MECHANISMS OF THIOBACILLUS FERROOXIDANS

DEPRESSING THE FLOATABILITY OF PYRITE

Dezhou Wei, Yimin Zhu, Haijun Liang and Zhifu Zhou

School of Resources and Civil Engineering, Northeastern University, Shenyang 110004, China.
E-mail: dzwei@mail.sy.ln.cn

ABSTRACT

It has been fairly well established by many experiments that Thiobacillus ferrooxidans (T. ferrooxidans),
also named Acidithiobacillus ferrooxidans(Kelly and Wood, 2000),can depress the floatability of pyrite.
However, the mechanisms governing the process have not been known completely. In the present
investigation, wettability and electrokinetic studies were carried out to examine the surface chemical
changes on pyrite before and after interaction with T. ferrooxidans or its secretions. Attachment of T.
ferrooxidans to pyrite was observed using a scanning electron microscope. The results of experiments
showed that, under certain conditions, T. ferrooxidans and its secretions would obviously change the surface
electricity of pyrite and result in the iso-electric point of pyrite shifting notably. However, the surface
wettability of pyrite hardly changed and its hydrophobicity increased little after interaction with T.
ferrooxidans and its secretions. Based on the results of the present investigation it can be reasoned that the
depression of floatability of pyrite is due to the reduction of surface free energy, rather than the increase of
surface hydrophilicity of pyrite. The decrease of surface free energy weakens the attachment of collector to
pyrite; during the process bacterial protein plays the most important role and the bacterial chemotaxis
strengthens the effect.

Keywords: Thiobacillus ferrooxidans, Pyrite, floatability, surface free energy.

INTRODUCTION

There have been many reports on research into the effects of microorganisms on the surfaces of mineral
particles (Santhiya et al, 2001; Zheng et al, 2001).The same kind of microorganism can have obviously
different effects on mineral particles, the mineral components of which are different. For example, the point
of zero charge of minerals can change a lot after interaction with microorganisms, or a chemical adsorption
with higher affinity is caused on the interface of microorganisms with minerals because some chemical
component of mineral particles (such as Mg2+) may interact with special components (such as teichoic acids)
on the surface of the microorganisms. A typical example of the latter case is that Bacillus sabtilis and
Marburg phlei depress dolomite in anionic floatation and improve the separating effect in floatation of
phosphorite and dolomite.
Pyrite is one of sulfide minerals that are distributed extensively in the earth. It is often necessary to
depress pyrite in the processes of flotation separation of sulfide minerals and coal flotation for
desulfurisation. However, the separation results are often not satisfying because of the small difference in the
floatability of different minerals to be separated. These problems are even more troublesome in the case of
coal flotation. In order to intensify the inhibition of pyrite flotation, many researchers have carried out a large
quantity of experiments with T. ferrooxidans (Yang et al, 2001; Zhou et al, 2000; Zhou et al, 2001; Wei et al,
2001). The results showed that the T. ferrooxidans habituated during the inoculation gives best results in the
depression of pyrite. After 10 minutes pretreatment, the removal rate of pyrite from coal is up to 80%~90%.
Research results of Yuan Xin et al (Yuan et al, 2000) showed that T. ferrooxidans could depress the
floatability of pyrite effectively and make the recovery of pyrite reduce from 95% down to 8%.
On the other hand, though the depressing function of T. ferrooxidans on pyrite has been confirmed by
many tests, the mechanism of depression of T. ferrooxidans on pyrite is seldom reported. Thus the systematic
experimental research has been carried through in this study.

Proceedings of the 22nd International Mineral Processing Congress (IMPC) 29 Sept 3 October 2003
Editors: Prof. L. Lorenzen and Dr Dee Bradshaw Cape Town, South Africa
ISBN: 0-958-46092-2 Produced by: Document Transformation Technologies
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EXPERIMENTAL

Materials

A pure mineral sample of pyrite was obtained from a mine in the southwest of China. Mineralogical
studies indicated that the sample was of high purity. Its X-ray diffraction spectrum is given in Figure 1. The
content of FeS2 is up to 99.3%. The sample was dry ground using a porcelain mill to 0.045mm for
adsorption studies. The 0.045mm size fraction was further ground in a porcelain mortar for the
electrokinetic studies.

Figure 1. XRD patterns of pyrite.

The strain of T. ferrooxidans used in this study was obtained from the resources and environmental
biotechnologicallaboratory of northeastern university, China. The bacterium was taken from a mine in the
southwest of China and was treated by separation and purification several times. The bacteria were
subcultured in the laboratory using modified 9 K medium (Table 1). The pH of the medium was adjusted to 2
using 5 mol/L sulfuric acid.

Table 1. 9 K cultivation medium.

Basal medium g/L

Ammonium sulfate 3.0
Magnesium sulfate 0.5
Potassium dihydrogen orthophosphate 0.5
Calcium nitrate 0.01
Pyrite powder 10.0

T. ferrooxidans secretion suspension is obtained by settling the T. ferrooxidans culture at a standstill for
15 minutes and then decanting 200mL supernatant liquid and filtering, following that settling the filtrate in
an ultra centrifuge for 15minutes at 10000 r/min. The supernatant liquid of the centrifuge tube obtained by
decantation is the T. ferrooxidans secretion suspension. The protein content of the secretion suspension
checked is 0.022g/L.

Methods
The cellular quantity of microorganisms adsorbed by mineral particles was expressed as the quantity of
proteins in the surface of mineral particles after interaction with microorganisms. The measurement of
proteins was carried out with a protein deposition method (Grary, 1977), which is based on the measurement
of Folin-hydrobenzene. During the measurement, in order to avoid the interference of colored ions in the
original solution the protein was precipitated with two kinds of reagents of DOCTCA and centrifuged in
an ultra centrifuge, then the settlings were taken into suspension with distilled water again and the content of
protein measured by thr Folin- hydrobenzene method. Before measuring the suspension,a calibration curve
was developed using the Folin- hydrobenzene method with solutions of bovine serum albumin in different
protein concentrations. The values of protein concentration were deduced from the measured results and
consulting the calibration curve.
The sugar measurement adopted the anthracene ketone colorimetry (Li et al, 1991)which is a sensitive,
fast, and simple way to measure the overall sugar quantity in samples.

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 As with the protein measurement, the calibration curve was determined according to several glucose
solutions with different known concentrations, then the colored iron ion was removed with alkalescence
hydrolyze method from the T. ferrooxidans solution to be tested and the sugar content was measured.
Eventually the value of sugar concentration was determined according to test results and consulting the
calibration curve.
Zeta potential measurements on pyrite and the cells, as well as bacterial secretion, before and after
interaction with each other, were carried out using a BDL-B surface potential meter. T. ferrooxidans culture
or T. ferrooxidans secretion suspension was put into a 100mL beaker. 0.3 g pyrite powder was added to the
beaker and mixed with a magnetic stirrer for 10 minutes. Then the beaker was placed at a standstill for 30
minutes. Finally the supernatant of the beaker was decanted for measuring Zeta potential.
In order to measure the contact angle of pyrite, the new surface of a pyrite block was prepared on
2000CCR carborundum paper, rinsed 3 times with distilled water and cleaned with cotton yarn. Then the
pyrite block was immersed into the medium immediately and an air bubble was injected on to the surface of
pyrite with a precision injector. After placing it at a standstill for 10minutes, the diameter and height of the
air bubble was observed with a microscope. Lastly the value of contact angle was calculated according to the
arithmetic average value of the testing results of ten air bubbles, the diameters of which ranged from 0.85mm
to 1.15mm.

RESULTS AND DISCUSSION

The solution containing T. ferrooxidans is a complicated system. Many researches have shown that the
surface properties of pyrite would be affected after adsorbing T. ferrooxidans and its secretion. This results
from the protein contained in the bacterium and its secretion. Protein is an amphoteric substance and its
electric properties will be changed when the pH of the system changes. Therefore, the electric properties of
T. ferrooxidans will alter with the pH of solutions. It is inevitable that the electric properties of pyrite would
be affected after adsorbing T. ferrooxidans. On the other hand, the surface properties of pyrite would be
affected after adsorbing T. ferrooxidans and its secretion because they possess different wettabilities.
Pyrite samples were reacted with two kinds of T. ferrooxidans cultures (the protein concentration is
0.0750.005g/L) and its secretion suspensions (one is at pH=1.97, the other is at pH=5.0) respectively in the
research. Then the adherence of T. ferrooxidans and its secretions on pyrite surface were measured and the
influence of the adherence on the Zeta potential and contact angle of pyrite were determined.

Adhesion on pyrite of T. ferrooxidans and its secretion

P.K. Sharma et al (Sharma et al, 2000)inoculated T. ferrooxidans with pyrite and found that their cell
walls were mainly composed of protein molecules by means of infrared spectrum measurement. Thus the
measurement of protein quantity adsorbed by pyrite surface can reflect the adsorption of T. ferrooxidans on
pyrite surface.
50mL T. ferrooxidans culture and its secretion suspension were poured into two 100mL beakers and 0.5
g pyrite powder (-0.045mm) was added into each beaker. The solution with different pH were stirred with a
magnetic stirrer for about 5 minutes. Then 10mL suspension was taken and filtered. After rinsing the filter
paper with distilled water 3 times, the filtrate solution was poured into a 50mL volumetric flask and the
volume of the solution was increased to the full scale by adding distilled water. Eventually, the protein
content of the solution was measured.
Adherent amount of bacterial proteins is calculated by the following formula:

Adherent amount of bacterial proteins (mg/g) = 50 (a-b)/ 0.5 (1)

where a is referred to the protein concentrations of T. ferrooxidans culture or its secretion suspensions (g/L);
b is the protein concentrations of filtrate after the adsorption (g/L).The measurement results are showed in
Figure 2.
It can be seen that the adhesion of T. ferrooxidans and its secretion on pyrite is very fast, the adherent
amount is already close to the max after stirring for 6 minutes and will seldom change any more in the case
of stirring time over 10 minutes. Therefore, it is suitable that stir and adsorption process lasts 10 minutes.

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 Figure 2. Attachment of bacterial protein on pyrite surfaces as a function of time.

Influence of T. ferrooxidans and its secretion on Zeta potential of pyrite

The measurement results of Zeta potential of pyrite at different conditions are shown in Figure3. The
results in Figure 3 show that the Zeta potential of pyrite is obviously changed because of the adherence of T.
ferrooxidans and its secretion to the surface of mineral particles. As the pH changes, the Zeta potential of
pyrite shows a great change, which is probably caused by the different quantity of electric charges possessed
by themselves, before T. ferrooxidans and its secretion adhere to the pyrite surface. According to the
measuring results of P..K.Sharma et al (Sharma et al, 2000), the iso-electric point of T. ferrooxidans is
pH=3.3 after equilibrating it with pyrite.

Figure 3. Zeta potential of pyrite before and after interaction with T. ferrooxidans and its secretion.

The results in Figure 3 also show that the bacterial chemotaxis (Zhang et al, 1993) and static gravitation
are the main causes of taking place adherence between T. ferrooxidans or its secretion and pyrite particles,
especially the special adsorption evocated by T. ferrooxidans or its secretion plays the key role within the
process.

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Influence of T. ferrooxidans and its secretion on contact angle of pyrite
The contact angle of pyrite is measured before and after reacting with T. ferrooxidans and its secretion
and the results are listed in Table 2.
The results in Table 2 show that the.change of wetting property of the pyrite surface is comparatively
complex after interaction with T. ferrooxidans and its secretion. The contact angle of pyrite reduces to some
degree after interaction with T. ferrooxidans and its secretion around the iso-electric point of pyrite. Away
from this point, the values of contact angle all increase to some degree. A.M.Raichur et al measured and
calculated the contact angle of pyrite in different mediums (Raichur et al 2001). The results showed that the
polar surface energy of pyrite is the lowest around the iso-electric point of pyrite and the interaction with
water molecules is weakened, which leads to the contact angle of pyrite increasing. The surface electric
properties of pyrite have obviously got to change after reacting with the bacterium and its secretion (see
Fig.3), which causes their contact angles to change apparently.
On the other hand, it is known from the results of Table 2 that T. ferrooxidans and its secretion cannot
increase the hydrophilicity of the surface of pyrite particles. Therefore, there must be other mechanisms for
depressing pyrite.

Table 2. Contact angle of pyrite before and after interaction with T. ferrooxidans and its secretion ( )

pH 3.06 4.03 5.41 6.10 7.63

17.62~ 16.43~20. 20.41~22. 26.19~30. 15.27~2

Range
21.44 08 29 67 0.27
Pyrite
Average value 19.85 18.13 21.59 29.04 17.56

19.55~ 20.22~24. 21.04~25. 20.52~24. 19.74~2

Range
Pyrite interaction with 23.93 75 36 37 3.10
T. ferrooxidans
pH= Average value 21.14 22.76 23.55 22.54 21.40
1.97
Pyrite interaction with 26.47~ 20.01~24. 23.43~28. 26.37~29. 26.03~3
Range
T. ferrooxidans 30.29 98 05 84 0.15
secretion
Average value 29.13 22.61 25.86 27.22 27.44

25.98~ 23.05~27. 21.32~25. 20.98~25. 20.05~2

Pyrite interaction with Range
30.14 87 09 46 3.94
T. ferrooxidans
Average value 28.85 24.04 22.83 23.01 22.05
pH=
5.0
Pyrite interaction with 28.47~ 24.72~28. 23.02~25. 21.47~25. 20.20~2
Range
T. ferrooxidans 32.43 85 89 36 4.96
secretion
Average value 29.23 26.72 24.42 23.47 22.96

Influence of T. ferrooxidans and its secretion on surface free energy of pyrite

Up to now, there is not a universal credible method to determine surface energy, surface free energy, or
surface tension on account of the complexity of the surface of the solid. In order to analyse the influence of
T. ferrooxidans and its secretion on surface free energy of pyrite we quote the formula for estimating the
surface free energy of a solid put forward based on Good-Girifalco liquid-liquid interfacial theory (Gu et al
2001):
S0 = [LV (1+cos ) + ]2/ (42LV) (2)

where S0 is the surface free energy of solid in the vacuum, LV is the liquid-gas interfacial tension, is the
contact angle of solid surface, is the reduction of solid surface free energy caused by adsorbing liquid
saturation vapor and is a parameter having a bearing on the solid-liquid interaction.

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 At the pyrite surface, because of > 0, can be neglected, so formula (2) can be simplified as:

S0 = LV (1+cos ) 2/ (42) (3)

From the results of measuring the contact angle of pyrite, the surface contact angle of pyrite becomes
larger in most cases after interaction of pyrite and T. ferrooxidans or its secretion. According to formula (3),
it is an inevitable outcome that the surface free energy of pyrite will reduce after interaction with T.
ferrooxidans or its secretion.

Interaction of pyrite and bovine serum albumin or cornstarch

Bovine serum albumin can dissolve in water and be deposited with saturated ammonium sulfate, so it is
extensively used in experimental research as a kind of standard protein. The iso-electric point of bovine
serum albumin is pH=4.7 according to Shens research ( Shen et al, 1980).
Starch is a kind of natural polysaccharide and can be made into soluble starch by reacting it gently with
acid. The final hydrolytic product of starch is glucose in an acidic solution. Cornstarch contains carboxyl and
can react with metal ions on the surface of minerals (Shen et al, 1980; Lu et al 1992).

Figure 4. Zeta potential of pyrite after interaction with bovine serum albumin and starch.

Two portions of 150mg bovine serum albumin were dissolved into 2000 mL distilled water respectively,
the pH of one solution was adjusted to 2.0 and that of another solution was adjusted to 5.0 with 0.1mol/L
diluted sulfuric acid. The two solutions of cornstarch were placed in an oscillator (250C, 100r/min) for 1 hour
to dissolve. The pH of the two solutions was measured and adjusted to 2.0 and 5.0 respectively again. The
process was repeated several times and was not stopped until the pH of the two solutions was stable at 2.0
and 5.0 respectively. Then the two solutions were filtered and the concentrations of sugar in filtrates were
measured. Finally, the two solutions were made into 1000mL solutions with a sugar concentration of
0.026g/L by adding distilled water with the same pH respectively.
The influence of bovine serum albumin and cornstarch on the Zeta potential of pyrite is shown in Figure
4. The influence of bovine serum albumin and cornstarch on the contact angle of pyrite surface is shown in Table 3.
It can be seen from the experimental results that bovine serum albumin has a stronger effect on the surface
electric properties of pyrite, the result of which is similar to that of T. ferrooxidans and its secretion, however
the change to the of iso-electric point is less. This is due to the special adsorption of T. ferrooxidans on pyrite
surface and the different iso-electric point of the cells and bovine serum albumin. The influence of bovine serum
albumin on the contact angle of pyrite is similar to that of T. ferrooxidans, but the degree of influence is
obviously less, which is mainly because bovine serum albumin can dissolve in water.

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 While the function of cornstarch is comparatively less, and the iso-electric point of pyrite is almost
unchanged, the change of Zeta potential of pyrite is also less, but it causes the decrease of contact angle on
the pyrite surface.

Table 3. Contact angle of pyrite after interaction with bovine serum albumin and corn starch ( 0 )

pH 3.06 4.03 5.41 6.10 7.63

18.72~2 17.92~2 18.91~2 21.02~2 18.02~2
bovine serum range
2.80 2.20 3.05 4.99 2.04
albumin
pH Average value 20.76 19.94 20.47 22.37 20.08
=2.0 15.33~1 15.31~1 16.24~2 20.01~2 16.06~2
range
Cornstarch 8.83 9.37 0.72 3.97 0.11
Average value 17.08 17.34 18.96 21.39 18.61
19.77~2 20.03~2 18.26~2 19.22~2 20.03~2
bovine serum range
4.04 4.38 2.44 4.92 4.84
albumin
pH Average value 22.77 22.06 20.35 21.07 21.03
=5.0 17.03~2 15.59~1 16.60~2 21.38~2 18.27~2
range
Cornstarch 0.48 8.75 0.34 5.76 1.93
Average value 19.03 17.67 18.94 23.58 20.49

The experimental results also show that the influence of T. ferrooxidans and its secretion on the surface
properties of pyrite is mainly the effect of the protein and polysaccharide, after they adhere to the pyrite
surface. The protein can increase the hydrophobicity slightly, but polysaccharide can increase the
hydrophilicity of pyrite to a greater extent.

Mechanisms of T. ferrooxidans depressing the floatability of pyrite

Protein is one of the most important compositions of T. ferrooxidans and its secretion, existing in the
form of compounds in the live tissue and cells. Every kind of cell contains thousands of kinds of proteins.
Sugar is an important constituent of the cells of bacteria, representing about 10%~30% in cell dry weight and
existing in the form of sugar or composite compounds with protein and fat. The composition of T.
ferrooxidans and its secretion is of course similar.

(a) (b)
Figure 5. Scanning electron micrographs of T. ferrooxidans adsorbing on pyrite surface.

Figure 5 is a scanning electron microscope image of the surface adsorption of T. ferrooxidans on pyrite .
Image (a) shows that T. ferrooxidans adhering uniformly onto the uneven and rough parts of the pyrite
surface, and from image (b) it can be seen that T. ferrooxidans and its secretion adhere to an assemblage of
pyrite grains.
It is clear by analyzing the experimental results, that the attachment of T. ferrooxidans and its secretion on
pyrite surfaces cannot increase their hydrophilicity but it causes the Zeta potential of the pyrite surface to
increase markedly and the contact angle of the pyrite surface to increase to some extent.

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 The increase of contact angle of the pyrite surface would cause a reduction of surface free energy of
pyrite. It is the increase of Zeta potential and reduction of surface free energy that inhibits the interaction of
collectors, such as xanthate and kerosene with pyrite surfaces, so that pyrite is depressed.

CONCLUSIONS

From researching the behaviour of T. ferrooxidans and its secretion with pyrite, we can draw the
following conclusions:
! The chemotaxis of T. ferrooxidans and static function are the main factors promoting attachment of T.
ferrooxidans to pyrite surfaces;
! The electric properties and wettability of pyrite surfaces change because of the adhesion of
T.ferrooxidans and its secretion. At the same time, the Zeta potential of pyrite will have a larger
excursion and hydrophobility of pyrite increase a little bit;
! The depression of T. ferrooxidans and its secretion on the floatability of pyrite is realized not through
increasing the hydrophilicity of pyrite surface, but mainly through decreasing the surface energy of
pyrite, as well as holding back the interaction of collector and pyrite surface. Bacterium protein and
polysaccharide play an important role in the process.

ACKNOWLEDGEMENTS

This research was supported with the National Natural Science Foundation of China (contribution No.
50174014), the Natural Science Foundation of Liaoning Province(contribution No.20022025) and the
Science and Technological Problem Cracking Program of Liaoning Province (contribution No.2001304024).

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