Sulfur Cycle of Chalcopyrite Bio-Oxidation Process by Acidithiobacillus Ferrooxidans

Lei Jiang, Yuruo Tao

School of environment and urban construction, Wuhan Textile University, Wuhan, China

Email: andy0920@163.com

Abstract: Bacteria of the sulfur cycle, in particular sulfide oxidizing and sulfate reducing bacteria, are of immense importance from the industrial and environmental point of views. As a typical sulfide oxidizing bacterium, Acidithiobacillus ferrooxidans has received increasing attentions from many researchers. This pa-per deals with the sulfur cycle of chalcopyrite bio-oxidation process by Acidithiobacillus ferrooxidans through the experiments of shake-flask. X-ray diffractometer results showed that, the intermediate product, elemental sulfur deposition was generated during the bio-oxidation processes. Transmission electron micro-scope (TEM) and energy dispersive spectrometer (EDS) analysis indicated that, sulfur particles in nanoscale existed within cells. This suggested that Acidithiobacillus ferrooxidans be directly concerned with the sulfur cycle. However, these sulfur particles were not sulfur globules as energy sources. This interesting phenome-non may be a biomineralization process of Acidithiobacillus ferrooxidans while the solution contained high concentration sulfur. The final product, sulfate increased during the whole bio-oxidation process, whereas some were precipitated from solution as a part of jarosite crystal.

Keywords: sulfur cycle; bio-oxdiation; Acidithiobacillus ferrooxidans 1 Introduction

Microorganisms play an important role in the global cy-cle of various elements such as sulfur, nitrogen, carbon and iron[1]. Sulfur occurs in variety of oxidation states with three oxidation states of −2 (sulfide and reduced organic sulfur), 0 (elemental sulfur) and +6 (sulfate) be-ing the most significant in nature. Chemical or biological agents contribute to transformation of sulfur from one state to another. A biogeochemical cycle which describes these transformations is comprised of many oxida-tion-reduction reactions. For instance, H2S, a reduced form of sulfur, can be oxidized to sulfur or sulfate by a variety of microorganisms. Sulfate, in turn, can be re-duced back to sulfide by sulfate reducing bacteria. The sulfur cycle consists of oxidative and reductive sides. The bacteria of the sulfur cycle, specifically sulfate re-ducing and sulfide oxidizing bacteria play an instrumen-tal role in many environmental and industrial settings. The sulfate reducing bacteria can be utilized in conjunc-tion with sulfide oxidizing bacteria to tackle the problem of acid mine drainage, a severe environmental challenge facing the mining industry. Apart from the contribution in biotreatment of acid mine drainage, sulfide oxidizers play a key role in bioleaching of refractory minerals, in situ removal of H2S from oil reservoirs and biological treatment of sour gases and waters contaminated with sulfide, with the latter being produced in large quantities in the enhanced oil recovery processes by water flood-ing[2]. As a typical sulfide oxidizer, Acidithiobacillus

ferrooxidans is highlighted owing to its ability to oxidize Fe2+ ions[3], elemental sulfur, hydrogen[4] and hydrogen sulfide[5-6] in acidic solution. With chemical analysis, X-ray Diffractometer analysis and in-situ chemical analyses (transmission electron microscope equipped with EDS), we will present in this paper sulfur cycle of chalcopyrite bio-oxidation process by Acidithiobacillus ferrooxidans through the experiments of shake-flask.

2 Experiments

2.1 Preparation of Mineral Samples

The chalcopyrite samples used for this study were ground in an agate mortar to the size of -80 meshes. The mineral grains were marinated in distilled ethanol for two hours and then rinsed twice with deionized water, and dried in a vacuum drying incubator at 40℃. Samples were ground further to the size of -200 meshes for chemical analysis, and the results are shown in Table 1.

2.2 Bacterial Strain and Media

A. ferrooxidans was cultured in 9K medium[7] that con-tained per liter: 3g (NH4)2SO4, 1g KCl, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.001g Ca(NO3)2, and 44.2g FeSO4·7H2O. The pH was adjusted to 2.0 with H2SO4. A. ferrooxidans was subcultured thrice before inoculation for experiments. The cultures were filtered through Whatman17 filter paper to remove the suspended solid material. The cells were then harvested from filtrate by centrifugation (2376×g) to eliminate residual ferric ion and washed twice with H2SO4 solution of pH 2.00. The suspending cells were counted using a haemacytometer.

Supported by the Science Foundation of Hubei Province (CDB348) and Hubei Province Department of Education project (Q20091703)

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The suspending bacteria concentration for the experiment was 4×108 cells/mL.

Table 1 . the chemical analysis of chalcopyrite (wt%)

composition/sample chalcopyrite

SiO2 7.90%

TiO2 0.18%

Al2O3 0.89%

Fe 27.23%

Pb 0.54%

Zn 0.92%

Cu 23.50%

MgO 5.67%

CaO 1.03%

Na2O 0.16%

S 31.48%

total 99.50%

2.3 Bioleaching Experiments

The experiments were conducted in a 250mL Erlenmeyer flask. The pulp density is 3.8% (4g of chalcopy-rite/chalcopyrite and 100mL of solution). Samples were put into 9K culture medium without Fe2+[7], added with A. ferrooxidans (inoculated amount of 10%), and pH was adjusted to 2.00 with H2SO4. The flasks were then placed into a 30°C constant-temperature incubator for 39 days. The concentration of Cu2+, total iron ion, and SO4

2- were measured at an interval of 3-4 days.

2.4 Analytical Methods

Measurements of ion concentration: During the ex-periment, the concentration of copper ion (Cu2+) and the total iron ion (Fe3+ and Fe2+) were measured with PE Plasma 2000 ICP-AES; the concentration of SO4

2- was measured with Dionex ICS-1500 Ion Chromatograph.

Reaction product analysis: After the samples were bio-oxidized for 39 days, solid minerals were collected, dried in a vacuum drying incubator at 60 , and qualit℃ a-tively analyzed using a Rigaku D/max-1200 X-ray Dif-fractometer.

Transmission electron microscope observation of bacteria: The minerals with 39-days-bio-oxidation of chalcopyrite were concreted by 0.4% agar solution, and cut into 0.15cm x 0.45cm slices. These slices were then fixed with 2% glutaraldehyde for 2 h, and washed by PBS thrice for 10 min each time. Subsequently, samples were immersed in 1% osmic acid at 4ºC for 2 h, and then were stepwise dehydrated in ethanol with concentration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% for 10 min respectively. After that, samples were transferred through epichlorohydrin and embedded in epoxy resin. Finally, samples were cut into ultrathin section and dyed with uranyl acetate and lead citrate. The

inner structure of bacteria and the relationship of mineral were observed under a JEM-2010HR Transmission Elec-tron Microscope with an accelerating voltage of 100kV, and chemical components in micro-area were analyzed using an OSFORD INCA Energy Dispersive Spectrome-ter equipped to the same TEM.

3 Results and Discussion

3.1 Reaction Solution Composition and Solid Product Identification

Table 2 shows the concentrations of solution ions in the bio-oxidation process of chalcopyrite. It can be found that the chalcopyrite oxidation rate is high. In general, the process for A. ferrooxidans to oxidize Fe2+ to Fe3+ is accompanied by deposition of jarosite[8] (Daoud and Karamanev, 2006). The deposition generally exists all through the bio-oxidation process of sulfides. With 3-days-bio-oxidation of chalcopyrite, yellowish deposi-tion appears in both reaction solutions, but the deposition changed to yellow brown after day 6. X-ray diffraction analysis revealed that the deposition phases include jarosite and elemental sulfur (Fig. 1).

Fig.1. X-ray diffractometer analysis of solid mineral of chalcopyrite

with 39-days-bio-oxidation

Table. 2. Time dependence of ion concentration in the bio-oxidation process of chalcopyrite

chalcopyrite

Time (days) / ion concentration

Total Fe(mmol/L)

SO42-

(mmol/L) Cu2+

(mmol/L)

0 0 38.90 0

3 4.12 35.79 1.07

6 0.54 26.90 2.34

9 0.54 32.97 5.68

12 1.09 37.06 10.28

15 0.65 41.25 15.34

18 0.78 50.22 21.28

22 1.81 59.96 29.33

26 2.75 61.80 38.27

30 4.16 73.36 46.42

34 10.08 91.72 48.94

39 19.63 108.12 50.75

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3.2 The Rate of Elemental Sulfur Oxidized to SO4

2- during the Bio-oxidation

The intermediate product, elemental sulfur, would be generated during the bio-oxidation of chalcopyrite, some of which would be oxidized to SO4

2- under the effect of A. ferrooxidans. With 39-days-bio-oxidation, the mol con-centration of total Fe ions increases by 19.63 mmol/L, that of Cu ions by 50.75 mmol/L (Table 2), and that of the added SO4

2- by 69.22 mmol/L (Table 2). As men-tioned before, the major chemical reaction during the bio-oxidation of chalcopyrite is as follows (Eq.1, Eq.2, Eq.3, Eq.4 and Eq.5):

S2O2HFeCuO4HCuFeS 222

22 (1)

H4SO2O3OH22S -2422

bacteria (2)

H6OHSOKFeOH6SO23FeK 6243224

3 ）（）（ (3)

OH24FeH4O4Fe 23

22 bacteria (4)

S2Fe5CuFe4CuFeS 2232 (5)

If impurity effect is ignored, Eq.2 completed and Eq.3 does not occur, theoretically the mol concentration of ions increasing in the solution should be Cu2+：Fet（total Fe）：SO4

2-=1: 1: 2. In fact, however, only part of the elemental sulfur would be oxidized by A. ferrooxidans and certain amount of SO4

2- has taken part in the forma-tion of jarosite. Based on the experimental data, the re-sults can be calculated as: Fe3+ mol concentration lost in deposition = (Cu2+ ion mol concentration – Fet（total Fe）mol concentration) = 31.12 mmol/L. Then, based on Eq.3, the SO4

2- mol concentration lost in deposition of jarosite can be calculated as 20.75 mmol/L. With this value and that got from the experiment, we can calculate the total SO4

2- mol concentration generating in Eq.2 as 89.97 mmol/L. The total elemental sulfur generating in Eq.1 and Eq.5 should be 101.50 mmol/L, twice the Cu ion mol concentration. Therefore, 88.86% of the elemental sulfur would be oxidized to SO4

2- in the process of experiment, and X-diffraction analysis after 39 days also proves that the reaction product contains un-reacted elemental sulfur (Fig. 1).

3.3 The Biological Microscopic Analyses of A. Ferrooxidans

The oxidation states of sulfur changed from −2 (chal-copyrite) to 0 (elemental sulfur) and then +6 (sulfate) during the bio-oxidation of chalcopyrite. What a role does A. ferrooxidans play during the oxidation process of sulfur? It is all known that A. ferrooxidans can accelerate the oxidation of sulfur, whereas our research indicates that A. ferrooxidans not only acts as a catalyzer. Fig. 2 shows the characteristics of such bacteria in large amount

Fig.2. TEM image of A. ferrooxidans with 39-days-bio-oxidation with 39-days-bio-oxidation. By analyzing microstructure of A. ferrooxidans deeply, we found that the bacterium was filled with particles in nanoscale. Contrasting the EDS analysis of the vacant area (Fig. 3A) to the particles (Fig. 3C), we found that the particles are elemental sulfur. Fig. 3B shows that the larger particle out of the bacte-rium is also elemental sulfur. Furthermore, there are some rhombi-structured crystals besides bacteria and elemental sulfur (Fig. 4). Contrasting the EDS analysis of the vacant area (Fig. 4A) to the crystals (Fig. 4B), it can be found that the elemental Fe, S and K in the rhombi-structured crystals (Fig. 4B) obviously increase. And these elements are right those constitute of jarosites. Therefore, with the result of X-ray diffraction analysis (Fig. 1), it can be found that the crystals are the jarosites formed under the effect of A. ferrooxidans in the bio-oxidation process of chalcopyrite.

Sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope, and this globule is called “sulfur globule”. Previous results indicated sulfur globule has obvious features. Xia et al isolated an acidophilic, rod-shaped Gram-negative sulfur oxidizing strain. Ultra-structural studies showed that the new bacteria possessed sulfur granules with clear membrane adhering to the cell innermembrane[9]. Prange et al indicated that the purple sulfur bacterium Allochromatium vinosum stored sulfur in the periplasm in the form of intracellular sulfur glob-ules during oxidation of reduced sulfur compounds, and the sulfur in the globules was enclosed by a protein en-velope[10]. It can be confirmed that the sulfur particles in nanoscale (Fig. 3) were not sulfur globules. These sulfur particles should attribute to the biomineralization during bio-oxidation process. The local supersaturated environ-ment of S element caused by A. ferrooxidans results in the crystallization of sulfur particles. The biomineraliza-

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References tion is an important geological process that has effect on the formation of microfossil and transfer of chemical elements. The microfossil of sulfur is really exciting, however, we don’t know how to explain its forming mechanism yet.

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4 Conclusions

The sulfur cycle of chalcopyrite bio-oxdiation process by A. ferrooxidans was studied with various methods. Ex-perimental results show that the intermediate product, elemental sulfur deposition was generated during the bio-oxidation processes, and sulfur particles in nanoscale existed within cells. This suggested that A. ferrooxidans be directly concerned with the sulfur cycle. 88.86% of the elemental sulfur was oxidized to sulfate in the proc-ess of experiment, whereas some sulfate was precipitated from solution as a part of jarosite crystal.

[3] Jiang, Lei, Zhou Huaiyang, Peng xiaotong, Ding zhonghao. The use of microscopy techniques to analyze microbial biofilm of the bio-oxidized chalcopyrite surface. Mineral engineering[J], 2009, 22(1):37-42

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[6] Quatrini, R., Appia-Ayme, C., Denis, Y., et al., Insights into the iron and sulfur energetic metabolism of Acidithiobacillus fer-rooxidans by microarray transcriptome profiling. Hydrometal-lurgy[J], 2006, 83: 263-272

[7] Sliverman, M., P., Lundgren, D., G., Studies on the chemoauto-trophic iron bacterium Ferrobacillus ferrooxidans I. An im-proved medium and a harvesting procedure for securing high cell yields. Journal of Bacteriology[J], 1959, 77, 642-647

[8] Daoud, J., Karamanev, D., Formation of jarosite during Fe2+ oxidation by Acidithiobacillus ferrooxidans. Minerals Engineer-ing, 2006, 19, 960-967

[9] Xia, J. L., Peng, A. A., Huan, H. E., YANG, Y., et al., A new strain Acidithiobacillus albertensis BY-05 for bioleaching of metal sulfides ores. Transactions of Nonferrous Metals Society of China, 2007, 17(1): 168-175

[10] Prange A, Engelhardt H, Truper HG, Dahl C. The role of the sulfur globule proteins of Allochromatium vinosum: mutagenesis of the sulfur globule protein genes and expression studies by real-time RT-PCR. Archives of Microbiology, 2004, 182:.2-3

Fig.3. TEM image and Energy spectrum analysis of A. ferrooxidans with 39-days-bio-oxidation

Fig.4. Energy spectrum analysis of the rhombi-structured bulk

crystals with 39-days-bio-oxidation

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