J. Cent. South Univ. Technol. (2009) 16: 0758-0762

DOI: 10.1007/s11771-009-0126-z

Bioleaching of soluble phosphorus from rock phosphate containing pyrite with DES-induced Acidithiobacillus ferrooxidans

CHI Ru-an(池汝安)^{1, 2}, HUANG Xiao-hui(黄晓慧)^{1, 2}, XIAO Chun-qiao(肖春桥)^{1, 2},

WU Yuan-xin(吴元欣)^{1, 2}, ZHANG Wen-xue(张文学)³

(1. Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology,

Wuhan 430073, China;

2. Hubei Key Laboratory of Novel Reactor and Green Chemical Technology, Wuhan Institute of Technology,

Wuhan 430073, China;

3. Yunnan Phosphate Chemical (Group) Co., Ltd., Kunming 650600, China)

Abstract: The effects of Acidithiobacillus ferrooxidans (At. f) mutated with diethyl sulfate (DES) as a mutagen on the bioleaching of soluble phosphorus (P) from rock phosphate (RP) were investigated. The results show that the oxidative activity of At. f is greatly improved by 1.0% (volume fraction) of DES. Correspondingly, the highest leaching rate of soluble P is also obtained to be 14.9% by the At. f mutated, which is 85.8% higher than that of the adapted At. f without mutation. In addition, the SEM images are significantly performed that the corrosion of RP residue surfaces leached by 1.0% DES-induced At.f is much worse than that of leached by the adapted At. f. All the above indicate that the leaching efficiency of soluble P from RP with pyrite can be greatly improved by using DES-induced At. f to a certain extent.

Key words: bioleaching; rock phosphate (RP); Acidithiobacillus ferrooxidans (At. f); diethyl sulfate; soluble phosphorus; mutation

1 Introduction

Phosphorus (P) plays a vital role in plant nutrition, but its concentration in soil solution is only approximately 0.05 mg/L [1]. For this reason, the possibility of the practical use of rock phosphate (RP) as a fertilizer has received significant interest in recent years [2-3]. Unfortunately, RP in plants is not available in soils with a pH range higher than 5.5-6.0, and even under optimal growth conditions, plant yields are lower than those obtained with soluble P [4].

Recently, the biological method on RP solubilization is more and more attractive due to its merits of environmental protection, low cost and high efficiency. At present, Acidithiobacillus ferrooxidans (At. f), as a kind of inorganic chemosynthetic autotrophy, is one of the mostly applied bacteria in biological oxidation, and recognized as a dominant strain to leach ores in acidic environment [5-7] . Its ability to oxidize Fe²⁺ to Fe³⁺ and elemental sulfur in acidic solution is well known [8]. Although bioleaching of low-grade sulfide minerals by At. f is successfully applied in mineral processing and hydrometallurgy, the use of At. f for leaching soluble P from RP has been only reported by a few researchers [9-12]. This bioleaching of soluble P is simple and economical, however, the leaching time from RP using At. f is longer, which is not suitable for application in industry.

At. f mutations by physical and chemical factors have also been increasingly applied to improving the bioleaching activity of strains due to their simple procedure and high efficiency [13]. Diethyl sulfate (DES) was chosen in this work due to the lower cost, more convenient and stable. Although At. f mutated by UV, microwave and nitrosoguanidine [14-18] has been successfully applied to ores leaching, there have been few reports on RP leaching by DES-induced At. f. Therefore, the concentration of DES mutation was optimized, and the leaching characteristics of soluble P by DES-induced At. f were investigated in this work, expecting to improve the leaching surroundings with pyrite by At. f mutated and increase leaching rate of soluble P.

2 Experimental

2.1 Materials

Culture medium of Leathern 9K consisted of 3 g/L (NH₄)₂SO₄, 0.5 g/L MgSO₄·7H₂O, 0.5 g/L K₂HPO₄,   0.1 g/L KCl and 0.01 g/L Ca(NO₃)₂ (sterilizating at   121 ℃ for 15 min), and 44.2 g/L FeSO₄·7H₂O. The pH of culture medium was adjusted to 2.5 by 20% H₂SO₄ solution. RP with particle size between 0.07 and 0.10 mm, which contained 49.8% CaO and 27.5% P₂O₅, was from Baokang Phosphate Mines in Hubei Province, China. Pyrite containing 48% sulfur was from Daye Iron Mines, China.

2.2 Activation of strain

At. f with inoculum concentration of 10% (volume fraction) was inoculated in flask containing 9K culture medium and then cultured in a shaker with a speed of 120 r/min at 30 ℃. 7 d later, the bacteria were inoculated again in the flasks containing pyrite so as to domesticate and elevate their adaptability to the surroundings [19]. Another 7 d later, all above were performed in duplicate. Lastly, the At. f activated was prepared for experiments after filtering while the concentration of At. f was adjusted to 1×10⁸ L^-1 at logarithmic phase.

2.3 DES mutation

The slurry of At. f with 1 mL DES of certain concentration added, was shaken for 15 min, and then the solution of sodium thiosulfate was used to end the induction. Lastly, the mutated At. f was kept in fridge at 4 ℃ for 12 h to enhance the effect of positive induction, and then cultured at 30 ℃. The prior strain was selected with the method suggested by ZHANG and WANG [20].

2.4 Bioleaching experiment

The domesticated and mutated At. f was inoculated in 40 mL iron-free 9K culture medium with 2.0 g RP and 2.0 g pyrite powders, respectively. The leaching rate of soluble P was measured continuously in the following  6 d. In addition, the leaching results by adapted At. f without mutation as the control were taken.

All the experiments were performed in triplicate.

2.5 Analytical methods

The concentrations of total iron and Fe²⁺ were determined by 1, 10-phenanthroline spectrophotometric method with a UV-Vis 8500 spectrophotometer at 510 and 506 nm, respectively [21-22]. The content of P₂O₅ in the leachate was measured using phosphomolybate method with a UV-Vis 8500 spectrophotometer at 420 nm [23], and the leaching rate of soluble P was expressed as the total content of P₂O₅ leached from 100 g RP. The pH was determined by an acidimeter (DELTA 320).

The oxidative activity of At. f was determined by the oxidation rate of Fe²⁺ [20]. Thereby, the oxidative activity was calculated by the following formula:

A=(C₀-C_t)/t                                  (1)

where  A, C₀ and C_t are the oxidative activity of At. f (g/(L·h)), the initial concentration of Fe²⁺ and the concentration of Fe²⁺ (g/L) by At. f for time t (h), respectively.

3 Results and discussion

3.1 Optimum concentration of DES

3.1.1 Optimum concentration of DES for oxidative activity of At. f

Using 0.2%, 0.4%, 0.6%, 0.8%, 1.0% and 1.2% (volume fraction) of DES as mutagens respectively, the effect of DES on oxidative activity of At. f was investigated. The oxidative activity of At. f was calculated on the basis of Eq.(1) by measurement of Fe²⁺ after being transferred for 3 generations. The results are shown in Fig.1.

Fig.1 Effect of DES concentration on oxidative activity of At. f after being treated at 120 r/min and 30 ℃ for 15 min

As shown in Fig. 1, obviously, with the increase of mutagen concentration from 0 to 1.0%, the oxidative activity of At. f. increases. The highest oxidative activity of At. f is 1.84 g/(L·h) when the concentration of DES is 1.0%, which is 3.8 times as large as that of the control. As the DES concentration reaches higher than 1.0%, the oxidative activity of At. f decreases.

3.1.2 Optimum concentration of DES for pH of medium with pyrite

After being mutated by 1.0% DES, the At. f was inoculated with 10% (volume fraction) of inoculum concentration into a flask that contains pyrite powders and 40 mL iron-free 9K culture medium with pH of 6.0 adjusted by H₂SO₄ solution, and then cultured in a shaker at a speed of 120 r/min and 30 ℃. The results of pH of liquid medium are shown in Fig.2.

It can be seen from Fig.2 that the pH decreases with the increase of culture time, and the lowest pH is obtained by 1.0% DES-induced At. f at the 5th day.

The main reactions of pyrite leached by At. f are as follows:

4FeS₂+15O₂+2H₂O2Fe₂(SO₄)₃+2H₂SO₄    (2)

FeS₂+Fe₂(SO₄)₃3FeSO₄+2S             (3)

4FeSO₄+O₂+2H₂SO₄2Fe₂(SO₄)₃+2H₂O    (4)

2S+3O₂+2H₂O2H₂SO₄                 (5)

Fig.2 Effects of DES concentration on pH of medium with pyrite at 120 r/min and 30 ℃

However, pH tends to be equilibrium after 5 d, that is to say, there is no more acid produced. Based on the above reactions, the main reasons are suggested that the content of iron as the growth energy source for bacteria has almost run out and then the use of At. f to sulfides (especially FeS₂) in pyrite is impacted, as shown in Fig.3. This indicates that, compared with the At. f without mutation, the amount of acid in the leachate by DES-induced At. f is enhanced under the same conditions.

Fig.3 Variations of total iron content in leachate by At. f during leaching at 120 r/min and 30 ℃

The main component of pyrite is FeS₂ companying with a few other sulfides and impurities, and that of RP is tricalcium phosphate. The mechanism of leaching soluble P from RP with pyrite by At. f is as follows.

Based on the oxidation of pyrite by At. f, Fe²⁺ from FeSO₄ is oxidized to Fe³⁺, producing energy for the growth of At. f, and the RP is then dissolved by H₂SO₄ from the former process, forming soluble P compounds:

3H₂SO₄+Ca₃(PO₄)₂→3CaSO₄+2H₃PO₄           (6)

Based on the above formula, the soluble P may be greatly impacted by the acidic surroundings for leaching. Therefore, the oxidative activity of At. f and the pH in the leachate were selected as references to find the proper concentration of DES.

Generally, considering the mechanism and the above results, the optimum concentration of DES of 1.0% was chosen for the following leaching experiments.

3.2 Dynamic characteristics of bioleaching soluble P by DES-induced At. f with pyrite

3.2.1 Variations of leaching rate of soluble P in leachate by DES-induced At. f

The At. f mutated by 1.0% DES was inoculated with 10% (volume fraction) of inoculum concentration into flasks containing 40 mL iron-free 9K culture medium, 2.0 g RP and 2.0 g pyrite powders, and then cultured in a shaker at a speed of 120 r/min and 30 ℃. The leaching rates of soluble P are shown in Fig.4.

Fig.4 Variations of leaching rate of soluble P in leachate by DES-induced At. f at 120 r/min and 30 ℃

It can be seen from Fig.4 that the leaching rate of soluble P increases continuously with the increase of leaching time. It reaches equilibrium after 5 d due to no more acid produced, which indicates the end of leaching. Compared with that of the control, the leaching efficiency of soluble P by 1.0% DES-induced At. f is prior. The corresponding leaching rate is 14.9%, which is 85.8% higher than that of the control at the end of leaching.

As for the leaching rate of soluble P, it almost gets to the maximum by bacteria induced in the 4th day as well as that of by bacteria adapted in the 5th day, so the leaching time is relatively shortened by 1 d. However, the highest leaching rates of the two appear in the 5th day due to the same growth periodicity of the bacteria which begins to be in the contabescence period in the 5th day. There is no more acid produced in both media at that time, which is the end of the leaching of soluble P.

3.2.2 Variations of pH by DES-induced At. f during leaching

Based on the leaching experiment mentioned in section 2.4, the variations of pH in the leachate were tested. The results are shown in Fig.5.

Fig.5 Variations of pH in leachate by DES-induced At. f at  120 r/min and 30 ℃

Clearly, the pH in the leachate inoculated with mutated At. f is much lower than that of the control during the leaching. That is to say, the acidic surrounding of the leachate with DES-induced At. f is prior to that of the control, which is beneficial to the leaching of soluble P from RP.

The acid should be generated by the bacteria and the pH in the leachate will decrease theoretically when the pyrite is oxidized by At. f. However, the leaching pH increases continuously with the increase of leaching  time, which is opposite to the former. There are probably two explanations. One is the main consumption of acid by leaching soluble P, which cannot maintain the optimum growth pH for the bacteria. The other is that the above results are caused by the physiological property of the bacterium which do not oxidize sulfur element immediately to produce sulfuric acid. Sulfur element is stored in the cells. Hence, the acidity of the leachate cannot meet the consumption rate of acid by leaching soluble P, and the pH in the leachate increases until acid in the leachate almost runs out after 5 d.

3.2.3 SEM analysis

The SEM images of RP residue surfaces are shown in Fig.6.

It can be seen from Fig.6 that the RP surface without treatment is smooth (Fig.6(a)), and the surfaces of RP residues leached by both the At. f without mutation and the DES-induced At. f are corroded to different extents as shown in Figs.6(b) and (c). Compared with the surface of RP residue in Fig.6(b), which is slightly eroded and covered by a lot of crystalline precipitate, the RP residue surface in Fig.6(c) is much scraggier, even producing some faults, which are seriously eroded by mutated At. f. The precipitate in Fig.6(b) can be dissolved into acid, so the lower the pH, the less the precipitate formed. All the above suggest that the enhancement of oxidative activity of bacterium by DES mutation is beneficial to the leaching of soluble P from RP.

Fig.6 SEM images of RP residue surfaces: (a) Control;      (b) Leached by adapted At. f; (c) Leached by 1.0% DES- induced At. f

4 Conclusions

(1) The results prove that At. f pretreated by DES mutation indirectly assists to leaching soluble P from RP, and the optimal concentration of DES to mutate At. f is 1.0% (volume fraction). The corresponding oxidative activity of mutated At. f is  1.84 g/(L·h), which is 3.8 times as large as that of the control, and the leaching rate of soluble P is 14.9%, which is 85.8% higher than that of the control.

(2) SEM image of RP residue surface by DES- induced At. f shows a more serious erosion than that of the At. f without mutation.

(3) Consequently, the leaching rate of soluble P from RP can be improved in the lower acidic surroundings with pyrite by DES-induced At. f. However, this improvement does still not meet the demands of industry, which needs to be probed further.

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(Edited by CHEN Wei-ping)