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Effect of ferric ions on the anaerobic bio-dissolution of jarosites by Acidithiobacillus ferrooxidans
Journal Pre-proof Effect of ferric ions on the anaerobic bio-dissolution of jarosites by Acidithiobacillus ferrooxidans
Yuankun Yang, Shu Chen, Bin Wang, Xinyu Wen, Hanke Li, Raymond Jianxiong Zeng PII:
S0048-9697(19)36330-2
DOI:
https://doi.org/10.1016/j.scitotenv.2019.136334
Reference:
STOTEN 136334
To appear in:
Science of the Total Environment
Received date:
3 November 2019
Revised date:
23 December 2019
Accepted date:
23 December 2019
Please cite this article as: Y. Yang, S. Chen, B. Wang, et al., Effect of ferric ions on the anaerobic bio-dissolution of jarosites by Acidithiobacillus ferrooxidans, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.136334
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© 2019 Published by Elsevier.
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Effect of ferric ions on the anaerobic bio-dissolution of jarosites by Acidithiobacillus ferrooxidans
Yuankun Yang a, b, Shu Chen b, Bin Wang b, Xinyu Wen b, Hanke Li b, Raymond Jianxiong Zeng a, c * CAS Key Laboratory for Urban Pollutant Conversion, Department of Applied
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a
Key Laboratory of Solid Waste Treatment and Resource Recycle, Ministry of
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b
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Chemistry, University of Science and Technology of China, Hefei 230026, China
Centre of Wastewater Resource Recovery, College of Resources and Environment,
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c
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Education, Southwest University of Science and Technology, Mianyang 621010, China
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Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
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*Corresponding author:
Raymond Jianxiong Zeng; E-mail address: [email protected], [email protected]
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Abstract Large amounts of jarosites are produced during zinc hydrometallurgy and bioleaching, as well as in acid sulfate soils and acid mine drainage environments. As such, understanding the behavior of jarosite dissolution is important for analyzing the iron cycle process and promoting the control and treatment of jarosites. In general,
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soluble ferric ions and jarosites coexist in acid environments; however, the
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relationship between soluble ferric ions and jarosites under anaerobic reductive
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conditions is still not well understood. In this study, the effect of adding Fe3+ on the
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promotion of the bio-dissolution of jarosites using Acidithiobacillus ferrooxidans is
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investigated. With the addition of 12 mM Fe3+, the efficiency and maximum rate of jarosite bio-dissolution were found to reach 84.1% and 2.66 mmol/(L∙d), respectively.
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The addition of Fe3+ at concentrations higher than 12 mM did not further improve the
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jarosite bio-dissolution. These results indicate that the mechanisms underlying these improvements include: (i) the reduction of the zeta potential due to the compression of the diffusion layer of the electric double layer by Fe3+; (ii) bacteria growth enhancement and the stabilization of the pH of cultures via the reduction of soluble Fe3+. Based on these observations, this study serves to promote the development of jarosite bio-dissolution using Acidithiobacillus ferrooxidans and challenges the idea that soluble Fe3+ suppresses the bio-dissolution reaction of solid Fe3+ substances such as jarosite when soluble ferric ions and jarosite coexist. Keywords: Jarosites; Anaerobic bio-dissolution; Ferric ions; Acidithiobacillus 2
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ferrooxidans; Waste management
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Journal Pre-proof 1. Introduction Jarosites play an important role in the iron cycle, such as that occurring in acid sulfate soils and acid mine drainage environments, and in deferrization, which occurs during zinc hydrometallurgy (Han et al., 2014; Johnston et al., 2011; Li et al., 2007). Additionally, jarosites are the main waste products generated in the bioleaching fields, and their generation can inhibit the leaching reaction (Johnson et al., 2017; Yang et al.,
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2014; Yu et al., 2011b). Jarosites possess relatively good coprecipitation and substitution
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properties and are composed of many kinds of metal ions, including valuable metals
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(Ag and Im) and harmful metals (Pb and As) (Asta et al., 2010; Courtin-Nomade et al.,
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2012; Martinez-Sanchez et al., 2019; Wegscheider et al., 2017). Therefore, the research
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of jarosite dissolution is of significant importance to the understanding of the iron cycle
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process in acidic environments, as is the research of the control and treatment of jarosites and their impact on bioleaching efficiency. Compared with other methods of
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jarosite dissolution, the bio-dissolution of jarosites via anaerobic reduction has great advantages for industrial applications, including lower costs, environmental friendliness, and a simple control and management process, among others (Castro et al., 2016; Gao et al., 2019; Gonzalez et al., 2015; Ouyang et al., 2014). Thus, further investigation of the bio-dissolution of jarosites is of great importance. During jarosite bio-dissolution, jarosites serve as terminal electron acceptors that are restored by electron donors, and this is accompanied by jarosite dissolution (González et al., 2016; Ouyang et al., 2014). Typically, both jarosites and soluble Fe3+ coexist in low-pH environments, such as those encountered in acid mine drainage (pH 1
Journal Pre-proof around 3 to 3.8) and bioleaching (Elwood Madden et al., 2012; Song et al., 2018b). Soluble Fe3+ can serve as a terminal electron acceptor in anaerobic environments, and relevant research has shown that soluble Fe3+ is more readily available to Fe3+ reductases as compared to solid jarosites (Nevin and Lovley, 2002). This means that soluble Fe3+ and jarosites will compete for electron donors during the anaerobic reductive bio-dissolution of jarosites. In addition, the hydrolysis reaction of jarosites
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(Eq. (1)) will be affected when soluble Fe3+ is present.(Baron and Palmer, 1996) Based
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on these understandings, it is posited that the efficiency of the bio-dissolution of
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jarosites will remain at a low level when coexisting with soluble Fe 3+ in low-pH
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environments.
(1)
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KFe3(SO4)2·(OH)6 + 6H+ ←→ K+ + 3Fe3+ + 2SO42- + 6H2O
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In a recent study by this research group, it was found that unsuitable pH environments are harmful to jarosite bio-dissolution (Yang et al., 2019). However, the
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process of jarosite bio-dissolution requires acid, regardless of whether the Fe3+ reduction pathway (Eq. (2)) or SO42- reduction pathway (Eq. (3)) is utilized (Gao et al., 2019; Gonzalez et al., 2015). Thus, maintaining the pH of the culture during bio-dissolution might greatly improve its efficiency. According to the reaction presented in Eq. (4), the process of soluble Fe3+ reduction by bacteria increases the production of H+, and this process might provide a possible way to maintain the pH of the culture within a suitable range via the bacterial autoregulation of soluble Fe3+ reduction. In this way, soluble Fe3+ ions could contribute to jarosite bio-dissolution in low-pH environments. Nevertheless, the relationship between soluble ferric ions and jarosites under anaerobic reductive 2
Journal Pre-proof conditions remains unclear, and the effect of soluble Fe3+ on jarosite bio-dissolution requires further study. KFe3(SO4)2·(OH)6 + 3e- → K+ + 3Fe2+ + 2SO42-+ 6OH-
(2)
KFe3(SO4)2·(OH)6 + 8H2O + 16e- → K+ + 3Fe3+ + 2S2-+ 22OH-
(3)
2Fe3+ + H2 + bacteria → 2Fe2+ + 2H+
(4)
According to the jarosite bio-dissolution reactions represented by Eqs. (2) and (3),
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for the bio-dissolution of 1 mol of jarosites, the Fe3+ reduction pathway needs 3 mol of
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electrons and 6 mol of H+. In contrast, the SO42- reduction pathway needs 16 mol of
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electrons and 22 mol of H+, indicating that more electron donors and acid will be
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consumed in the SO42- reduction pathway than in the Fe3+ reduction pathway (Yang et
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al., 2019). Therefore, the Fe3+ reduction pathway is superior in regards to jarosite
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bio-dissolution. Correlational research has shown that soluble Fe3+ can be restored by Acidithiobacillus ferrooxidans (A. ferrooxidans) by using hydrogen or S0 as an electron
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donor in anaerobic environments (Das et al., 1992; Ohmura et al., 2002). Recently, the results of a study by the present research group also demonstrated that solid jarosites can be bio-dissolved by A. ferrooxidans by using hydrogen as the electron donor (Eq. (5)) in anaerobic conditions (Yang et al., 2019). This means that A. ferrooxidans can be used to help explore the relationship between soluble Fe3+ and jarosites in anaerobic reduction environments. Specifically, A. ferrooxidans activity in aerobic environments, such as in bioleaching or acid mine drainage, is one of the main factors resulting in jarosite generation. In this regard, research on the bio-dissolution of jarosites by A. ferrooxidans is important for the understanding of the iron cycle in natural 3
Journal Pre-proof environments, as well as for the in-situ disposal and reuse of jarosites during bioleaching. 2KFe3(SO4)2·(OH)6 + 6H+ + 3H2
A. ferrooxidans
2K+ + 6Fe2+ + 4SO42- + 12H2O (5)
This study aims to explore the effects of ferric ions on the mechanisms of the anaerobic bio-dissolution of jarosites by A. ferrooxidans. Changes of Fe2+ levels, Fe3+ levels, pH values, zeta potential, and adenosine triphosphate (ATP) concentrations were
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evaluated in batch experiments, and the surface morphologies of jarosites and
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extracellular polymeric substance (EPS) of A. ferrooxidans were also examined. These
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analyses will be beneficial for the development of jarosite bio-dissolution technologies,
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and will help promote the large-scale control and treatment of jarosite in acid mine
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environments and bioleaching in the future.
2. Materials and methods
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2.1 Preparation of jarosites
Jarosites were produced by using A. ferrooxidans (GenBank accession number: KJ094412) (Chen et al., 2015; Yang et al., 2014) in a 9K medium containing 45.00 g FeSO4∙7H2O as the energy source, and 3.00 g (NH4)2SO4, 0.50 g K2HPO4, 0.10 g KCl, 0.01 g Ca(NO3)2, and 0.50 g MgSO4∙7H2O in 1.0 L distilled water (Yang et al., 2019; Zhu et al., 2011). After 5 days at 30 °C and 170 rpm in a rotary shaker, the solid resultants of the Erlenmeyer flask were collected and washed by ultrapure water (pH = 2.00) three times, and then dried with a vacuum. 2.2 Adaptation phase in anaerobic conditions 4
Journal Pre-proof It was necessary to adapt the utilized bacteria to the presence of the jarosites. A. ferrooxidans was cultured using an anaerobic mineral medium containing 132 mg (NH4)2SO4, 52 mg KCl, 490 mg MgSO4∙7H2O, 41 mg K2HPO4, 9 mg CaCl2∙2H2O, 2 mg CuSO4∙5H2O, 0.5 mg NaMoO4∙5H2O, 1 mg Na2SeO4∙10H2O, 1 mg ZnSO4∙7H2O, 1 mg MnSO4∙H2O, 0.5 mg CoCl2∙6H2O, and 1 mg NiCl2∙2H2O in 1.0 L distilled water (Ohmura et al., 2002). As described in detail in a previous work (Yang et al., 2019),
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after deoxygenation (using high-purity nitrogen), pH adjustment (using dilute sulphuric
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acid (5 M)), and autoclave sterilization (at 121 °C for 30 min), jarosites (30 mM in Fe3+)
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and a 1 mL inoculum of A. ferrooxidans were then added into a serum bottle containing
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49 mL of anaerobic medium. Subsequently, 70 mL of mixed gas with a V(H2):V(CO2)
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ratio of 4:1 was filled into the bottle using a disposable sterilized syringe after removing
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the headspace with a vacuum pump (Ohmura et al., 2002). The bottle was incubated at 30 °C (170 rpm) in a rotary shaker. After several sub-cultivation processes, the
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supernatant of the bottle (late logarithmic phase) was applied as the inoculum. 2.3 Jarosite bio-dissolution testing Batch experiments were conducted in serum bottles with a total volume of 120 mL to evaluate the effect of adding Fe3+ during jarosite bio-dissolution using A. ferrooxidans. The variables for each of the groups are listed in Table 1. For each group, the additions of Fe3+ (ferric sulfate), jarosites (30 mM in Fe3+), and inoculums (1 mL) to the bottles were performed in an anaerobic glove box. The pH of the cultures was measured after 10 min. For the control groups, jarosites (30 mM in Fe3+) and inoculums (1 mL) were added to the bottles, and the pH of cultures was then adjusted to either 1.68 5
Journal Pre-proof (the same pH as the group with 12 Fe3+) using dilute sulphuric acid (5 M) or maintained at 2.00. Subsequently, the headspaces of all the serum bottles were removed with a vacuum pump, and 70 mL of mixed gas at a V(H2):V(CO2) ratio of 4:1 was added. All the bottles were cultivated in a rotary shaker at 30 °C (170 rpm). All experiments were performed in triplicate. During the experiments, supernatant samples (4 mL) were collected to analyze the
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soluble Fe2+, pH values, and total soluble iron ions (including Fe2+ and Fe3+ irons),
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while mix samples (4 mL) were collected to analyze the zeta potential, ATP, and total
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Fe(II) via sacrificed sampling, and each bottle was sampled only one time. For total Fe2+
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measurements, 0.5 mL of mixed samples were added into a polytetrafluoroethylene
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(PTFE) vessel in the anaerobic glove box, and 4.5 mL of hydrochloric acid solution (50%
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v/v) was then added to dissolve the samples. Finally, the solution was transferred to a 50-mL volumetric flask and brought to volume with deionized water for total Fe2+
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determination after 1 hour (Castro et al., 2017). When the jarosite bio-dissolution reactions had ended, the bio-dissolution residues were collected to assess their morphologies. The mixed samples were also collected to evaluate the EPS of A. ferrooxidans. For EPS assessment, 20 mL of the mixed samples were added into a centrifuge tube and shaken for 30 seconds. Subsequently, the supernatant samples were collected after centrifugation (at 4000 rpm and 4 °C for 3 min), and the supernatant was then discarded after a second centrifugation (at 8000 rpm and 4°C for 10 min). A total of 10 mL mineral medium (pH = 2.00) was then added into the centrifuge tube and treated for 10 min via ultrasound (200 W and 40 KHz). Finally, 6
Journal Pre-proof the supernatant was used for EPS analysis after a third centrifugation (at 11000 rpm and 4°C for 10 min) (Yu et al., 2011a). 2.4 Analytical methods The total soluble iron ions were measured with an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 8300, PerkinElmer, United States). The Fe2+ ion concentration was measured using the ferrozine method.(Stookey, 2002)
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The Fe3+ ion concentration is the concentration of the total soluble iron ions minus the
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concentration of Fe2+ ions. The ATP levels of A. ferrooxidans were analyzed via
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chemiluminescence using an A095-2 kit (Nanjing Jiancheng Bioengineering Institute,
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Nanjing, China) and a multiscan spectrum (SpectraMax iD3, Molecular Devices, United
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States). The pH and zeta potential were measured using a digital pH meter (SevenMult
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S40, Mettler Toledo, Germany) and zeta potential meter (Zetasizer Nano ZS90, Malvern, UK), respectively. The EPS characteristics of A. ferrooxidans were analyzed via
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three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy (QuantaMaster-40, PTI, United States). Finally, the surface morphologies of the jarosites were analyzed via scanning electron microscopy (Ultra 55, ZEISS, Germany).
3. Results and discussion 3.1 Changes in the Fe2+ and Fe3+ concentrations The changes in the Fe2+ and Fe3+ concentrations during the jarosite bio-dissolution process are presented in Figs. 1(A-D). On the whole, there was no significant difference between the soluble Fe2+ concentration (Fig. 1(A)) and the total Fe2+ concentration (Fig. 7
Journal Pre-proof 1(B)) in any of the groups. This indicates that the concentrations of other forms of Fe2+, such as Fe2+ adsorbed to the cell surface and ferrous minerals, were very low in these experiments. Considering that soluble Fe2+ ions can be reused directly in bioleaching fields, the bio-dissolution of jarosites under low-pH conditions is beneficial for jarosite treatment and recovery in the form of Fe2+. Additionally, it was apparent that the concentration of soluble Fe3+ ions (Fig. 1(C)) was rapidly decreased and remained at a
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low level after 12 days in the groups with added Fe3+. This result implies that almost all
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soluble Fe3+ was restored to Fe2+ by A. ferrooxidans. Moreover, the concentrations of
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Fe2+ (soluble or total Fe2+) continued to increase during the bio-dissolution process, and
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were higher than the initial concentrations of added Fe3+. This signifies that the
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concurrent.
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reduction of the added Fe3+ and the occurrence of jarosite bio-dissolution were
More specifically, with the addition of 12 mM Fe3+, the total Fe2+ concentration
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reached 37.2 mM after 24 days of bio-dissolution (Fig. 1(B)). This implies that most of the total Fe2+ was generated via jarosite bio-dissolution when using A. ferrooxidans. Therefore, it was necessary to assess the Fe2+ concentrations during jarosite bio-dissolution. In theory, the paths for the generation of Fe2+ ions include the reduction of soluble Fe3+ and jarosite bio-dissolution in the groups with added Fe3+. As such, the generation of Fe2+ ions via jarosite bio-dissolution can be calculated by knowing the amount of added Fe3+ that is reduced. However, the hydrolysis of jarosites (Eq. (1)) in low-pH environments can produce some Fe3+ ions, which increases the difficulty of determining the amount of added Fe3+ that is reduced. In the authors’ prior study, it was 8
Journal Pre-proof found that the rate of Fe3+ generation by jarosite hydrolysis was lower than the rate of Fe3+ reduction by A. ferrooxidans (Yang et al., 2019). In addition, the hydrolysis of jarosites (Eq. (1)) and Fe3+ reduction (Eq. (4)) is one of the routes of jarosite bio-dissolution (i.e., an indirect pathway). Based on this, it was assumed that the soluble Fe3+ concentration in the culture could represent the remaining concentration of the
could be estimated by Eqs. (6) and (7), respectively.
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added Fe3+. In this case, the Fe2+ generated by jarosite bio-dissolution and the efficiency
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Fe2+ generated by jarosites (mM) = concentration of total Fe2+ −
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(concentration of added Fe3+ − concentration of soluble Fe3+)
(6)
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Jarosite bio-dissolution (%) = Fe2+ generated by jarosites (mM)/ (7)
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30 (mM, jarosites in Fe3+)
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The estimated data regarding the Fe2+ concentrations and jarosite bio-dissolution efficiency during jarosite bio-dissolution are presented in Fig. 1(D). In the control
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groups without added Fe3+, the jarosite bio-dissolution efficiencies reached only 34.5% (pH 2.00) and 37.5% (pH 1.68). In the groups with the addition of 12 mM Fe3+, the jarosite bio-dissolution efficiency was increased to 84.1%; the jarosite bio-dissolution efficiency was clearly improved greatly by the addition of 12 mM Fe3+. In addition, the jarosite bio-dissolution efficiency was enhanced from 67.2% to 84.1% when the added Fe3+ concentration was increased from 6 mM to 12 mM. When the added Fe3+ concentration was increased from 12 mM to 24 mM, the jarosite bio-dissolution efficiency rapidly decreased from 84.1% to 59.2%. This result indicates that the higher Fe3+ concentration did not further improve the dissolution efficiency. 9
Journal Pre-proof Table 2 summarizes the maximum generation rates of Fe2+ as estimated from the experimental results. In the jarosite bio-dissolution pathway, the maximum generation rate of Fe2+ reached 2.66 mmol/(L∙d) with the addition of 12 mM Fe3+. In contrast, the maximum generation rates of Fe2+ reached 0.97 mmol/(L∙d) (pH 2.00) and 0.88 mmol/(L∙d) (pH 1.68) without the addition of Fe3+. This suggests that the rate of jarosite bio-dissolution could be improved by adding Fe3+. In addition, the maximum generating
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rate of Fe2+ (including the soluble Fe3+ reduction and jarosite bio-dissolution paths)
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began to reduce when the addition of Fe3+ exceeded 18 mM, while the maximum
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generating rate of Fe2+ (jarosite bio-dissolution path) had already started to decrease
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when the addition of Fe3+ exceeded 12 mM. This implies that high concentrations of
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additional Fe3+ first affect the rate of jarosite bio-dissolution by A. ferrooxidans, and the
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rate of soluble Fe3+ reduction then also decreases when the concentration of additional Fe3+ continues to increase.
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3.2 Zeta potential and jarosite morphology Soluble Fe3+, a multivalent ion, may compress the electric double layers of jarosite particles when added to the reaction (Ma and Pierre, 1999). To investigate the effects, the zeta potential and jarosite morphology during the bio-dissolution process were assessed. As presented in Fig. 2, the zeta potentials were reduced gradually in the two control groups without added Fe3+. This was because, as compared with the initial culture, there were more divalent ions (Fe2+ and SO42-) in the culture as the jarosite bio-dissolution progressed. Subsequently, the diffusion layer was compressed by divalent ions. However, with the addition of Fe3+, the zeta potentials exhibited an initial 10
Journal Pre-proof rapid decrease that occurred with the increase in the Fe3+ concentration, and then a slow increase. Moreover, the initial zeta potential decreased from 8.21 mV to 6.36 mV when the initial pH was decreased from 2.00 to 1.68 in the control groups. Additionally, the initial zeta potential in the group with 12 mM Fe3+ was 4.86 mV, which was lower than the value of 6.36 mV measured for the control group (pH 1.68), although the initial pH of each culture was the same. On the one hand, this indicates that the addition of Fe3+
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was more effective than adjusting the initial pH in regards to the zeta potential. On the
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other hand, the main effect of the decrease of the zeta potential in the groups with added
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Fe3+ was the decrease in the concentration of Fe3+ rather than the low initial pH due to
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Fe3+ hydrolysis (Eq. (8)) (Ebrahimi et al., 2003).
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When the diffusion layer is compressed, the mass transfer process between
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jarosites and bacteria may be affected, leading to alterations of the jarosite surface. The effects of Fe3+-assisted bio-dissolution on the surfaces of the jarosite samples are
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depicted in Fig. 3. Without the addition of Fe3+, the surfaces of the jarosites in the control groups (pH 2.00 and 1.68) were smooth. With the addition of Fe3+, many particulate structures on the surfaces of the jarosites were apparent, indicating that the bacterial erosion of the surfaces was increased after the addition of Fe3+. Fe3+ + H2O → Fe(OH)2+ + H+
(8)
3.3 ATP quantity and EPS alterations In general, the ATP quantity of a single cell is relatively fixed. For example, the ATP level of A. ferrooxidans grown with Fe2+ was found to be 1.16 amol per cell (Pakostova et al., 2013). Thus, the concentration of ATP can reflect the relative quantity 11
Journal Pre-proof of microorganisms. As determined by the experiments, the ATP concentration in all the groups first increased and then decreased, as presented in Fig. 4(A). In the control groups, the concentration of ATP decreased with the decrease in the initial pH from 2.00 to 1.68. In contrast, the concentration of ATP increased with the addition of Fe3+ although the initial pH decreased from 1.76 to 1.61 (Table 1). Additionally, the ATP concentration of the group with 12 mM Fe3+ was higher than that of the control group
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(pH 1.68), although the initial of pH values for these two groups were the same. These
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effect of a low pH on bacteria growth.
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results demonstrate that the addition of Fe3+ can boost bacteria growth and eliminate the
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In addition, the EPS of A. ferrooxidans after bio-dissolution was characterized via
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three-dimensional EEM fluorescence spectroscopy. As presented in Fig. 5, with the
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addition of Fe3+, the fluorescence peak locations in all the groups were located at Ex = 400 nm (Em = 430 nm). However, the fluorescence intensity and area coverage were
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diminished along with the increase of the concentration of added Fe3+ from 6 mM to 24 mM. Moreover, the same phenomena were also observed in the control groups. In the pH 2.00 control group, two fluorescence peaks were located at Ex = 400 nm (Em = 430 nm) and Ex = 280 nm (Em = 500 nm), whereas the fluorescence intensity and area coverage were diminished in the pH 1.68 control group. In the EEM spectra, the locations of the fluorescence peaks related to the different components of the EPS were as follows: tyrosine and aromatics in protein-like components (Ex = 220-250 nm, Em = 280-380 nm); tryptophan in protein-like components (Ex > 250 nm, Em = 280-380 nm); fulvic acid-like components (Ex = 220-250 nm, Em > 380 nm); humic acid-like 12
Journal Pre-proof components (Ex > 250 nm, Em > 380 nm).(Chen et al., 2003; Song et al., 2018a) These results suggest that the main constituents of the A. ferrooxidans EPS under these dissolution conditions were associated with humic acid-like components. This indicates that with the addition of Fe3+ and/or the adjustment of the initial pH, both the EPS components and EPS contents were reduced. 3.4 Changes in pH values
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Fig. 4(B) presents the changes in pH that occurred during the experimental process.
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In these experiments, the factors influencing pH included jarosite bio-dissolution (Eq.
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(5)), Fe3+ reduction (Eq. (4)), and Fe3+ hydrolysis (Eq. (8)). With the addition of Fe3+,
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the initial pH values in all groups with added Fe3+ were lower than that of the control
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group (pH 2.00) due to Fe3+ hydrolysis. Subsequently, the pH values in all these groups
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continued to increase with the progression of the jarosite bio-dissolution reaction (Eq. (5)). In addition, after the completion of the jarosite bio-dissolution reactions, the pH
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value (2.24) in the group with 12 mM Fe3+ was lower than the pH value (3.34) in the control group (pH 2.00). However, in contrast, the jarosite bio-dissolution efficiency in the group with 12 mM Fe3+ was more than twice that in the control group (pH 2.00). Clearly, the Fe3+ reduction process (Eq. (5)) served to stabilize the low-pH environment of the culture. In the authors’ previous study, it was found that bacteria activity was inhibited when the culture pH was less than 2.0 (Yang et al., 2019); however, the mechanism underlying this pH effect is not clear. In the present study, the initial culture pH in all the groups was less than 2.00, except for the control group (pH 2.00). The variation in EPS 13
Journal Pre-proof tended to be the same among the groups when the initial pH was less than 2.00; however, the amounts of A. ferrooxidans of these groups were significantly different, indicating that a low pH (under 2.00) mainly affected the EPS of A. ferrooxidans during jarosite bio-dissolution. Related research has shown that the EPS components and contents of A. ferrooxidans have an obvious impact on the interaction between the bacteria and minerals (Yu et al., 2011a). In bioleaching processes, the EPS secreted by
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attached cells is significantly greater than that secreted by free cells (Yu et al., 2014; Yu et
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al., 2017). As such, a reduction in the EPS components and contents of A. ferrooxidans
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is disadvantageous to jarosite bio-dissolution, which reflects that the amount of attached
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cells in anaerobic bio-dissolution environments is decreased. These results explain why
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the jarosite bio-dissolution efficiency cannot be improved by reducing the initial pH of
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the cultures. However, with the addition of Fe3+, the relatively higher amounts of
contents.
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bacteria likely counterbalanced the negative impact of the EPS components and
3.5 Mechanisms underlying the efficiency of jarosite bio-dissolution First, with the addition of Fe3+, the initial pH and zeta potential of the culture were reduced. The jarosite bio-dissolution efficiency could not be improved under a relatively low initial pH, as determined by the comparison of the jarosite bio-dissolution efficiencies of the experimental group (with the addition of 12 mM Fe3+) and control group (pH 1.68). This implies that the jarosite bio-dissolution efficiency is related to the zeta potential. In general, the surfaces of the dispersed jarosite particles are covered with an electric double layer, and the diffusion layer will be compressed with the 14
Journal Pre-proof addition of Fe3+ and become thinner with the increase of the Fe3+ concentration (Ostolska and Wisniewska, 2014; Yuan et al., 2018). In this case, the interaction and mass transfer between jarosites and bacteria will be easier as compared with those samples without the addition of Fe3+. Therefore, the bacterial erosion of the jarosite surfaces was strengthened, and the jarosite bio-dissolution efficiency was improved. Secondly, it was determined from the ATP results that the addition of Fe3+ could
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substantially boost bacterial growth. In the presence of Fe3+, the energy capture of A.
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ferrooxidans via the reduction of soluble Fe3+ is earlier and superior to that of the
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jarosites. Hence, it appears that the addition of Fe3+ provides an additional energy
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capture path to A. ferrooxidans, allowing the bacterial count to improve along with the
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increase in the Fe3+ concentration. In the authors’ prior study, it was found that the
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jarosite bio-dissolution efficiency is related to bacterial activity in the bio-dissolution system (Yang et al., 2019). Evidently, increased numbers of bacteria can serve to
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improve the contact probability between jarosites and bacteria; thus, this contact can be improved, ultimately enhancing the efficiency of the dissolution process. Moreover, the reduction reaction induced by adding Fe3+ can stabilize the culture pH during jarosite bio-dissolution by bacteria. In this regard, the increasing trend of the culture pH during jarosite bio-dissolution becomes slower, and this contributes to efficient jarosite bio-dissolution. The mechanisms influencing these improvements in jarosite bio-dissolution efficiency are presented in Fig. 6. On the one hand, the interaction and mass transfer between the jarosites and bacteria will be more efficient with the addition of Fe3+ 15
Journal Pre-proof because the diffusion layer of the electric double layer will be compressed and the zeta potential of the dispersed jarosite particles will be reduced. On the other hand, the amount of A. ferrooxidans is also improved by the addition of Fe3+, as the energy capture of A. ferrooxidans occurs earlier in the presence of Fe3+. Finally, the pH of the cultures can be stabilized by the reduction reaction induced by soluble Fe3+ (Eq. (4)) in the presence of Fe3+. Under the combined action of these factors, the rate and efficiency
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of jarosite bio-dissolution are increased. However, it is important to note that high
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concentrations of additional Fe3+ can limit the rate of Fe3+ reduction by A. ferrooxidans.
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3.6 Implications
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In this study, it was apparent that the addition of soluble Fe3+ ions contributed to
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jarosite bio-dissolution. Considering that there is a coexistence between jarosites and
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soluble Fe3+ in low-pH environments, it is not necessary to separate soluble Fe3+ and jarosites when performing dissolution under anaerobic conditions. Understandably, it is
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important to reuse jarosites in situ during acid mine drainage and bioleaching processes.(Wei et al., 2019) Moreover, in the case of jarosites, it is very simple to acquire soluble Fe3+ via Fe2+ leachate oxidation (Yang et al., 2018). In addition, the total Fe2+ concentrations of the samples only presented small differences when the concentration of the additional Fe3+ was greater than or equal to 12 mM. This implies that the total Fe2+ concentration in the group with the addition of 12 mM Fe3+ was close to the maximum possible amount of Fe3+ reduction in these batch experiments. Possible constraints affecting these results include the presence of hydrogen (low concentration levels and mass transfer rate in water) and the use of a 16
Journal Pre-proof mineral medium. As such, jarosite bio-dissolution in hollow-fiber membrane biofilm reactors may achieve superior results (Shen et al., 2018; Wu et al., 2017; Zhou et al., 2018), and further study of such reactions using hollow-fiber membrane biofilm reactors is required for large-scale applications.
Conclusions
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The addition of Fe3+ has an important influence on jarosite bio-dissolution using A.
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ferrooxidans. With the addition of Fe3+, the zeta potential can be reduced, as Fe3+ serves
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to compress the diffusion layer of the electric double layer. Additionally, the amount of
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A. ferrooxidans can be increased by the Fe3+ reduction pathway, and the pH of the
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cultures can also be maintained via this pathway. Considering these factors, the rate and
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efficiency of jarosite bio-dissolution can be significantly accelerated and improved. Furthermore, only limited amounts of Fe3+ are required for jarosite bio-dissolution. This
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study proposed a new method for improving the efficiency of jarosite bio-dissolution, and could help promote the use of jarosite bio-dissolution using A. ferrooxidans. However, more studies regarding the large-scale application of this approach are still needed.
Acknowledgements The authors would like to acknowledge the financial support from National Natural Science Foundation of China (Grant No.: 51478447 and 51878175), the Program for Innovative Research Team in Science and Technology in Fujian Province University 17
Journal Pre-proof (IRTSTFJ), and the Key Research and Development Program (2019YFS0055) and the Key Science and Technology Special Project (2018SZDZX0026) in Sichuan Province of China.
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waste printed circuit boards by mixed culture of acidophilic bacteria. J. Hazard. Mater. 2011; 192: 614-9.
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Figure Captions Fig. 1. Changes in iron ion concentrations during jarosite bio-dissolution Fig. 2. Changes in the zeta potential during jarosite bio-dissolution. Fig. 3. SEM images of jarosites after jarosite bio-dissolution. Fig. 4. Changes in the (A) ATP concentrations and (B) pH values during jarosite
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bio-dissolution.
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Fig. 5. The EEM of A. ferrooxidans EPS after jarosite bio-dissolution.
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Fig. 6. The underlying mechanisms of the improvement of jarosite bio-dissolution
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efficiency with the addition of Fe3+.
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Table Legends Table 1. The variables designed for the different groups.
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Table 2. The maximum generating rates of Fe2+.
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Journal Pre-proof Declaration of interests
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Groups
Medium pH
Added Fe3+
Adjusted pH
Initial pH of cultures
6 mM Fe3+
2.00
6 mM
--
1.76
12 mM Fe3+
2.00
12 mM
--
1.68
18 mM Fe3+
2.00
18 mM
--
1.64
24 mM Fe3+
2.00
24 mM
--
1.61
Control (pH 2.00)
2.00
--
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Table 1. The variables designed for the different groups.
1.68
Control (pH 1.68)
2.00
--
--
2.00
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1.68
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Journal Pre-proof Table 2. The maximum generating rates of Fe2+. The maximum generating rates of Fe2+ (mmol/(L∙d)) Only jarosite
and jarosite bio-dissolution +
bio-dissolution path++
6 mM Fe3+
2.96 (days 4-8)
2.28 (days 4-8)
12 mM Fe3+
4.23 (days 4-8)
2.66 (days 4-8)
18 mM Fe3+
4.60 (days 4-8)
24 mM Fe3+
4.35 (days 4-8)
Control (pH 2.00)
--
Control (pH 1.68)
--
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Including soluble Fe3+ reduction
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1.97 (days 8-12)
1.33 (days 12-15) 0.97 (days 4-8) 0.88 (days 4-8)
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Groups
(“+”: the maximum generating rate of Fe2+ including soluble Fe3+ reduction and
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jarosite bio-dissolution calculated from Fig. 1(B); “++”: the maximum generating rate
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of Fe2+ (only jarosite bio-dissolution path) calculated from Fig. 1(D).)
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Journal Pre-proof Graphical abstract
Highlights: Jarosite bio-dissolution efficiency is improved by adding soluble Fe3+
The diffusion layer of electric double layers on jarosites is compressed by Fe3+
Low pH (<2.0) can affect the extracellular polymeric substance of A. ferrooxidans
The pH of cultures can be stabilized by soluble ferric ions reduction
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Yuankun Yang, Shu Chen, Bin Wang, Xinyu Wen, Hanke Li, Raymond Jianxiong Zeng PII:
S0048-9697(19)36330-2
DOI:
https://doi.org/10.1016/j.scitotenv.2019.136334
Reference:
STOTEN 136334
To appear in:
Science of the Total Environment
Received date:
3 November 2019
Revised date:
23 December 2019
Accepted date:
23 December 2019
Please cite this article as: Y. Yang, S. Chen, B. Wang, et al., Effect of ferric ions on the anaerobic bio-dissolution of jarosites by Acidithiobacillus ferrooxidans, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.136334
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
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Effect of ferric ions on the anaerobic bio-dissolution of jarosites by Acidithiobacillus ferrooxidans
Yuankun Yang a, b, Shu Chen b, Bin Wang b, Xinyu Wen b, Hanke Li b, Raymond Jianxiong Zeng a, c * CAS Key Laboratory for Urban Pollutant Conversion, Department of Applied
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a
Key Laboratory of Solid Waste Treatment and Resource Recycle, Ministry of
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b
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Chemistry, University of Science and Technology of China, Hefei 230026, China
Centre of Wastewater Resource Recovery, College of Resources and Environment,
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c
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Education, Southwest University of Science and Technology, Mianyang 621010, China
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Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
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*Corresponding author:
Raymond Jianxiong Zeng; E-mail address: [email protected], [email protected]
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Abstract Large amounts of jarosites are produced during zinc hydrometallurgy and bioleaching, as well as in acid sulfate soils and acid mine drainage environments. As such, understanding the behavior of jarosite dissolution is important for analyzing the iron cycle process and promoting the control and treatment of jarosites. In general,
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soluble ferric ions and jarosites coexist in acid environments; however, the
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relationship between soluble ferric ions and jarosites under anaerobic reductive
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conditions is still not well understood. In this study, the effect of adding Fe3+ on the
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promotion of the bio-dissolution of jarosites using Acidithiobacillus ferrooxidans is
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investigated. With the addition of 12 mM Fe3+, the efficiency and maximum rate of jarosite bio-dissolution were found to reach 84.1% and 2.66 mmol/(L∙d), respectively.
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The addition of Fe3+ at concentrations higher than 12 mM did not further improve the
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jarosite bio-dissolution. These results indicate that the mechanisms underlying these improvements include: (i) the reduction of the zeta potential due to the compression of the diffusion layer of the electric double layer by Fe3+; (ii) bacteria growth enhancement and the stabilization of the pH of cultures via the reduction of soluble Fe3+. Based on these observations, this study serves to promote the development of jarosite bio-dissolution using Acidithiobacillus ferrooxidans and challenges the idea that soluble Fe3+ suppresses the bio-dissolution reaction of solid Fe3+ substances such as jarosite when soluble ferric ions and jarosite coexist. Keywords: Jarosites; Anaerobic bio-dissolution; Ferric ions; Acidithiobacillus 2
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ferrooxidans; Waste management
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Journal Pre-proof 1. Introduction Jarosites play an important role in the iron cycle, such as that occurring in acid sulfate soils and acid mine drainage environments, and in deferrization, which occurs during zinc hydrometallurgy (Han et al., 2014; Johnston et al., 2011; Li et al., 2007). Additionally, jarosites are the main waste products generated in the bioleaching fields, and their generation can inhibit the leaching reaction (Johnson et al., 2017; Yang et al.,
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2014; Yu et al., 2011b). Jarosites possess relatively good coprecipitation and substitution
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properties and are composed of many kinds of metal ions, including valuable metals
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(Ag and Im) and harmful metals (Pb and As) (Asta et al., 2010; Courtin-Nomade et al.,
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2012; Martinez-Sanchez et al., 2019; Wegscheider et al., 2017). Therefore, the research
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of jarosite dissolution is of significant importance to the understanding of the iron cycle
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process in acidic environments, as is the research of the control and treatment of jarosites and their impact on bioleaching efficiency. Compared with other methods of
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jarosite dissolution, the bio-dissolution of jarosites via anaerobic reduction has great advantages for industrial applications, including lower costs, environmental friendliness, and a simple control and management process, among others (Castro et al., 2016; Gao et al., 2019; Gonzalez et al., 2015; Ouyang et al., 2014). Thus, further investigation of the bio-dissolution of jarosites is of great importance. During jarosite bio-dissolution, jarosites serve as terminal electron acceptors that are restored by electron donors, and this is accompanied by jarosite dissolution (González et al., 2016; Ouyang et al., 2014). Typically, both jarosites and soluble Fe3+ coexist in low-pH environments, such as those encountered in acid mine drainage (pH 1
Journal Pre-proof around 3 to 3.8) and bioleaching (Elwood Madden et al., 2012; Song et al., 2018b). Soluble Fe3+ can serve as a terminal electron acceptor in anaerobic environments, and relevant research has shown that soluble Fe3+ is more readily available to Fe3+ reductases as compared to solid jarosites (Nevin and Lovley, 2002). This means that soluble Fe3+ and jarosites will compete for electron donors during the anaerobic reductive bio-dissolution of jarosites. In addition, the hydrolysis reaction of jarosites
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(Eq. (1)) will be affected when soluble Fe3+ is present.(Baron and Palmer, 1996) Based
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on these understandings, it is posited that the efficiency of the bio-dissolution of
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jarosites will remain at a low level when coexisting with soluble Fe 3+ in low-pH
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environments.
(1)
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KFe3(SO4)2·(OH)6 + 6H+ ←→ K+ + 3Fe3+ + 2SO42- + 6H2O
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In a recent study by this research group, it was found that unsuitable pH environments are harmful to jarosite bio-dissolution (Yang et al., 2019). However, the
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process of jarosite bio-dissolution requires acid, regardless of whether the Fe3+ reduction pathway (Eq. (2)) or SO42- reduction pathway (Eq. (3)) is utilized (Gao et al., 2019; Gonzalez et al., 2015). Thus, maintaining the pH of the culture during bio-dissolution might greatly improve its efficiency. According to the reaction presented in Eq. (4), the process of soluble Fe3+ reduction by bacteria increases the production of H+, and this process might provide a possible way to maintain the pH of the culture within a suitable range via the bacterial autoregulation of soluble Fe3+ reduction. In this way, soluble Fe3+ ions could contribute to jarosite bio-dissolution in low-pH environments. Nevertheless, the relationship between soluble ferric ions and jarosites under anaerobic reductive 2
Journal Pre-proof conditions remains unclear, and the effect of soluble Fe3+ on jarosite bio-dissolution requires further study. KFe3(SO4)2·(OH)6 + 3e- → K+ + 3Fe2+ + 2SO42-+ 6OH-
(2)
KFe3(SO4)2·(OH)6 + 8H2O + 16e- → K+ + 3Fe3+ + 2S2-+ 22OH-
(3)
2Fe3+ + H2 + bacteria → 2Fe2+ + 2H+
(4)
According to the jarosite bio-dissolution reactions represented by Eqs. (2) and (3),
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for the bio-dissolution of 1 mol of jarosites, the Fe3+ reduction pathway needs 3 mol of
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electrons and 6 mol of H+. In contrast, the SO42- reduction pathway needs 16 mol of
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electrons and 22 mol of H+, indicating that more electron donors and acid will be
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consumed in the SO42- reduction pathway than in the Fe3+ reduction pathway (Yang et
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al., 2019). Therefore, the Fe3+ reduction pathway is superior in regards to jarosite
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bio-dissolution. Correlational research has shown that soluble Fe3+ can be restored by Acidithiobacillus ferrooxidans (A. ferrooxidans) by using hydrogen or S0 as an electron
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donor in anaerobic environments (Das et al., 1992; Ohmura et al., 2002). Recently, the results of a study by the present research group also demonstrated that solid jarosites can be bio-dissolved by A. ferrooxidans by using hydrogen as the electron donor (Eq. (5)) in anaerobic conditions (Yang et al., 2019). This means that A. ferrooxidans can be used to help explore the relationship between soluble Fe3+ and jarosites in anaerobic reduction environments. Specifically, A. ferrooxidans activity in aerobic environments, such as in bioleaching or acid mine drainage, is one of the main factors resulting in jarosite generation. In this regard, research on the bio-dissolution of jarosites by A. ferrooxidans is important for the understanding of the iron cycle in natural 3
Journal Pre-proof environments, as well as for the in-situ disposal and reuse of jarosites during bioleaching. 2KFe3(SO4)2·(OH)6 + 6H+ + 3H2
A. ferrooxidans
2K+ + 6Fe2+ + 4SO42- + 12H2O (5)
This study aims to explore the effects of ferric ions on the mechanisms of the anaerobic bio-dissolution of jarosites by A. ferrooxidans. Changes of Fe2+ levels, Fe3+ levels, pH values, zeta potential, and adenosine triphosphate (ATP) concentrations were
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evaluated in batch experiments, and the surface morphologies of jarosites and
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extracellular polymeric substance (EPS) of A. ferrooxidans were also examined. These
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analyses will be beneficial for the development of jarosite bio-dissolution technologies,
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and will help promote the large-scale control and treatment of jarosite in acid mine
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environments and bioleaching in the future.
2. Materials and methods
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2.1 Preparation of jarosites
Jarosites were produced by using A. ferrooxidans (GenBank accession number: KJ094412) (Chen et al., 2015; Yang et al., 2014) in a 9K medium containing 45.00 g FeSO4∙7H2O as the energy source, and 3.00 g (NH4)2SO4, 0.50 g K2HPO4, 0.10 g KCl, 0.01 g Ca(NO3)2, and 0.50 g MgSO4∙7H2O in 1.0 L distilled water (Yang et al., 2019; Zhu et al., 2011). After 5 days at 30 °C and 170 rpm in a rotary shaker, the solid resultants of the Erlenmeyer flask were collected and washed by ultrapure water (pH = 2.00) three times, and then dried with a vacuum. 2.2 Adaptation phase in anaerobic conditions 4
Journal Pre-proof It was necessary to adapt the utilized bacteria to the presence of the jarosites. A. ferrooxidans was cultured using an anaerobic mineral medium containing 132 mg (NH4)2SO4, 52 mg KCl, 490 mg MgSO4∙7H2O, 41 mg K2HPO4, 9 mg CaCl2∙2H2O, 2 mg CuSO4∙5H2O, 0.5 mg NaMoO4∙5H2O, 1 mg Na2SeO4∙10H2O, 1 mg ZnSO4∙7H2O, 1 mg MnSO4∙H2O, 0.5 mg CoCl2∙6H2O, and 1 mg NiCl2∙2H2O in 1.0 L distilled water (Ohmura et al., 2002). As described in detail in a previous work (Yang et al., 2019),
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after deoxygenation (using high-purity nitrogen), pH adjustment (using dilute sulphuric
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acid (5 M)), and autoclave sterilization (at 121 °C for 30 min), jarosites (30 mM in Fe3+)
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and a 1 mL inoculum of A. ferrooxidans were then added into a serum bottle containing
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49 mL of anaerobic medium. Subsequently, 70 mL of mixed gas with a V(H2):V(CO2)
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ratio of 4:1 was filled into the bottle using a disposable sterilized syringe after removing
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the headspace with a vacuum pump (Ohmura et al., 2002). The bottle was incubated at 30 °C (170 rpm) in a rotary shaker. After several sub-cultivation processes, the
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supernatant of the bottle (late logarithmic phase) was applied as the inoculum. 2.3 Jarosite bio-dissolution testing Batch experiments were conducted in serum bottles with a total volume of 120 mL to evaluate the effect of adding Fe3+ during jarosite bio-dissolution using A. ferrooxidans. The variables for each of the groups are listed in Table 1. For each group, the additions of Fe3+ (ferric sulfate), jarosites (30 mM in Fe3+), and inoculums (1 mL) to the bottles were performed in an anaerobic glove box. The pH of the cultures was measured after 10 min. For the control groups, jarosites (30 mM in Fe3+) and inoculums (1 mL) were added to the bottles, and the pH of cultures was then adjusted to either 1.68 5
Journal Pre-proof (the same pH as the group with 12 Fe3+) using dilute sulphuric acid (5 M) or maintained at 2.00. Subsequently, the headspaces of all the serum bottles were removed with a vacuum pump, and 70 mL of mixed gas at a V(H2):V(CO2) ratio of 4:1 was added. All the bottles were cultivated in a rotary shaker at 30 °C (170 rpm). All experiments were performed in triplicate. During the experiments, supernatant samples (4 mL) were collected to analyze the
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soluble Fe2+, pH values, and total soluble iron ions (including Fe2+ and Fe3+ irons),
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while mix samples (4 mL) were collected to analyze the zeta potential, ATP, and total
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Fe(II) via sacrificed sampling, and each bottle was sampled only one time. For total Fe2+
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measurements, 0.5 mL of mixed samples were added into a polytetrafluoroethylene
lP
(PTFE) vessel in the anaerobic glove box, and 4.5 mL of hydrochloric acid solution (50%
na
v/v) was then added to dissolve the samples. Finally, the solution was transferred to a 50-mL volumetric flask and brought to volume with deionized water for total Fe2+
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determination after 1 hour (Castro et al., 2017). When the jarosite bio-dissolution reactions had ended, the bio-dissolution residues were collected to assess their morphologies. The mixed samples were also collected to evaluate the EPS of A. ferrooxidans. For EPS assessment, 20 mL of the mixed samples were added into a centrifuge tube and shaken for 30 seconds. Subsequently, the supernatant samples were collected after centrifugation (at 4000 rpm and 4 °C for 3 min), and the supernatant was then discarded after a second centrifugation (at 8000 rpm and 4°C for 10 min). A total of 10 mL mineral medium (pH = 2.00) was then added into the centrifuge tube and treated for 10 min via ultrasound (200 W and 40 KHz). Finally, 6
Journal Pre-proof the supernatant was used for EPS analysis after a third centrifugation (at 11000 rpm and 4°C for 10 min) (Yu et al., 2011a). 2.4 Analytical methods The total soluble iron ions were measured with an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 8300, PerkinElmer, United States). The Fe2+ ion concentration was measured using the ferrozine method.(Stookey, 2002)
of
The Fe3+ ion concentration is the concentration of the total soluble iron ions minus the
ro
concentration of Fe2+ ions. The ATP levels of A. ferrooxidans were analyzed via
-p
chemiluminescence using an A095-2 kit (Nanjing Jiancheng Bioengineering Institute,
re
Nanjing, China) and a multiscan spectrum (SpectraMax iD3, Molecular Devices, United
lP
States). The pH and zeta potential were measured using a digital pH meter (SevenMult
na
S40, Mettler Toledo, Germany) and zeta potential meter (Zetasizer Nano ZS90, Malvern, UK), respectively. The EPS characteristics of A. ferrooxidans were analyzed via
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three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy (QuantaMaster-40, PTI, United States). Finally, the surface morphologies of the jarosites were analyzed via scanning electron microscopy (Ultra 55, ZEISS, Germany).
3. Results and discussion 3.1 Changes in the Fe2+ and Fe3+ concentrations The changes in the Fe2+ and Fe3+ concentrations during the jarosite bio-dissolution process are presented in Figs. 1(A-D). On the whole, there was no significant difference between the soluble Fe2+ concentration (Fig. 1(A)) and the total Fe2+ concentration (Fig. 7
Journal Pre-proof 1(B)) in any of the groups. This indicates that the concentrations of other forms of Fe2+, such as Fe2+ adsorbed to the cell surface and ferrous minerals, were very low in these experiments. Considering that soluble Fe2+ ions can be reused directly in bioleaching fields, the bio-dissolution of jarosites under low-pH conditions is beneficial for jarosite treatment and recovery in the form of Fe2+. Additionally, it was apparent that the concentration of soluble Fe3+ ions (Fig. 1(C)) was rapidly decreased and remained at a
of
low level after 12 days in the groups with added Fe3+. This result implies that almost all
ro
soluble Fe3+ was restored to Fe2+ by A. ferrooxidans. Moreover, the concentrations of
-p
Fe2+ (soluble or total Fe2+) continued to increase during the bio-dissolution process, and
re
were higher than the initial concentrations of added Fe3+. This signifies that the
na
concurrent.
lP
reduction of the added Fe3+ and the occurrence of jarosite bio-dissolution were
More specifically, with the addition of 12 mM Fe3+, the total Fe2+ concentration
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reached 37.2 mM after 24 days of bio-dissolution (Fig. 1(B)). This implies that most of the total Fe2+ was generated via jarosite bio-dissolution when using A. ferrooxidans. Therefore, it was necessary to assess the Fe2+ concentrations during jarosite bio-dissolution. In theory, the paths for the generation of Fe2+ ions include the reduction of soluble Fe3+ and jarosite bio-dissolution in the groups with added Fe3+. As such, the generation of Fe2+ ions via jarosite bio-dissolution can be calculated by knowing the amount of added Fe3+ that is reduced. However, the hydrolysis of jarosites (Eq. (1)) in low-pH environments can produce some Fe3+ ions, which increases the difficulty of determining the amount of added Fe3+ that is reduced. In the authors’ prior study, it was 8
Journal Pre-proof found that the rate of Fe3+ generation by jarosite hydrolysis was lower than the rate of Fe3+ reduction by A. ferrooxidans (Yang et al., 2019). In addition, the hydrolysis of jarosites (Eq. (1)) and Fe3+ reduction (Eq. (4)) is one of the routes of jarosite bio-dissolution (i.e., an indirect pathway). Based on this, it was assumed that the soluble Fe3+ concentration in the culture could represent the remaining concentration of the
could be estimated by Eqs. (6) and (7), respectively.
of
added Fe3+. In this case, the Fe2+ generated by jarosite bio-dissolution and the efficiency
ro
Fe2+ generated by jarosites (mM) = concentration of total Fe2+ −
-p
(concentration of added Fe3+ − concentration of soluble Fe3+)
(6)
re
Jarosite bio-dissolution (%) = Fe2+ generated by jarosites (mM)/ (7)
lP
30 (mM, jarosites in Fe3+)
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The estimated data regarding the Fe2+ concentrations and jarosite bio-dissolution efficiency during jarosite bio-dissolution are presented in Fig. 1(D). In the control
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groups without added Fe3+, the jarosite bio-dissolution efficiencies reached only 34.5% (pH 2.00) and 37.5% (pH 1.68). In the groups with the addition of 12 mM Fe3+, the jarosite bio-dissolution efficiency was increased to 84.1%; the jarosite bio-dissolution efficiency was clearly improved greatly by the addition of 12 mM Fe3+. In addition, the jarosite bio-dissolution efficiency was enhanced from 67.2% to 84.1% when the added Fe3+ concentration was increased from 6 mM to 12 mM. When the added Fe3+ concentration was increased from 12 mM to 24 mM, the jarosite bio-dissolution efficiency rapidly decreased from 84.1% to 59.2%. This result indicates that the higher Fe3+ concentration did not further improve the dissolution efficiency. 9
Journal Pre-proof Table 2 summarizes the maximum generation rates of Fe2+ as estimated from the experimental results. In the jarosite bio-dissolution pathway, the maximum generation rate of Fe2+ reached 2.66 mmol/(L∙d) with the addition of 12 mM Fe3+. In contrast, the maximum generation rates of Fe2+ reached 0.97 mmol/(L∙d) (pH 2.00) and 0.88 mmol/(L∙d) (pH 1.68) without the addition of Fe3+. This suggests that the rate of jarosite bio-dissolution could be improved by adding Fe3+. In addition, the maximum generating
of
rate of Fe2+ (including the soluble Fe3+ reduction and jarosite bio-dissolution paths)
ro
began to reduce when the addition of Fe3+ exceeded 18 mM, while the maximum
-p
generating rate of Fe2+ (jarosite bio-dissolution path) had already started to decrease
re
when the addition of Fe3+ exceeded 12 mM. This implies that high concentrations of
lP
additional Fe3+ first affect the rate of jarosite bio-dissolution by A. ferrooxidans, and the
na
rate of soluble Fe3+ reduction then also decreases when the concentration of additional Fe3+ continues to increase.
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3.2 Zeta potential and jarosite morphology Soluble Fe3+, a multivalent ion, may compress the electric double layers of jarosite particles when added to the reaction (Ma and Pierre, 1999). To investigate the effects, the zeta potential and jarosite morphology during the bio-dissolution process were assessed. As presented in Fig. 2, the zeta potentials were reduced gradually in the two control groups without added Fe3+. This was because, as compared with the initial culture, there were more divalent ions (Fe2+ and SO42-) in the culture as the jarosite bio-dissolution progressed. Subsequently, the diffusion layer was compressed by divalent ions. However, with the addition of Fe3+, the zeta potentials exhibited an initial 10
Journal Pre-proof rapid decrease that occurred with the increase in the Fe3+ concentration, and then a slow increase. Moreover, the initial zeta potential decreased from 8.21 mV to 6.36 mV when the initial pH was decreased from 2.00 to 1.68 in the control groups. Additionally, the initial zeta potential in the group with 12 mM Fe3+ was 4.86 mV, which was lower than the value of 6.36 mV measured for the control group (pH 1.68), although the initial pH of each culture was the same. On the one hand, this indicates that the addition of Fe3+
of
was more effective than adjusting the initial pH in regards to the zeta potential. On the
ro
other hand, the main effect of the decrease of the zeta potential in the groups with added
-p
Fe3+ was the decrease in the concentration of Fe3+ rather than the low initial pH due to
re
Fe3+ hydrolysis (Eq. (8)) (Ebrahimi et al., 2003).
lP
When the diffusion layer is compressed, the mass transfer process between
na
jarosites and bacteria may be affected, leading to alterations of the jarosite surface. The effects of Fe3+-assisted bio-dissolution on the surfaces of the jarosite samples are
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depicted in Fig. 3. Without the addition of Fe3+, the surfaces of the jarosites in the control groups (pH 2.00 and 1.68) were smooth. With the addition of Fe3+, many particulate structures on the surfaces of the jarosites were apparent, indicating that the bacterial erosion of the surfaces was increased after the addition of Fe3+. Fe3+ + H2O → Fe(OH)2+ + H+
(8)
3.3 ATP quantity and EPS alterations In general, the ATP quantity of a single cell is relatively fixed. For example, the ATP level of A. ferrooxidans grown with Fe2+ was found to be 1.16 amol per cell (Pakostova et al., 2013). Thus, the concentration of ATP can reflect the relative quantity 11
Journal Pre-proof of microorganisms. As determined by the experiments, the ATP concentration in all the groups first increased and then decreased, as presented in Fig. 4(A). In the control groups, the concentration of ATP decreased with the decrease in the initial pH from 2.00 to 1.68. In contrast, the concentration of ATP increased with the addition of Fe3+ although the initial pH decreased from 1.76 to 1.61 (Table 1). Additionally, the ATP concentration of the group with 12 mM Fe3+ was higher than that of the control group
of
(pH 1.68), although the initial of pH values for these two groups were the same. These
-p
effect of a low pH on bacteria growth.
ro
results demonstrate that the addition of Fe3+ can boost bacteria growth and eliminate the
re
In addition, the EPS of A. ferrooxidans after bio-dissolution was characterized via
lP
three-dimensional EEM fluorescence spectroscopy. As presented in Fig. 5, with the
na
addition of Fe3+, the fluorescence peak locations in all the groups were located at Ex = 400 nm (Em = 430 nm). However, the fluorescence intensity and area coverage were
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diminished along with the increase of the concentration of added Fe3+ from 6 mM to 24 mM. Moreover, the same phenomena were also observed in the control groups. In the pH 2.00 control group, two fluorescence peaks were located at Ex = 400 nm (Em = 430 nm) and Ex = 280 nm (Em = 500 nm), whereas the fluorescence intensity and area coverage were diminished in the pH 1.68 control group. In the EEM spectra, the locations of the fluorescence peaks related to the different components of the EPS were as follows: tyrosine and aromatics in protein-like components (Ex = 220-250 nm, Em = 280-380 nm); tryptophan in protein-like components (Ex > 250 nm, Em = 280-380 nm); fulvic acid-like components (Ex = 220-250 nm, Em > 380 nm); humic acid-like 12
Journal Pre-proof components (Ex > 250 nm, Em > 380 nm).(Chen et al., 2003; Song et al., 2018a) These results suggest that the main constituents of the A. ferrooxidans EPS under these dissolution conditions were associated with humic acid-like components. This indicates that with the addition of Fe3+ and/or the adjustment of the initial pH, both the EPS components and EPS contents were reduced. 3.4 Changes in pH values
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Fig. 4(B) presents the changes in pH that occurred during the experimental process.
ro
In these experiments, the factors influencing pH included jarosite bio-dissolution (Eq.
-p
(5)), Fe3+ reduction (Eq. (4)), and Fe3+ hydrolysis (Eq. (8)). With the addition of Fe3+,
re
the initial pH values in all groups with added Fe3+ were lower than that of the control
lP
group (pH 2.00) due to Fe3+ hydrolysis. Subsequently, the pH values in all these groups
na
continued to increase with the progression of the jarosite bio-dissolution reaction (Eq. (5)). In addition, after the completion of the jarosite bio-dissolution reactions, the pH
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value (2.24) in the group with 12 mM Fe3+ was lower than the pH value (3.34) in the control group (pH 2.00). However, in contrast, the jarosite bio-dissolution efficiency in the group with 12 mM Fe3+ was more than twice that in the control group (pH 2.00). Clearly, the Fe3+ reduction process (Eq. (5)) served to stabilize the low-pH environment of the culture. In the authors’ previous study, it was found that bacteria activity was inhibited when the culture pH was less than 2.0 (Yang et al., 2019); however, the mechanism underlying this pH effect is not clear. In the present study, the initial culture pH in all the groups was less than 2.00, except for the control group (pH 2.00). The variation in EPS 13
Journal Pre-proof tended to be the same among the groups when the initial pH was less than 2.00; however, the amounts of A. ferrooxidans of these groups were significantly different, indicating that a low pH (under 2.00) mainly affected the EPS of A. ferrooxidans during jarosite bio-dissolution. Related research has shown that the EPS components and contents of A. ferrooxidans have an obvious impact on the interaction between the bacteria and minerals (Yu et al., 2011a). In bioleaching processes, the EPS secreted by
of
attached cells is significantly greater than that secreted by free cells (Yu et al., 2014; Yu et
ro
al., 2017). As such, a reduction in the EPS components and contents of A. ferrooxidans
-p
is disadvantageous to jarosite bio-dissolution, which reflects that the amount of attached
re
cells in anaerobic bio-dissolution environments is decreased. These results explain why
lP
the jarosite bio-dissolution efficiency cannot be improved by reducing the initial pH of
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the cultures. However, with the addition of Fe3+, the relatively higher amounts of
contents.
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bacteria likely counterbalanced the negative impact of the EPS components and
3.5 Mechanisms underlying the efficiency of jarosite bio-dissolution First, with the addition of Fe3+, the initial pH and zeta potential of the culture were reduced. The jarosite bio-dissolution efficiency could not be improved under a relatively low initial pH, as determined by the comparison of the jarosite bio-dissolution efficiencies of the experimental group (with the addition of 12 mM Fe3+) and control group (pH 1.68). This implies that the jarosite bio-dissolution efficiency is related to the zeta potential. In general, the surfaces of the dispersed jarosite particles are covered with an electric double layer, and the diffusion layer will be compressed with the 14
Journal Pre-proof addition of Fe3+ and become thinner with the increase of the Fe3+ concentration (Ostolska and Wisniewska, 2014; Yuan et al., 2018). In this case, the interaction and mass transfer between jarosites and bacteria will be easier as compared with those samples without the addition of Fe3+. Therefore, the bacterial erosion of the jarosite surfaces was strengthened, and the jarosite bio-dissolution efficiency was improved. Secondly, it was determined from the ATP results that the addition of Fe3+ could
of
substantially boost bacterial growth. In the presence of Fe3+, the energy capture of A.
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ferrooxidans via the reduction of soluble Fe3+ is earlier and superior to that of the
-p
jarosites. Hence, it appears that the addition of Fe3+ provides an additional energy
re
capture path to A. ferrooxidans, allowing the bacterial count to improve along with the
lP
increase in the Fe3+ concentration. In the authors’ prior study, it was found that the
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jarosite bio-dissolution efficiency is related to bacterial activity in the bio-dissolution system (Yang et al., 2019). Evidently, increased numbers of bacteria can serve to
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improve the contact probability between jarosites and bacteria; thus, this contact can be improved, ultimately enhancing the efficiency of the dissolution process. Moreover, the reduction reaction induced by adding Fe3+ can stabilize the culture pH during jarosite bio-dissolution by bacteria. In this regard, the increasing trend of the culture pH during jarosite bio-dissolution becomes slower, and this contributes to efficient jarosite bio-dissolution. The mechanisms influencing these improvements in jarosite bio-dissolution efficiency are presented in Fig. 6. On the one hand, the interaction and mass transfer between the jarosites and bacteria will be more efficient with the addition of Fe3+ 15
Journal Pre-proof because the diffusion layer of the electric double layer will be compressed and the zeta potential of the dispersed jarosite particles will be reduced. On the other hand, the amount of A. ferrooxidans is also improved by the addition of Fe3+, as the energy capture of A. ferrooxidans occurs earlier in the presence of Fe3+. Finally, the pH of the cultures can be stabilized by the reduction reaction induced by soluble Fe3+ (Eq. (4)) in the presence of Fe3+. Under the combined action of these factors, the rate and efficiency
of
of jarosite bio-dissolution are increased. However, it is important to note that high
ro
concentrations of additional Fe3+ can limit the rate of Fe3+ reduction by A. ferrooxidans.
-p
3.6 Implications
re
In this study, it was apparent that the addition of soluble Fe3+ ions contributed to
lP
jarosite bio-dissolution. Considering that there is a coexistence between jarosites and
na
soluble Fe3+ in low-pH environments, it is not necessary to separate soluble Fe3+ and jarosites when performing dissolution under anaerobic conditions. Understandably, it is
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important to reuse jarosites in situ during acid mine drainage and bioleaching processes.(Wei et al., 2019) Moreover, in the case of jarosites, it is very simple to acquire soluble Fe3+ via Fe2+ leachate oxidation (Yang et al., 2018). In addition, the total Fe2+ concentrations of the samples only presented small differences when the concentration of the additional Fe3+ was greater than or equal to 12 mM. This implies that the total Fe2+ concentration in the group with the addition of 12 mM Fe3+ was close to the maximum possible amount of Fe3+ reduction in these batch experiments. Possible constraints affecting these results include the presence of hydrogen (low concentration levels and mass transfer rate in water) and the use of a 16
Journal Pre-proof mineral medium. As such, jarosite bio-dissolution in hollow-fiber membrane biofilm reactors may achieve superior results (Shen et al., 2018; Wu et al., 2017; Zhou et al., 2018), and further study of such reactions using hollow-fiber membrane biofilm reactors is required for large-scale applications.
Conclusions
of
The addition of Fe3+ has an important influence on jarosite bio-dissolution using A.
ro
ferrooxidans. With the addition of Fe3+, the zeta potential can be reduced, as Fe3+ serves
-p
to compress the diffusion layer of the electric double layer. Additionally, the amount of
re
A. ferrooxidans can be increased by the Fe3+ reduction pathway, and the pH of the
lP
cultures can also be maintained via this pathway. Considering these factors, the rate and
na
efficiency of jarosite bio-dissolution can be significantly accelerated and improved. Furthermore, only limited amounts of Fe3+ are required for jarosite bio-dissolution. This
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study proposed a new method for improving the efficiency of jarosite bio-dissolution, and could help promote the use of jarosite bio-dissolution using A. ferrooxidans. However, more studies regarding the large-scale application of this approach are still needed.
Acknowledgements The authors would like to acknowledge the financial support from National Natural Science Foundation of China (Grant No.: 51478447 and 51878175), the Program for Innovative Research Team in Science and Technology in Fujian Province University 17
Journal Pre-proof (IRTSTFJ), and the Key Research and Development Program (2019YFS0055) and the Key Science and Technology Special Project (2018SZDZX0026) in Sichuan Province of China.
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Figure Captions Fig. 1. Changes in iron ion concentrations during jarosite bio-dissolution Fig. 2. Changes in the zeta potential during jarosite bio-dissolution. Fig. 3. SEM images of jarosites after jarosite bio-dissolution. Fig. 4. Changes in the (A) ATP concentrations and (B) pH values during jarosite
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bio-dissolution.
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Fig. 5. The EEM of A. ferrooxidans EPS after jarosite bio-dissolution.
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Fig. 6. The underlying mechanisms of the improvement of jarosite bio-dissolution
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efficiency with the addition of Fe3+.
22
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Table Legends Table 1. The variables designed for the different groups.
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Table 2. The maximum generating rates of Fe2+.
23
Journal Pre-proof Declaration of interests
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
24
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Groups
Medium pH
Added Fe3+
Adjusted pH
Initial pH of cultures
6 mM Fe3+
2.00
6 mM
--
1.76
12 mM Fe3+
2.00
12 mM
--
1.68
18 mM Fe3+
2.00
18 mM
--
1.64
24 mM Fe3+
2.00
24 mM
--
1.61
Control (pH 2.00)
2.00
--
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Table 1. The variables designed for the different groups.
1.68
Control (pH 1.68)
2.00
--
--
2.00
Jo ur
na
lP
re
-p
ro
1.68
25
Journal Pre-proof Table 2. The maximum generating rates of Fe2+. The maximum generating rates of Fe2+ (mmol/(L∙d)) Only jarosite
and jarosite bio-dissolution +
bio-dissolution path++
6 mM Fe3+
2.96 (days 4-8)
2.28 (days 4-8)
12 mM Fe3+
4.23 (days 4-8)
2.66 (days 4-8)
18 mM Fe3+
4.60 (days 4-8)
24 mM Fe3+
4.35 (days 4-8)
Control (pH 2.00)
--
Control (pH 1.68)
--
of
Including soluble Fe3+ reduction
re
-p
ro
1.97 (days 8-12)
1.33 (days 12-15) 0.97 (days 4-8) 0.88 (days 4-8)
lP
Groups
(“+”: the maximum generating rate of Fe2+ including soluble Fe3+ reduction and
na
jarosite bio-dissolution calculated from Fig. 1(B); “++”: the maximum generating rate
Jo ur
of Fe2+ (only jarosite bio-dissolution path) calculated from Fig. 1(D).)
26
Journal Pre-proof Graphical abstract
Highlights: Jarosite bio-dissolution efficiency is improved by adding soluble Fe3+
The diffusion layer of electric double layers on jarosites is compressed by Fe3+
Low pH (<2.0) can affect the extracellular polymeric substance of A. ferrooxidans
The pH of cultures can be stabilized by soluble ferric ions reduction
Jo ur
na
lP
re
-p
ro
of
27
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6