Investigation of Elemental Sulfur Speciation ... - Springer Link

Curr Microbiol (2009) 58:300–307 DOI 10.1007/s00284-008-9330-6

Investigation of Elemental Sulfur Speciation Transformation Mediated by Acidithiobacillus ferrooxidans Huan He Æ Cheng-Gui Zhang Æ Jin-Lan Xia Æ An-An Peng Æ Yi Yang Æ Hong-Chen Jiang Æ Lei Zheng Æ Chen-Yan Ma Æ Yi-Dong Zhao Æ Zhen-Yuan Nie Æ Guan-Zhou Qiu

Received: 2 June 2008 / Accepted: 11 November 2008 / Published online: 16 December 2008 Ó Springer Science+Business Media, LLC 2008

Abstract The speciation transformation of elemental sulfur mediated by the leaching bacterium Acidithiobacillus ferrooxidans was investigated using an integrated approach including scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, and X-ray absorption near edge spectroscopy (XANES). Our results showed that when grown on elemental sulfur powder, At. ferrooxidans ATCC23270 cells were first attached to sulfur particles and modified the surface sulfur with some amphiphilic compounds. In addition, part of the elemental sulfur powder might be converted to polysulfides. Furthermore, sulfur globules were accumulated inside the cells. XANES spectra of these cells suggested

Huan He and Cheng-Gui Zhang made equal contributions to this paper. H. He C.-G. Zhang J.-L. Xia (&) A.-A. Peng Y. Yang Z.-Y. Nie G.-Z. Qiu Key Laboratory of Biometallurgy of Ministry of Education of China, School of Minerals Processing & Bioengineering, The South Road of YueLu Mountain, Central South University, Changsha, Hunan 410083, China e-mail: [email protected] H. He e-mail: [email protected] H.-C. Jiang Geomicrobiology Laboratory, State Key Laboratory of Geological Processes and Mineral Resources, School of Earth Sciences and Resources, China University of Geosciences, Beijing 100083, China L. Zheng C.-Y. Ma Y.-D. Zhao Institute of High Energy Physics, Chinese Academy of Science, Beijing Synchrotron Radiation Facility, Beijing 100049, China

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that these globules consisted of elemental sulfur bound to thiol groups of protein.

Introduction Acidithiobacillus ferrooxidans is the most widely studied acidophilic bioleaching bacterium, which obtains energy by oxidizing of ferrous ions, elemental sulfur, and reduced sulfur compounds [16]. Bio-oxidation of elemental sulfur and reduced sulfur compounds by Acidithiobacillus spp. typically is not only beneficial for recovering valuable metals from metal sulfides ores and removing heavy metals from specific locally polluted sites, but also causes serious environment problems by forming typical acidic rock/mine drainages, leading to leakage of heavy metals and extremely acidity of local soils [17]. To find some suitable measures to control sulfur bio-oxidation, it is necessary to understand the detailed biochemical reactions in terms of sulfur speciation and transformation. Several sulfur species including elemental sulfur and reduced sulfur compounds can be produced when acidsoluble and nonsoluble metals sulfides are oxidized to sulfate by Acidithiobacillus via the thiosulfate or polysulfide pathway [16]. Unlike soluble sulfur compounds, elemental sulfur is hydrophobic and generally prone to form sulfur granules that may further deposit onto the surface of minerals and hinder the bioleaching process [1, 4]. In some cases, it exists in the form of colloid globules in vitro and/or in vivo, which must be related to the sulfur oxidation of typical Acidithiobacillus spp. [7, 19]. Although a lot of enzymes related to elemental sulfur oxidation have been characterized, the activation and transportation of elemental sulfur by cells remain an open

H. He et al.: Elemental Sulfur Speciation Transformation Mediated by At. ferrooxidans

problem [17]. Hence, study of the speciation states of elemental sulfur and further elucidation of its transfer route during bioleaching seem meaningful. However, biological sulfur speciation is too complex to be analyzed in situ by traditional methods based on chemical titration analysis, electrochemistry, or chromatography. To date, synchrotron-based sulfur K-edge X-ray absorption near-edge spectroscopy (XANES) seems to be the only efficient technique for characterizing sulfur speciation in nature samples. With the technique of XANES, electronic structure and chemical forms of sulfur in samples can be deduced by comparing the absorption spectra of samples to those of reference compounds [11–13]. The objective of this study was to investigate and analyze the chemical speciation transformation of elemental sulfur mediated by At. ferrooxidans grown on elemental sulfur.

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through filter paper (pore size 10 lm) to remove the suspended elemental sulfur. Cells in the filtrate were then collected by centrifugation at 10,000 rpm for 20 min, followed by washing three times with carbon disulfide and centrifuging at 10,000 rpm for 20 min. Cell pellets were then resuspended with basic medium and transferred to formaldehyde (25%, v/v). Samples were additionally fixed with OsO4, dehydrated, embedded, and sectioned using standard procedures. They were stained with lead citrate and uranyl acetate and then deposited on copper grids covered with carbon membrane. The components of intracellular inclusions on the thin section were examined with a TEM (FEI Tecnai G2 20AEM) equipped with an EDS analytical system operating at 80 keV, which is capable of measuring all elements heavier than lithium. Fourier Transform Infrared Spectroscopy (FT-IR)

Materials and Methods Bacterial Strain and Culture Conditions Acidithiobacillus ferrooxidans ATCC23270 was purchased from the American Type Culture Collection, and it was cultured in basal medium supplemented with, respectively, 5.0 g L-1 elemental sulfur powder and 44.5 g L-1 ferrous sulfate. The basal medium contained (g L-1 distilled water): (NH4)2SO4, 3.0; MgSO47H2O, 0.5; KCl, 0.1; K2HPO4, 0.5; and Ca (NO3)2, 0.01. The pH of the medium was adjusted with 1 M sulfuric acid to 2.5 and 1.7, respectively. The culture of At. ferrooxidans was grown in 500-ml flasks containing 300 mL medium and incubated at 30°C with shaking at 170 rpm. Scanning Electronic Microscopy (SEM) At. ferrooxidans cells grown on elemental sulfur powder were harvested in the exponential phase. The sampled culture was allowed to settle for half an hour until the majority of elemental sulfur powders were deposited. Then a 100-lL suspension culture was transferred to a 1.5-mL tube containing 1 mL formaldehyde (25%, v/v). Fixed samples were dehydrated, plated, and then introduced into the SEM (JEOL JSM-6360 LV) chamber for SEM observation. Transmission Electronic Microscopy (TEM) and Energy Dispersive X-Ray Spectroscopy (EDS) At. ferrooxidans cells grown in elemental sulfur were harvested in the exponential phase according to the following procedure: the sampled culture was filtered twice

The elemental sulfur powder modified by cells was obtained when cells reached the late exponential phase using the following procedure: the suspended elemental sulfur powder was filtered and resuspended with deionized water. The modified sulfur samples were vortexed and centrifuged at 3000 rpm for 5 min, and then the supernatant was decanted. This process was repeated multiple times until no cells were detected under light microscopy (Olympus CX31). Then the washed modified sulfur powder was freeze-dried. FT-IR spectra of modified and original sulfur powder were obtained using a Fourier transform spectrometer (Nicolet Nexus 670) with diffuse reflectance attachment. X-Ray Absorption Spectroscopy Aliquots of At. ferrooxidans cells were harvested in the exponential phase by centrifuging at 10,000 rpm for 20 min, followed by washing three times with carbon disulfide and centrifuging at 10,000 rpm for 20 min. The washed cell pellets were freeze-dried and then stored at -20°C until further analysis. X-ray absorption spectra were recorded at 4B7A beamline (medium X-ray beamline, 2100–6000 eV) using synchrotron radiation at the Beijing National Synchrotron Radiation Facility, Institute of High Energy Physics of China. The storage ring was operated at the energy of 2.5 GeV with electron currents of 80–100 mA. The synchrotron radiation was monochromatized with a monochromator equipped with Si (111) double crystals. Measurements were performed in fluorescence mode and spectra were recorded by monitoring the X-ray fluorescence using a fluorescent ion chamber Si (Li) detector (PGT LS30135). Spectra were scanned at step widths of 0.3 eV in the region between 2420 and 2520 eV. The X-ray energy was calibrated with reference to the

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spectrum of the highest resonance energy peak of ZnSO4 at 2480.4 eV. Reference Compounds To explore different sulfur oxidation states (0 to ?6) and chemical structures potentially present in the sulfur transformation mediated by At. ferrooxidans, solid S8 and a series of typical sulfur compounds (potassium persulfate, zinc sulfate, potassium tetrathionate, thiosulfate, sodium sulfite, methyl sulfone, methionine, reduced glutathione, oxidized glutathione, cystine, and cysteine) were selected as sulfur references according to Prange et al. [12] and Pickering et al. [11]. All the compounds listed were of reagent grade and were purchased from Sigma. Polymeric sulfur (insoluble sulfur) was purchased from Flexsys (Crystex HD OT 20), washed 10 times with acetone and carbon disulfide to remove any possible organic substance and soluble sulfur, centrifuged, and then freeze-dried. Solid compounds were grounded well and mounted on vacuum adhesive film (sulfur free). The sulfur K-edge XANES spectra of the reference compounds were used to deduce the chemical speciation transformation of solid S8 modified by At. ferrooxidans and detailed chemical speciation of sulfur globules present in cells of At. ferrooxidans.

Fig. 1 Comparison of sulfur K-edge XANES spectra of reference compounds: (a) potassium persulfate, (b) zinc sulfate, (c) potassium tetrathionate, (d) thiosulfate, (e) sodium sulfite, (f) methionine, (g) reduced glutathione, (h) oxidized glutathione, (i) cystine, (j) elemental sulfur (S8), and (k) cysteine. a.u., arbitrary units

Results and Discussion Sulfur K-Edge XANES Spectra of Reference Compounds XANES spectra of selected representative organic and inorganic sulfur reference compounds are shown in Fig. 1, which vary almost linearly with the oxidation state of the sulfur atoms. Organic sulfur compounds with different functional groups show distinct spectra (Fig. 2a and b). The intense absorption position of cysteine originating from the transition S 1 s ? r*(S-H) was at 2.4714 keV, whereas that of cystine S 1 s ? r*(S-S) was at 2.4705 keV (Fig. 2a) [12]. Clear differences also exist between reduced glutathione and oxidized glutathione (Fig. 2b). In comparison, organic sulfur compounds with the same functional groups show a similar peak shape and absorption edge position. For example, the most intense absorptions on the sulfur K-edge XANES spectra are similar to each other for pairs of cysteine and reduced glutathione (containing –HS group) (Fig. 2c) and of cystine and oxidized glutathione (containing S-S group) (Fig. 2d), respectively. When the energy position is [5 eV larger than the strong resonance peak, however, obvious differences appear. Such differences are caused by the more distant atoms and multiple scattering effects [20].

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Micrographs of Absorbed Cells and Spectra of Elemental Sulfur Interactions with Cells It is well known that during the oxidation of elemental sulfur, At. ferrooxidans are attached to the surface of solid sulfur particles and that the extracellular polymeric substances (EPSs) of At. ferrooxidans play a crucial role in mediating the attachment between cells and the surface of elemental sulfur or metal sulfide minerals [10, 21]. Hydrophobic elemental sulfur is modified by special compounds secreted by At. ferrooxidans cells, so the sulfur surface tends to be hydrophilic and have dispersive characteristics in water. On the other hand, the electrokinetic behavior and hydrophobicity of elemental sulfur were altered significantly after interaction with bacteria [3, 23]. SEM images show that a few cells are accumulated on the surface of solid sulfur particles (Fig. 3a), on which there are some visible pits (Fig. 3b). We speculated that the structure of sulfur crystals might be destroyed by the attached cells. Figure 4 shows the FT-IR spectra of original and modified sulfur particles. The latter (Fig. 4b) becomes much more complex than the former (Fig. 4a). The wide absorption bands at 3600–2500 cm-1 are related to –NH and –OH groups. The strong absorption peak at 1650 cm-1 represents the –C=O group, and the absorption peaks at


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Fig. 2 Comparison of sulfur Kedge XANES spectra of a cysteine (solid line) and cystine (dotted line), b reduced glutathione (dotted line) and oxidized glutathione (solid line), c cysteine (dotted line) and reduced glutathione (solid line), and d cystine (solid line) and oxidized glutathione (dotted line). a.u., arbitrary units

Fig. 3 SEM micrographs of solid sulfur powder and adsorbed cells

1210 and 1051 cm-1 may be assigned to –CH3 and –CH2 groups, respectively. So it can be concluded that some amphiphilic protein molecules [22], are attached to the

surface of elemental sulfur particles, suggesting that the characteristics of elemental sulfur are remarkably altered by At. ferrooxidans cells.

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Fig. 4 FT-IR spectra of a original sulfur powder and b cell modified sulfur powder

The sulfur K-edge XANES spectrum of At. ferrooxidans modified sulfur powder is shown in direct comparison with that of solid S8 and polymeric sulfur (Fig. 5a). The strong resonance peak of these spectra is at 2.4705 keV, but the peak width and the shape spectra are slightly different. This indicates that part of the original sulfur powder may be converted to polymeric sulfur [13]. In the present study, we also found the following interesting phenomenon. When we used carbon disulfide to dissolve the cell-modified sulfur, the majority of elemental sulfur was dissolved into the organic phase containing carbon disulfide, but partial elemental sulfur floated in the organic phase. In contrast,

Fig. 5 Sulfur K-edge XANES spectra of a elemental sulfur modified by At. ferrooxidans (solid line), elemental sulfur (S8) (dotted line), and polymeric sulfur (dashed line) and b cell modified sulfur powder after treatment with carbon disulfide (dashed line), polymeric sulfur (S8; solid line), and potassium tetrathionate (dotted line). a.u., arbitrary units

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the same weight of original elemental sulfur can be completely dissolved in the same volume of carbon disulfide. We extracted those floating sulfur particles by centrifuging three times, washing with deionized water, then analyzing using X-ray absorption spectroscopy. Figure 5b shows sulfur K-edge XANES spectra of floating sulfur in comparison with those of polymeric sulfur and tetrathionate. The XANES spectra indicated that the floating sulfur powder may be converted to polysulfide, because the spectrum of the former is perfectly coincident with that of the latter. Rohwerder and Sand [18] proposed that elemental sulfur (S8) is first activated by thiol-bearing proteins to persulfides, but this activation process has not been confirmed yet. Our results support that the elemental sulfur must be activated first and then a part of it may be converted to polysulfides before it is transported into the cell. Ultrastructural Studies and Spectra of Cells Thin sections of At. ferrooxidans show some particles of high electron transmission (black arrow in Fig. 6a). The size of these particles is *50 nm. EDS analysis of these particles indicates that they are mainly composed of sulfur (Fig. 6b). It had been reported that colloidal sulfur was distributed over the organic membrane when At. ferrooxidans was cultivated with synthetic pyrite, thiosulfate, and tetrathionate [7, 19]. Our TEM images definitely show that sulfur particles are accumulated inside the cells grown on solid sulfur powder.


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Fig. 6 a TEM image of At. ferrooxidans cells. Inset An enlargement of the sulfur inclusion. b The energy dispersive spectrum derived from the observed sulfur inclusion (black circle). The large C and Cu peaks are derived from the supporting grid. The N and Cl peaks are derived from cell components. The Os peak is derived from the fixation reagent OsO4. (Original magnifications: a, 950,000; inset, 9100,000.)

At. ferrooxidans cells cultured with ferrous sulfate served as a negative control and the sulfur K-edge XANES spectrum was directly compared with those of methionine, cysteine, and zinc sulfate (Fig. 7a). According to the spectra of reference compounds, the absorption peak at 2.4714 keV is mainly derived from C-S bonds in sulfurcontaining amino acids of cells. The peak at 2.4804 keV is the specific spectrum for sulfate, which may originate from the culture medium or the intermediate metabolic products inside the cells. So it can be concluded that there are no sulfur globules accumulated in control cells. In comparison, cells grown on elemental sulfur powder have unique absorption peaks at 2.4705 and 2.4741 keV, corresponding to accumulated sulfur globules inside cells and the S-H Rydberg transition, respectively (Fig. 7b) [12, 14]. So protein- or enzyme-containing thiols (S-H) may be

involved in elemental sulfur oxidation by At. ferrooxidans cells. Although intracellular globules have been found in many sulfur-oxidizing microbes, their detailed chemical forms and mechanism of formation are debatable and incompletely understood. At present, there are several proposed points of views about the chemical speciation of sulfur in bacterial sulfur globules. For example, Pickering and coworkers claimed that sulfur was presented in the form of solid S8 [6, 11]. In comparison, it was revealed that there were at least three species of sulfur globules: cyclooctasulfur in Beggiatoa alba and Thiomargarita namibiensis grown on sulfide, sulfur chain in purple and green sulfur bacteria grown on sulfide, and polythionate in extracellular globules of At. ferrooxidans grown on thiosulfate [13]. Another study proposed that intracellular

Fig. 7 Sulfur K-edge XANES spectra of a At. ferrooxidans grown on ferrous sulfate (solid line), methionine (dashed line), cysteine (dotted line), and zinc sulfate (dashed-dotted line) and b At. ferrooxidans grown on sulfur powder (solid line), reduced glutathione (dotted line), and elemental sulfur (dashed line). a.u., arbitrary units

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sulfur globules of Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes grown on thiosulfate were mainly composed of sulfur chains with organic residuals [9]. So it seems that the obvious microbes fed with different substrates will result in different sulfur speciation in sulfur globules produced. In this study, the substrate for At. ferrooxidans is different from that of phototrophic sulfur bacteria (elemental sulfur powder vs. sulfide). Thus, it is reasonable that the chemical speciation in intracellular sulfur globules of At. ferrooxidans is different from that of phototrophic bacteria listed above (polymeric sulfur vs. solid S8). Various proteins or enzymes may be involved in sulfur speciation in sulfur globules of different microorganisms [2, 5, 15]. Recent studies showed that different elemental sulfur metabolisms may be employed in phototrophic bacteria versus acidophilic sulfur bacteria [17]. A possible pathway for elemental sulfur oxidation by Acidithiobacillus spp. has been proposed: (1) extracellular rhombic elemental sulfur (S0) has to be activated as thiol-bound sulfane sulfur atoms (R-SSnH) [18] and then transported to the periplasmic space, where it is oxidized to SO32– by sulfur dioxygenase (SDO) [24]; (2) sulfane sulfur can also be reduced to hydrogen sulfide and then oxidized back to S0 by sulfide:quinone oxidoreductase (SQR) [25]; and (3) S0 can react abiotically with the above sulfite to form thiosulfate and then can be oxidized to tetrathionate by thiosulfide:quinone oxidoreductase (TQO) [17], which is then degraded by tetrathionate hydrolase (TTH) [8]. Obviously, S0 can be produced at each of three stages in the aboveproposed pathways: sulfur activation process, SQR metabolism, and TQO metabolism. Our present results suggest that the sulfur globules originating from At. ferrooxidans cells grown on elemental sulfur powder might be produced as intermediate products in the sulfur activation process. The observed sulfur globules might be bound to thiol groups of proteins such as the reported sulfur sulfane of polysulfides of At. ferrooxidans cells [18]. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 50674101), the National Basic Research Program of China (No. 2004CB619201), and the Chinese Science Foundation for Distinguished Groups (No. 50621063). The authors are indebted to the staff at the Beijing Synchrotron Radiation Facility (BSRF) medium-energy beam-line station for their generous assistance. Measurements at BSRF were supported by the public user program.

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