2.1 Microorganism, medium, and strain domestication

Four acidophilic strains, A. ferrooxidans (30 ℃), L. ferrooxidans (30 ℃), L. ferriphilum YSK (45 ℃) and S. thermosulfidooxidans (45 ℃) were obtained from the Key Laboratory of Biometallurgy of Ministry of Education, China. Strains were grown in 9 K medium ((NH₄)₂SO₄ 3 g/L, KCl 0.1 g/L, K₂HPO₄ 0.5 g/L, MgSO₄·7H₂O 0.5 g/L, Ca(NO₃)₂ 0.01 g/L) at 180 r/min and their specific optimal temperatures. Additional 0.02% yeast extract was added when growing S. thermosulfidooxidans. The medium was adjusted to pH 2.0 and autoclaved for 20 min at 121 ℃. Since the presence of excessive Cl^-, NH₄⁺ or Fe³⁺ could inhibit ferrous oxidation and growth of the bacteria [9, 31, 32], bacteria acclimation to FeCl₂·4H₂O, FeCl₂·4H₂O and (NH₄)₂Fe(SO₄)₂·6H₂O was conducted. Each strain was first cultured with initial concentration of 50 g/L FeSO₄·7H₂O, 20 g/L FeCl₂·4H₂O, and 50 g/L (NH₄)₂Fe(SO₄)₂·6H₂O respectively. Then the concentration of the substrate gradually increased to acclimate the bacteria until the concentration reached 150, 59.6, and 150 g/L, respectively. Acclimated bacteria were cultivated for 2 more generations until Fe²⁺ oxidation rate was stable.

2.2 Sample preparation

Acclimated bacteria were harvested at the end of exponential phase (about four days). Cultures were first filtered by 0.45 μm filter paper to remove the precipitate. Then the filtrate was centrifuged at 12000 r/min for 20 min to harvest the cells. Cells were washed twice and resuspended in distilled water. Then each strain cultured with different substrate was added into 200 mL double distilled water (pH 2.0) containing only 30 g FeSO₄·7H₂O, 11.92 g FeCl₂·4H₂O, and 30 g (NH₄)₂Fe(SO₄)₂·6H₂O respectively. The cell density in these cultures was adjusted to , determined by microscopic counting prior to incubation. Under these conditions, the bacteria cannot proliferate but still possess ferrous oxidation capability. By the seventh day of incubation, the precipitate was collected with 0.45 μm filter paper through filtration, washed twice with distilled water and then dried at room temperature.

2.3 Cr(VI) adsorption experiment procedures

Adsorption experiments were conducted in 50 mL centrifuge tubes with Cr(VI) concentration (potassium dichromate) at 50 mg/L consisting of 0.01 mol/L NaNO₃ as background electrolyte. The tubes were added with 0.1 g adsorbent and 20 mL pH 7.5 potassium dichromate solutions correspondingly. Then tubes were incubated for 240 min at 180 r/min, 30 ℃. For each tube, a total of 12 supernatant samples (200 µL each) were taken from 5 min to 240 min. Cr(VI) concentration in the supernatant was detected by spectrophotometer after 5 min following the 1,5-diphenyl-carbazide method. Adsorption isotherm batch experiments were conducted over a range of Cr(VI) concentrations from 5 to 250 mg/L. All centrifuge tubes were shaken for 240 min.

2.4 Analytical methods

Scanning electron microscope (SEM) analysis was performed with a JSM-6360LV instrument operated at 15 kV accelerating voltage. Energy dispersive spectrum (EDS) analysis was applied to the samples to quantitatively identify the major elements with rough estimates of composition based on relative peak intensities. X-ray diffraction analysis was conducted with Cu Kα radiation (40 kV/250 mA) in a RINT2000 vertical goniometer. Samples were scanned from 5° to 80° with a step increment at 0.02° and 4 s counting time. The FT-IR spectra were taken on a Nicolet Nexus670 Fourier transform spectrometer at a resolution of 4 cm^-1 using a KBr beamsplitter, a DTGS detector with KBr window, and a sample shuttle for the transmittance measurements. The background was taken on a disk made from 400 mg KBr. XANES analyses of the Cr-K edges and the Fe-K edges were performed in transmission mode at the BL14W1 XAFS beam line at the Shanghai Synchrotron Radiation Facility (SSRF). Data were analyzed using the Athena program.

The experimental data were fitted with kinetics models containing pseudo first-order, pseudo second-order, and intra-diffusion particle models.

(1)

(2)

(3)

(4)

where q_e and qt are the amount (mg/g) of adsorbed Cr(VI) on materials at equilibrium and at time t, respectively; k₁ is the first order rate constant (min^-1); k₂ (g/(mg·min)) is the rate constant of pseudo-second-order; h (mg/(g·min)) is the initial sorption rate; k_i is the intraparticle diffusion rate constant (mg/(g·min^0.5)), which can be calculated from the slop of the linear plots of qt versus t^0.5.

The batch experimental data were fitted by the Langmuir and Freundlich isotherm models represented mathematically as follows.

(5)

(6)

where Q_e is the amount of Cr(VI) adsorbed on materials at different concentrations; q_m is the saturated adsorption capacity; K_L is a constant of the Langmuir isotherm and C_e is the equilibrium Cr(VI) concentration remained in the solution; K_F is Freundlich constant, which indicates the relative adsorption capacity of the adsorbent; 1/n is the heterogeneity factor and is known as Freundlich coefficient.

3.1 Characterization of biosynthesized iron-minerals

We constructed secondary minerals synthesizing systems using different acidophilic bacteria and detected the variation in pH and ORP, which reflected the dynamic change process of precipitate formation (Figure S1). The rising trend of pH value observed in the early stage can be attributed to the acid-consuming Fe(II) oxidation process. The subsequent dropping trend is due to the Fe(III) hydrolysis and precipitation. However, the pH in the Akaganéite formation system mediated A. ferrooxidans and L. ferrooxidans is inconsistent with others. The ORP values display a steady uptrend, which illustrates the effective oxidation of ferrous ions. These changes indicate that different bacteria mediate similar iron oxidation processes, but the oxidation rate may differ, leading to the distinction in synthetic precipitation.

The SEM images and corresponding EDS data of the precipitate samples are presented in Figures 1 and S2. Sh-Af(30) and Sh-Lf(30) showed similar structures; both of the precipitates were spherical aggregates and covered by “pincushions”. This type of loose structure insures high surface area of the precipitate. Surface structure of Sh-Lf(45) was much more regular. Irregular cubic existed in Sh-St(45), which could be caused by the higher culture temperature and greater ferrous oxidation ability, as both lead to a faster growth of the precipitate and inhibit the formation of the hierarchy structure [31]. Akaganéite formed by different bacteria all presented as rod shaped crystal. Samples Ak-Lf(30) and Ak-Lf(45) showed as aggregated clusters. Ak-St(45) was smaller and longer than the others and presented as needle crystal. All four samples of ammoniojarosite were cubic shaped while the size of Ja-Lf(45) and Ja-St(45) was more angular and bigger which may also be the result of faster precipitate growth.

Figure 1全屏查看

The SEM images of the precipitate: Schwertmannite (a-d), Akaganéite (e-h) and ammoniojarosite (i-l) biosynthesized by A. ferrooxidans (30 ℃), L. ferrooxidans (30 ℃), L. ferriphilum YSK (45 ℃), and S. thermosulfidooxidans (45 ℃) respectively

Furthermore, EDS analysis revealed the specific composition of the precipitates (Table S1 and Figure S2). Schwertmannite is generally described as the chemical formula Fe₈O₈(OH)_8-2x(SO₄)x, in which x is determined by Fe/S molar ratio [33]. The compositional formula of each precipitate produced in our study was consistent with prior study except for Sh-St(45), which was smaller than 4.3, a value usually considered as the lower limit [9, 33]. The chemical formula of Akaganéite can be summarized as Fe₈O₈(OH)_8-xClx. Although differences in ferric, chloride and oxygen content between materials are not significant, diversity in shape, size, and crystal size still presented, which difference may be rooted in bacteria. As to the inner structure of Akaganéite, the tunnel is partly occupied by Cl^- which is essential for its stability [34]. This tunnel position was replaced by SO₄^2- instead of Cl^- in analogous Schwertmannite and caused the difference in elemental composition, color and crystallinity [35]. Since report on chemical structure of ammoniojarosite is not available yet, here we refer ammoniojarosite as Fe₈O₈(OH)₈₊y_-2x(NH₄)y(SO₄)x (the relative value x, y depended on mole ratio of Fe/S and Fe/N). As shown in Table 1, the ferric content of ammoniojarosite was lower than that of Schwertmannite and Akaganéite, which could be an important factor causing the difference in its structure and adsorption performance. The chemical form of Ja-Lf(45) and Ja-St(45) can be summarized as Fe₈O₈(OH)_3.01(NH₄)_3.14(SO₄)_4.06 and Fe₈O₈(OH)_0.76(NH₄)_3.76(SO₄)_5.50 respectively. Compared to Ja-Af(30) and Ja-Lf(30) (Fe₈O₈(OH)_4.85(NH₄)_5.62(SO₄)_4.38, Fe₈O₈(OH)_4.55(NH₄)_6.31(SO₄)_4.88), Ja-Lf(45) and Ja-St(45) have much lower content of —OH, —NH₄, which may contribute to their bigger crystal structure.

XRD patterns (Figure 2(a)) of Schwertmannite formed in the pure FeSO₄ system are quite similar; no single, sharp and well recognizable peaks were observed, except the precipitate formed by L. ferriphilum YSK (45 ℃) and S. thermosulfidooxidans (45 ℃), which had better crystallinity due to the higher formation temperature and heterogeneity of bacteria. Schwertmannite belongs to non-crystal substances, so there is no specific data on grain size. Patterns of Akaganéite indicated a lower crystalline compared with previous reports [9]. This difference may be the result of different substrate compositions since only pure FeCl₂ with no other monovalent cation existed in the formation system. XRD pattern and its corresponding peak number (Figures 2(b), (c) and Table S1 XRD peak) suggest that ammoniojarosite (peak numbers 13, 16, 19, 18) had a much better crystallinity than Akaganéite (peak numbers 6, 2, 4, 2). XRD peaks of ammoniojarosite were sharp and well recognizable, and the grain size was greater than that of others (390, 411, 694, 499 nm vs 381, 179, 358, 167 nm).

Figure 2全屏查看

XRD patterns (a, b, c) and FT-IR spectra of the precipitates (d, e, f)

The above results indicate that bacteria affect biosynthesized iron minerals’ atomic composition, crystallinity and molecular structure, which may affect their adsorption performance. Thus, a series of relevant experiments were carried out to verify this speculation.

3.2 Cr(VI) adsorption capacity of biosynthesized iron-minerals

The adsorption capacity of Schwertmannite, Akaganéite and ammoniojarosite formed by A. ferrooxidans, L. ferrooxidans, L. ferriphilum YSK, and S. thermosulfidooxidans is presented in Figure 3. The average adsorption capacities of Akaganéite, Schwertmannite, and ammoniojarosite were 25.2, 13.8, and 4.10 mg/g, respectively, which are tightly related with the nature of the materials that Akaganéite, Schwertmannite have tunnel structure while ammoniojarosite has crystal structure without tunnel structure. In addition, it is worth noting that the absorption performance of the same type of biosynthesized mineral varied when produced by different bacteria. Schwertmannite, ammoniojarosite formed by L. f(30) (15.7, 4.37 mg/g) exhibited the highest adsorption capacity compared with A. f (14.9, 4.22 mg/g), L. f(45) (13.4, 3.95 mg/g), S. t (11.3, 3.86 mg/g). In contrast, Akaganéite formed by S. t (27.6 mg/g) exhibited the excellent adsorption capacity compared with A. f (23.4 mg/g), L. f(45) (24.4 mg/g), L. f(30) (25.3 mg/g). Moreover, compared with Sh-St(45), Sh-Lf(45) possessed better adsorption capacity owing to its weaker crystallinity. These results indicated that the difference of Cr(VI) adsorption capacity is related to the composition, morphology, size, structure, and crystallinity disparity of the minerals, and initially, it may be influenced by the biosynthesizing subject the bacteria.

Figure 3全屏查看

Adsorption capacity (Q_e) of the biosynthesized iron minerals by different bacteria

3.3 Cr(VI) adsorption kinetic of biosynthesized iron-minerals

Schwertmannite, Akaganéite and ammoniojarosite formed by A. ferrooxidans were used to investigate adsorption kinetics and mechanism. As can be seen from Figure 4, among three types of material, ammoniojarosite reached the adsorption equilibrium first, and its initial sorption rate of 0.696 was the lowest among these materials. As for the other two types of material, the removal efficiency was 25.0% and 32.9% for Schwertmannite and Akaganéite after 5 min adsorption, while removal efficiency of 64.9% and 85.6% was obtained after 60 min adsorption, respectively. Moreover, the initial sorption rate reached 0.972 and 1.26 for Schwertmannite and Akaganéite, respectively. The difference in Cr(VI) adsorption kinetics among the three materials may be attributed to their crystal structure. ammoniojarosite is known to exhibit hexagonal, pseudo-cubic structure with no tunnel structure [19, 36]. Thus, we propose that Cr(VI) adsorption occurs on the surface sites which leads to the fast adsorption equilibrium and relatively low adsorption capacity, which is consistent with its tunnel structure and elemental composition. However, the adsorption equilibrium of Cr(VI) on Schwertmannite and Akaganéite is on the order of hours, indicating an alternative adsorption mechanism or formation of a novel chemical bond between the Cr(VI) species and adsorption surface and tunnel structures [37].

Figure 4全屏查看

Kinetics of Cr(VI) removal by biogenic Schwertmannnite, Akaganéite and ammoniojarosite: Pseudo first-order (solid red lines) and pseudo second-order (dash blue lines) kinetic plots (a), intra-diffusion particle models (b)

The internal micro-porous process of adsorbate diffusion into adsorbent porous takes time, and the intra-particle diffusion model can be applied to further explain the assumption [16, 38, 39]. According to the intra-diffusion particle model in Table 1, the order of adsorption rate was K_i1>K_i2. At the beginning, Cr(VI) was adsorbed by the exterior surface of the materials, and the first stage was the instantaneous diffusion period (k_i1). When exterior surface reached saturation, the heavy metal ions entered into the mesopores on the second stage. With Cr(VI) diffusing into the pores, the diffusion resistance increased, resulting in the decrease of diffusion rates (k_i2). The intra-particle diffusion rate constant of Akaganéite and Schwertmannite on second stage was 1.360 and 0.278 mg/(g·min^0.5) respectively, while the constant of ammoniojarosite was only 0.0430 mg/(g·min^0.5), which illustrated the adsorption on second stage not significant [40]. This suggests that the deficiency of tunnel structures determined inferior adsorption capacity of ammoniojarosite.

3.4 Cr(VI) adsorption isotherm of biosynthesized iron-minerals

It was clearly seen from Figure 5 that the values of Q_e of three materials became significantly larger with increased Cr(VI) concentration and then nearly reached the platform. Among them, ammoniojarosite and Schwertmannite reached the platform at the Cr(VI) concentration of 31.5 and 124.7 mg/L, respectively, and the increase of q_e values of Akaganéite nearly reached the platform until the Cr(VI) concentration of 133.1 mg/L. Moreover, the order of three materials’ best Cr(VI) adsorption capacity is Akaganéite > Schwertmannite > ammoniojarosite. Then, the batch experimental data were fitted by the Langmuir and Freundlich isotherm models, which were represented mathematically. Table 2 presents the parameters of the Langmuir and Freudlich isotherm. Three materials’ Cr(VI) adsorption process is satisfactory for the Langmuir isotherm, with all correlation coefficients (R²) being over 0.97. The description of Freudlich isotherm on Schwertmannite is relatively poor (R²=0.842) but well with other materials (R²=0.975). Thus, it is clear that Langmuir isotherm better described the adsorption process of Schwertmannite and both models fit well with Akaganéite and ammoniojarosite, and all the fitting curves meet the detection data’s expectations. The magnitude of the Freundlich coefficient 1/n is an index of the favorability of the adsorption reaction [41], and the value of 1/n<1.0 generally represents an advantageous adsorption condition [42]. The corresponding values of the three types of materials are 0.350, 0.333 and 0.113, which indicate favorable Cr(VI) adsorption occurred for all three materials. The fractional value of 1/n is assigned due to the heterogeneous nature of the adsorbent surface. Moreover, the maximal adsorption capacities of Akaganéite, Schwertmannite and ammoniojarosite were 26.6, 17.5 and 3.90 mg/g, respectively, exceeding many iron minerals as shown in Table 3, especially Akaganéite.

Figure 5全屏查看

Langmuir and Freundlich non-linear plots of adsorption isotherms for Cr(VI) on Schwertmannnite, Akaganéite and ammoniojarosite

The Langmuir isotherm equation and the Freundlich equation both represent different adsorption forms. The former simulates monolayer adsorption of adsorbate onto a homogeneous adsorbing surface without lateral interaction between adsorbed molecules, and the latter postulates a multilayer adsorption process onto heterogeneous adsorption sites [43]. Therefore, their fitting results suggest that the adsorption capacities of Akaganéite, Schwertmannite and ammoniojarosite are determined by their composition as analyzed above and highly related to their crystal structures. Both Akaganéite and Schwertmannite have well-organized tunnel structures [44], allowing chromium to penetrate through the tunnel structure and facilitate the adsorption process with enhanced adsorption capacity. Correspondingly, the poor adsorption performance of ammoniojarosite was due to its highly crystalline, cubic shape, low specific surface binding area and non-tunnel structure. Based on the analysis above, we can conclude that molecular structure is the primary determining factor for adsorption performance.

3.5 Adsorption mechanism of Cr(VI) on biosynthesized Iron-minerals

The XPS spectra of S 2p and Cr 2p in Figure S3 show that SO₄^2- was the sulfur component in the three minerals, and the valence state of chromate had not changed after adsorbed to minerals. In addition, the atomic concentration of Cr on ammoniojarosite surface area was the highest, which is completely opposite to its adsorption capacity. According to the XPS spectra of Fe 2p, the atomic concentration of Fe on the material surfaces reached 16.8%, 12.6% and 9.73% in Akaganéite, Schwertmannite and ammoniojarosite respectively (Figure S3(c), Table S2). Moreover, their Cr(VI) adsorption capacity (Table 2) was consistent with the Fe atomic concentration. Therefore, we hypothesized that Fe plays a critical role in Cr(VI) removal through processes such as adsorption [45, 46]. Although all materials had considerable surface carbon content, ammoniojarosite in particular had the highest, suggesting that bacteria absorbed on the mineral surface and mediated the formation process (Figure S3(d)). Since extracellular polymeric substances or metabolites of bacteria may play a critical role in material nucleation, grading and growth, biosynthesized materials would be varied when different bacteria are involved.

The FT-IR spectra of minerals of minerals before and after adsorption of Cr(VI) are shown in Figure 2. The intense absorption observed in the region 3000 to 3400 cm^-1 can be attributed to O—H stretching, which were present in all of the twelve samples and is important for the stability of iron oxyhydroxides and subsequent adsorption process. The band observed around 1610 cm^-1 indicates H—O—H deformation. Other peaks existed in Schwertmannite and ammoniojarosite including bands at 1200, 1070 cm^-1 and 630, 680 cm^-1 are due to v₃ and v₄ vibration mode of the sulfate [47-49]. Note that sulfate peak transmissivity of sample Sh-St(45) was only 20%, which indicate the lower sulfate content in Sh-St(45) that could lead to the formation of cubic in the precipitate. The bands around 850 and 670 cm^-1 are vibration modes of the two O—H···Cl hydrogen bonds present, which are characteristic of chloride containing Akaganéite [9]. An obvious adsorption band around 1429 cm^-1 was observed in sample ammoniojarosite, confirming the presence of NH₄⁺. However, the band intensity of O—H, sulfate and O—H···Cl of both materials (Figure S4) with tunnel structure obviously reduced after adsorption of Cr(VI), this is the direct evidence that the adsorption occurred through a combination of surface complexation with active hydroxyl groups, exchange with both surface adsorbed, structural sulfate sites and O—H···Cl sites. These results suggest that the mechanism of Cr(VI) adsorption on these materials involves ligand exchange, similar to the arsenic adsorption mechanism on Schwertmannite and Akaganéite [11, 37].

To further verify the above speculation, XAFS was used for in-depth analysis. XAFS analysis of Cr showed that there were obvious Cr(VI)—O covalent bonds on the surfaces of three minerals after adsorption (Figures 6(a) and (b)), which may be due to the adsorption of chromate on the mineral surface consistent with the XPS data of Cr 2p or may be formed by the combination of chromate and O—H on the mineral surface according to the FT-IR spectra. In addition, the XAFS analysis of Fe in minerals also can be explored. The data in k-space and R-space prove that the adsorption of Cr(VI) has no effect on minerals about the formation of new covalent bonds and the disappearance of the original covalent bonds (Figures 6(c) and (d)), indicating that the adsorption pathways without the destruction of mineral structure, such as electrostatic adsorption and ligand exchange, were the main contributors to the adsorption of Cr(VI) [50-52]. Moreover, the components of iron minerals before and after adsorption were also analyzed (Figure 6(e), Table 4). Akaganéite has Fe(III)-O (99.6%) and Fe(III)-Cl (0.6%), while the adsorption of Cr(VI) makes the Fe(III)-Cl disappears completely, indicating that the O—H‧Cl sites in Akaganéite may be replaced by chromate. FeSO₄^2- is the Fe-components of ammoniojarosite, which will partly change to Fe(III)-O (6.90%) and Fe-OH (12.9%) after adsorption of Cr(VI), demonstrating that SO₄^2- in ammoniojarosite was partially replaced, moreover, the mineral was partly dissolved and r e-precipitated to form FeOOH. Similar changes have also been discovered in the system of adsorption of Cr(VI) by Schwertmannite. The FeSO₄^2- in Schwertmannite decreased from 48.4% to 36.5%, while Fe(III)-O and Fe-OH increased from 51.5%, 0 to 56.2%, 7.30%, respectively. These results indicate that the replacement of SO₄^2- with CrO₄^2-, the dissolution and reprecipitation of the minerals also occur in the system of adsorption of Cr(VI) by Schwertmannite.

Figure 6全屏查看

K3-weighted chi spectra (a) and Fourier-transformed real part (b) of Cr; K3-weighted chi spectra (c) and Fourier-transformed real part (d) of Fe; Fe K-edge XANES spectra of minerals (blue line), best fit (red line), and reference standards (e). Abbreviations: Ak=Akaganéite (Ja=ammoniojarosite, Sh=Schwertmannite, Ak (pristine)=primitive Akaganéite, Ak (adsorb)=Akaganéite adsorbed with Cr(VI))

Based on the above results, the schematic illustration of the Cr(VI) adsorption mechanism by ammoniojarosite, Akaganéite, and Schwertmannite is shown in Figure 7. The tunnel free crystal structure of ammoniojarosite makes its adsorption of Cr(VI) only have one stage, which occurs in the early stage of the reaction. On the surface of ammoniojarosite, the hydrogen bonding between Cr(VI) and O—H, the electrostatic interaction between Cr(VI) and mineral, and the ligand exchange between CrO₄^2- and SO₄^2- contributed to the adsorption of Cr(VI). The tunnel structure of Akaganéite brings two stages of Cr(VI) adsorption: on the surface of Akaganéite, hydrogen bonding, electrostatic interaction, and ligand exchange between CrO₄^2- and O—H·Cl leads to the adsorption of Cr(VI), and then, Cr(VI) will enter into the mesopores for new adsorption when exterior surface reached saturation. Owing to the tunnel structure similar to that of Akaganéite, Schwertmannite also have two stages of adsorption of Cr(VI) on the mineral surface and mesopores through hydrogen bonding, electrostatic interaction, and ligand exchange between CrO₄^2- and SO₄^2-.

Figure 7全屏查看

Schematic illustration of the Cr(VI) adsorption mechanism by ammoniojarosite (a), Akaganéite (b), and Schwertmannite (c)