factor of 10, the Cr-immobilization ratio (45) Cheung, K. H.; Gu, J. D. Int. Biodeterior. Biodegrad. 2007, 59, 8–15. (46) Appenroth, K. J.; Bischoff, M.; Gabrys, H.; Stoeckel, J.; Swartz, H. M.; Walczak, T.; Winnefeld, K. J. Inorg. Biochem. 2000, 78, 235–242.

Cr(VI) Uptake Mechanism of Ochrobactrum anthropi

apparently increased to 70%. If we try to avoid almost all of the organic components in the buffer solution, then the Crimmobilization efficiency can reach 95%. We hope the example in this work will provide related information for help in achieving the immobilization of chromium with high efficiency by microbes.

5. Conclusions The present work investigated the Cr(VI) uptake mechanism by living Ochrobactrum anthropi both in Tris-HCl buffer and LB medium. Noticeable changes were found on the bacterial surfaces. A Cr(VI) uptake mechanism considering both the surface immobilization and intracellular accumulation of Cr(III) in living Ochrobactrum anthropi was suggested. A comparison of the bacterial surface situations in Tris-HCl buffer and LB medium

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reveals that the immobilization efficiency of Cr(III) was affected by the bacterial culture medium. Acknowledgment. Financial support for this study was provided by the National Basic Research Program of China (973 program, no. 2007CB815601), the National Natural Science Foundation of China (20501020, 40772034), the President Foundation of CAS, the Nanoscience Foundation of China (90406024), YIF (2007F3120) of Fujian Province, the Natural Science Foundation of Fujian Province (no. X0650094/2006J0383), and the Special Project on Science and Technology of Fujian Province (2005YZ1026). Supporting Information Available: Cr(VI) reduction and total Cr uptake by living Ochrobactrum anthropi in LB medium. This material is available free of charge via the Internet at http://pubs.acs.org. LA801851H