Materials Science & Engineering C 111 (2020) 110755

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Materials Science & Engineering C

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Formation and stability of biogenic tooeleite during Fe(II) oxidation by T

Acidithiobacillus ferrooxidans
Qingzhu Lia,b,c, Mengxue Zhanga, Jinqin Yanga, Qianwen Liua, Guanshi Zhanga, Qi Liaoa,b,c,
⁎
Hui Liua,b,c, Qingwei Wanga,b,c,d,
a
School of Metallurgy and Environment, Central South University, Changsha, 410083, China
b
Chinese National Engineering Research Center for Control & Treatment of Heavy Metal Pollution, Changsha 410083, China
c
Water Pollution Control Technology Key Lab of Hunan Province, Changsha 410083, China
d
Shandong Humon Smelting Co., Ltd., Yantai, 264109, China

A R T I C LE I N FO A B S T R A C T

Keywords: Tooeleite is the only known ferric arsenite sulfate mineral and has environmental significance for arsenic re-
Arsenic mediation. This study investigated the formation and stability of biogenic tooeleite in Fe(II)-As(III)-SO42− en-
Biogenic tooeleite vironment using Acidithiobacillus ferrooxidans under the ambient conditions. The results show that bacteria fa-
Acidithiobacillus ferrooxidans cilitated the formation and crystallization of tooeleite owing to the microbial oxidation of Fe(II) to Fe(III). Due to
Ferrous oxidation
the better growth of bacteria, the higher removal of As(III) by tooeleite formation was achieved under
Stability
8.978–10.806 g/L initial Fe(II) concentration and 2.00–3.00 initial pH, and the highest efficiency was ~95%. Fe
(III) and As(III) precipitated simultaneously into two types of tooeleite. The relatively stable tooeleite is featured
by the developed (020) crystal face and the bulk-like structure with thick flakes. This study yields a better
understanding of biogenic tooeleite, and the importance of tooeleite formation in As(III)-rich environment for
arsenic remediation.

1. Introduction
Tooeleite (Fe6(AsO3)4(SO4)(OH)4·4H2O) is the first and unique ar-
senite-sulfate mineral known, with an arsenic content of 25 wt%, and
Fe/As molar ratio of 1.50 [1,2]. The mineral occurs naturally as a
secondary As-bearing phase with pyrite, arsenopyrite, scorodite, and
jarosite in the oxidizing environments, such as ore deposits, waste
dumps, slag localities, and acid mine drainages [1,3–5]. It is potentially
regarded as an attractive material for arsenic immobilization, due to a
low iron demand and a high As(III) removal efficiency during its for-
mation [6].
The occurrence of tooeleite in nature, at Gold Hill District, Tooele
County, Utah, USA, was firstly described by Nolan in 1935 [7]. In 1992,
Cesborn and Willams reported the physical, chemical, and optical
characteristics of the field samples [1]. Tooeleite was initially assumed
as an As(V) mineral [1], but later confirmed as As(III)-bearing phase by
XANES, high-resolution synchrotron XRD, Rietveld refinement, TG-
DTA, SEM and XPS analyses, based on the field and chemogenic sam-
ples [2,5,6]. Efforts were also put to investigate the mineral features,
including spectra (infrared, Raman) [8] and thermodynamic properties
[9]. More importantly, field tooeleite found in the waste dumps re-
mained unchanged under severe weathering conditions over time,
which may support the mineral as a potential arsenic disposal alter-
native [6].
Chemogenic tooeleite can be easily obtained under ambient condi-
tions. Samples were synthesized by mixing Fe(III) and As(III) stock
solutions directly under a temperature (T) of 25–90 °C and an initial pH
of 1.8–3.0 for 1–30 days [6], or presented as the dominant mineral
phase when agitated under a Fe(III)/As(III) molar ratio of 1.0–2.0 and
constant pH of 2.0–4.0 for 1 h (T = 25 °C) [10]. Up to 99% As(III) was
removed by forming chemogenic tooeleite under conditions of
pH 1.8–4.5, initial As(III) concentration > 0.75 g/L, and Fe/As 0.8–2
at room temperature [11]. However, the As leaching toxicity of this
mineral is difficult to reach the standard. The As leached from tooeleite

Abbreviations: AMD, acid mine drainage; DO, dissolved oxygen; EDS, energy dispersive X-ray spectroscopy; EPS, extracellular polymeric substances; FTIR, Fourier
transformed infrared; ICP-OES, inductively coupled plasma-optical emission spectroscopy; OD, optical density; SEM, scanning electron microscopy; TCLP, Toxicity
Characteristic Leaching Procedure; TG-DTA, thermogravimetric-differential thermal analysis; XANES, X-ray absorption near edge structure; XPS, X-ray photoelectron
spectroscopy; XRD, X-ray diffraction
⁎
Corresponding author at: School of Metallurgy and Environment, Central South University, Changsha 410083, China.
E-mail address: qw_wang@csu.edu.cn (Q. Wang).

https://doi.org/10.1016/j.msec.2020.110755
Received 21 November 2018; Received in revised form 6 January 2020; Accepted 15 February 2020
Available online 18 February 2020
0928-4931/ © 2020 Elsevier B.V. All rights reserved.
Q. Li, et al.
was above 100 mg/L, much higher than the arsenic permitted con-
centration in the leachate (5 mg/L) [12]. Our previous work and Opio
also showed a high TCLP As leachability of chemogenic tooeleite
[10,11]. To solve this problem, tooeleite was calcinated under 600 °C
[10], or coated by silica using hydrothermal method under 120 °C [13].
The high temperature and pressure applied in these methods are not
applicable to industrial treatment. Moreover, as the phase after calci-
nation changed into ferric arsenate calcine, the leaching of calcine is
not the real leachability of tooeleite. The unsatisfactory As leachability
of chemogenic tooeleite raised the question whether this mineral can be
applied in the arsenic remediation.
Biogeochemical processes are believed to have environmental sig-
nificance and control the fate of arsenic [14–18]. Several mechanisms
(single or combined) have been invoked to explain interactions between
arsenic and microbes. These include uptake, precipitation, extrusion,
methylation, oxidation, reduction, and even assimilation into biomo-
lecules [19–22]. The biologically-induced mineralization of As-bearing
iron minerals often occurs in Fe-As-rich environments [19,21,23]. In
Regious Creek (France), 20–60% of arsenic was removed over the years
due to the formation of tooeleite and other arsenic-bearing minerals
[24]. Bacterial formation of tooeleite was confirmed to play an im-
portant role in As(III) removal in the wet season, because micro-
organisms were able to selectively oxidize Fe(II) to Fe(III), but do not
oxidize As(III) to As(V) [5,9]. The precipitation may be controlled by
the oxidation rate of Fe(II) mediated by various Acidithiobacillus fer-
rooxidans strains [25]. Since the field minerals found in AMD are linked
to bacterial activities [5,26], similar characteristics between field and
biogenic tooeleite may exist (e.g. morphology [5]); whereas few con-
tributions reported the synthesis and stability of biogenic tooeleite,
whether the As leachability of biogenic tooeleite is acceptable is still
unknown.
Thus, the objective of this study is to investigate the formation and
stability of biogenic tooeleite by Acidithiobacillus ferrooxidans ATCC
23270. The role of bacteria, formation conditions and stability of bio-
genic tooeleite are described in detail. The characteristics of chemo-
genic and two biogenic minerals are compared. The scheme of biogenic
tooeleite formation is proposed.
2. Materials and methods
2.1. Strains domestication and culture conditions
Acidithiobacillus ferrooxidans ATCC 23270 (A. ferrooxidans) were
obtained from the American Type Culture Collection. The bacteria were
grown on modified 9 K medium, which consists of 4.47% (w/v)
FeSO4·7H2O solution and 10% (v/v) basal salt solution. The basal salt
solution contains: 5 g/L (NH4)2SO4, 5 g/L MgSO4·7H2O, 5 g/L KH2PO4,
and 1 g/L KCl. It reduced the content of (NH4)2SO4 and removed Ca
(NO3)2, to avoid/decrease the production of jarosite-type precipitates,
because jarosite easily forms in the monovalent cation-rich water
[27–29]. The basal salt solution was autoclaved with deionized water at
121 °C for 15 min, cooled at room temperature, and mixed with the Fe
(II) solution before inoculation. Bacterial cell concentration was de-
termined by counting using Thoma cells [25]. The concentration of A.
ferrooxidans after inoculation was 105 cells/mL. The pH of the solution
was adjusted to 2.00 with H2SO4 after adding As(III).
Sodium arsenite (NaAsO2) was used as As(III) source. Fe(II) oxida-
tion rate is an indicator of A. ferrooxidans growth [27]. To ensure the
survival and reproduction of bacteria in the arsenic-containing solution,
the Fe(II) oxidation was monitored during the domestication. Before
being inoculated into the higher level of As(III), A. ferrooxidans were
cultivated repeatedly until Fe(II) oxidation completed within 3 days.
The concentration of As(III) gradually increased from 0.288 g/L to
4.5 g/L. Finally, the domesticated bacteria can oxidize Fe(II) within
3 days under the As(III) concentration up to 4.5 g/L.
All chemicals in this study were analytical reagent grade or
2
Materials Science & Engineering C 111 (2020) 110755
equivalent analytical purity. All reagents were purchased from
Sinopharm Group Chemical Reagent Co., Ltd., except for NaAsO2 from
Shuikoushan Mining Bureau of Hengyang Industrial Company in Hunan
Province. Deionized water was used. The Fe(II), Fe(III) and As(III) stock
solutions were prepared by FeSO4·7H2O, Fe2(SO4)3 and NaAsO2, re-
spectively. All Fe(II) solution and As(III) solution were filter-sterilized
by 0.22 μm membrane before use. All the pH values were adjusted by
H2SO4 and NaOH. All cultivations (domestications and batch assays)
were aerobically grown at 30 °C in 500 mL Erlenmeyer flasks on a
horizontal rotary shaker at 170 rpm.
2.2. Batch experiments and analytic methods
Three series of laboratory batch assays (designated as batch assay 1,
2, 3) and one chemical control experiment were performed. The ex-
perimental procedure and conditions are summarized in Fig.S1 and
Table S1. In this study, chemogenic tooeleite is referred to the solid
formed by chemical methods without the participation of active mi-
crobial metabolism; biogenic sample represents the solid collected from
the process dominated by microorganisms.
The first batch assay was designed to confirm the role of bacteria in
the formation of tooeleite in a synthetic Fe(II)-As(III)-rich water. The
concentration of bacteria was 0 or 105 cells/mL after inoculation, la-
beled as “Control” and “A. ferrooxidans”, respectively. The initial so-
lution contained 8.978 g/L Fe(II) and 2.997 g/L As(III), having a pH of
2.50. The media were cultivated for 5 days.
The other two batch assays were conducted to investigate biogenic
tooeleite formation, As(III) removal and the stability of tooeleite under
different Fe(II) concentrations and pH values. The concentration of A.
ferrooxidans was 105 cells/mL after inoculation. The initial Fe(II) con-
centrations in batch assay 2 were 3.595, 5.383, 7.191, 8.978 and
10.806 g/L, respectively. The initial pH values in batch assay 3 were
1.60, 1.80, 2.00, 2.20, 2.50 and 3.00, respectively.
The chemical control experiment was to study the influence of slow
supply rate of Fe(III) during tooeleite formation. To imitate the release
of Fe(III), the Fe(III) solution was pumped into the As(III)-containing
solution by peristaltic pump for 3 days (rate: 2.993 g/(L·d)). 10%(v/v)
culture suspension (after incubation) was added into the As-containing
solution before reaction, to eliminate the effect resulted from other ions
in the medium. The cells were killed by ultraviolet. The culture sus-
pension without cells was prepared by filtration using 0.22 μm mem-
brane. The initial pH of all stock solutions was set at 2.00. The mixed
solution was stirred at 170 rpm under room temperature during the Fe
(III) pumping.
All bacteria used in this study were prepared in advance. They were
cultivated under the condition of 8.978 g/L Fe(II), 0.2884 g/L As(III)
and pH 2.00 until the Fe(II) oxidation was completed (i.e. bacteria
entered into the stationary stage, ~3 days). 0.2884 g/L As(III) was to
maintain the bacteria arsenic-resistant performance. The culture sus-
pension (10%(v/v)) was inoculated into flask after being filtered by
Whatman filter paper to remove precipitates. The concentration of Fe
(III) and As(III) introduced by the inoculation were taken into the
calculation of removal efficiency.
Experimental processes were monitored by sampling at intervals
and samples were filtered immediately. The filtrates were acidified to
pH 1.0 and stored at 4 °C until analysis. The concentration of total As
and total Fe was tested by ICP-OES (ICAP 7000). Dissolved Fe(II) was
measured by potassium dichromate titration. pH value was monitored
with a PHSJ-4F equipped with a pH sensor. OD was measured at
600 nm using 752 S spectrometer. The filter residues were washed with
deionized water for three times, dried under vacuum at 60 °C and then
ground for further analyses.
XRD, FTIR, XPS, SEM, Mössbauer spectra and particle size dis-
tribution were performed for the characterization of solid samples. XRD
was scanned from 5° to 70° with a step increment of 0.02° and 0.12 s
counting time. Data were collected using Rigaku D/Max-RB
Q. Li, et al.
diffractometer with Cu-Kα radiation (λ = 0.15406 nm, 40 kV, 250 mA)
[30]. Microscopic observations were conducted with SEM (JSM-
IT300LA), equipped with an EDS (Genesis 60S). The valence of the
arsenic was identified with XPS on a Thermo Fisher Scientific K-Alpha
1063 using Al Kα X-ray as the excitation source. All XPS spectra were
referenced to the standard C 1s peak at 284.5 eV [6]. The functional
groups of solid samples were distinguished with FTIR (NicoletIS10) at a
resolution of 4 cm−1. Mössbauer spectroscopy was employed to study
the chemical state of the iron atom in samples, which was recorded in
transmission mode by WISSEL (Starnberg, Germany) and using 57Co as
a Mössbauer source. The particle size was determined by a laser particle
size analyzer (LS-pop(6)) under the shading ratio of around 12%.
2.3. Stability evaluation
The short-term stability of solid samples obtained from batch assay
2 and 3 were evaluated by TCLP test [31], using an extraction fluid of
pH 4.93 ± 0.05. The extraction fluid consists of 5.7 mL of acetic acid
(CH3CH2COOH) and 64.3 mL of 1 M NaOH, and has a final volume of
1000 mL. The solid samples were mixed with the extraction fluid in the
solid-to-liquid ratio of 1:20 in 50 mL centrifuge tubes, and then con-
tinuously shaken on a 30 rpm tumble shaker at 25 °C for 18 ± 2 h.
Final suspensions were filtered using 0.45 μm membrane. The con-
centration of total As and total Fe in the leachate was analyzed by ICP-
OES.
3. Results and discussion
3.1. Role of Acidithiobacillus ferrooxidans in the formation of biogenic
tooeleite
A comparison experiment in the presence and absence of bacteria
was conducted to investigate the role of A. ferrooxidans during the
formation of tooeleite. XRD patterns are shown in Fig.S2 (a). The dif-
fraction peaks in XRD patterns at 9.06° and 27.79° are two main peaks
of tooeleite, corresponding to the (020) and (200) face of the mineral.
The solids formed with A. ferrooxidans (Fig.S2 (a), A. ferrooxidans 3d
and 5d) were well-crystalline tooeleite (PDF#44-1468); while that of
control had relatively weaker peaks (Fig.S2 (a), Control 5d). This re-
veals that A. ferrooxidans contributed to a better crystallization of
tooeleite in the Fe(II)-As(III)-SO42− system.
The pH value, the concentration of Fe(II), the removal efficiency of
total As and total Fe are demonstrated in Fig. 1(a–d). In the A. fer-
rooxidans run, pH decreased dramatically from 2.50 to ~2.00 during
0–0.5 d, and slowly to just above 1.80 in the following days. The Fe(II)
concentration figure of A. ferrooxidans fell obviously in the first 2 days
and rapidly to 0 in the third day. The removal efficiency of total As
increased correspondingly to ~40% in the first 2 days, sharply to ~90%
in the third day, and finally stood at 95.81%. The removal efficiency of
total Fe experienced a similar profile, where the figure rose to ~10%,
just below 30% and 30.28%, respectively. In the control, these patterns
were almost constant during the entire period.
Generally, in the medium containing Fe(II) for A. ferrooxidans spe-
cies cultivation, two series of reactions determine the solution pH value.
One is the oxidation of Fe(II) (Eq. (1)), which provides energy for the
bacteria growth [32]. This is an acid-consuming process, leading to the
pH increase, which would dominate in the early stage of the incubation
[27,33,34]. Another is the formation of solids. The solids could vary
(e.g. Eq. (2)–(5)), depending on the concentrations and species of ca-
tions and anions in the solution [27–29]. In our experiment, decreasing
the monovalent cations in 9 K medium reduces the formation of jar-
osite. Hence, tooeleite turns out to be the dominant mineral (Eq. (5),
Fig. S2). The solid formation produces H+, thus causes the decrease of
pH. It is expected that pH decreased to a relatively low value (~1.8) in
the A. ferrooxidans cultivation.
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Materials Science & Engineering C 111 (2020) 110755
A . ferrooxidans
4Fe 2 + + O2 + 4H+ → 4Fe3 + + 2H2 O (1)
Fe3 + + 3H2 O → Fe (OH )3 + 3H+ (2)
3Fe3 + + (K+, Na+, NH4+) + 2SO42 − + 6H2 O → (K+, Na+, NH4+) F
e3 (SO4 )3 (OH )6 + 6H+ (3)
3Fe 3 + + 2SO42 − + 7H2 O → (H3 O ) Fe3 (SO4 )3 (OH )6 + 5H+ (4)
6Fe 3 + + 4AsO33 − + SO42 − + 8H2 O → Fe6 (AsO3 )4 (SO4 )(OH )4⋅4H2 O + 4H+
(5)
The consistent pattern of various parameters in the control run in-
dicates abiotic Fe(II) oxidation played a minor role during the process.
In extreme acidic conditions (pH 1.5–3), the Fe(II) oxidation strongly
depends on acidophilic iron-oxidizing bacteria [35,36]. The biotic
oxidation rate is around 10−6–10−7 mol/(L·s), which is 105–106 times
faster than the abiotic action [37]. The similar figure of As and Fe re-
moval in the A. ferrooxidans run shows the two elements precipitated
simultaneously during the bioprocess. The corresponding pattern of
removal efficiency and Fe(II) concentration means the precipitation
was closely related to the bio-oxidation of Fe(II).
Batch assay 1 proves that the process with A. ferrooxidans is mi-
crobial-activity dominant, and the precipitates produced belong to
biogenic tooeleite. A. ferrooxidans contribute not only to the crystal-
lization of tooeleite but also the effective removal of As and Fe. The key
of the bioprocess lies in the oxidation of Fe(II) induced by bacteria.
3.2. Biogenic tooeleite formation regulated by Fe(II) concentration and pH
value
Fe(II) concentration and pH are important factors closely related to
the growth of bacteria as well as the characteristic of precipitates [38].
The function of initial Fe(II) concentration (3.595–10.806 g/L) and pH
(1.60–3.00) was investigated in detail.
XRD patterns (Fig.S2 (b–c)) show that all precipitates obtained from
batch assay 2–3 were tooeleite. FTIR (Fig.S3 (a–b)) and XPS (Fig.S3 (c))
results further confirm that the solids were tooeleite and the valence of
As was +3, which is consistent with the previous reports [11,13]. These
show that biogenic tooeleite precipitated easily under initial Fe(II)
concentration of 3.595–10.806 g/L and pH of 1.60–3.00.
The properties of solids formed on the fifth day were analyzed
(Table 1). The yield of solids increased with the initial Fe(II) con-
centration and pH. The weight of product grew from 5.406 g/L to
8.998 g/L when initial Fe(II) concentration increased from 3.595 g/L to
10.806 g/L, while that rose from 9.575 g/L to 13.567 g/L when pH
changed from 1.60 to 3.00. Fe/As molar ratio of solids in batch assay 2
and 3 was 1.53–1.62, and 1.56–1.65, respectively. Table 1 shows that
higher initial Fe(II) concentration and pH were favorable for more
tooeleite formation, and Fe/As molar ratio of the solids was slightly
higher than the theoretical one (1.50).
The parameters in solution (pH, the concentration of Fe(II), removal
of As and Fe) of batch assay 2 and 3 were recorded (Fig. 1 (e-h) and (i-
l)). The tendency of parameters and the relationship among them in
batch assay 2 and 3 were basically similar to that in batch assay 1. In
batch assay 2, pH values fell dramatically in the first 3 days after
peaking at ~2.2. The oxidation of Fe(II) also occurred in this period,
causing most of As and Fe removed correspondingly. In theory, all As
(III) is supposed to be removed by forming tooeleite under the initial Fe
(II) concentration of 3.595 g/L, because (1) initial Fe/As = 1.88 >
1.50; (2) Fe(II) was completely oxidized to Fe(III) after 3 days
(Fig. 1(f)). Whereas the real removal efficiency of As observed was
61.97% (Fig. 1(g)). The growth of bacteria has an impact on the pre-
cipitation, such as morphology and metal removal [27,39]. As a con-
sequence, the low removal of As can be explained by the insufficient
growth of bacteria (Table 1, final OD = 0.047). Namely, the better
growth of A. ferrooxidans contributed to the formation of biogenic
Q. Li, et al. Materials Science & Engineering C 111 (2020) 110755

Fig. 1. pH, the concentration of Fe(II) and the removal of total As and total Fe in batch assay 1 (a–d), batch assay 2 (e–h), and batch assay 3 (i–l), respectively.

tooeleite and the removal of As(III), which can be better-mediated by more significant to the removal of As, rather than the growth of A.
higher initial Fe(II) concentration. The initial Fe(II) in the range of ferrooxidans. The pH range of 2.00–3.00 was suitable for better As(III)
8.978–10.806 g/L was appropriate to achieve a better As(III) removal. removal.
In batch assay 3 (Fig. 1(i–l)), pH value fell gradually when the initial
pH > 2.00, while a sudden increase occurred in the first 2 days when
3.3. Stability evaluation and formation mechanism of biogenic tooeleite
the initial pH < 2.00. This is due to the balance among Fe(II) oxida-
tion and solid formation influenced by different initial pH. Fe(II) was
3.3.1. Stability evaluation of the precipitates
completely oxidized on the third day like other batch assays. When the
Identifying and quantifying the solid toxicity is the primary step to
initial pH increased from 1.60 to 3.00, the removal efficiency of As and
assess a new potential arsenic remediation candidate [40,41]. TCLP test
Fe varied from 76.34 to 93.10%, and 40.26 to 49.60%, respectively
of all samples in batch assay 2 and 3 was performed (Fig. 2). In the
(Fig. 1(k–l)). The final OD was 0.092–0.107 (Table 1), which suggests
TCLP test, arsenic leaching toxicity higher than the standard limit
that there was no significant difference among the growth of A. fer-
(5 mg/L) is unacceptable, while there is no limit of Fe [42]. In each
rooxidans. Consequently, the influence caused by the initial pH was
sample, the leaching concentration of As and Fe experienced the

Table 1
Properties of solids formed by A. ferrooxidans on the fifth day and the OD gained at the final of the incubation.
Label Initial Fe(II) concentration(g/L) Initial pH Final OD Yield of Solid (Dry, g/L) Elemental composition (wt%) Fe/As

As Fe Molar ratio

Fe_3.595 3.595 2.00 0.047 5.406 19.23 22.14 1.54

Fe_5.383 5.383 2.00 0.068 7.133 19.14 21.79 1.53
Fe_7.191 7.191 2.00 0.081 8.361 18.86 21.75 1.55
Fe_8.978 8.978 2.00 0.115 8.866 18.74 22.07 1.58
Fe_10.806 10.806 2.00 0.149 8.998 18.72 22.62 1.62
pH_1.60 8.978 1.60 0.104 9.575 18.69 21.72 1.56
pH_1.80 8.978 1.80 0.101 11.122 18.98 22.62 1.60
pH_2.00 8.978 2.00 0.107 12.140 17.62 20.99 1.60
pH_2.20 8.978 2.20 0.101 12.896 17.91 21.66 1.62
pH_2.50 8.978 2.50 0.092 13.190 18.54 22.46 1.62
pH_3.00 8.978 3.00 0.101 13.567 18.08 22.27 1.65

4
Q. Li, et al. Materials Science & Engineering C 111 (2020) 110755

Fig. 2. TCLP leaching concentration of As and Fe from samples in batch assay 2 (a) and 3 (b). The dash line is the standard limit for arsenic (5 mg/L).

opposite result. The samples were divided into two types according to Table 2
their leaching behavior: (1) samples releasing more As than Fe. The Mössbauer spectra parameters of solids obtained under pH 1.60 (a) and 2.50 (b)
released As generally exceeded the criteria (5 mg/L). Samples belong to in batch assay 3.
this type: Fe_3.595, Fe_5.383, Fe_7.191, pH_1.60; (2) samples releasing Sample IS(mm/s) QS(mm/s) Г/2(mm/s) Area
more Fe than As. The leached As was generally below 5 mg/L. Samples
belong to this type: Fe_8.978, pH_1.80, pH_2.00, pH_2.20, pH_2.50, pH_1.60 (release more As) 0.36 1.04 0.26 0.342
0.39 0.76 0.32 0.658
pH_3.00.
pH_2.50 (release more Fe) 0.36 1.05 0.28 0.352
In batch assay 2, increasing initial Fe(II) concentration caused less 0.39 0.76 0.14 0.648
As leached (Fig. 2 (a)), probably due to the better growth of bacteria
(Table 1). Batch assay 3 provided sufficient energy for bacteria growth,
and tooeleite produced under pH 1.80–3.00 had low As leachability isomer shift (IS) of 0.36 and 0.39 mm/s. Small differences were seen in
(Fig. 2 (b)). However, high As leaching concentration (as high as the quadrupole splittings (QS), half-line widths (Г/2) and area between
~250 mg/L) was observed in chemogenic tooeleite [10–12,43]. As a two samples (Table 2). The areas for pH_1.60 (34% and 66%) and for
consequence, biogenic tooeleite had better As fixing performance than pH_2.50 (35% and 65%) were well consistent with the site occupancy in
chemogenic samples. Therefore, giving sufficient energy to bacteria, the crystal model [2]. The Mössbauer spectra in our study are in
biogenic tooeleite was stable under pH of 1.80–3.00. Samples releasing agreement with the previous observations [9,13]. The doublet in the
more Fe than As were favorable for the stability. Mössbauer spectra shows that all Fe existed in the form of tooeleite
rather than other amorphous Fe phases. Besides, the high leaching Fe
concentration of Fe-releasing samples is not due to the existence of
3.3.2. Formation and stability mechanism of biogenic tooeleite amorphous Fe-bearing phases.
To understand the stability mechanism of biogenic tooeleite, further Samples having different release behavior were chosen for the
comparisons were conducted. Fe(III) was the only iron state at the end morphology comparison (Fig. 4, Fig.S4, and Table S1). Samples re-
of incubation in the solution. The high Fe/As ratio in the precipitates leasing more As had thin flakes loosely combined and sometimes ag-
(Table 1) and the high leaching Fe concentration in the TCLP test were gregated into sphere- or multi-sphere-like structures. The hollow
also observed. However, whether amorphous Fe phases existed in the structure was observed in this kind of sample (Fig. 4 (d)), which may
precipitates was unknown, because XRD patterns are unable to tell contribute to a higher As leachability [13]. Samples releasing more Fe
much information about amorphous solids. Hence, Mössbauer spectra were thick platy particles combined tightly as agglomerates. The ag-
of samples having different leaching behavior were carried out glomerates exhibited bulk-like structure, with an agglomerate size
(pH_1.60: release more As than Fe; pH_2.50: release more Fe than As). ranging from 30 to around 180 μm (in diameter). According to the TCLP
In the crystal structure model of tooeleite, there are two types of Fe, results, the bulk-like structure in Fe releasing samples was favorable for
named Fe1 and Fe2, having a site occupancy of 33.3:66.7 [2]. Our the stabilization of tooeleite.
Mössbauer spectra (Fig. 3) exhibited one quadrupole doublet with

Fig. 3. Mössbauer spectra of solids obtained under

pH 1.60 (a) and 2.50 (b) in batch assay 3. Mössbauer
spectra were measured at room temperature. The
measured data are given as points and individual
fitting components as red and green lines. (For in-
terpretation of the references to colour in this figure
legend, the reader is referred to the web version of
this article.)

5
Q. Li, et al. Materials Science & Engineering C 111 (2020) 110755

Fig. 4. SEM images of the precipitates obtained under different initial Fe(II) concentrations and pH values: (a) Fe(II)_3.595, (b) Fe(II)_7.191, (c) Fe(II)_8.978, (d)
pH_1.60, (e) pH_2.00 and (f) pH_2.50. (a, b, d): samples releasing more As; (c, e, f): samples releasing more Fe. The magnification is ×10,000.

In other observations, the chemogenic tooeleite formed under am-

bient condition or elevated temperature had loose sphere structure
aggregated by thin platy particles [13,43,44]. The loose sphere struc-
ture was generally distinct and around 5–10 μm (in diameter), de-
pending on the synthetic method and formation time. Hollow structure
was observed in some cases [13]. Therefore, laboratory prepared
tooeleite samples were made up by flakes, which exhibited different
structures: (1) chemogenic tooeleite tends to have loose sphere struc-
ture with thin flakes; (2) unstable biogenic tooeleite has loose sphere-
or multi-sphere-like structure with thin flakes; (3) stable biogenic
tooeleite displays bulk-like structure with thick flakes. The agglomerate
size is as follows: stable biogenic tooeleite > unstable biogenic tooe-
leite > chemogenic tooeleite.
Fe(III) was added directly as the iron source at the beginning of the
reaction in the previous chemical syntheses [8,11,13,43,44]. While in
this study, Fe(III) was supplied continuously during the first 3 days due
to the bacterial Fe(II) oxidation. It is like a way of releasing Fe(III)
slowly. Vital ion supply rate would influence the results of precipitation
[45]. It controls the rate and way of crystal growth. Chemical experi- Fig. 5. XRD patterns of batch assay, chemical control with dead cells and
ment was conducted to know the influence of low supply rate of Fe(III) without cells.
during the tooeleite formation. The results are shown in Fig. 5, Table 3
and Fig.S5. All obtained precipitates were well-crystalline tooeleite.
However, the two chemical runs exhibited low removal efficiency

6
Q. Li, et al.
Table 3
The comparison between chemical control with dead cells and without cells.
The particle size is D(4,3) determined by laser particle size analyzer.
Chemical Yield of Particle Removal Leaching
control solid size (μm) efficiency (%) concentration (mg/L)
(Dry, g/L)
As Fe As Fe
With dead 1.5645 36.34 29.71 34.68 32.45 0.72
cells
Without 1.3880 29.63 28.18 31.84 37.04 0.72
cells
(~30% for As(III)) and high As leachability. The removal efficiency is
very similar with the results by mixing As(III) and Fe(III) directly under
pH 2.0 (after mixing, As(III) = 0.1 mol/L, Fe(III) 0.2 mol/L) [11]. It
means acidic pH value is not favorable for As removal by forming
tooeleite under chemical conditions, and the slow supply of Fe(III) is
not the essential factor for the formation of stable minerals.
The obtained chemogenic tooeleite were typical samples releasing
more As than Fe. The crystallization, yield, particle size, removal effi-
ciency and leaching concentration, got from the chemical control with
dead cells were slightly better than those without cells. The existence of
dead cells enhanced the As removal and stability. This can be explained
by the adsorption capability of A. ferrooxidans mediated by the EPS
[46,47], with the cells acting as a concentrated pool or nucleation sites
for precipitation [48,49]. Since tooeleite formation is an acid-producing
process, the pH decreased along the time. When pH < 2.0, the EPS
surface charge of cells grown on Fe(II) medium was positively charged
[50,51]. This is not beneficial for the capture of As(III) and Fe(III), as
they occur as neutral and positive form, respectively [52]. This may
account for the limited improvement when adding dead cells. As a
consequence, living cells run performed the best crystallization, As re-
moval and stability, followed by the chemical control with dead cells
and without cells. The great performance was mainly due to the active
microbial activities.
Field tooeleite crystal minerals were characterized by inclined
planes [1], but the precipitates gained in the laboratory were flakes.
The difference in crystal growth direction and time may be the reasons
for this phenomenon [53]. Preferred orientation occurred in the growth
of tooeleite, as shown in XRD patterns (Fig. S2). In the two main faces of
tooeleite, the development of (020) face is favorable for mineral sta-
bility.
The formation of biogenic tooeleite and the relationship between
crystal growth and mineral stability are described in Fig. 6. Firstly, Fe
(II) is oxidized into Fe(III) under the action of A. ferrooxidans and the
process provides the energy for the bacteria growth in return. The
oxidation happens during 0–3 days and completes when bacteria grow
into the stationary stage. Fe(III) precipitates with As(III) simultaneously
Fig. 6. Scheme of biogenic tooeleite formation mediated by A. ferrooxidans and the relationship between crystal growth and mineral stability. The (020) and (200)
faces are two main crystal planes of tooeleite.
7
Materials Science & Engineering C 111 (2020) 110755
mainly during the same period. Two forms of biogenic tooeleite, sphere-
like and bulk-like, therefore, are produced as well as the removal of As
(III). When (200) crystal plane is developed, unstable tooeleite is pro-
duced with a sphere-like/multi-sphere-like structure formed by ultra-
thin flakes. The agglomerates sometimes show obvious hollow struc-
tures. When (020) crystal plane is developed, the precipitate is
characterized by bulk-like structure with thick flakes aggregated
tightly. The toxic leaching concentration of the latter structure met the
standard and was regarded relatively stable.
4. Conclusion
The formation of biogenic tooeleite under the action of A. ferroox-
idans is an effective method to remove As(III), which is closely related
to the microbial Fe(II) oxidation during the growth of bacteria. Biogenic
tooeleite can be easily obtained under initial Fe(II) concentration of
3.595–10.806 g/L and pH of 1.60–3.00. Up to ~95% of As(III) can be
removed with initial Fe(II) concentration of 8.978–10.806 g/L and in-
itial pH of 2.00–3.00. The relatively stable tooeleite is featured by the
developed (020) crystal face and the bulk-like structure with thick
flakes.
CRediT authorship contribution statement
Qingzhu Li: Supervision, Conceptualization, Methodology.
Mengxue Zhang: Methodology, Investigation, Data curation, Writing -
original draft. Jinqin Yang: Investigation, Visualization. Qianwen Liu:
Visualization. Guanshi Zhang: Investigation, Visualization. Qi Liao:
Supervision, Visualization. Hui Liu: Investigation, Visualization.
Qingwei Wang: Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by National Natural Science Foundation of
China (51974379), National Key Research and Development Program
of China (2017YFC0210401), the Key Project of National Natural
Science Foundation of China (51634010), Major Science and
Technology Program for Water Pollution Control and Treatment
(2017ZX07402004) and Postdoctoral Science Foundation of Central
South University.
Q. Li, et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.msec.2020.110755.
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