Fe(II) oxidation by Acidithiobacillus ferrooxidans in pure and mixed cultures in the presence of arsenate

Abstract

Fe²⁺ oxidation by Acidithiobacillus ferrooxidans in pure and mixed cultures was investigated in batch cultures in the presence of arsenate. The pH value was periodically monitored and Fe²⁺ content was analyzed by the 1,10-phenanthroline method. ICP-AES was employed for the analysis of As(V) concentration in the solution phase. Precipitates were collected and analyzed by X-ray diffraction. Slight enhancement of iron bio-oxidation was observed in mixed cultures with the two greatest As(V) concentrations (1.0 and 5.0 mg/L As), which were enriched from sediment samples in an abandoned copper mine site. As(V) concentrations decreased with time, indicating either the co-precipitation with or the adsorption by jarosite, the major sink of solid phase. Our data suggest that biogenically synthesized jarosite may play an important role in the attenuation of soluble arsenate in natural aquatic environments.

Keywords

Acidithiobacillus ferrooxidans / arsenate / jarosite / bio-oxidation / geomicrobiology

Introduction

Acidithiobacillus ferrooxidans is commonly found in acid mine drainage receiving waters and sulfate soils (Duquesne et al., 2003), and is widely exploited in the bioleaching and biomining industries (Tuovinen et al., 1971; Hutchins et al., 1986; Leduc and Ferroni, 1994) due to its ability to oxidize sulfide minerals and its resistance to many heavy metals. To date, much attention has been paid to the influence of heavy metals on the activity of A. ferrooxidans. In particular, the tolerance of A. ferrooxidans to arsenic has been confirmed in the bioleaching of arsenopyrite (Ehrlich, 1963; 1964; Collinet and Morin, 1990; Mandl et al., 1992; Morin et al., 2003), and the organisms can be readily enriched from the effluents with great concentration of arsenic (Duquesne et al., 2003; Dave et al., 2008). Arsenic resistance genes are present on the chromosome of several A. ferrooxidans strains (Butcher et al., 2000), and the tolerance to As can be greatly enhanced by introducing the antiarsenical plasmid into A. ferrooxidans by conjugation (Xu et al., 1995).

A. ferrooxidans can greatly enhance the oxidation of sulfide minerals and result in the formation of secondary hydroxysulfate (jarosite and schwertmannite) in natural aquatic environments. Formation of jarosite [MFe₃(SO₄)₂(OH)₆] is dependent on the aqueous activities of monovalent cations (such as K⁺, Na⁺, and NH₄⁺) and sulfate at pH 1.5-3 (Alpers et al., 1989; Bigham et al., 1996). The three crystallographic structural cation sites in jarosite can provide different coordination positions for various metals such as Cr, Cd, Cu, Hg, Pb, and As. Thus, during jarosite precipitation toxic metals may be removed from the solution phase by sorption or substitution (Wang et al., 2006a).

In natural environments, jarosite-rich precipitates can contain up to ~2000 ppm As in the fine grain fraction, such as at the southern Mother Lode gold district of California (Savage et al., 2000). Amorphous Fe(III)-sulfoarsenates could form a matrix with jarosite inclusions inside (Gieré et al., 2003). Arsenic speciation in a synthetic jarosite system indicated a preferential uptake of arsenate over sulfate for both Na and K-jarosites at low temperatures in the presence of 67 mM As in the starting solutions (Dutrizac and Jambor, 1987). Arsenate can also occupy up to approximately 30% of the tetrahedral sites normally filled by sulfate in jarosite (Savage et al., 2005). Thus, jarosite might serve as a potential sink for arsenic, and stabilize the toxic metal. An investigation on arsenic behavior during the bacterial oxidation of Fe²⁺ may further provide an insight into the biogeochemical processes involving arsenic speciation in natural environmental situations such as acid mine drainage. The purpose of this study was to investigate Fe²⁺ oxidation by A. ferrooxidans in pure and mixed cultures in the presence of different As(V) concentrations. The transformation processes of Fe(III) and As(V) in liquid and solid phases were also elucidated.

Materials and methods

Cultures and experimental conditions

A pure culture of A. ferrooxidans (strain PU1) was a gift from Prof. Lu Anhuai (School of Earth and Space Sciences, Peking University). Enriched cultures of A. ferrooxidans were derived from water (denoted as W3) and sediments (S3) collected from an abandoned copper mine in Tongling, Anhui Province, eastern China. The medium contained (in 4.05 mM H₂SO₄) 1.6 mM MgSO₄·7H₂O, 3.0 mM (NH₄)₂ SO₄, and 2.9 mM of KH₂PO₄ with 120 mM Fe²⁺ as the energy source (added as FeSO₄·7H₂O) (Wang et al., 2006b). The A. ferrooxidans strain PU1, W3, and S3 were inoculated in 200 ml medium and incubated in 500 ml shake flasks at 230 rpm and 30°C. Cultures in the exponential stage were transferred to fresh media with As(V) of different concentrations being added (as Na₃AsO₄·12H₂O) to study Fe²⁺ oxidation and As(V) behavior. Two sets of experiments were set up. The first included the test cultures in the presence of 0, 0.01, and 0.1 mg/L As(V). In the second set, the enriched culture S3 was incubated in the presence of 0.1, 0.5, 1.0, and 5.0 mg/L As(V).

Analytical methods

The oxidation of ferrous ion was measured by the 1, 10-phenanthroline method (Vogel, 1989). The concentration of As(V) in the solution was analyzed by ICP-AES (IRIS Intrepid II, Thermal Electron Corporation, USA). Precipitates were harvested by centrifugation with a centrifugal force of 9,056 g for 15 min and dried at 50°C for 12 h. The precipitates were analyzed by powder X-ray diffraction (XRD), which was conducted with CuKα radiation and a Philips PW1070 goniometer equipped with a diffracted-beam monochrometer and a q-compensating slit. Samples were scanned from 3° to 80° 2q with a step increment of 0.05° 2q and a 4-s counting time.

Results

Fe²⁺ oxidation by A. ferrooxidans

The color of enriched cultures of S3 and W3 turned orange within 20 h incubation followed by the formation of yellow Fe(III) precipitate. It took 36 h to show the color change and the formation of precipitates in the pure culture of A. ferrooxidans.

The pH values of S3 and W3 increased to about 2.5 in the first 30 h and then decreased to 1.9 (Figs. 1(b), 1(c)), lower than those at the starting points, suggesting that Fe(III) hydrolysis leading to the precipitation was a dominant reaction. No obvious difference in the pH values was observed in the mixed cultures, either the control (without As) or the As amended cultures. The pH values in mixed cultures changed in a pattern different from those observed in pure A. ferrooxidans cultures. The pH values in pure culture decreased to 2.1 within the first 30 h, followed by an increase and some fluctuation, with the final pH values close to the initial values (Fig. 1(a)).

Fe²⁺ was oxidized completely within 49 to 56 h, depending on the initial concentration of As(V) added to the cultures of S3 and W3 (Figs. 1(e), 1(f)). A different oxidative behavior by pure A. ferrooxidans was observed. In the control pure A. ferrooxidans culture, Fe²⁺ was oxidized at an even rate in the first 43 h, and the oxidation sped up till the completion of the process. However, in the As(V)-treated pure culture systems, there existed a plateau of Fe²⁺ concentration which lasted for about 20 h (Fig. 1(d)). Then Fe²⁺ was completely oxidized within 68 h.

Fe²⁺ oxidation by S3 in the presence of elevated As(V) concentrations

The influence of elevated As(V) concentrations (0.1, 0.5, 1.0, and 5.0 mg/L ) on the bio-oxidation of Fe²⁺ by S3 was further studied. The pH values increased to about 2.6 followed by a decrease to about 2.0 (Fig. 2(a)). The time period to reach the greatest pH value varied with the As(V) concentration in the cultures. The bio-oxidation capacity of S3 was enhanced by the elevated concentrations of As(V) (Fig. 2(b)). In the culture with an As(V) concentration of 0.1 mg/L, it took 73 h to reach the complete oxidation of Fe²⁺, in contrast with the 56 h in the cultures with 0.5, 1.0, and 5.0 mg/L As(V) content. The bio-oxidation rates were similar in the cultures with an As(V) concentration between 0.5 and 5.0 mg/L (Fig. 2(b)).

Transformation of As(V) between liquid and solid phases

Observed changes in dissolved As(V) concentrations with time indicated (Table 1) the presence of As(V) transformation into the solid phase. Alternatively, As(V) was incorporated into the crystal structure or adsorbed by the solid surface. At the end of the experiments, it was estimated that 100%, 40%, and 70% As(V) entered the solid phase in the cultures with an initial As(V) concentration of 0.1, 0.5, and 5.0 mg/L, respectively. Only about 2% As(V) was observed to transfer into the solid phase in the culture with the initial 1.00 mg/L As(V) content, clearly much lower than those found in the other three cultures.

Solid products of bio-oxidation

X-ray diffractograms of the precipitates showed that jarosite was the major sink of Fe³⁺ during the bio-oxidation (Fig. 3). The increases in the baselines and the weakened peaks in the XRD patterns indicated the changes in the crystallinity of jarosite, possibly due to the presence of the incorporated As or other undetectable poorly crystallized material in the solid phase.

Discussion

In all experiments with mixed cultures, the pH values were featured by an increase, followed by a decrease. This change reflected the oxidation of Fe²⁺, which is a proton-consuming reaction (Eq. (1)), and the formation of jarosite, an acid-forming reaction (Eq. (2)).

(1)\mathrm{4}\mathrm{F}{\mathrm{e}}^{\mathrm{2}+}+{\mathrm{O}}_{\mathrm{2}}+\mathrm{4}{\mathrm{H}}^{+}\to \mathrm{4}\mathrm{F}{\mathrm{e}}^{\mathrm{3}+}+\mathrm{2}{\mathrm{H}}_{\mathrm{2}}\mathrm{O}

(2){\mathrm{K}}^{+}+\mathrm{3}\mathrm{F}{\mathrm{e}}^{\mathrm{3}+}+\mathrm{6}{\mathrm{H}}_{\mathrm{2}}\mathrm{O}+\mathrm{2}\mathrm{S}{\mathrm{O}}_{}^{-}\to \mathrm{K}\mathrm{F}{\mathrm{e}}_{\mathrm{3}}{(\mathrm{S}{\mathrm{O}}_{\mathrm{4}})}_{\mathrm{2}}{(\mathrm{O}\mathrm{H})}_{\mathrm{6}}+\mathrm{6}{\mathrm{H}}^{+}

In the pure A. ferrooxidans culture systems, the pH values changed oppositely, characterized by a decrease followed by an increase with some fluctuation. Compared with the mixed cultures, the pH values varied in a small range in the pure cultures. The oxidation of Fe²⁺ in pure culture systems also showed a pattern different from that in the mixed cultures. All of these differences might suggest different behavior of A. ferrooxidans in the pure and the mixed cultures during the oxidation of Fe²⁺.

The fact that dissolved As decreased in concentration with time indicated the presence of As in the solid phase. One possible explanation is either by the adsorption or co-precipitation with jarosite, the only detected solid phase by XRD in our system. Since no change was observed in any XRD spectrum of jarosite, rather than the uplift of the baseline and the decrease of the relative abundance of the peaks, we concluded that the decrease of dissolved As in the solution is possibly caused by the adsorption of arsenate by jarosite.

Another possible explanation of the decrease of arsenate content in the solution might result from the formation of amorphous ferric arsenate hydrate. It was reported that arsenic may be sequestered from the solutions during the hydrolysis of soluble iron, resulting in the formation of a poorly crystalline hydrous ferric oxide (FeAsO₄·2H₂O) (Waychunas et al., 1993; Fuller et al., 1993; Mamtaz and Bache, 2001; Pichler et al., 1999; Tahija and Huang, 2000). The poorly crystalline ferric arsenate could strongly adsorb arsenate anions, and it appears to be stable under slightly acidic pH and oxidizing conditions (Riveros et al., 2001). Therefore, amorphous iron oxyhydroxides co-precipitating with arsenic may be responsible for the increases in the baselines and the weakened peaks in the XRD patterns of jarosite. One concern should be pointed out here is that we use very low arsenate concentration due to the high toxic chemical control regulation of the University. The low As concentration might limit the formation of hydrous ferric oxide (FeAsO₄·2H₂O). Therefore, jarosite is most possibly responsible for the decrease of arsenate concentration in the liquid phase.

Conclusions

Slight enhancement of iron bio-oxidation by A. ferrooxidans was observed in the mixed cultures with the two greatest As(V) concentrations (1.0 and 5.0 mg/L As). Changes in pH values and Fe²⁺ oxidation rate by A. ferrooxidans were clearly observed, but showed different patterns between the pure and the mixed cultures. Jarosite was the only solid product detected in the experiments, proposed to be the result of Fe²⁺ oxidation and Fe³⁺ hydrolysis. As(V) was adsorbed by or incorporated in the solid phase during the Fe²⁺ oxidation, which was supported by the decrease in dissolved arsenate concentration over time.

References

[1] Alpers C N, Nordstrom D K, Ball J W (1989). Solubility of jarosite solid solutions precipitated from acid mine waters, Iron Mountain, California, USA. Sci Géol Bull, 42: 281-298

[2] Bigham J M, Schwertmann U, Traina, S J, Winland R L, Wolf M (1996). Schwertmannite and the chemical modeling of iron in acid sulfate waters. Geochim Cosmochim Acta, 60: 2111-2121

[3] Butcher B G, Deane S M, Rawlings D E (2000). The chromosomal arsenic resistance genes of Thiobacillus ferrooxidans have an unusual arrangement and confer increased arsenic and antimony resistance to Escherichia coli. Appl Environ Microbiol, 66: 1826-1833

[4] Collinet M N, Morin D (1990). Characterization of arsenopyrite oxidizing Thiobacillus.Tolerance to arsenite, arsenate, ferrous and ferric iron. Antonie van Leeuwenhoek, 57: 237-244

[5] Dave S R, Gupta K H, Tipre D R (2008). Characterization of arsenic resistant and arsenopyrite oxidizing Acidithiobacillus ferrooxidans from Hutti gold leachate and effluents. Bioresource Technology, 99: 7514-7520

[6] Duquesne K, Lebrun S, Casiot C, Bruneel O, Personné J-C, Leblanc M, Elbaz-Poulichet F, Morin G, Bonnefoy V (2003). Immobilization of arsenite and ferric iron by Acidithiobacillus ferrooxidans and its relevance to acid mine drainage. Appl Environ Microbiol, 69: 6165-6173

[7] Dutrizac J E, Jambor J L (1987). The behavior of arsenic during jarosite precipitation: arsenic precipitation at 97 oC from sulfate or chloride media. Can Metall Q, 26: 91-101

[8] Ehrlich H L (1963). Bacterial action on orpiment. Econ Geol, 58: 991-994

[9] Ehrlich H L (1964). Bacterial oxidation of arsenopyrite and enargite. Econ Geol, 59: 1306-1312

[10] Fuller C C, Davis J A, Waychunas G A (1993). Surface chemistry of ferrihydrite: part 2. Kinetics of arsenate adsorption and coprecipitation. Geochim Cosmochim Acta, 57: 2271-2282

[11] Gieré R, Sidenko N V, Lazareva E V (2003). The role of secondary minerals in controlling the migration of arsenic and metals from high-sulfide wastes (Berikul gold mine, Siberia). Appl Geochem, 18: 1347-1359

[12] Hutchins S R, Davidson M S, Brierley J A, Brierley C L (1986). Microorganisms in reclamation of metals. Annu Rev Microbiol, 40: 311-336

[13] Leduc L G, Ferroni G D (1994). The chemolithotrophic bacterium Thiobacillus ferrooxidans. FEMS Microbiol Revs, 14: 103-119

[14] Mamtaz R, Bache D H (2001). Reduction of arsenic in groundwater by coprecipitation with iron. J Water Supply Res Technol, 50: 313-324

[15] Mandl M, Matulova P, Docekalova H (1992). Migration of AsIII during bacterial oxidation of arsenopyrite in chalcopyrite concentrates by Thiobacillus ferrooxidans. Appl Microbiol Biotechnol, 38: 429-431

[16] Morin G, Juillot F, Casiot C, Bruneel O, Personné J-C, Elbaz-Poulichet F, Leblanc M, Ildefonse P, Calas G (2003). Bacterial formation of tooeleite and mixed As(III)/(V)-Fe(III) gels in the Carnoulés acid mine drainage, France. A XANES, XRD and SEM study. Environ Sci Technol, 37: 1705-1712

[17] Pichler T, Veizer J, Hall G E M (1999). Natural input of arsenic into a coral-reef ecosystem by hydrothermal fluids and its removal by Fe(III) oxyhydroxides. Environ Sci Technol, 33: 1373-1378

[18] Riveros P A, Dutrizac J E, Spencer P (2001). Arsenic disposal practices in the metallurgical industry. Can Metall Q, 40: 395-420

[19] Savage K S, Bird D K, Ashley R P (2000). Legacy of the California gold rush: environmental geochemistry of arsenic in the southern Mother Lode Gold District. Int Geol Rev, 42: 385-415

[20] Savage K S, Bird D K, O’Day P A (2005). Arsenic speciation in synthetic jarosite. Chem Geol, 215: 473-498

[21] Tahija D, Huang H H (2000). Factors influencing arsenic coprecipitation with ferric hydroxide. In: Young C, ed. Proceeding of Minor Elements 2000: Processing and Environmental Aspects of As, Sb, Se, Te, and Bi, Society for Mining, Metallurgy, and Exploration. Littleton CO, 149-155

[22] Tuovinen O H, Niemelä S I, Gyllenberg H G (1971). Tolerance of Thiobacillus ferrooxidans to some metals. Antonie van Leeuwenhoek, 37: 489-496

[23] Vogel A I (1989). Vogel’s Textbook of Quantitive Chemical Analysis. 5th ed. London: London Group Ltd

[24] Wang C, Ma S, Lu A (2006a). A preliminary study on the Cr(Ⅳ) removing from wastewater by precipitation of jarosite group minerals. Bulletin of Mineralogy, Petrology and Geochemistry, 25(4): 335-338(in Chinese with English abstract)

[25] Wang H, Bigham J M, Tuovinen O H (2006b). Formation of schwertmannite and its transformation to jarosite in the presence of acidophilic iron-oxidizing microorganisms. Mater Sci Eng, 26: 588-592

[26] Waychunas G A, Rea B A, Fulle C C, Davis J A (1993). Surface chemistry of ferrihydrite: Part 1. EXAFS studies of the geometry of coprecipitated and adsorbed arsenate. Geochim Cosmochim Acta, 57: 2251-2269

[27] Xu H, Yan W, Liu Z, Luo X, Wang Z (1995). Construction of antiarsenical vector and expression in Thiobacillus ferrooxidans. Chin J Appl Environ Biol, 1: 238-243 (in Chinese with English abstract)

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg