Arsenic Precipitation in the Bioleaching of Realgar Using Acidithiobacillus ferrooxidans

1 Gansu Institute of Business and Technology (GIBT), Yannan Road No. 18, Lanzhou 730010, China 2 School of Pharmacy, Lanzhou University, West Donggang Road No. 199, Lanzhou 730020, China 3 College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China 4College of Chemistry and Chemical Engineering, Lanzhou University, Tianshui Road No. 222, Lanzhou 730000, China


Introduction
Realgar, which has a chemical formula of -As 4 S 4 , is a sulfide mineral with high arsenic content (over 90% As 4 S 4 ) and has been widely used as a traditional Chinese herbal medicine [1].However, realgar is not soluble in water, and thus, pretreatment is frequently required to enhance its solubility and/or to prepare it in an appropriate form for clinical use.Recent considerable efforts have focused on developing protocols to increase the solubility and bioavailability of realgar.Among the protocols, bioleaching, which is a simple and effective technology in hydrometallurgy, has attracted extensive interests [2].For instance, our group previously succeeded in developing a bio-arsenic aqueous solution from realgar particles by taking advantage of an arsenicresistant indigenous strain of A. ferrooxidans BY-3 using the bioleaching method and confirmed the anticancer activities of the developed bioarsenic aqueous solution in vitro and in vivo [3].
On the other hand, arsenic is known to be poisonous for many organisms [4].A high concentration of arsenic in the leaching solution could inhibit bacterial growth and activity, which have negative effects on realgar bioleaching.Based on our previous observations during the entire bioleaching process [3], the concentration of arsenic interestingly increased to a peak value at first and then decreased, instead of increasing continuously, but there have been no thorough studies on this reason for this phenomenon.The decrease in arsenic concentration may be attributed to the arsenic adsorption and/or precipitation.So far little is known about the arsenic adsorption and/or precipitation in the bioleaching of realgar.
Previous studies have shown that arsenic ions in aqueous solutions can be adsorbed on iron-based adsorbents (via chemical, electrochemical, and physicochemical processes) [5,6] or on microorganisms (via biosorption, bioprecipitation, bioaccumulation, and biotransformation) [7,8].Even though bacterial cells are known to play important roles in bioleaching, the surface properties of A. ferrooxidans with reference to arsenic tolerance and their function under high arsenic concentrations remain unclear.In addition, there is little information on arsenic mobility during the bioleaching of realgar using A. ferrooxidans in the presence of ferrous iron.Therefore, the current study aims to investigate arsenic precipitation using the combination of A. ferrooxidans and ferric iron compounds and to investigate the surface properties of A. ferrooxidans cell response to arsenic.

Microorganism.
The native strain of A. ferrooxidans, which was isolated from an abandoned copper mine in Baiyin, Gansu Province, China, was named BY-3 (CCTCC-M203071).The identities of the isolates were confirmed by phylogenetic analyses of 16S rRNA gene sequences and the 16S-23S intergenic spacer regions (GenBank accession numbers are DQ676505).The wild strain of A. ferrooxidans was adapted to successive increasing concentrations of the realgar in 9 K medium in a continuous mode [10].As a consequence, the arsenic-adapted strain A. ferrooxidans BY-3, which was able to tolerate up to 680 mg/L of As (III), was used to convert raw realgar into an aqueous solution [3].Bioleaching was performed in a 250 mL Erlenmeyer flask containing 100 mL distilled water and 4.469 g of FeSO 4 ⋅7H 2 O and 0.5 g of realgar with an initial pH value of 1.7 on a rotary shaker at 150 rpm and a constant temperature of 30 ∘ C. The control experiment was performed under similar conditions without realgar.

Analysis.
The experiments lasted for 30 days.Arsenic concentrations were measured by an inductively coupled plasma atomic emission spectroscope (ICP-AES, IRIS Advantage, Thermo Jarrell Ash Corporation, USA).The sample preparation method for ICP-AES analysis was previously described [3].All experiments were performed in duplicate at a minimum, and the average values were reported.The cell surface properties of A. ferrooxidans coated with gold after bioleaching were identified through JEOL field-emission scanning electron microscopy (FESEM, JSM-6701F, Japan) operated at 5 kV to 10 kV, and the images were captured using an image slave software.The microscopic features of A. ferrooxidans were observed through JEOL transmission electron spectroscopy (TEM; JEM-1230, Japan) operated at 100 kV and an FEI high-resolution transmission electron microscopy (HR-TEM; Tecnai F30, Japan) operated at 300 kV.The sample preparation method for the TEM analysis was previously described [11].The powder XRD analyses of A. ferrooxidans in the media with and without realgar were conducted on a multipurpose X-ray diffraction system (X'Pert-Pro MPD, Philips) with Cu K radiation ( = 0.15406 nm) and operated at 40 kV and 40 mA.The vibrational frequency changes on the surface of biomass in the media with and without adsorbed arsenic were detected using a Fourier transform infrared spectrometer (FT-IR) according to the procedures reported by Yan et al. [12].

Results and Discussion
3.1.Bioleaching of Realgar Using A. ferrooxidans.The realgar bioleaching mechanism was investigated to determine the direct or indirect nature of realgar.Even though defining the predominance of a mechanism is not easy, the importance of cell attachment to the particles to perform bioleaching is widely accepted [13].Several groups have reported the adhesion of A. ferrooxidans to sulphur and mineral surfaces [13,14].In the current study, the SEM image of the surfaces of the realgar particles after bioleaching demonstrated the adhesion behavior of A. ferrooxidans, as shown in Figure 1.Moreover, the cell adhesion caused by the interactions of minerals with A. ferrooxidans BY-3 has been shown to induce either the hydrophilicity or the hydrophobicity of the minerals and render them floatable or nonfloatable, respectively [14], which is consistent with the results of the current study.In addition, the precipitation of jarosite was visually observed in the process, and the SEM and XRD solid residue patterns confirmed the presence of jarosites.The jarosites on the substrate were expected to prevent direct bacterial action and/or iron (III) attacks.The results above confirmed that A. ferrooxidans is essentially attached to the minerals prior to the occurrence of the direct action.Therefore, metabolic energy would be obtained from the constituents released during the dissolution of a solid mineral and subsequently supplied to the suspended bacteria in the solution.The attached and free bacteria might exert combined action on the surfaces of realgar.

Arsenic Precipitation Using Living A. ferrooxidans Biomass.
Following an arsenic precipitation process, the A. ferrooxidans biomass was characterized using TEM (Figure 2).The physical integrity of the bacterial cells was maintained at a total arsenic concentration of 680 mg/L.A mass of electron-dense precipitates was clearly observed around the bacterial cells (Figures 2(b) and 2(c)).Further XRD analysis of the isolated precipitates revealed their abundance in arsenic (Figure 3).The above-mentioned results confirmed that arsenicbearing precipitates are attached to bacterial cells, which can adsorb arsenic from a solution.Therefore, these results may explain the concentration reduction of arsenic in the leachate during the bioleaching of realgar.Although the biosorptions of arsenite [iAs (III)], monomethyl arsenate [MMA (V)], and dimethylarsinic acid [DMA (V)] from aqueous solutions have been reported before [12], the arsenic precipitation using living A. ferrooxidans biomass during the bioleaching of realgar that led to arsenic ion accumulation is reported for the first time in the current study.On the other hand, A. ferrooxidans was found capable to efficiently enhance realgar dissolution and remove arsenic from the aqueous solution.Remarkably, these findings are promising for the development of a low-cost realgar pretreatment protocol as a substitute for the existing methodologies.

Arsenic Precipitation on Ferric Compound. Figure 3(a)
shows the XRD patterns of the ferric iron compounds derived from the oxidation of iron (II) using A. ferrooxidans BY-3 in the medium without realgar.Magnetite, jarosite, hydroniumjarosite, and ammoniojarosite were found in the arsenic precipitates.Iron or iron compounds (iron oxides, oxyhydroxides, and hydroxides, including amorphous hydrous ferric oxide [FeO-OH], goethite [-FeO-OH], magnetite [-Fe 3 O 4 ], and hematite [-Fe 2 O 3 ]) are the most widely used adsorbents exhibiting high arsenic removal efficiency [15].Consequently, realgar, mackinawite, ferric arsenate, potassium arsenate, and sodium hydrogen arsenite were observed in the medium with realgar (Figure 3(b)).The changes of arsenic concentration with time in the presence of ferrous iron are shown in Figure S1 (see Supplementary Material available online at http://dx.doi.org/10.1155/2013/424253).During the first 15 days, the arsenic concentrations of the medium with A. ferrooxidans (121 mg/L) were 6.9 times higher than those of the sterile control (17.59 mg/L).From the 15th to the 30th day, however, a decrease in arsenic content over time was observed (from 121 mg/L to 25.56 mg/L).These results suggest that the decrease in the extraction of arsenic was due to the uptake of arsenic by adsorbents. A. ferrooxidans efficiently enhances realgar dissolution under experimental conditions, corresponding to the catalysis described in (1).With the direct action of A. ferrooxidans used, the dissolution of elemental arsenic was observed during the bioleaching of realgar.Consequently, As (III) mainly exists in the form of H 3 AsO 3 , which can be easily oxidized to As (V) by A. ferrooxidans by the reaction described in (2).These assays can be described by the following equations: (1) This decrease was due to the generation of iron (III) by the abiotic oxidation of iron (II) described in (3).Furthermore, increasing the concentration of iron (III) enhanced the stimulative effect of iron (III) on realgar oxidation, shown in (3), resulting in the transformation of As (III) into As (V), shown in (2).The above results suggest that the reactions may have occurred as described in (2) and the following: Coprecipitation of Fe (III) with arsenic was the main process by which soluble arsenic was partially resorbed by jarosite and magnetite, apart from the biosorption of arsenic from aqueous solution by A. ferrooxidans BY-3 in the bioleaching process.These provide a reasonable explanation as to why the concentration of arsenic was reduced [3].
Therefore, these results indicate that increasing the concentration of ferric iron can enhance the stimulating effect of ferric iron on realgar oxidation, resulting in the transformation of arsenic acid (H 3 AsO 4 ) into ferric arsenate (FeAsO 4 ).Ferric arsenate, which is characterized by two broad diffuse peaks on the XRD pattern, is a common precipitate used in metallurgical industries to control arsenic in effluents [16].In the current study, ferric arsenate was the major existing form of arsenic precipitation on the ferric compounds in the realgar solutions.Therefore, the variations in the arsenic concentrations in the solutions were due to the occurrence of arsenic precipitation during the bioleaching.

Surface Properties of A. ferrooxidans with Reference to
Arsenic Tolerance.FT-IR measurements were further performed to investigate the main functional groups involved in the arsenic biosorption.The peaks at 3301, 2926, 1655, 1539, 1452, 1236, and 1079cm −1 in the FT-IR spectrum of unloaded biosorbent were assigned to the -OH, -NH groups, and -CH 2 , C=O stretching, -NH stretching, -CH, -SO 3 stretching, and -CN stretching vibration groups, respectively (Figure 4(a)).Figure 4(b) shows the changes in the FT-IR spectrum of the biomass after arsenic adsorption.However, the peaks shifted to 3413, 2962, 1640, 1541, 1421, 1196, and 1083 cm −1 , respectively, after arsenic loading.Since FT-IR spectra exhibited fingerprints of microenvironmental variations, the observed spectral changes vividly reflected that the corresponding functional groups were closely involved in the arsenic precipitation, especially the -OH and -NH groups, which experienced dramatic shifts as large as 112 cm −1 .The interaction between the functional groups on the surface of the biomass and realgar is considered as a critical factor that avoided significant bacterial inhibition and facilitated the bioprecipitation process.

Conclusion
SEM, TEM, XRD, and FT-IR analysis were used to reveal the characteristics of arsenic precipitation.All the results confirmed that precipitation of arsenic with A. ferrooxidans and iron compound can decrease arsenic concentration in solution.Thus, arsenic concentrations in leaching solution of realgar are thought to be reduced through the precipitation of arsenic.This study demonstrates that the combined action of biologic and chemical absorbents may play important roles in arsenic precipitation during the process of leaching realgar by A. ferrooxidans.

Figure 1 :
Figure 1: SEM image of the surfaces of realgar interactions with A. ferrooxidans BY-3 after bioleaching of realgar (scale bar = 100 nm).

Figure 4 :
Figure 4: FT-IR difference spectra of A. ferrooxidans BY-3.The FT-IR spectra were recorded using a FT-IR (NEXUS 670, Thermo Nicolet, USA) over the wave number range of 500 cm −1 to 4000 cm −1 under ambient conditions.a: Pristine: A. ferrooxidans BY-3 in the medium without realgar.b: As-loaded: A. ferrooxidans BY-3 in the medium with realgar.