Effect of energy source and leaching method on bio-leaching of rock phosphates by Acidithiobacillus ferrooxidans

    Effect of energy source and leaching method on bio-leaching of rock phosphates by Acidithiobacillus ferrooxidans Lingling Li, Zaosheng Lv, Zhenyu Zuo, Zhonghua Yang, Xiangli Yuan PII: DOI: Reference:

S0304-386X(16)30372-3 doi: 10.1016/j.hydromet.2016.06.018 HYDROM 4384

To appear in:

Hydrometallurgy

Received date: Revised date: Accepted date:

25 February 2016 23 May 2016 21 June 2016

Please cite this article as: Li, Lingling, Lv, Zaosheng, Zuo, Zhenyu, Yang, Zhonghua, Yuan, Xiangli, Effect of energy source and leaching method on bio-leaching of rock phosphates by Acidithiobacillus ferrooxidans, Hydrometallurgy (2016), doi: 10.1016/j.hydromet.2016.06.018

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Effect of energy source and leaching method on

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bio-leaching of rock phosphates by Acidithiobacillus ferrooxidans

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Lingling Li*, Zaosheng Lv, Zhenyu Zuo, Zhonghua Yang, Xiangli Yuan

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College of Chemical Engineering and Technology, Wuhan University of Science and Technology, Wuhan, Hubei Province, 430081, PR China

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Corresponding author*: Lingling Li; Affiliation: College of Chemical Engineering and Technology, Wuhan University of Science and Technology, Wuhan, PR China; Permanent address: Wuhan University of Science and Technology, 947 Heping Avenue, Qingshan District, Wuhan, Hubei, 430081, PR China； E-mail address: [email protected]; Tel: +86 027 85967458;

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Zaosheng LV: E-mail address: [email protected]；

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Zhenyu Zuo: E-mail address: [email protected]； Zhonghua Yang； E-mail address: [email protected]； Xiangli Yuan： E-mail address: [email protected]；

ACCEPTED MANUSCRIPT Abstract The effects of energy source and leaching method on improving the bio-leaching of rock phosphates by Acidithiobacillus ferrooxidans CK were investigated. The dissolution of rock

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phosphates by strain CK in response to ferrous iron and elemental sulfur as the sole and mixed energy source was studied in a series of bio-leaching experiments without and with pre-cultivation

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of the microorganism. The mixture of ferrous iron and sulfur was found to be optimal for bio-leaching of rock phosphates. Bio-leaching of rock phosphates with pre-grown culture was observed to be enhanced significantly in comparison to that obtained without pre-cultivation under

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the condition of the same energy source. The presence of rock phosphates exerts a negative effect on bacterial oxidative activity. Phosphorous solubilization from rock phosphates relies on protons

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to proceed. The enhanced bio-leaching of rock phosphates coincided with the increased generation of gypsum crystals and the decreased formation of jarosite precipitates due to the relatively lower

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pH values achieved by the simultaneous release of protons from the precipitation of ferric ions and

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bio-oxidization of sulfur when both energy sources were available as well as the delayed addition of ore samples under pre-grown culture leaching.

1 Introduction

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Keywords: Acidithiobacillus; rock phosphates; jarosite; gypsum; bio-leaching; pre-cultivation

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Bio-leaching is an economical and environmentally friendly method for extracting metals from minerals, especially low-grade ores. Acidithiobacillus ferrooxidans (A.ferrooxidans) is one of the most important autotrophic bacterium involved in bio-leaching. This microorganism has been employed successfully in bio-leaching of low-grade sulfide minerals (Suzuki, 2001) and has also been used to dissolve nonmetallic elements, for example, to recover arsenic from medicinal realgar (Zhang et al., 2007) and solubilize phosphorous from rock phosphates (Priha et al., 2014; Xiao et al.,2013; Bhatti and Yawar,2010; Chi et al., 2006). Compared to leaching with sulphuric acid, bio-leaching of rock phosphates by A.ferrooxidans strain is expected to be an efficient, ecologically safe, low-cost way for comprehensive utilization of low-grade phosphorite. The A.ferrooxidans strain can derive energy from oxidizing elemental sulfur, ferrous iron and metal sulfide ores. The oxidized products, namely ferric iron and sulfuric acid respectively, can be thus released for the dissolution of ores. Therefore, ferrous iron and elemental sulfur as energy

ACCEPTED MANUSCRIPT sources have an important influence on mineral dissolution by A.ferrooxidans. It’s worthy to investigate the extent of mineral dissolution using A.ferrooxidans strain in the presence of ferrous iron and elemental sulfur. Previous studies have revealed that both ferrous iron and sulfur can

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effectively enhance the bio-leaching of sulfide ores (Sand et al., 2001). Ferrous iron was also found to be more effective than elemental sulfur for bio-leaching of realgar by this microorganism

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(Chen et al., 2011). However, little is known about the effects of ferrous iron and elemental sulfur on bio-leaching of rock phosphates by A.ferrooxidans as the sole and mixed energy source. Furthermore, some studies have investigated that different leaching methods exert great effects on

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metal recovery, showing that finding an effective process for the bio-leaching is very useful (Yan et al., 2014).

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In the present work, the effects of energy source and leaching method on improving the bio-leaching of rock phosphates by A.ferrooxidans were investigated. The dissolution of rock

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phosphates by strain CK with the sole and mixed energy source (ferrous iron or/and elemental

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sulfur) was studied in a series of bio-leaching experiments without and with pre-cultivation of the microorganisms. X-ray diffraction (XRD), scanning electron microscopy (SEM) and elemental

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analysis were analyzed to elucidate the changes in structural morphology, mineral surface features and chemical composition of leached residues for different energy sources and leaching methods, respectively.

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2 Materials and method 2.1 Mineral sample

Mineral samples of rock phosphates used in this study were obtained from Yichang phosphorite (Hubei, PR China). The ore samples were ground and sieved to 200-mesh size. The chemical composition of samples is shown as following: 21.98% P2O5, 33.55% CaO, 24.94% SiO2, 1.27% MgO, 5.08% Al2O3, 2.52% Fe2O3, 1.45% S, 0.86% Na2O, 2.42% K2O, 0.11% BaO, 0.025% MnO, 0.34% TiO2, 2.09%F, 0.003% As, 0.0001% Mo ,0.00014% Hg. 2.2 Culture medium 9K basal salt medium ((NH4)2SO4 3.0 g/L, K2HPO4 0.5 g/L, KCl 0.1 g/L, MgSO4·7H2O 0.5 g/L, Ca(NO3)2 0.01 g/L, pH adjusted to 2.0 with 1:1 H2SO4) was supplemented with ferrous iron (9K-Fe medium, 44.7 g/L of FeSO4.7H2O), sulfur powders (9K-S medium, 10.0 g/L of elemental sulfur) or the mixture of ferrous iron and sulfur (9K-Fe+S medium, 44.7 g/L of FeSO4.7H2O and

ACCEPTED MANUSCRIPT 10.0 g/L of elemental sulfur) for bacterial culture and bio-leaching (Zhang et al., 2013). 2.3 Bacterial culture A pure culture of A.ferrooxidans CK was isolated from an acid mine drainage of Yichang

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phosphorite (Hubei, China) (Li et al., 2013). To improve leaching efficiency of the isolated strain CK, the cells were domesticated in 9K-Fe medium by continuous transfer domestication which the

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concentration of rock phosphates gradually added was 0, 5, 10, 15, 20 g/L, respectively. The bacteria were then cultured for inoculum in 250-mL flasks, on an orbital shaker at 150 r/min and

0.7) and ninety mL of liquid 9K-Fe medium. 2.4 Bio-leaching experiments

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constant temperature of 30ºC. Each flask contained ten mL of inocula of active CK cells (OD600

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All bio-leaching experiments were performed in a 250-mL flask on a rotary shaker (at 150 r/min) incubated at 30ºC. The 9K-Fe cultured bacterium suspension was filtered out through

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Whatman No.4 filter paper (Φ≥ 20-25 μm) to remove jarosite precipitates (Wang and Zhou, 2012).

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The cells in the filtrate were harvested by centrifugation at 10,000 rpm for 10 min. The cells pellets were washed twice with 9K basal salt medium and re-suspended in basal salt solution with

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the final concentration of ~6.0×107 cells/mL to use as 10% inocula in the experiments. The experiments consisted of two leaching methods: bio-leaching without and with pre-cultivation of the microorganisms. Each method was further divided into three groups: the first group was 9K-Fe

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medium, the second group was 9K-S medium, the third group was 9K-Fe+S medium. For bio-leaching without pre-cultivation, 10.0 g/L of rock phosphates were initially added to these media with the inocula. For bio-leaching with pre-cultivation, however, 10.0 g/L of rock phosphates were added into the pre-grown cultures after 20-days of incubation. Sterile abiotic control was used in each set of experiments to determine the contribution of acid leaching. To calculate the fraction of phosphorus leached from rock phosphates, leaching solution (1 mL) was sampled from each flask at regular intervals to determine the supernatant content of P2O5 by phosphomolybdate method with a UV-2000 spectrophotometer at 420 nm (Chi et al., 2006). The pH value of each sample was measured with a pH probe. Each measurement was repeated three times and the average values were reported. The water lost by evaporation and sampling was compensated by the addition of sterilized K2HPO4-free 9K basal salt medium. The residues after leaching were washed three times using sterilized water and then dried for

ACCEPTED MANUSCRIPT analyses

by

scanning

electron

microscopy

(SEM,

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Nova400NanoSEM),

energy

dispersive X-ray spectrometry (EDS, Bruker Nano xFlash Detector 410-M), X-ray diffraction (XRD, Philips X’pert Pro MPD X-ray diffractometer) and elemental analysis (Elementar Vario EL

respectively.

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2.5 The effect of rock phosphates added on bacterial growth

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III) to examine their surface features, structural morphology and chemical composition,

Bacterial growth in liquid 9K media (9K-Fe, 9K-S or 9K-Fe+S medium) with or without the addition of 10.0 g/L of rock phosphates was monitored by directly counting the cells with

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Petroff-Hausser counting chamber in an optical microscope. The concentration of ferrous ions in 9K-Fe and 9K-Fe+S cultures and the content of sulfate in 9K-S culture were also measured at a

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certain intervals. The concentration of ferrous ions was determined by titration with potassium dichromate (Zhang et al., 2008). The sulfate concentration was determined by barium sulfate

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turbidimetry (Chesnin and Yien, 1950).

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2.6 RNA manipulation

A.ferrooxidans CK cells from 9K-S/ 9K-Fe+S culture without or with the addition of rock

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phosphates were collected at exponential growth phase, washed twice using sterile RNase-free ddH2O and treated with RNA protect® Bacteria Reagent (Qiagen) (Chen et al., 2012). The total RNA of CK cells was extracted using RNeasy Mini Kit (Qiagen). RNA concentration and purity

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was measured with NanoDrop 8000 UV-Vis spectrophotometer (Thermo Fisher). RT-PCR was performed using Toyobo ReverTra Ace qRCR RT Master Mix with gDNA remover (Toyobo). Q-RT-PCR was performed according to the manufacturer’s instruction (SsoAdvanced™ SYBR® Green Supermix, Biorad) in Biorad TQ™5 Multicolor Real-time RCR Detection System (Biorad) with the oligonucleotides listed in table 1.

Table 1 Oligonucleotides used in this study. Gene

Primer

Sequence

hdrA-L

5’-AGGAAATTTCTCCGAGTACC-3’

hdrA-R

5’-GTGTTTTTCCAGACCGATAC-3’

hdrA

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5’-CAATTACATGGTCCACCACA-3’

cycoB-R

5’-AGGGTATACACCACGATACC-3’

sqr-L

5’-AGACCGGTTATATGATCGAA-3’

sqr-R

5’-ATCATCTTGAACAGCACCTT-3’

tetH-L

5’-GGCATGTCCCAACCAAAG-3’

tetH-R

5’-GGAATCGCCATGATCCAG-3’

16SPF

5’-TGCAGGTCGACGATTGAGAGT-3’

16SPR

5’-GGTATTAGCCCAAGTTTCCCTG-3’

cycoB

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sqr

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tetH

rrs

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3 Results and discussion

3.1 Effect of rock phosphates on bacterial growth under different energy sources

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To study bacterial growth with ferrous iron and elemental sulfur as the sole and mixed energy source, the change of cell concentration with respect to time was monitored in 9K-Fe, 9K-Fe+S

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and 9K-S media. In 9K-Fe medium, A.ferrooxidans CK entered the log phase at about 18 h,

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sustained the exponential growth for 18 h and reached the highest cell concentration of 16.25× 107 cells/mL (Fig.1a). By comparison, the log growth phase of 9K-S cultured strain CK started at

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the 2nd day, and last over about 6 days to the highest cell concentration of 10.66×108 cells/mL (Fig.1c). The extended period of lag phase was observed in 9K-S medium due to the adaptation of cells to utilize sulfur which was always domesticated in 9K-Fe leaching medium. Despite the

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longer log growth phase and higher cell concentration due to the relatively abundant energy sources in 9K-S medium, the specific growth rate constant (µ) of strain CK cultured in 9K-S medium was lower than that of 9K-Fe cultured cells (0.0285 h-1 versus 0.1003 h-1, Table 2). A.ferrooxidans could derive energy for growth from the oxidation of ferrous ions and elemental sulfur. Some A.ferrooxidans strains have been reported to prefer to oxidize ferrous iron prior to sulfur when both substrates are available, while others oxidize the two substrates simultaneously (Ponce et al., 2012; Zhang et al., 2013). Strain CK oxidized ferrous ions immediately in the presence of both ferrous ions and elemental sulfur as electron donor. The CK cells cultured in 9K-Fe+S medium entered into logarithmic growth (Fig.1e) as soon as the fraction of Fe(II) oxidized initially increased (Fig.1f) at ~12 h. Noteworthy, the cells were still growing after the complete oxidization of ferrous ions at 40 h, indicating strain CK derived energy for this

ACCEPTED MANUSCRIPT growth from the utilization of sulfur. In order to investigate the effect of rock phosphates added in different leaching systems on bacterial growth, the change of cell concentration over time with rock phosphates added was

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studied. The logarithmic growth phase of the 9K-Fe cultured CK cells in the presence of rock

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phosphates started at 24 h, ended at 44 h with the highest cell concentration of 14.77×107

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cells/mL (Fig.1a). When rock phosphates were added into 9K-S medium, the CK cells entered the log phase at the 4th days and reached the highest cell concentration of 22.12×107 cells/mL at the 10th days (Fig.1c). The cell concentration in 9K-Fe+S culture was also decreased with the addition

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of rock phosphates in comparison with that without rock phosphates added (Fig.1e). Furthermore, the specific growth rate constant (µ) of strain CK was greatly reduced (9K-Fe: 0.0894 h-1 versus

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0.1003 h-1; 9K-S: 0.0136 h-1 versus 0.0285 h-1, Table 2) due to the inhibition of some elements (e.g. F) in rock phosphates on cellular biological activity (Peng et al., 2013；Li et al., 2013). Therefore,

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the presence of rock phosphates has a negative influence on the growth of CK cells, especially

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when cultured with elemental sulfur as the sole energy donor. The inhibition of rock phosphates on the growth of CK cells cultured with ferrous ions as the sole and mixed energy source was

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lessened compared to that with sulfur as the sole energy donor, which may result from the domestication of strain CK in Fe2+-containing leaching medium at all times. A.ferrooxidans could oxidize ferrous ions and elemental sulfur to ferric ions and sulfate,

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respectively. In order to study the effect of rock phosphates added on bacterial activity in the cultures with ferrous ions and elemental sulfur as the sole and mixed energy source, the content of ferrous ions or sulfate was monitored and compared between without and with the addition of rock phosphates. The comparison with abiotic control reveals that ferrous ions and elemental sulfur were oxidized markedly in the presence of the A.ferrooxidans strains. The bio-oxidation of ferrous iron were relatively slow in the first 12~18 h and afterwards increased dramatically regardless of the existence of rock phosphates in 9K-Fe and 9K-Fe+S cultures (Fig.1b and Fig.1f). Ferrous iron was completely oxidized at ～36 h without rock phosphates. However, the delay of the complete oxidization of ferrous ions was observed when rock phosphates were added. In 9K-S culture, the content of sulfate initially scarcely changed during lag phase and then continuously increased due to the bio-oxidation of sulfur powders by A.ferrooxidans CK. In the presence of rock phosphates, however, an initial slight drop of sulfate content was observed due to the consumption of sulfuric

ACCEPTED MANUSCRIPT acid (added for pH adjusting) during phosphorous solubilization when CK cells were at their lag phase. The concentration of sulfate then continued to increase progressively from bio-oxidation of sulfur powders by strain CK after the 4-days of lag phase. Furthermore, the increase extent of

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sulfate concentration was significantly reduced due to the addition of rock phosphates (Fig.1d).

Fig.1. The changes of bacterial populations and oxidative activity of A.ferrooxidans CK with or without the addition of rock phosphates (a) the change of bacteria populations in 9K-Fe culture; (b) the change of the fraction of Fe2+ oxidized in 9K-Fe culture; (c) the change of bacteria populations in 9K-S culture; (d) the change of sulfate content in 9K-S culture; (e) the change of bacteria populations in 9K-Fe+S culture; (f) the change of the fraction of

ACCEPTED MANUSCRIPT Fe2+ oxidized in 9K-Fe+S culture.

Table 2 Specific growth rate constant and generation time of strain CK cultured

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in 9K-Fe and 9K-S medium with or without the addition of rock phosphates. specific growth rate constant

generation time

(µ, h-1)

(G, h)

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medium

0.1003

9.97

9K-Fe (rock phosphates added)

0.0894

11.19

9K-S

0.0285

35.04

0.0136

73.53

9K-S (rock phosphates added)

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9K-Fe

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To determine the effect of rock phosphates added on the expression of some genes involved in reduced inorganic sulfur compounds (RISC) oxidation in the 9K-S and 9K-Fe+S cultured cells,

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the transcripts corresponding to the genes involved in S0 oxidation were quantified by using

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real-time quantitative RT-PCR (Q-RT-PCR) from 9K-S and 9K-Fe+S cultures under the cases without and with the addition of rock phosphates (Fig.2). The rrs gene coding for 16S rRNA was

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used as the reference standard. The fold increase in mRNA level between the culture without the addition of rock phosphates and that with ore samples added is represented in Fig.2. The hdrA, sqr, tetH, cycoB genes were less expressed in the CK cells grown with elemental sulfur as the sole

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energy source when rock phosphates added. These data indicated that the genes involved in RISC oxidation were highly repressed when rock phosphates were applied in 9K-S culture. However, the expression pattern of these sulfur-oxidation genes in 9K-Fe+S culture showed little distinct differences between without and with the addition of rock phosphates. Therefore, bio-oxidation of sulfur was greatly inhibited due to the addition of rock phosphates only when the CK cells were cultured with elemental sulfur in the absence of alternative other energy donors. The 9K-Fe+S cultured cells initially continued to grow from the oxidization of ferrous ions and reached their first plateau phase until the exhaustion of ferrous ions. When the 9K-Fe+S cultured cells at plateau phase turned to utilize elemental sulfur, they were more resistance to unfavorable elements in the niche than the 9K- S cultured cells which were just at lag phase.

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Fig.2. Q-RT-PCR experiments of genes involved in reduced inorganic sulfur compounds (RISC) oxidation. The data (S/S+P or Fe+S/Fe+S+P) represent the ratio of the amount of transcripts determined by Q-RT-PCR from 9K-S

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(a) or 9K-Fe+S culture (b) without the addition of rock phosphates versus that with rock phosphates added. The sqr, tetH, hdrA, cycoB genes are involved in RISC oxidation and encode sulfide quinone reductase, tetrathionate hydrolase, subunit A of heterodisulfide reductase and cytochrome oxidase bo 3, respectively.

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3.2 Bio-leaching of rock phosphates under different energy sources and leaching methods

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For bioleaching with pre-cultivation of the microorganism, rock phosphates were added into the pre-grown culture on the twentieth day. Fig. 3a shows the changes in pH values of different

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media during this process. The pH values of 9K-Fe and 9K-Fe+S cultures initially increased before the first days and then progressively decreased prior to the addition of rock phosphates. However, the decrease of pH value was observed in 9K-S culture all the time. The lowest pH

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values of 1.19, 0.99 and 1.14 for 9K-Fe, 9K-Fe+S and 9K-S cultures were attained after 20 days of incubation, respectively. After rock phosphates were added, pH values increased again due to acid consumption during phosphorous solubilization. The initial increases of pH value in the cultures with ferrous ions as the sole and the mixed energy source may be due to the oxidation of ferrous iron. In 9K-Fe and 9K-Fe+S media, ferrous ions were bio-oxidized by A.ferrooxidans strain into ferric ions with the consumption of H+ (Eq.(1)). Subsequently, ferric ions could be hydrolyzed in aqueous solutions (Eqs. (2), (3) and (4)) or be converted to basic jarosite, MFe3(SO4)2(OH)6 (Eq. (5)) (Wang et al., 2013). During this process, acid was produced and pH value gradually decreased. 4FeSO4+ O2+ 2H2SO4  2Fe2(SO4)3+ 2H2O

Eq.（1）

Fe2(SO4)3+2H2O  2FeOH2++H2SO4+2SO42-

Eq.（2）

A. ferrooxidans

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Eq.（3）

 2Fe(OH)3+3H2SO4 Fe2(SO4)3+6H2O 

Eq.（4）

2M++3Fe2(SO4)3+SO42-+12H2O  2MFe3(SO4)2(OH)6+6H2SO4

Eq.（5）

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(M may be H3O+, K+, Na+ or NH4+)

In 9K-S medium, elemental sulfur was bio-oxidized by A.ferrooxidans strain to sulfuric acid

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(Eq. (6)) (Leahy and Schwarz, 2009). Consequently, pH value of 9K-S culture kept decreasing. 2S+3O2+2H2O  2H2SO4 A. ferrooxidans

Eq.（6）

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In 9K-Fe+S medium, the decrease of pH values results from the simultaneous release of H+ in the bio-oxidization of elemental sulfur and related to Fe3+ precipitates. Thus, the 9K-Fe+S

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leaching solution is the most acidic among three types of leaching systems. The changes in the fraction of phosphorous leached with pre-cultivation of the microorganism over time are shown in Fig. 3b. After 4 days of leaching, the maximum fraction of

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phosphorous leached in 9K-Fe, 9K-Fe+S and 9K-S media reached 57.07%, 76.75% and 58.13%,

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respectively. The mixed energy source of ferrous ions and elemental sulfur was optimal for bio-leaching of rock phosphates by A.ferrooxidans CK. Phosphorous solubilization from rock

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phosphates was an acid-consuming reaction. The pH of the leachate has a significant effect on the extent of rock phosphate dissolution. The lowest pH value attained in 9K-Fe+S leaching solution results in the maximum dissolution of rock phosphates.

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Under the condition of bio-leaching without pre-cultivation, rock phosphates was initially added to the media with the inocula. The changes observed in pH values over time are presented in Fig. 3c. The pH values in 9K-Fe, 9K-Fe+S and 9K-S media increased during the first 2 days and then gradually decreased until the 20th day, reaching their lowest values of 1.45, 1.21 and 1.68, respectively. The initial increases of pH value in these cultures may be mainly due to acid consumption of phosphorous solubilization. The oxidation of ferrous ions also contributed to acid consumption in 9K-Fe and 9K-Fe+S leaching systems. By comparison, the pH value of 9K-S medium was greatly higher than those of 9K-Fe and 9K-Fe+S media. The changes of pH value for different media were compared under the conditions of two different leaching methods. For a given leaching medium, pH value of the leachate under pre-grown culture leaching was lower than that of bio-leaching without pre-cultivation, especially for 9K-S medium. Furthermore, the

ACCEPTED MANUSCRIPT decreased degree of pH value in 9K-S medium could hardly overtake that of 9K-Fe medium under the condition of bio-leaching without pre-cultivation while it was even a little higher than that of 9K-Fe medium under pre-grown culture leaching. The genes involved in RISC oxidation were

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repressed in the presence of rock phosphates when CK cells cultured with elemental sulfur as the sole energy. Consequently, the coexistence of rock phosphates and sulfur powders could hinder the

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bio-oxidation of sulfur and thereby elevate pH value of 9K-S leachate in the bio-leaching system without pre-cultivation. In pre-grown culture leaching system sulfur powders, however, could have been efficiently bio-oxidized to produce acids prior to the addition of ores, explaining the

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relatively lower pH value of this leaching solution.

Fig. 3d shows the changes in the fraction of phosphorous leached under the condition of

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bio-leaching without pre-cultivation. After leaching for 22 days, the maximum fraction of phosphorous leached in 9K-Fe, 9K-Fe+S and 9K-S media was 41.76%, 58.89% and 25.16%,

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respectively. The maximum fractions of bio-leached phosphorus with pre-cultivation constitute

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increases of 36.66%, 30.33% and 131.04% over those achieved by bio-leaching without pre-cultivation, respectively. Hence, the delayed supplementing of rock phosphates under

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pre-grown culture leaching can elevate leaching acidity and consequently accelerate the

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bio-leaching, especially in 9K-S leaching system.

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Fig.3. Effect of energy sources on the bio-leaching of rock phosphates under different leaching methods. (a) the

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changes of pH value over time during bio-leaching with pre-cultivation in different media (10.0 g/L of rock phosphates were added after 20 days’ culture); (b) the changes of the fraction of phosphorous leached over time

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during bio-leaching with pre-cultivation in different media; (c) the changes of pH value over time during bio-leaching without pre-cultivation in different media (10.0 g/L of rock phosphates were initially supplemented into each leaching medium); (d) the changes of the fraction of phosphorous leached over time during bio-leaching

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without pre-cultivation in different media.

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A.ferrooxidans can oxidize ferrous iron, elemental sulfur to produce sulfuric acid, which

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creates an acidic environment for the solubilization of rock phosphates (Xiao et al., 2011). In previous scientific studies, this autotrophic bacterium has been shown to solubilize phosphorous from rock phosphates or fluorapatite ores (Priha et al., 2014; Xiao et al., 2013; Bhatti and Yawar,

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2010; Chi et al., 2006). The percentage of phosphorous recovery from rock phosphates in this study was comparable to the previously reported P solubilized data as shown in table 3. Ferrous iron and elemental sulfur together as energy sources were optimal for bio-leaching of rock phosphates. In addition, fraction of phosphorous leached from rock phosphates with pre-grown culture were higher than that obtained without pre-incubation of bacteria under the condition of the same energy source. Although iron addition might have technological and economical unfavorable impact on the production of wet process phosphoric acid (e.g. the change of viscosity, P2O5 losses due to iron-phosphate precipitation and inferior fertilizer products) (El-Bayaa et al., 2011; El-Asmy et al., 2008), the larger amount of phosphorous was dissolved from rock phosphates in the presence of ferrous ions than sulfur as the sole energy donor in the bio-leaching experiments without pre-cultivation of microorganism. This difference of bio-leaching resulted from not the optimization of bio-leaching step due to the addition of ferrous iron but the inhibition

ACCEPTED MANUSCRIPT of rock phosphates on bio-oxidization of sulfur, which could be proved by the result that percentage of phosphorous recovery from rock phosphates with ferrous iron was almost the same as that with sulfur powders only under pre-grown culture leaching.

Leaching method

source sulfur-mud

bio-leaching with

Leaching

Dosage of rock

Percentage recovery

time (days)

phosphates (g/L)

of phosphorous (%)

40

100

41.8

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Energy

bio-leaching without

3.5

pre-cultivation bio-leaching without

14

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pyrite

pre-cultivation bio-leaching with

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pre-cultivation

21

Fe2+

bio-leaching without

10

Reference

Bhatti and Yawar, 2010

11.8

Chi et al., 2006

26.1

Xiao et al., 2013

10

25

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Fe2+ and S0

1

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pre-cultivation pyrite

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Table 3 The comparison of P solubilized (%) from rock phosphates by A.ferrooxidans in this paper and references

Priha et al., 2014

22

10

41.76

4

10

57.07

22

10

25.16

4

10

58.13

22

10

58.89

4

10

76.75

CE P

pre-cultivation Fe2+

bio-leaching with pre-cultivation

bio-leaching without

AC

S0

pre-cultivation

S0

bio-leaching with

this paper

pre-cultivation Fe2+ and S0

bio-leaching without pre-cultivation

Fe2+ and S0

bio-leaching with pre-cultivation

3.3 The morphology and structural changes of residues from different bio-leaching experiments The comparison of X-ray diffraction of rock phosphates with standard diffraction maxima for

ACCEPTED MANUSCRIPT fluorapatite

(ICDD

15-0876,

Ca5(PO4)3F),

carbonate-fluorapatite

(ICDD

21-0141,

Ca10(PO4)5CO3F1.5(OH0.5)), dolomite (ICDD11-0078, CaMg(CO3)2), pyrite (ICDD-24-0076, FeS2) and quartz (ICDD 01-0649, SiO2), indicated that crude ores mainly consist of fluorapatite,

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carbonate-fluorapatite and quartz associated with pyrite and dolomite (Fig. 4a).

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When comparing XRD analyses of all the residues leached in Fe2+-containing leaching

SC R

systems with the reported diffractograms for ammoniojarosite (ICDD 26-1014) and hydroniumjarosite (ICDD 21-0932), there are a number of peaks coinciding with ammoniojarosite and hydroniumjarosite (Fig. 4c, d, e, f). The structure of jarosite formed upon the residues can be

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also identified by SEM observation of these samples. There were many clusters of round and granular jarosite particles without sharp edges (Fig.5c-2, d-2, e-1) (Gunneriusson et al., 2009;

MA

Henao and Godoy, 2010). Another type of jarosite precipitates was still found to form euhedral compounds with well-developed sharp edges upon the residues (Fig.5e-2) (Wang et al., 2006;

D

Henao and Godoy, 2010). Furthermore, jarosite precipitates with a regular, smooth, round and

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crystalline appearance had also formed on the surface of minerals (Fig.5f-2) (Grishin et al., 1988; Wang et al., 2006). Jarosite precipitation is dependent on pH values, ionic composition and the

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concentration of leaching solution (Grishin et al., 1988). Since the concentration of ammonium ions (0.0455M) was far more than that of potassium ions (0.007M) in 9K medium, jarosites precipitates were mainly composed of ammoniojarosite and hydronium jarosite rather than

AC

potassium jarosite.

In XRD patterns of leached residues in 9K-Fe+S (Fig.4e and Fig.4f) and 9K-S media (Fig.4g and Fig.4h), the spectral line of elemental sulfur appeared significantly. Sulfur powders were also observed in SEM images to be precipitated in these residues (Fig.5g-3 and Fig.5h-2). Comparing to the standard file of database (ICDD 06-0047), X-ray diffractogram of bio-leached residues exhibited a number of characteristic peaks at 7.65 Ǻ and 4.28 Ǻ related to gypsum except that bio-leached in 9K-S medium without pre-cultivation (Fig.4c, d, e, f, h). The formation of gypsum in these inoculated leached samples was also verified by SEM observations. Gypsum crystals with prismatic morphology showed very little interlocking and were dispersed in these residues (Fig.5c-3, d-3, e-3, f-3, h-2). The acicular or needle-shape crystals with a high degree of interlocking were also observed in the residues leached in 9K-Fe+S medium with pre-cultivation (Fig. 5f-3).

ACCEPTED MANUSCRIPT Counts/s h

T

1100

900

NU

800

f

MA

700

e

TE

D

600

d

CE P

500

c

AC

400

g

SC R

IP

1000

300

200

b

100 a

0 20

30

40

50

60

Position [2ºTheta] fluorapatite (ref.no:15-0876)

carbonate-fluorapatite (ref.no:21-0141) pyrite (ref.no:24-0076)

ammoniojarosite (ref.no:26-1014) sulfur (ref.no:08-0247)

hydronium jarosite (ref.no:21-0932) dolomite(ref.no:11-0078) quartz (ref.no:01-0649) gypsum (ref.no:06-0047)

Fig.4. XRD patterns of (a) crude phosphorus, (b) residues in abiotic control, (c) residues bio-leached in 9K-Fe

ACCEPTED MANUSCRIPT medium with pre-cultivation, (d) residues bio-leached in 9K-Fe medium without pre-cultivation, (e) residues bio-leached in 9K-Fe+S medium without pre-cultivation, (f) residues bio-leached in 9K-Fe+S medium with pre-cultivation, (g) residues bio-leached in 9K-S medium without pre-cultivation and (h) residues bio-leached in

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T

9K-S medium with pre-cultivation.

The formation of gypsum crystals in the residues may be due to acid attack on the

A.ferrooxidans strains via several following steps:

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fluorapatite, carbonate-fluorapatite and dolomite in rock matrix through bacterial activity of

(1) The microbial metabolism provides H+ ions to solubilize rock phosphates. There are

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different possible reactions for producing sulfuric acid in leaching systems with ferrous iron and sulfur as the sole and mixed energy source as shown in (Eq. (1)~(6)).

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(2) During the solubilization of rock phosphates, H+ ions are consumed and soluble phosphorus increases. Fluorapatite, carbonate-fluorapatite and dolomite were the major

D

acid-consuming minerals in rock phosphates. Gypsum was produced due to acid attack on these

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minerals (Eq. (7), (8) and (9)). In this process, phosphoric acid was formed (Eq. (7) and (8)) which would further react with fluorapatite and carbonate-fluorapatite to produce soluble calcium

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dihydrogen phosphate (Ca(H2PO4)2) (Eq. (10) and (11)). This soluble phosphate further reacted with sulfuric acid to produce gypsum and phosphoric acid (Eq. (12)) (Bhatti and Yawar, 2010). Ca5(PO4)3F+5H2SO4+10H2O→3H3PO4+5CaSO4.2H2O+HF

Eq. (7)

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Ca10(PO4)5CO3F1.5(OH0.5)+10H2SO4+18.5H2O  10CaSO4.2H2O+CO2+1.5HF+H++5H3PO4 Eq. (8)

CaMg(CO3)2+2H2SO4→CaSO4.2H2O+MgSO4+2CO2

Eq. (9)

Ca5(PO4)3F+7H3PO4→5Ca(H2PO4)2+HF

Eq. (10)

Ca10(PO4)5CO3F1.5(OH0.5)+15H3PO4→10Ca(H2PO4)2+CO2+1.5HF+1.5H2O+H+ Eq. (11) Ca(H2PO4)2+H2SO4+2H2O→CaSO4.2H2O+2H3PO4

Eq. (12)

Given the dissolution of rock phosphates relies on the protons to proceed (Dorozhkin, 2002), the extent of phosphorous solubilization was directly related to the concentration of acids produced in leaching systems. SEM observation of the residues could further reveal the extent of rock phosphate dissolution in different leaching systems as shown in Fig.5. Morphology of rock phosphates prior to leaching observed under SEM (Fig.5a) indicate that the surface of crude ores was almost smooth and clear with some grinded scraps. After 20 days of leaching, there were only

ACCEPTED MANUSCRIPT a few insignificant erosive crevices in abiotic control (Fig.5b). On the surface of the leached residues in 9K-Fe medium, shown in Fig.5c and 5d, massive cracked blocks were observed in the bio-leached residues without pre-cultivation (Fig.5c-1), while many erosive pores were visible on

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T

the mineral surface obtained by pre-grown culture leaching (Fig.5d-1). In addition, large quantities of erosive pores were visible on the surface of the residues leached in 9K-Fe+S medium,

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especially those obtained by bio-leaching with pre-cultivation, exhibiting pronounced leaching features (Fig.5e-1 and Fig.5f-1). Many erosive pores were observed on the residues obtained by bio-leaching with pre-cultivation in 9K-S medium (Fig.5h-1), whereas intact crude ores appeared

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to be still dispersed around in the bio-leached residues without pre-cultivation (Fig. 5g-1), showing the relatively lower efficiency of phosphorous solubilization without pre-cultivation of

a

AC

c-1

CE P

TE

D

b

MA

A.ferrooxidans CK cultured with elemental sulfur only.

d-1

c-3

c-2

G

J

d-3

d-2

G J

ACCEPTED MANUSCRIPT

e-2

e-1

e-3

G

SC R

IP

T

J

f-2

f-3

G

TE

D

MA

J

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f-1

g-2

g-3

CE P

g-1

S

AC

P

h-1

h-2

S

G

Fig.5. SEM images of (a) rock phosphates, (b) residues leached by H2SO4, (c) residues bio-leached in 9K-Fe medium without pre-cultivation, (d) residues bio-leached in 9K-Fe medium with pre-cultivation, (e) residues bio-leached in 9K-Fe+S medium without pre-cultivation, (f) residues bio-leached in 9K-Fe+S medium with

ACCEPTED MANUSCRIPT pre-cultivation, (g) residues bio-leached in 9K-S medium without pre-cultivation, (h) residues bio-leached in 9K-S medium with pre-cultivation. (P: rock phosphates; J: jarosite; G: gypsum; S: sulfur powders.)

Although XRD is not a quantitative method, relative peak heights can be used as indicators of

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T

changes in the abundance of minerals (Tuovinen et al., 1994). The extent of dissolution could be denoted by the intensity ratio of the gypsum peak (d=7.56Å) and the fluorapatite peak (d=2.80Å).

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As shown in table 4, the intensity ratio of the gypsum peak and the fluorapatite peak in XRD patterns of bio-leached residues with pre-cultivation of the microorganism was largely increased when compared to that without pre-cultivation, indicating larger amounts of fluorapatite in rock

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phosphates were dissolved and converted to gypsum under pre-grown culture leaching. Elemental analyses of all the residues were also conducted to measure the percentage of dissolved

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phosphorous. Based on elemental analyses of rock phosphates prior to leaching and of the leached residues, dissolved phosphorus in all leaching systems was calculated. As Table 5 shows, for the

D

same leaching medium, phosphorus was dissolved in larger amounts with pre-cultivation of the

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microorganism than that without pre-cultivation. Furthermore, a larger amount of phosphorus was dissolved from rock phosphates in 9K-Fe+S medium than in 9K-Fe and 9K- S media.

CE P

Jarosite precipitation can lead to the generation of a passivation layer on the mineral surface, which can strongly inhibit ore erosion in 9K-Fe and 9K-Fe+S leaching systems. In order to examine the role of jarosite precipitates in the passivation of phosphorous ores during the

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bio-leaching, the intensities of the jarosite peak (d=5.12Å) and the fluorapatite peak (d=2.80Å) were monitored. As table 4 shown, the intensity ratio of the jarosite peak and the fluorapatite peak in the bio-leached samples with pre-cultivation was less than that without pre-cultivation. The significant decrease of jarosite precipitates on the residues leached in 9K-Fe+S medium was also observed in comparison with those in 9K-Fe medium. The proportion of jarosite precipitates in the leached residues was thus determined by measuring the content of elemental iron (Li et al., 2013). It can be seen in table 5 that in all the Fe2+-containing leaching systems, the lower weight ratio of jarosite precipitates and leached phosphorus, the greater dissolution of phosphorus from rock phosphates by strains CK. Therefore, the declined formation of jarosite precipitates as a passivation layer on the mineral surface coincided with the increased generation of gypsum crystals and the enhanced bio-leaching of rock phosphates in these leaching systems.

ACCEPTED MANUSCRIPT Table 4 Relative intensity of XRD peaks of leached residues under different conditions of energy source as well as leaching method.

Samples

Relative peak height (%)

jarosite

gypsum

(d=2.80Å)

(d=5.12Å)

(d=7.56Å)

crude ores

842.00

-

sulfuric acid leached residues

619.61

14.91

140.42

SC R

4.77

628.87

781.82

80.44

556.77

1033.84

1389.09

74.43

2100.86

579.00

1135.00

51.01

922.01

85.10

866.37

1868.70

46.36

2195.89

355.47

-

10.20

-

2.87

87.97

3.50

1375.37

0.25

1563.45

MA

66.12

NU

bio-leached residues with pre-cultivation (9K-Fe) bio-leached residues without

123.10

D TE

pre-cultivation (9K-Fe+S)

-

50.42

pre-cultivation (9K-Fe)

bio-leached residues with

gypsum fluorapati te

29.57

bio-leached residues without

pre-cultivation (9K-Fe+S)

jarosite fluorapatite

IP

fluorapatite

T

Peak height (cts)

CE P

bio-leached residues without pre-cultivation (9K-S)

AC

bio-leached residues with pre-cultivation (9K-S)

ACCEPTED MANUSCRIPT Table 5 Element analysis of leached residues under different conditions of energy source as well as leaching method (molecular mass of jarosite MFe3(SO4)2(OH)6 is determined by NH4Fe3(SO4)2(OH)6). leached P

Fe2O3

S

(wt.%)

(wt.%)

(wt.%)

(wt.%)

p

(mol/mol) (wt.%)

2.42

9.6

2.52

1.45

2.35

8.56

2.46

1.35

1.69

4.85

29.00

6.51

1.36

2.03

-

0.73

-

-

4.75

1.78

58.00

12.21

9.67

7.57

1.37

66.16

8.74

MA

pre-cultivation (9K-Fe)

AC

(9K-Fe+S)

CE P

bio-leached residues

pre-cultivation

33.08

TE

(9K-Fe)

without

D

bio-leached residues with pre-cultivation

1.04

NU

without

(wt./wt.) -

residues bio-leached residues

/leached P

(wt.%)

-

sulfuric acid leached

-

SC R

crude ores

jarosite

IP

Samples

jarosite Fe/S

T

K2O

1.14

2.10

23.49

17.37

7.50

0.54

46.98

6.26

0.96

0.91

24.43

16.81

8.69

0.58

48.86

5.62

1.60

5.80

6.67

20.76

3.80

0.13

-

-

1.43

3.02

6.33

24.48

6.58

0.10

-

-

bio-leached residues with pre-cultivation (9K-Fe+S)

bio-leached residues without pre-cultivation (9K-S) bio-leached residues with pre-cultivation (9K-S)

ACCEPTED MANUSCRIPT The main parameter affecting jarosite formation was pH value and the low pH value could hinder the formation of jarosite precipitates (Fu et al., 2008). The enhanced bio-leaching of rock phosphates when both energy sources were available may be due to the relatively lower pH

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T

achieved by the bio-oxidization of additional sulfur powders besides the precipitation of ferric ions and the consequent declined formation of jarosite precipitates on the surface of phosphorous

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ores when compared to that with ferrous ions in the absence of alternative other energy sources. Furthermore, phosphorous was more efficiently dissolved in the presence of ferrous ions than sulfur as the sole energy donor during bio-leaching without pre-cultivation while ferrous iron was

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almost as effective as sulfur for bio-leaching of rock phosphates under pre-grown culture leaching. The presence of rock phosphates in leaching system with elemental sulfur as the sole energy donor

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resulted in the inhibition of sulfur oxidation and slowing down the decrease of pH value due to the repression of the genes involved in sulfur oxidation. Under the condition of pre-grown culture

D

leaching method the relatively higher acidity, however, had formed prior to the addition of ores,

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which could further facilitate the dissolution of ores. Hence, the significant enhancement of bio-leaching by the delayed addition of rock phosphates under pre-grown culture leaching results

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from the lessened unfavorable impact of minerals on the bio-oxidation of energy source and the consequent elevated leaching acidity as well as the decreased precipitation of jarosite. 4 Conclusions

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The effects of energy source and leaching method on the bio-leaching of rock phosphates were explored in this work. The dissolution of rock phosphates was associated with acid production. The mixed energy source of ferrous ions and elemental sulfur was optimal for phosphorous solubilization due to the simultaneous production of acid from both the precipitation of ferric ions and bio-oxidation of sulfur. In the process of bio-leaching with pre-cultivation, the supplement of ore samples into leaching system later would lessen the unfavorable impact of some mineral elements (e.g. F) on bacterial activity, resulting in elevating the acidity of leachate and consequently enhancing bio-leaching of rock phosphates in comparison with that without pre-cultivation. Acknowledgement The work was supported by National Natural Science Foundation of China (grant number 21376184) and Natural Science Foundation of Hubei province of China (grant number

ACCEPTED MANUSCRIPT 2009CDA006). Reference Bhatti, T.M., Yawar, W., 2010. Bacterial solubilization of phosphorus from phosphate rock containing sulfur-mud.

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Hydrometallurgy 103(1-4), 54-59.

Chen, L.X., Ren, Y.L., Lin, J.Q., Liu, X.M., Pang, X., Lin, J.Q., 2012. Acidithiobacillus caldus sulfur oxidation

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by Acidithiobacillus ferrooxidans using ferrous iron and elemental sulfur as the sole and mixed energy source. Bioresource Technology 102(3), 3260~3267.

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Chesnin, L., Yien, C.H., 1951. Turbidimetric determination of available sulfates. Soil Science Society of America Journal 15 (1), 149-151.

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Chi, R., Xiao, C., Gao, H., 2006. Bio-leaching of phosphorous from rock phosphate containing pyrites by

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Acidithiobacillus ferroxidans. Minerals Engineering 19(9), 979-981. Dorozhkin, S.V., 2002. A review on the dissolution models of calcium apatites. Progress in crystal growth and

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El-Bayaa, A.A., Badawy, N.A., Gamal, A.M., Zidan, I.H., Mowafy, A.R., 2011. Purification of wet process phosphoric acid by decreasing iron and uranium using white silica sand. Journal of Hazardous Materials 190 (1-3), 324-329.

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ACCEPTED MANUSCRIPT Leahy, M.J., Schwarz, M.P., 2009. Modelling jarosite precipitation in isothermal chalcopyrite bio-leaching columns. Hydrometallurgy 98(1-2), 181-191.

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ferrooxidans. International Biodeterioration & Biodegradation 85(7), 156-165.

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Li, L.L., Lv, Z.S., Yuan, X.L., 2013. Effect of L-glycine on bio-leaching of collophanite by Acidithiobacillus

Peng, Z.J., Yu, R.L., Qiu, G.Z., Qin, W.Q., Gu, G.H., Wang, Q.L., Li, Q., Liu, X.D., 2013. Really active form of

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fluorine toxicity affecting Acidithiobacillus ferrooxidans activity in bio-leaching uranium. Transactions of Nonferrous Metals Society of China 23(3), 812-817.

Ponce, J.S., Moinier, D., Byrne, D., Amouric, A., Bonnefoy, V., 2012. Acidithiobacillus ferrooxidans oxidizes

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ferrous iron before sulfur likely through transcriptional regulation by the global redox responding RegBA signal transducing system. Hydrometallurgy 127-128(18), 187-194.

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Priha, O., Sarlin, T., Blomberg, P., Wendling, L., Mäkinen, J., Arnold, M., Kinnunen, P., 2014. Bioleaching phosphorus from fluorapatites with acidophilic bacteria. Hydrometallurgy 150, 269-275.

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Sand, W., Gehrke, T., Jozsa, P.G., Schippers, A., 2001. (Bio)chemistry of bacterial leaching—direct vs. indirect

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Tuovinen, O.H., Bhatti, T.M., Bigham, J.M., Hallberg, K.B., Garcia, O., Lindström, E.B., 1994. Oxidative dissolution of arsenopyrite by mesophilic and moderately thermophilic acidophiles. Applied and environmental microbiology 60(9), 3268-3274.

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Wang, Y.J., Li, H.Y., Li, D.P., 2013. Biosynthesis of natrojarosite by immobilized iron-oxidizing bacteria. International Journal of Mineral Processing 120(15), 35-38. Wang, M., Zhou, L.X., 2012. Simultaneous oxidation and precipitation of iron using jarosite immobilized Acidithiobacillus ferrooxidans and its relevance to acid mine drainage. Hydrometallurgy 125-126 (8), 152-156. Xiao, C.Q., Chi, R.A., Fang, Y.J., 2013. Effect of Acidiphilium cryptum on biosolubilization of rock phosphate in the presence of Acidithiobacillus ferrrooxidans. Trans. Nonferrous Met. Soc. China 23(7), 2153-2159. Xiao, C.Q., Chi, R.A., Li, W.S., Zheng, Y., 2011. Biosolubilization of phosphorus from rock phosphate by moderately thermophilic and mesophilic bacteria. Minerals Engineering 24(8), 956-958. Yan, H., Chai, L.Y., Peng, B., Li, M., Peng, N., Hou, D.K., 2014. A novel method to recover zinc and iron from

ACCEPTED MANUSCRIPT zinc leaching residue. Minerals Engineering 55, 103-110. Zhang, J.H., Zhang, X., Ni, Y.Q., Yang, X.J., Li, H.Y., 2007.Bio-leaching of arsenic from medicinal realgar by pure and mixed cultures. Process Biochemistry 42(9), 1265-1271.

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Zhang, Y.F., Yang, Y., Liu, J.S., Qiu, G.Z., 2013. Isolation and characterization of Acidithiobacillus ferrooxidans strain QXS-1 capable of unusual ferrous iron and sulfur utilization. Hydrometallurgy 136(4), 51-57.

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Zhang, Y.S., Qin, W.Q., Wang, J., Zhen, S.J., Yang, C.R., Zhang, J.W., Nai, S.S., Qiu, G.Z., 2008. Bio-leaching of

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chalcopyrite by pure and mix culture. Transactions of Nonferrous Metals Society of China 18(6), 1491-1496.

ACCEPTED MANUSCRIPT

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Highlights

The mixture of Fe2+ and S0 was optimal for bio-leaching of rock phosphates (RPs).



Bio-leaching of RPs could be significantly enhanced with pre-cultivation.



The mechanism involved in the enhanced bio-leaching of RPs was studied.



The mechanism relies on the lower pH and the consequent decreased jarosite.

AC

CE P

TE

D

MA

NU

SC R

IP

