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Cr(VI) uptake mechanism of Bacillus cereus
Chemosphere 87 (2012) 211–216
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Cr(VI) uptake mechanism of Bacillus cereus Zhi Chen a,b, Zhipeng Huang b, Yangjian Cheng a, Danmei Pan a, Xiaohong Pan a,b, Meijuan Yu c, Zhiyun Pan d, Zhang Lin a,⇑, Xiong Guan b,⇑, Ziyu Wu c,d,⇑ a
State Key Lab of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, PR China Key Lab of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Ministry of Education, Fuzhou, Fujian 350002, PR China c Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, PR China d National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029, PR China b
a r t i c l e
i n f o
Article history: Received 6 July 2011 Received in revised form 19 December 2011 Accepted 19 December 2011 Available online 4 January 2012 Keywords: Chromium Reduction Immobilization Valence states Microscopy Bacillus cereus
a b s t r a c t In this study, we investigated the Cr(VI) uptake mechanism in an indigenous Cr(VI)-tolerant bacterial strain – Bacillus cereus through batch and microscopic experiments. We found that both the cells and the supernatant collected from B. cereus cultivation could reduce Cr(VI). The valence state analysis revealed the complete transformation from Cr(VI) into Cr(III) by living B. cereus. Further X-ray absorption fine structure and Fourier transform infrared analyses showed that the reduced Cr(III) was coordinated with carboxyl and amido functional groups from either the cells or supernatant. Scanning electron microscopy and atomic force microscopy observation showed that noticeable Cr(III) precipitates were accumulated on bacterial surfaces. However, Cr(III) could also be detected in bacterial inner portions by using transmission electron microscopy thin section analysis coupled with energy dispersive X-ray spectroscopy. Through quantitative analysis of chromium distribution, we determined the binding ratio of Cr(III) in supernatant, cell debris and cytoplasm as 22%, 54% and 24%, respectively. Finally, we further discussed the role of bacterium-origin soluble organic molecules to the remediation of Cr(VI) pollutants. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The Cr(VI) compounds are extremely toxic to human, animals and plants. The continuous discharge of Cr(VI)-containing wastewater from industries, such as electroplating, tanning or pigment, has already caused severe pollution of Cr(VI) in environment. Traditional physical and chemical strategies for dealing with Cr(VI)containing wastewater include reverse osmosis, ion exchange and reduction–precipitation. However, they have the disadvantages of high cost, recontamination, operational complexity and low efficiency (Ahluwalia and Goyal, 2007; Cheung and Gu, 2007; Demir and Arisoy, 2007). Bioremediation of Cr(VI)-containing wastewater with bacteria, algae and fungi has been regarded as low-cost, promising new technology (Rawlings et al., 2003; Prigione et al., 2009; Wang and Chen, 2009). Since the late 1970s, a large number of bacterial strains have been reported with the potential of reducing Cr(VI). Moreover, much attention has been paid to the Cr(VI) uptake mechanism in bacteria, which would provide crucial insights into the application ⇑ Corresponding authors. Address: Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, PR China (Z. Wu). Tel./fax: +86 591 83705445 (Z. Lin). E-mail addresses: [email protected] (Z. Lin), [email protected] (X. Guan), [email protected] (Z. Wu). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.12.050
of bioremediation. Currently, investigations on the interaction between Cr(VI)-reducing bacteria and Cr(VI) mainly focus on four aspects as follows: (i) The reduction mechanism. The enzymemediated reduction mechanism is regarded as the most common one by studying the reduction in several bacterial systems, such as Thermus scotoductus, Bacillus megaterium, Vibrio harveyi and Pseudomonas ambigua (Horitsu et al., 1987; Kwak et al., 2003; Cheung et al., 2006; Opperman and Van Heerden, 2007). (ii) The valence states and the related transformation process of chromium. The transformation process from Cr(VI) to Cr(III) in several Cr(VI)-reducing bacterial systems has been investigated, such as P. ambigua, Arthrobacter oxydans and Spirodela polyrhiza, revealing that prior to the formation of Cr(III) end product, Cr(IV) and Cr(V) can be the intermediate states (Suzuki et al., 1992; Appenroth et al., 2000; Kalabegishvili et al., 2003; Codd et al., 2006). (iii) The accumulation situation of Cr(III). By atomic force microscopy (AFM), scanning/transmission electron microscopy (SEM/ TEM) observations coupled with energy dispersive X-ray spectroscopy (EDS) analysis, it has been found that the accumulation of Cr(III) in bacteria can be both intracellularly (inside the cell membrane) or extracellularly (outside the cell membrane) (Daulton et al., 2002; Srivastava and Thakur, 2007; Yang et al., 2007; Li et al., 2008). (iv) The immobilization mechanism. More and more evidence shows that water soluble Cr(III) end product can be formed (Puzon et al., 2002, 2005; Bencheikh-Latmani et al., 2007;
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Li et al., 2008), instead of forming insoluble Cr(OH)3 precipitates directly (Barnhart, 1997). Thus, in order to more effectively remove Cr(III), particular attentions have been paid to study the preferential coordination structures of Cr(III). In several bacterial strains, such as Chlorella miniata, Ochrobactrum anthropi and Pseudomonas aeruginosa, amino and carboxyl groups may involve the coordination of Cr(III) (Han et al., 2007; Kang et al., 2007; Cheng et al., 2010). Based on the understanding of the Cr(VI)-microbe interaction mechanism, it was anticipated that suggestive strategies regarding both centralized treatment or on-site remediation of Cr(VI) could be proposed, not only for accelerating the Cr(VI) reduction ability, but also for enhancing the Cr(III) immobilization efficiency. However, due to the diversity and complexity of Cr(VI)-tolerant bacteria, the Cr(VI)-microbe interaction mechanism can be distinct in different bacterial systems. For example, our previous studies on two Gram-negative bacteria show that when interacting with Cr(VI), Enterobacter cloacae can prevent the permeability of Cr(VI) into the cell by forming compact convex patches on the cell surface, whereas O. anthropi can reduce Cr(VI) to Cr(III) and mostly accumulate Cr(III) on the cell surface (Yang et al., 2007; Cheng et al., 2010). Thus, the cell surface of living bacteria plays an important role in the Cr(III) immobilization process. Hence, it is necessary to investigate different bacteria from other classes to understand the common rules of the Cr immobilization mechanisms and test the feasibility of the bacterial strategy. Moreover, for other bacterial strains, systematic survey is still not well conducted on the transformation process and the accumulation situation of chromium during Cr(VI) uptake using microscopic methods and spectroscopic techniques. In the present work, since the cell surfaces of Gram-positive and Gram-negative bacteria are obviously different, we selected an indigenous non-pathogenic Gram-positive strain (Bacillus cereus) as the research object. Through a series of batch and microscopic experiments, dominant role of cell debris during Cr(III) immobilization was found. We also obtained a comprehensive view on the binding sites and valence states of chromium, the possible coordination situations and the key factors to affecting both Cr(VI) reduction and Cr(III) immobilization processes. We then discuss the role of the as-produced soluble organic molecules during bacterium cultivation for both accelerating Cr(VI) reduction and Cr(III) coordination. Our study provide novel insights into the development of feasible bioremediation strategies of Cr(VI)-containing wastewater. 2. Materials and methods 2.1. Bacteria The strain was isolated from a chromate slag heap in Pingnan, Fujian Province, China (26.894° N, 118.978° E), and it was identified as B. cereus based on its biochemical properties and 16S rDNA sequence homology analysis. The details could be found in Section 1.1 in Supplementary Material (SM). Bacteria were cultured in Luria Bertani (LB) liquid medium at 37 °C with shaking at 180 rpm. The composition of medium was as follows (g L 1): NaCl (10), tryptone (10), yeast extract (5), pH 7.2. 2.2. The Cr(VI)-reducing experiment The Cr(VI) stock solution was prepared by dissolving K2Cr2O7 (AR) in deionized-distilled water. The initial concentration of K2Cr2O7 ranged from 0 to 200 mg L 1 according to the different experimental design. All the Cr(VI)-reducing experiments were conducted in 5 g L 1 glucose solution (pH 7.2) at 37 °C.
Intact cells and supernatant were used for the Cr(VI)-reducing experiment. For the intact cells, B. cereus cells were cultured in LB medium overnight and harvested by centrifugation at 5000 g for 5 min. Then cell pellet was transferred into glucose solution (5 g L 1) to produce a final cell concentration of 0.01 g mL 1 (dry weight). Moreover, the supernatant was obtained in the absence of Cr(VI). Briefly, B. cereus cells were transferred into glucose solution (5 g L 1) and cultured at 37 °C for 24 h with shaking at 180 rpm. The supernatant was collected by centrifugation at 5000 g for 5 min, filtered through 0.22 lm filters and stored at 4 °C until use. 2.3. Valence state analysis of the reduced chromium Intact cells (0.01 g mL 1, dry weight) were reacted with 2 mM Cr(VI) (104 mg L 1) at 37 °C for 24 h. The cells were then centrifuged at 5000 g for 10 min and collected as ‘‘Cr-loaded bacteria’’. The supernatant was collected as ‘‘Cr-supernatant’’, and further valence state analysis was performed using extended X-ray absorption fine structure (EXAFS). The bacterial sample was washed three times with deionized-distilled water and dried at 60 °C for 6 h. The valence state of chromium on the bacteria was analyzed by X-ray photoelectron spectroscopy (XPS) using an ESCALAB MK II spectrometer (VG Scientific, UK). 2.4. Analysis of Cr(III) binding situation ‘‘Cr-loaded bacteria’’ and ‘‘Cr-supernatant’’ were analyzed by XAFS spectroscopy. XAFS measurements of chromium K-edge were performed at the BL14W1 beamline of Shanghai Synchrotron Radiation Facility, China. The typical energy of the storage ring was 3.5 GeV with a maximum current of 300 mA. A Si (1 1 1) double crystal monochromator and a harmonic rejection mirror were used to minimize the high harmonics content. Furthermore, these samples were also measured at the U7C beamline of the National Synchrotron Radiation Facility (NSRL), China. The typical energy of NSRL was 0.8 GeV with a maximum stored current of 250 mA. A Si (1 1 1) double crystal monochromator and high purity 7-element Ge array detectors were used. The absolute energy positions in the two beamlines were both calibrated using a chromium metal foil. Moreover, the chromium K-edge of K2Cr2O7, Cr2O3, Cr(OH)3 and Cr(Glycine)3H2O (Cr-Gly) were also recorded as the model compounds. Data analysis of all the experimental XAFS spectra was performed using WinXAS3.1 (Ressler, 1998). 2.5. Analysis of chromium distribution The quantitative analysis of the reduced Cr(III) was performed in our study. Briefly, intact cells (0.01 g mL 1, dry weight) were reacted with 2 mM Cr(VI) at 37 °C for 24 h. The supernatant was obtained from the cells by centrifugation at 7000 g for 5 min. The cell pellet was washed three times, re-suspended and then homogenized into cell debris and cytoplasm by sonication (200 W, 9 s pulses at 1 s intervals for 35 min). The chromium content in the supernatant and cytoplasm was detected by inductively coupled plasma optical emission spectroscopy (ICP–OES) (Ultima2). The total immobilization ratio (%) = (1 (total chromium in supernatant/initial Cr(VI) concentration)) 100%. Therefore, the cytoplasm immobilization ratio (%) = (chromium in cytoplasm/total immobilized chromium) 100%, whereas the cell debris immobilization ratio (%) = 1 cytoplasm immobilization ratio. Before AFM observation, the cell pellets were washed three times and re-suspended with 1 mM Tris–HCl buffer (pH 7.0). Samples were observed by AFM (Veeco Multimode NS3A-02 Nanoscope III) under the tapping-mode, and the cantilever of the tip is silicon. All experiments were conducted within 1 h. For
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SEM observation, the cell pellets were first fixed by 2% glutaraldehyde for 24 h, then washed three times with 0.1 mM sodium dimethylarsenate buffer for 3 min and finally post-fixed with 1% osmic acid for 2 h. Samples were washed three times with sodium dimethylarsenate (0.1 mM). Subsequently, they were dehydrated step by step with 35%, 50%, 70%, 80%, 95% and 100% acetone in deionized-distilled water for 3 min, respectively. SEM investigations were performed using a JEOL JSM-6700F SEM coupled with an EDS (Oxford). The preparation of TEM samples was as same as that of SEM samples from the harvest to dehydration steps, and then the bacterial cells were embedded in resin. Ultrathin sections of embedded bacteria were obtained using ultramicrotome (Leica EM UC6, Germany) and stained with uranyl acetate and lead citrate. Finally, the samples were observed using a JEM-2010 TEM coupled with an EDS (Oxford) system at 200 kV. For the chromium analysis, a colorimetric method of 1,5-diphenylcarbazide spectrophotometry (Urone, 1955) was used to analyze the Cr(VI) concentration in the supernatant with UV/Vis spectrometer (Perkin–Elmer Lamda 35, USA) at 540 nm. Before each measurement, the cell suspension was centrifuged at 20,000 g for 20 min (4 °C). Consequently, the Cr(VI) and water soluble Cr(III) remained in the supernatant. The Cr(VI) concentration could be directly detected with UV/Vis spectrometer method. The concentration of total chromium in the supernatant was determined after all the soluble chromium forms were oxidized into Cr(VI) by acidic potassium permanganate at high temperature.
3. Results 3.1. Reduction and immobilization situation of chromium Previous work has shown that the preferential coordination of Cr(III) to the soluble organic molecules in LB culture medium can inhibit an effective immobilization of Cr(III) on the O. anthropi cells. In glucose medium, the Cr(III) binding ability of saccharide is lower than that of the cells. Therefore, over 96% of the Cr(III) is effectively immobilized (Cheng et al., 2010). In this study, we investigated the capability of Cr(VI) uptake of B. cereus in LB medium (providing nitrogen source and carbon source) and glucose solution (only providing carbon source), respectively. Fig. 1 shows that 104 mg L 1 of Cr(VI) could be completely reduced within several hours in LB medium. However, the total Cr immobilization ratio of B. cereus was only around 25%. The results were consistent with the previous study (Cheng et al., 2010), suggesting that small soluble molecules, such as amino acids and their derivatives, or multi-carboxyl compounds in LB medium had stronger coordination ability with reduced Cr than B. cereus cells. Interestingly, when using glucose solution as the bacterial culture medium, where all the nitrogen sources were eliminated, the immobilization ratio of Cr was only about 70%. This implied that the existence of some soluble organic molecules in the supernatant might have a competitive coordination effect on the Cr immobilization. In fact, in the preliminary study, we found different results from B. cereus compared with other Cr(VI)-tolerant bacterial strains, such as O. anthropi, Planococcus citreus and E. cloacae, in previous studies (Yang et al., 2007; Cheng et al., 2010). When B. cereus cells were cultured in glucose solution, the colorless culture medium changed into light yellow in 24 h. The yellow supernatant, collected by centrifugation and filtered through 0.22 lm filters, was used for the Cr(VI)-reducing experiment. As shown in Fig. 1c, the Cr(VI) could be completely reduced by the supernatant in about 120 h, indicating that the supernatant of B. cereus also had strong ability to reduce Cr(VI). This suggested that the supernatant of B. cereus cells could affect the Cr(VI) reduction and Cr(III) immobilization process.
Fig. 1. Cr(VI) reduction and total chromium immobilization by living B. cereus (a) in LB medium; (b) in 5 g L 1 glucose solution. The original concentration of Cr(VI) was 104 mg L 1; (c) The reducing capability of intact cells and the supernatant of B. cereus cells on Cr(VI). The final cell concentration was 0.01 g mL 1 (dry weight).
3.2. The valence and binding state of chromium After incubating living B. cereus with 104 mg L 1 Cr(VI) in glucose for 24 h, we analyzed the valence state of chromium both with the bacteria and supernatant. The valence state of chromium on the bacteria was identified by XPS spectra. Since XPS is inappropriate for analyzing liquid samples, we determined the valence state of chromium in the supernatant using XAFS spectroscopy. Fig. 2a shows that two distinct peaks were obtained from the Cr-loaded bacteria sample at 585.0–588.0 eV (Cr 2p1/2) and 576.0–578.0 eV (Cr 2p3/2), respectively. It was consistent with the spectrum of Cr2O3 (Li et al., 2008), showing that the chromium immobilized by B. cereus was at Cr(III) state. Fig. 2b shows that, via comparing with the standard Cr(III) and Cr(VI) compounds, the X-ray absorption near-edge structure (XANES) of the Cr-supernatant sample exhibited a typical feature of Cr(III), whereas no representative peaks of Cr(VI) were found. Therefore, the XPS and XAFS analyses revealed that Cr(VI) was completely transformed into Cr(III) by living B. cereus.
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peak at 1731.6 cm 1 was transferred to higher frequency, whereas the peak at 1458.3 cm 1 moved to lower frequency. This indicated that Cr(III) could possibly be coordinated with carbonyl and amino. Fig. SM-1b shows the functional groups of supernatant. We found that the supernatant collected from B. cereus cultivation contained amino and carboxyl groups similar to those of cell debris. Therefore, it could chemically involve in the coordination of Cr(III) (the details could be found in Section 2 in SM). 3.3. Cr(III) distribution
Fig. 2. Spectroscopic analyses of the valence and binding state of chromium. (a) XPS spectra of Cr-loaded bacteria, Cr2O3 and K2Cr2O7; (b) XANES signal of Cr-supernatant, Cr-loaded bacteria and standards; (c) EXAFS signal of samples and standards. Cr-Gly represents Cr(Glycine)3H2O.
We quantitatively analyzed the Cr(III) distribution in above systems. Table 1 shows that the soluble chromium in the supernatant was about 23 mg L 1, indicating only 78% of the reduced Cr(III) was immobilized by the bacteria. ICP–OES analysis revealed that the chromium in the cytoplasm was about 26.5 mg L 1. Therefore, the binding ratio of Cr(III) by the supernatant, cell debris and cytoplasm was 22%, 54% and 24%, respectively. This revealed that cell debris (including cell wall and some insoluble bio-macromolecules) was the dominant site of Cr(III) immobilization. In our present study, we performed the SEM, AFM and TEM microscopic investigations to elucidate the morphologic changes and detailed distribution situation of Cr in living B. cereus cells. SEM observation revealed that the intact B. cereus cells exhibited plump and smooth surfaces (Fig. 3a). After the treatment of 104 mg L 1 Cr(VI) for 24 h, the bacterial surfaces became rough (Fig. 3b). EDS spectra confirmed that a certain amount of chromium was accumulated on the bacterial surfaces (Fig. 3d). AFM could more directly and realistically image the characteristic of bacterial surfaces due to its simpler sample preparation process. Similar to SEM observation, we found that the intact bacteria exhibited long-rod shapes and smooth surfaces (Fig. 3e). After the treatment of Cr(VI) for 24 h, the bacterial surfaces became rough (Fig. 3f). Via investigating an area of 500 nm 500 nm from 10–15 bacteria, we found that the numerical estimates of rootmean-square roughness were increased from 5.6 ± 0.5 to 17.8 ± 3.4 nm after Cr(VI) treatment. These data were consistent with the results of SEM observations. To investigate the changes inside the bacteria, bacteria with ultra-thin section were further observed by TEM. Fig. 3g shows that the section shape of the intact bacteria was round or oval according to the different section angles. The cell wall could be clearly observed. After the treatment of Cr(VI) for 24 h, the bacterial edges became irregular or rough (Fig. 3h). However, no obvious chromium precipitates around intracellular membrane region were observed as shown in B. megaterium strain (Cheung et al., 2006). EDS analysis revealed that chromium signal could be detected from the inside of bacteria (Fig. 3j), indicating that the chromium could be evenly distributed in the cytoplasm. 4. Discussion
In order to determine the coordination situation of Cr(III) with cells and supernatant of B. cereus cultivation, we analyzed the Crloaded bacteria and Cr-supernatant samples using XAFS spectroscopy. Fig. 2c shows the corresponding EXAFS signals of all the spectra. We found that the Cr-loaded bacteria and Cr-supernatant samples exhibited the similar oscillation peaks, which were very similar to those of Cr-Gly. This indicated that the chromium in the supernatant and in the bacteria probably existed with the six O/N coordination style as Cr-Gly (Cheng et al., 2010). In order to clarify the active functional groups coordinating with Cr(III), we performed fourier transform infrared (FT-IR) analysis in the living cell as well as in the supernatant. Fig. SM-1a shows the FT-IR spectra of the intact and Cr(VI)-loaded bacteria, and Table SM-2 summarizes the corresponding absorption bands. After interacted with Cr(VI), the
Bacterial strategy is a new idea for reducing highly toxic Cr(VI) and recycle Cr(III) on the remediation of Cr(VI) industrial wastes. The ideal Cr(VI)-reducing bacteria can not only reduce Cr(VI) into less toxic Cr(III), but also completely immobilize the reduced Cr(III). Therefore, water soluble Cr(III) end products are not generated. However, it is the preferential coordination of Cr(III) to the soluble amino acids in the bacterial culture medium that inhibits an effective immobilization of Cr(III) on O. anthropi and P. citreus cells. Based on this notion, in order to avoid the nitrogen source during Cr(VI) treatment, we proposed a strategy with two-step medium control, which achieved the successful immobilization of Cr(VI) as Cr(III) in the centralized treatment of Cr(VI) (Cheng et al., 2010). Moreover, caution should be paid to the bacterial sys-
Z. Chen et al. / Chemosphere 87 (2012) 211–216 Table 1 Distribution of Cr(III) after interacting with Cr(VI) in glucose solution for 24 h. Initial Cr(VI) (mg L 1)
106.9 ± 1.4
Soluble chromium (mg L 1)
The immobilized chromium Chromium on cell debris (mg L 1)
Chromium in cytoplasm (mg L
23.0 ± 1.6
57.4 ± 0.3
26.5 ± 0.7
1
)
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wastewater with bacteria. We speculated that the active components of the supernatant might be bacterium-origin soluble molecules from extracellular secretions, metabolic end products or the released cytoplasm of B. cereus. Although these bacterium-origin soluble molecules in the supernatant could affect Cr(III) immobilization in bacteria in the centralized Cr(VI) treatment, it cannot be simply regarded as one disadvantage of the Cr(VI)-reducing bacteria for the following reasons. First, these bacterium-origin soluble molecules are very complex macromolecules, so they could have migration properties in the contaminated soils totally different from those small organic ligands. Indeed, many studies have reported the interactions between extracellular secretion of microorganisms and heavy metal ions (Salehizadeh and Shojaosadati, 2003). They diffuse in the surrounding of microorganisms and play an important role in the sequestration and biomineralization process of Fe, Mn and Au (Ghiorse, 1984; Frankel and Bazylinski, 2003; Slocik and Wright, 2003; Bazylinski et al., 2007). These components arrest dissolved metallic cations through chelating coordination or electrostatic attraction using their negative-charge multi-organic functional groups, such as amino acids, the derivatives of amino acids or multi-carboxyl compounds, and then they mediate metal fixation on clay particles in soils (Corzo et al., 1994) or gradually deposit during the long and complicate geological processes. Second, for B. cereus-Cr(VI) system, the soluble Cr(III) in the supernatant totally disappeared after about two months in the test tube, implying that the soluble Cr(III) with high molecular weight could be either simply precipitated or further immobilized by bacteria via ligand-exchange. Although more efforts are required, our results suggested that the deposition of Cr(III)-supernatant was not as difficult as other Cr(III) complex with small molecular weights. The coordination of these bacterium-origin soluble molecules with Cr(III) in the supernatant could finally facilitate the immobilization of Cr(III). Last but not the least, the bacterium-origin soluble molecules in the supernatant increases the Cr(VI) tolerant ability of the bacteria. It cannot only reduce a considerable amount of Cr(VI) before this toxic species penetrates into the cell membrane, but also directly decrease the enrichment of Cr(III) in the bacteria. This would be helpful for the survival and continuous propagation of Cr(VI)reducing bacteria in the Cr(VI) contaminated environment. 5. Conclusions
Fig. 3. Microscopic investigations of B. cereus. (a and c) SEM and corresponding EDS spectrum of intact bacteria; (e) AFM amplitude image of intact bacteria; (g and i) TEM and corresponding EDS spectrum of intact bacteria (white circular portion); (b and d) SEM and corresponding EDS spectrum of Cr-loaded bacteria; (f) AFM amplitude image of Cr-loaded bacteria; (h and j) TEM and corresponding EDS spectrum of Cr-loaded bacteria (white circular portion); Pb and U were from the chemical agents and used for staining the cells.
tems. We found that even when the culture medium was strictly restricted to carbon source, the reduced Cr(III) still could not be effectively immobilized by B. cereus. Moreover, we found the supernatant collected from B. cereus cultivation could reduce Cr(VI) to Cr(III) and coordinate with the reduced Cr(III) through carboxyl and amido functional groups. The formation of soluble organico– metal complexes leads to the decrease of the immobilizing efficiency of Cr(III) during the treatment of Cr(VI) contaminated
In this study, we investigated the Cr(VI)-microbe interaction of a Cr(VI)-tolerance bacterial strain-B. cereus. For the first time, we found the supernatant collected from B. cereus cultivation is the active components for reducing Cr(VI) and coordinating Cr(III). The reduced Cr(III) could coordinate with the supernatant, cell debris and cytoplasm through carboxyl and amido functional groups. Acknowledgments This study was supported by the National Basic Research Program of China (973 Program) (No. 2007CB815601, 2010CB933 501), National Natural Science Foundation of China (40902097, 20803082, 40772034, 31071745), the Knowledge Innovation Project of the Chinese Academy of Sciences (KJCX2.YW.W01), the Outstanding Youth Fund (10125523, 21125730). YIF (2007F3120) of Fujian Province, the National Natural Science Foundation for Distinguished Young Scholars (40902097), Ministry of Education and the Specialized Research Fund for the Doctoral Program of Higher Education (20093515110010), the National High Technology Research and Development Program (‘‘863’’Program) of China (2011AA10A203).
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Horitsu, H., Futo, S., Miyazawa, Y., Ogai, S., Kawai, K., 1987. Enzymatic reduction of hexavalent chromium by hexavalent chromium tolerant Pseudomonas ambigua G-1. Agric. Biol. Chem. 51, 2417–2420. Kalabegishvili, T.L., Tsibakhashvili, N.Y., Holman, H., 2003. Electron spin resonance study of chromium(V) formation and decomposition by basalt-inhabiting bacteria. Environ. Sci. Technol. 37, 4678–4684. Kang, S.Y., Lee, J.U., Kim, K.W., 2007. Biosorption of Cr(III) and Cr(VI) onto the cell surface of Pseudomonas aeruginosa. Biochem. Eng. J. 36, 54–58. Kwak, Y.H., Lee, D.S., Kim, H.B., 2003. Vibrio harveyi nitroreductase is also a chromate reductase. Appl. Environ. Microb. 69, 4390–4395. Li, B., Pan, D.M., Zheng, J.S., Cheng, Y.J., Ma, X.Y., Huang, F., Lin, Z., 2008. Microscopic investigations of the Cr(VI) uptake mechanism of living Ochrobactrum anthropi. Langmuir 24, 9630–9635. Opperman, D.J., Van Heerden, E., 2007. Aerobic Cr(VI) reduction by Thermus scotoductus strain SA-01. J. Appl. Microbiol. 103, 1907–1913. Prigione, V., Zerlottin, M., Refosco, D., Tigini, V., Anastasi, A., Varese, G.C., 2009. Chromium removal from a real tanning effluent by autochthonous and allochthonous fungi. Bioresource Technol. 100, 2770–2776. Puzon, G.J., Petersen, J.N., Roberts, A.G., Kramer, D.M., Xun, L.Y., 2002. A bacterial flavin reductase system reduces chromate to a soluble chromium(III)-NAD+ complex. Biochem. Bioph. Res. Co. 294, 76–81. Puzon, G.J., Roberts, A.G., Kramer, D.M., Xun, L.Y., 2005. Formation of soluble organo-chromium(III) complexes after chromate reduction in the presence of cellular organics. Environ. Sci. Technol. 39, 2811–2817. Rawlings, D.E., Dew, D., Du Plessis, C., 2003. Biomineralization of metal-containing ores and concentrates. Trends Biotechnol. 21, 38–44. Ressler, T., 1998. WinXAS: a program for X-ray absorption spectroscopy data analysis under MS-Windows. J. Synchrotron. Radiat. 5, 118–122. Salehizadeh, H., Shojaosadati, S.A., 2003. Removal of metal ions from aqueous solution by polysaccharide produced from Bacillus firmus. Water Res. 37, 4231– 4235. Slocik, J.M., Wright, D.W., 2003. Biomimetic mineralization of noble metal nanoclusters. Biomacromolecules 4, 1135–1141. Srivastava, S., Thakur, I.S., 2007. Evaluation of biosorption potency of Acinetobacter sp for removal of hexavalent chromium from tannery effluent. Biodegradation 18, 637–646. Suzuki, T., Miyata, N., Horitsu, H., Kawai, K., Takamizawa, K., Tai, Y., Okazaki, M., 1992. NAD(P)H-dependent chromium(VI) reductase of Pseudomonas ambigua G1 – a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III). J. Bacteriol. 174, 5340–5345. Urone, P.F., 1955. Stability of colorimetric reagent for chromium, sdiphenylcarbazide, in various solvents. Anal. Chem. 27, 1354–1355. Wang, J.L., Chen, C., 2009. Biosorbents for heavy metals removal and their future. Biotechnol. Adv. 27, 195–226. Yang, C.P., Cheng, Y.J., Ma, X.Y., Zhu, Y., Holman, H.Y., Lin, Z., Wang, C., 2007. Surfacemediated chromate-resistant mechanism of Enterobacter cloacae bacteria investigated by atomic force microscopy. Langmuir 23, 4480–4485.
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Cr(VI) uptake mechanism of Bacillus cereus Zhi Chen a,b, Zhipeng Huang b, Yangjian Cheng a, Danmei Pan a, Xiaohong Pan a,b, Meijuan Yu c, Zhiyun Pan d, Zhang Lin a,⇑, Xiong Guan b,⇑, Ziyu Wu c,d,⇑ a
State Key Lab of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, PR China Key Lab of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Ministry of Education, Fuzhou, Fujian 350002, PR China c Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, PR China d National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029, PR China b
a r t i c l e
i n f o
Article history: Received 6 July 2011 Received in revised form 19 December 2011 Accepted 19 December 2011 Available online 4 January 2012 Keywords: Chromium Reduction Immobilization Valence states Microscopy Bacillus cereus
a b s t r a c t In this study, we investigated the Cr(VI) uptake mechanism in an indigenous Cr(VI)-tolerant bacterial strain – Bacillus cereus through batch and microscopic experiments. We found that both the cells and the supernatant collected from B. cereus cultivation could reduce Cr(VI). The valence state analysis revealed the complete transformation from Cr(VI) into Cr(III) by living B. cereus. Further X-ray absorption fine structure and Fourier transform infrared analyses showed that the reduced Cr(III) was coordinated with carboxyl and amido functional groups from either the cells or supernatant. Scanning electron microscopy and atomic force microscopy observation showed that noticeable Cr(III) precipitates were accumulated on bacterial surfaces. However, Cr(III) could also be detected in bacterial inner portions by using transmission electron microscopy thin section analysis coupled with energy dispersive X-ray spectroscopy. Through quantitative analysis of chromium distribution, we determined the binding ratio of Cr(III) in supernatant, cell debris and cytoplasm as 22%, 54% and 24%, respectively. Finally, we further discussed the role of bacterium-origin soluble organic molecules to the remediation of Cr(VI) pollutants. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The Cr(VI) compounds are extremely toxic to human, animals and plants. The continuous discharge of Cr(VI)-containing wastewater from industries, such as electroplating, tanning or pigment, has already caused severe pollution of Cr(VI) in environment. Traditional physical and chemical strategies for dealing with Cr(VI)containing wastewater include reverse osmosis, ion exchange and reduction–precipitation. However, they have the disadvantages of high cost, recontamination, operational complexity and low efficiency (Ahluwalia and Goyal, 2007; Cheung and Gu, 2007; Demir and Arisoy, 2007). Bioremediation of Cr(VI)-containing wastewater with bacteria, algae and fungi has been regarded as low-cost, promising new technology (Rawlings et al., 2003; Prigione et al., 2009; Wang and Chen, 2009). Since the late 1970s, a large number of bacterial strains have been reported with the potential of reducing Cr(VI). Moreover, much attention has been paid to the Cr(VI) uptake mechanism in bacteria, which would provide crucial insights into the application ⇑ Corresponding authors. Address: Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, PR China (Z. Wu). Tel./fax: +86 591 83705445 (Z. Lin). E-mail addresses: [email protected] (Z. Lin), [email protected] (X. Guan), [email protected] (Z. Wu). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.12.050
of bioremediation. Currently, investigations on the interaction between Cr(VI)-reducing bacteria and Cr(VI) mainly focus on four aspects as follows: (i) The reduction mechanism. The enzymemediated reduction mechanism is regarded as the most common one by studying the reduction in several bacterial systems, such as Thermus scotoductus, Bacillus megaterium, Vibrio harveyi and Pseudomonas ambigua (Horitsu et al., 1987; Kwak et al., 2003; Cheung et al., 2006; Opperman and Van Heerden, 2007). (ii) The valence states and the related transformation process of chromium. The transformation process from Cr(VI) to Cr(III) in several Cr(VI)-reducing bacterial systems has been investigated, such as P. ambigua, Arthrobacter oxydans and Spirodela polyrhiza, revealing that prior to the formation of Cr(III) end product, Cr(IV) and Cr(V) can be the intermediate states (Suzuki et al., 1992; Appenroth et al., 2000; Kalabegishvili et al., 2003; Codd et al., 2006). (iii) The accumulation situation of Cr(III). By atomic force microscopy (AFM), scanning/transmission electron microscopy (SEM/ TEM) observations coupled with energy dispersive X-ray spectroscopy (EDS) analysis, it has been found that the accumulation of Cr(III) in bacteria can be both intracellularly (inside the cell membrane) or extracellularly (outside the cell membrane) (Daulton et al., 2002; Srivastava and Thakur, 2007; Yang et al., 2007; Li et al., 2008). (iv) The immobilization mechanism. More and more evidence shows that water soluble Cr(III) end product can be formed (Puzon et al., 2002, 2005; Bencheikh-Latmani et al., 2007;
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Li et al., 2008), instead of forming insoluble Cr(OH)3 precipitates directly (Barnhart, 1997). Thus, in order to more effectively remove Cr(III), particular attentions have been paid to study the preferential coordination structures of Cr(III). In several bacterial strains, such as Chlorella miniata, Ochrobactrum anthropi and Pseudomonas aeruginosa, amino and carboxyl groups may involve the coordination of Cr(III) (Han et al., 2007; Kang et al., 2007; Cheng et al., 2010). Based on the understanding of the Cr(VI)-microbe interaction mechanism, it was anticipated that suggestive strategies regarding both centralized treatment or on-site remediation of Cr(VI) could be proposed, not only for accelerating the Cr(VI) reduction ability, but also for enhancing the Cr(III) immobilization efficiency. However, due to the diversity and complexity of Cr(VI)-tolerant bacteria, the Cr(VI)-microbe interaction mechanism can be distinct in different bacterial systems. For example, our previous studies on two Gram-negative bacteria show that when interacting with Cr(VI), Enterobacter cloacae can prevent the permeability of Cr(VI) into the cell by forming compact convex patches on the cell surface, whereas O. anthropi can reduce Cr(VI) to Cr(III) and mostly accumulate Cr(III) on the cell surface (Yang et al., 2007; Cheng et al., 2010). Thus, the cell surface of living bacteria plays an important role in the Cr(III) immobilization process. Hence, it is necessary to investigate different bacteria from other classes to understand the common rules of the Cr immobilization mechanisms and test the feasibility of the bacterial strategy. Moreover, for other bacterial strains, systematic survey is still not well conducted on the transformation process and the accumulation situation of chromium during Cr(VI) uptake using microscopic methods and spectroscopic techniques. In the present work, since the cell surfaces of Gram-positive and Gram-negative bacteria are obviously different, we selected an indigenous non-pathogenic Gram-positive strain (Bacillus cereus) as the research object. Through a series of batch and microscopic experiments, dominant role of cell debris during Cr(III) immobilization was found. We also obtained a comprehensive view on the binding sites and valence states of chromium, the possible coordination situations and the key factors to affecting both Cr(VI) reduction and Cr(III) immobilization processes. We then discuss the role of the as-produced soluble organic molecules during bacterium cultivation for both accelerating Cr(VI) reduction and Cr(III) coordination. Our study provide novel insights into the development of feasible bioremediation strategies of Cr(VI)-containing wastewater. 2. Materials and methods 2.1. Bacteria The strain was isolated from a chromate slag heap in Pingnan, Fujian Province, China (26.894° N, 118.978° E), and it was identified as B. cereus based on its biochemical properties and 16S rDNA sequence homology analysis. The details could be found in Section 1.1 in Supplementary Material (SM). Bacteria were cultured in Luria Bertani (LB) liquid medium at 37 °C with shaking at 180 rpm. The composition of medium was as follows (g L 1): NaCl (10), tryptone (10), yeast extract (5), pH 7.2. 2.2. The Cr(VI)-reducing experiment The Cr(VI) stock solution was prepared by dissolving K2Cr2O7 (AR) in deionized-distilled water. The initial concentration of K2Cr2O7 ranged from 0 to 200 mg L 1 according to the different experimental design. All the Cr(VI)-reducing experiments were conducted in 5 g L 1 glucose solution (pH 7.2) at 37 °C.
Intact cells and supernatant were used for the Cr(VI)-reducing experiment. For the intact cells, B. cereus cells were cultured in LB medium overnight and harvested by centrifugation at 5000 g for 5 min. Then cell pellet was transferred into glucose solution (5 g L 1) to produce a final cell concentration of 0.01 g mL 1 (dry weight). Moreover, the supernatant was obtained in the absence of Cr(VI). Briefly, B. cereus cells were transferred into glucose solution (5 g L 1) and cultured at 37 °C for 24 h with shaking at 180 rpm. The supernatant was collected by centrifugation at 5000 g for 5 min, filtered through 0.22 lm filters and stored at 4 °C until use. 2.3. Valence state analysis of the reduced chromium Intact cells (0.01 g mL 1, dry weight) were reacted with 2 mM Cr(VI) (104 mg L 1) at 37 °C for 24 h. The cells were then centrifuged at 5000 g for 10 min and collected as ‘‘Cr-loaded bacteria’’. The supernatant was collected as ‘‘Cr-supernatant’’, and further valence state analysis was performed using extended X-ray absorption fine structure (EXAFS). The bacterial sample was washed three times with deionized-distilled water and dried at 60 °C for 6 h. The valence state of chromium on the bacteria was analyzed by X-ray photoelectron spectroscopy (XPS) using an ESCALAB MK II spectrometer (VG Scientific, UK). 2.4. Analysis of Cr(III) binding situation ‘‘Cr-loaded bacteria’’ and ‘‘Cr-supernatant’’ were analyzed by XAFS spectroscopy. XAFS measurements of chromium K-edge were performed at the BL14W1 beamline of Shanghai Synchrotron Radiation Facility, China. The typical energy of the storage ring was 3.5 GeV with a maximum current of 300 mA. A Si (1 1 1) double crystal monochromator and a harmonic rejection mirror were used to minimize the high harmonics content. Furthermore, these samples were also measured at the U7C beamline of the National Synchrotron Radiation Facility (NSRL), China. The typical energy of NSRL was 0.8 GeV with a maximum stored current of 250 mA. A Si (1 1 1) double crystal monochromator and high purity 7-element Ge array detectors were used. The absolute energy positions in the two beamlines were both calibrated using a chromium metal foil. Moreover, the chromium K-edge of K2Cr2O7, Cr2O3, Cr(OH)3 and Cr(Glycine)3H2O (Cr-Gly) were also recorded as the model compounds. Data analysis of all the experimental XAFS spectra was performed using WinXAS3.1 (Ressler, 1998). 2.5. Analysis of chromium distribution The quantitative analysis of the reduced Cr(III) was performed in our study. Briefly, intact cells (0.01 g mL 1, dry weight) were reacted with 2 mM Cr(VI) at 37 °C for 24 h. The supernatant was obtained from the cells by centrifugation at 7000 g for 5 min. The cell pellet was washed three times, re-suspended and then homogenized into cell debris and cytoplasm by sonication (200 W, 9 s pulses at 1 s intervals for 35 min). The chromium content in the supernatant and cytoplasm was detected by inductively coupled plasma optical emission spectroscopy (ICP–OES) (Ultima2). The total immobilization ratio (%) = (1 (total chromium in supernatant/initial Cr(VI) concentration)) 100%. Therefore, the cytoplasm immobilization ratio (%) = (chromium in cytoplasm/total immobilized chromium) 100%, whereas the cell debris immobilization ratio (%) = 1 cytoplasm immobilization ratio. Before AFM observation, the cell pellets were washed three times and re-suspended with 1 mM Tris–HCl buffer (pH 7.0). Samples were observed by AFM (Veeco Multimode NS3A-02 Nanoscope III) under the tapping-mode, and the cantilever of the tip is silicon. All experiments were conducted within 1 h. For
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SEM observation, the cell pellets were first fixed by 2% glutaraldehyde for 24 h, then washed three times with 0.1 mM sodium dimethylarsenate buffer for 3 min and finally post-fixed with 1% osmic acid for 2 h. Samples were washed three times with sodium dimethylarsenate (0.1 mM). Subsequently, they were dehydrated step by step with 35%, 50%, 70%, 80%, 95% and 100% acetone in deionized-distilled water for 3 min, respectively. SEM investigations were performed using a JEOL JSM-6700F SEM coupled with an EDS (Oxford). The preparation of TEM samples was as same as that of SEM samples from the harvest to dehydration steps, and then the bacterial cells were embedded in resin. Ultrathin sections of embedded bacteria were obtained using ultramicrotome (Leica EM UC6, Germany) and stained with uranyl acetate and lead citrate. Finally, the samples were observed using a JEM-2010 TEM coupled with an EDS (Oxford) system at 200 kV. For the chromium analysis, a colorimetric method of 1,5-diphenylcarbazide spectrophotometry (Urone, 1955) was used to analyze the Cr(VI) concentration in the supernatant with UV/Vis spectrometer (Perkin–Elmer Lamda 35, USA) at 540 nm. Before each measurement, the cell suspension was centrifuged at 20,000 g for 20 min (4 °C). Consequently, the Cr(VI) and water soluble Cr(III) remained in the supernatant. The Cr(VI) concentration could be directly detected with UV/Vis spectrometer method. The concentration of total chromium in the supernatant was determined after all the soluble chromium forms were oxidized into Cr(VI) by acidic potassium permanganate at high temperature.
3. Results 3.1. Reduction and immobilization situation of chromium Previous work has shown that the preferential coordination of Cr(III) to the soluble organic molecules in LB culture medium can inhibit an effective immobilization of Cr(III) on the O. anthropi cells. In glucose medium, the Cr(III) binding ability of saccharide is lower than that of the cells. Therefore, over 96% of the Cr(III) is effectively immobilized (Cheng et al., 2010). In this study, we investigated the capability of Cr(VI) uptake of B. cereus in LB medium (providing nitrogen source and carbon source) and glucose solution (only providing carbon source), respectively. Fig. 1 shows that 104 mg L 1 of Cr(VI) could be completely reduced within several hours in LB medium. However, the total Cr immobilization ratio of B. cereus was only around 25%. The results were consistent with the previous study (Cheng et al., 2010), suggesting that small soluble molecules, such as amino acids and their derivatives, or multi-carboxyl compounds in LB medium had stronger coordination ability with reduced Cr than B. cereus cells. Interestingly, when using glucose solution as the bacterial culture medium, where all the nitrogen sources were eliminated, the immobilization ratio of Cr was only about 70%. This implied that the existence of some soluble organic molecules in the supernatant might have a competitive coordination effect on the Cr immobilization. In fact, in the preliminary study, we found different results from B. cereus compared with other Cr(VI)-tolerant bacterial strains, such as O. anthropi, Planococcus citreus and E. cloacae, in previous studies (Yang et al., 2007; Cheng et al., 2010). When B. cereus cells were cultured in glucose solution, the colorless culture medium changed into light yellow in 24 h. The yellow supernatant, collected by centrifugation and filtered through 0.22 lm filters, was used for the Cr(VI)-reducing experiment. As shown in Fig. 1c, the Cr(VI) could be completely reduced by the supernatant in about 120 h, indicating that the supernatant of B. cereus also had strong ability to reduce Cr(VI). This suggested that the supernatant of B. cereus cells could affect the Cr(VI) reduction and Cr(III) immobilization process.
Fig. 1. Cr(VI) reduction and total chromium immobilization by living B. cereus (a) in LB medium; (b) in 5 g L 1 glucose solution. The original concentration of Cr(VI) was 104 mg L 1; (c) The reducing capability of intact cells and the supernatant of B. cereus cells on Cr(VI). The final cell concentration was 0.01 g mL 1 (dry weight).
3.2. The valence and binding state of chromium After incubating living B. cereus with 104 mg L 1 Cr(VI) in glucose for 24 h, we analyzed the valence state of chromium both with the bacteria and supernatant. The valence state of chromium on the bacteria was identified by XPS spectra. Since XPS is inappropriate for analyzing liquid samples, we determined the valence state of chromium in the supernatant using XAFS spectroscopy. Fig. 2a shows that two distinct peaks were obtained from the Cr-loaded bacteria sample at 585.0–588.0 eV (Cr 2p1/2) and 576.0–578.0 eV (Cr 2p3/2), respectively. It was consistent with the spectrum of Cr2O3 (Li et al., 2008), showing that the chromium immobilized by B. cereus was at Cr(III) state. Fig. 2b shows that, via comparing with the standard Cr(III) and Cr(VI) compounds, the X-ray absorption near-edge structure (XANES) of the Cr-supernatant sample exhibited a typical feature of Cr(III), whereas no representative peaks of Cr(VI) were found. Therefore, the XPS and XAFS analyses revealed that Cr(VI) was completely transformed into Cr(III) by living B. cereus.
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peak at 1731.6 cm 1 was transferred to higher frequency, whereas the peak at 1458.3 cm 1 moved to lower frequency. This indicated that Cr(III) could possibly be coordinated with carbonyl and amino. Fig. SM-1b shows the functional groups of supernatant. We found that the supernatant collected from B. cereus cultivation contained amino and carboxyl groups similar to those of cell debris. Therefore, it could chemically involve in the coordination of Cr(III) (the details could be found in Section 2 in SM). 3.3. Cr(III) distribution
Fig. 2. Spectroscopic analyses of the valence and binding state of chromium. (a) XPS spectra of Cr-loaded bacteria, Cr2O3 and K2Cr2O7; (b) XANES signal of Cr-supernatant, Cr-loaded bacteria and standards; (c) EXAFS signal of samples and standards. Cr-Gly represents Cr(Glycine)3H2O.
We quantitatively analyzed the Cr(III) distribution in above systems. Table 1 shows that the soluble chromium in the supernatant was about 23 mg L 1, indicating only 78% of the reduced Cr(III) was immobilized by the bacteria. ICP–OES analysis revealed that the chromium in the cytoplasm was about 26.5 mg L 1. Therefore, the binding ratio of Cr(III) by the supernatant, cell debris and cytoplasm was 22%, 54% and 24%, respectively. This revealed that cell debris (including cell wall and some insoluble bio-macromolecules) was the dominant site of Cr(III) immobilization. In our present study, we performed the SEM, AFM and TEM microscopic investigations to elucidate the morphologic changes and detailed distribution situation of Cr in living B. cereus cells. SEM observation revealed that the intact B. cereus cells exhibited plump and smooth surfaces (Fig. 3a). After the treatment of 104 mg L 1 Cr(VI) for 24 h, the bacterial surfaces became rough (Fig. 3b). EDS spectra confirmed that a certain amount of chromium was accumulated on the bacterial surfaces (Fig. 3d). AFM could more directly and realistically image the characteristic of bacterial surfaces due to its simpler sample preparation process. Similar to SEM observation, we found that the intact bacteria exhibited long-rod shapes and smooth surfaces (Fig. 3e). After the treatment of Cr(VI) for 24 h, the bacterial surfaces became rough (Fig. 3f). Via investigating an area of 500 nm 500 nm from 10–15 bacteria, we found that the numerical estimates of rootmean-square roughness were increased from 5.6 ± 0.5 to 17.8 ± 3.4 nm after Cr(VI) treatment. These data were consistent with the results of SEM observations. To investigate the changes inside the bacteria, bacteria with ultra-thin section were further observed by TEM. Fig. 3g shows that the section shape of the intact bacteria was round or oval according to the different section angles. The cell wall could be clearly observed. After the treatment of Cr(VI) for 24 h, the bacterial edges became irregular or rough (Fig. 3h). However, no obvious chromium precipitates around intracellular membrane region were observed as shown in B. megaterium strain (Cheung et al., 2006). EDS analysis revealed that chromium signal could be detected from the inside of bacteria (Fig. 3j), indicating that the chromium could be evenly distributed in the cytoplasm. 4. Discussion
In order to determine the coordination situation of Cr(III) with cells and supernatant of B. cereus cultivation, we analyzed the Crloaded bacteria and Cr-supernatant samples using XAFS spectroscopy. Fig. 2c shows the corresponding EXAFS signals of all the spectra. We found that the Cr-loaded bacteria and Cr-supernatant samples exhibited the similar oscillation peaks, which were very similar to those of Cr-Gly. This indicated that the chromium in the supernatant and in the bacteria probably existed with the six O/N coordination style as Cr-Gly (Cheng et al., 2010). In order to clarify the active functional groups coordinating with Cr(III), we performed fourier transform infrared (FT-IR) analysis in the living cell as well as in the supernatant. Fig. SM-1a shows the FT-IR spectra of the intact and Cr(VI)-loaded bacteria, and Table SM-2 summarizes the corresponding absorption bands. After interacted with Cr(VI), the
Bacterial strategy is a new idea for reducing highly toxic Cr(VI) and recycle Cr(III) on the remediation of Cr(VI) industrial wastes. The ideal Cr(VI)-reducing bacteria can not only reduce Cr(VI) into less toxic Cr(III), but also completely immobilize the reduced Cr(III). Therefore, water soluble Cr(III) end products are not generated. However, it is the preferential coordination of Cr(III) to the soluble amino acids in the bacterial culture medium that inhibits an effective immobilization of Cr(III) on O. anthropi and P. citreus cells. Based on this notion, in order to avoid the nitrogen source during Cr(VI) treatment, we proposed a strategy with two-step medium control, which achieved the successful immobilization of Cr(VI) as Cr(III) in the centralized treatment of Cr(VI) (Cheng et al., 2010). Moreover, caution should be paid to the bacterial sys-
Z. Chen et al. / Chemosphere 87 (2012) 211–216 Table 1 Distribution of Cr(III) after interacting with Cr(VI) in glucose solution for 24 h. Initial Cr(VI) (mg L 1)
106.9 ± 1.4
Soluble chromium (mg L 1)
The immobilized chromium Chromium on cell debris (mg L 1)
Chromium in cytoplasm (mg L
23.0 ± 1.6
57.4 ± 0.3
26.5 ± 0.7
1
)
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wastewater with bacteria. We speculated that the active components of the supernatant might be bacterium-origin soluble molecules from extracellular secretions, metabolic end products or the released cytoplasm of B. cereus. Although these bacterium-origin soluble molecules in the supernatant could affect Cr(III) immobilization in bacteria in the centralized Cr(VI) treatment, it cannot be simply regarded as one disadvantage of the Cr(VI)-reducing bacteria for the following reasons. First, these bacterium-origin soluble molecules are very complex macromolecules, so they could have migration properties in the contaminated soils totally different from those small organic ligands. Indeed, many studies have reported the interactions between extracellular secretion of microorganisms and heavy metal ions (Salehizadeh and Shojaosadati, 2003). They diffuse in the surrounding of microorganisms and play an important role in the sequestration and biomineralization process of Fe, Mn and Au (Ghiorse, 1984; Frankel and Bazylinski, 2003; Slocik and Wright, 2003; Bazylinski et al., 2007). These components arrest dissolved metallic cations through chelating coordination or electrostatic attraction using their negative-charge multi-organic functional groups, such as amino acids, the derivatives of amino acids or multi-carboxyl compounds, and then they mediate metal fixation on clay particles in soils (Corzo et al., 1994) or gradually deposit during the long and complicate geological processes. Second, for B. cereus-Cr(VI) system, the soluble Cr(III) in the supernatant totally disappeared after about two months in the test tube, implying that the soluble Cr(III) with high molecular weight could be either simply precipitated or further immobilized by bacteria via ligand-exchange. Although more efforts are required, our results suggested that the deposition of Cr(III)-supernatant was not as difficult as other Cr(III) complex with small molecular weights. The coordination of these bacterium-origin soluble molecules with Cr(III) in the supernatant could finally facilitate the immobilization of Cr(III). Last but not the least, the bacterium-origin soluble molecules in the supernatant increases the Cr(VI) tolerant ability of the bacteria. It cannot only reduce a considerable amount of Cr(VI) before this toxic species penetrates into the cell membrane, but also directly decrease the enrichment of Cr(III) in the bacteria. This would be helpful for the survival and continuous propagation of Cr(VI)reducing bacteria in the Cr(VI) contaminated environment. 5. Conclusions
Fig. 3. Microscopic investigations of B. cereus. (a and c) SEM and corresponding EDS spectrum of intact bacteria; (e) AFM amplitude image of intact bacteria; (g and i) TEM and corresponding EDS spectrum of intact bacteria (white circular portion); (b and d) SEM and corresponding EDS spectrum of Cr-loaded bacteria; (f) AFM amplitude image of Cr-loaded bacteria; (h and j) TEM and corresponding EDS spectrum of Cr-loaded bacteria (white circular portion); Pb and U were from the chemical agents and used for staining the cells.
tems. We found that even when the culture medium was strictly restricted to carbon source, the reduced Cr(III) still could not be effectively immobilized by B. cereus. Moreover, we found the supernatant collected from B. cereus cultivation could reduce Cr(VI) to Cr(III) and coordinate with the reduced Cr(III) through carboxyl and amido functional groups. The formation of soluble organico– metal complexes leads to the decrease of the immobilizing efficiency of Cr(III) during the treatment of Cr(VI) contaminated
In this study, we investigated the Cr(VI)-microbe interaction of a Cr(VI)-tolerance bacterial strain-B. cereus. For the first time, we found the supernatant collected from B. cereus cultivation is the active components for reducing Cr(VI) and coordinating Cr(III). The reduced Cr(III) could coordinate with the supernatant, cell debris and cytoplasm through carboxyl and amido functional groups. Acknowledgments This study was supported by the National Basic Research Program of China (973 Program) (No. 2007CB815601, 2010CB933 501), National Natural Science Foundation of China (40902097, 20803082, 40772034, 31071745), the Knowledge Innovation Project of the Chinese Academy of Sciences (KJCX2.YW.W01), the Outstanding Youth Fund (10125523, 21125730). YIF (2007F3120) of Fujian Province, the National Natural Science Foundation for Distinguished Young Scholars (40902097), Ministry of Education and the Specialized Research Fund for the Doctoral Program of Higher Education (20093515110010), the National High Technology Research and Development Program (‘‘863’’Program) of China (2011AA10A203).
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