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CHAPTER THREE
CHAPTER THREE:
RESULTS
RESULTS 91
CHAPTER THREE
This study is focused on investigating the ability and role of native bacteria on the
bioleaching of zinc and optimization of their bioleaching activity by manipulation different
parameters. To achieve these objectives the potential of a new strain of Acidithiobacillus
ferrooxidans isolated from complex zinc and lead sulfide mine was investigated. The
identification of organism has been done with the conventional and specialized techniques.
Further, the effects of pH, Fe2+ concentration, temperature, ammonium sulfate
concentration and magnesium concentration on growth and biooxidation efficiency of
bacterium was evaluated. The bioleaching ability of this bacterium was studied at two
different levels (small scale in flask and large scale in 450 l column bioreactor). The
optimization of growth conditions for higher extraction of zinc was performed at both
scales to find out the best conditions for bioleaching of zinc and to compare these results
with previous results for optimization of growth and oxidation efficiency of bacterium.
The response of a bacterial strain to heavy metal toxicity is described in this chapter.
The isolate was studied for its tolerance to nine heavy metals, i.e., zinc, manganese, nickel,
cobalt, copper, arsenate, chromium, lead and mercury. The minimum inhibitory
concentration (MIC) of each metal was determined. The growth response of the isolate to
metals was carried out by growing the bacteria in seventeen different concentrations of
those metals.
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Proteins are involved in the various pathways. In any response of an organism towards its
surrounding environment proteins are bound to be involved. Global analysis of the proteins
is therefore most essential. This has been done by employing a proteomic approach. The
detail results are presented below.
3.1 MINE ANALYSIS AND SAMPLING
In the present study, Iranian zinc and lead sulfide complex mine was selected for
bioleaching propose. This mine is a low grade sulfide mine with more than 120 million
tons of concentrate which is one of the largest zinc sulfide mines of the world. The mine is
located in central of Iran in Yazd province. The bioleaching process has not been used
industrially for extracting of metals from Iranian zinc and lead mines (Report of Iranian
Ministry of Mining and Industry). The main product of these mines mostly is concentrate
and even the pyrometallurgical methods are not used for extraction the precious metals
from these mines. One of these mines is Koshk zinc and lead mine in Yazd central of Iran.
The mine is sulfide base mine and one of the main problem of such kind of mine is
environmental contamination due to burn the mine while extraction with pyrometallurgical
methods. Hence to solve this problem, in this study we have studied the ability of native
bacteria for bioleaching of zinc from the mine.
The samples were collected from different sites of the mine, including the different
parts of surface and the depth of the mine (190 m). Samples were included the rocks of the
RESULTS 93
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mine and concentrates in the form of soil. After sampling the chemical and mineralogical
analysis of the ore by XRD and XRF was carried out. As can be seen from Table 3.1.,
XRD analysis of mine sample revealed that Pyrite, Calcite, Dolomite, Gypsum and
Sphalerite are the main components of the mine and Pyrite, Gypsum and Sphalerite are the
sulfide parts of mine. The XRF analysis of mine also showed that Fe, S, Zn and Pb with
concentration of 24.24, 14.8, 3.1 and 1.27 % respectively having highest concentration of
different elements in the mine. The other properties of mine like temperature of different
sites of mine, size of the mine, water availability and type of water (salinity and other
properties) were detected. At the time of sampling the temperature of the surface of the
mine was 38 - 40 oC and at 190 M depth of mine we had 55 oC temperatures.
Fig. 3.1. Mine samples, collected from Koshk Zn-Pb mine.
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Table 3.1. Composition of the mine sample was analyzed by (A) XRD and (B) XRF
(A) XRD analysis:
Component % Component %
Pyrite (FeS2) 35 Asphalerite (ZnS) 4
Calcite (CaCO3) 24 Quarts (SiO2) 2
Dolomite(CaMg(CO3)2) 21 Others 1
Gypsum (CaSO4.2H2O) 13 Total 100
(B) XRF analysis:
Component % Component % Component %
Fe 24.24 MgO 4.52 CuO 0.008
S 14.8 MnO 0.512 SiO2 2.83
Zn 3.1 Na2O 0.53 TiO2 0.169
Pb 1.27 BaO 0.065 Y2O2 0.003
Cl 0.11 K2O 0.15 Al2O3 0.59
Co3O4 0.015 P2O6 0.12 Nb2O3 <0.001
CeO2 <0.001 MoO3 <0.001 V2O5 <0.001
Rb2O <0.001 NiO3 <0.001 ThO2 <0.001
ZnO2 <0.001 WO3 <0.001 U3O8 <0.001
RESULTS 95
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3.2. ISOLATION AND IDENTIFICATION OF BACTERIA
A total of five acidophilic bacterial strains and one fungus were isolated from the mine
sample by using three different media viz. TK medium (Tuovinen, et al 1973) Leathen
(Leathen., et al. 1956) and 9-K (Silverman, et al. 1959). The results of bacterial isolation
indicated that 9-K medium is the best medium for isolation of acidophilic microorganisms
from mine samples. Because among the six isolates, four of them were isolated in the 9-K
medium and also the amount of iron oxidation was highest in 9-k medium as compared to
other media. The cultures were purified by using modified 9-K solid 2:2 medium. As we
wanted to make the solid media with lower pH (2.5) we used 0.6 % agarose (Agarose for
routine work from Sigma) instead of noble agar and we could solidify the medium at pH
2.5. Fig 3.2 shows the colonies of bacterial isolate in 9-k 2:2 solid medium. The primary
identification of the bacteria was performed by study the various parameters. The results
are given in Table 3.2
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Fig. 3.2. Colonies of bacterial isolate. The left side plate is the control without inoculum
and the right side plate is showing the colonies and growth of the isolate. As can be seen
due to oxidation of iron (ferrous to ferric) the color of medium has changed to red.
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Table 3.2. Primary identification of isolates by studies the important parameters.
M.Y.1 M.Y.2 M.Y.3 M.Y.4 M.Y.5
Gram staining - - - - -
Motility + + + + -
Oxidation of
iron
+ + _ + +
Oxidation of
sulfur
+ + + - +
pH range 1.2-3.3 1.2-3 1.5-3.2 1- 2.8 1.5- 3.6
Growth on
organic
compounds
_ - - - -
Temperature
range (oC)
10-37 10-40 15-40 4-35 4-35
Anaerobic
growth
- - + - -
catalase + + + + +
oxidize + + _ + +
Growth on agar + + _ _ +
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For screening the isolates the iron oxidation efficiency of each one were evaluated. The
amount of oxidation of ferrous iron to ferric iron was measured with colorimetric method.
On the basis of these results we have selected the best bacterium with maximum iron
oxidation efficiency (Fig 3.3)
0
10
20
30
40
50
60
70
Fe (I
I) o
xida
tion
(%)
M.Y.2 M.Y.3 M.Y.4 M.Y.5
Bcterial Codes
Fig. 3.3. The amount of iron oxidation in the 9-k medium by different bacterial
isolates.
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The selected bacterium was used for further identification by 16S ribotyping. For this
propose we should isolate the bacterial DNA, but the presence of iron precipitation in the
medium was interfering in the DNA isolation process so we were used different
concentrations of EDTA to remove irons and 150 mM EDTA was the best concentration
for the same. Fig. 3.4 shows the analysis of isolated DNA and plasmids from different
isolates. The 16S ribotyping and BLAST-n analysis of the 753 base pairs was done at the
National Center for Biotechnology Information (NCBI) sever which confirmed the identity
of organism. The results confirmed that the isolated organism is a new strain of At.
ferrooxidans, and we have named it as an Acidithiobacillus ferrooxidans DF1. At.
ferrooxidans belongs to the group of chemolithotrophic organisms, which are rod-shaped,
non-spore forming, gram-negative, motile, and single pole flagellated.
Fig.3.4. Analysis of DNA and plasmids isolated from different strains by Agarose
Electrophoresis.
RESULTS 100
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Sequence of the 16S r RNA gene fragment (753 Base pairs)
GTCGGTCGGT CGTTGATCAT GCTTGTCGAG GGTAACAGCT CTTCGGATGC
TGACGAGTGG CGAACGGGTG AGTAATGCGT AGGAATCTGT CTTTTAGTGG
GGGACAACCC AGGGAAACTT GGGCTAATAC CGCATGAGCC CTGAGGGGGA
AAGCGGGGGA TCTTCGGACC TCGCGCTAAG AGAGGAGCCT ACGTCCGATT
AGCTAGTTGG CGGGGTAAAG GCCCACCAAG GCGACGATCG GTAGCTGGTC
TGAGACGAGG ACCAGCTACA CTGGGACTGA TACACGGCCC AGACTCCTAC
GGGAGGCAGC AGTGGGGAAT TTTTCGCAAT GGGGGCAACC CTGACGAAGC
AATGCCGCGT GCATGAAGAA GGCCTTCGGT TTGTAAAGTC CATTCGTGGA
GGACGAAAAG GTGGGTTCTA ATACAATCTG CTATTGACGT GAATCCAAGA
AGAAGCACCG GCTAACTCCG TGCCAGCAGC CGCGGTAATA CGGGTGGTGC
AAGCGTTAAT CGGAATCACT GGGCGTAAAG GGTGCGTAGG CTGTAGTTAG
GTCTGTCGTG AAATCCCCGG GCTCAACCTG GGAATGGCGG TGGAAACCGG
TGTACTAGAG TATGGGAGAG GGTGGTGTAA TTCCAGGTGT AGCGGTGAAA
TGCGTAGAGA TCTGGAGGAA CATCAGTGGC GAAGGAGGTC ACCTGGCCCA
ATACTGACGC TGAGGCACGA AAGCATGCTG GAGCACACAG GATTAGATCC
GGG
RESULTS 101
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3.3 PROCESS OPTIMIZATION To achieve the best conditions for growth and activity of bacteria the composition of
medium and growth conditions were optimized by varying five different factors and each
one were studied in four different levels; pH (1.4, 1.6, 1.8 and 2); temperature (25, 30, 35
and 40 oC); Fe2+ concentration (2, 4, 6, and 8 g/l), (NH4)2SO4 (1, 2, 3 and 4 g/l) and Mg2+
concentration (20, 40, 60 and 80 mg/l). The effect of each factor was studied on the growth
and activity of bacterium separately, because we wanted to find out that whether the
optimum conditions for the growth are same as optimum conditions required for maximum
oxidation. The increase in the number of free bacterial cells in solution was considered as
growth improvement and the increase in concentration of ferric iron (oxidation of ferrous
iron) was also considered for indicator of improvement of activity of bacterium as the main
role of microbes in bioleaching process is to oxidize the ferrous iron and producing ferric
ion which can ultimately oxidize the metal sulfides of the mine.
The concentration of free bacteria in solution was determined by direct counting using a
Thoa chamber with an optical microscope (X1000), and ferric ion concentration was
measured spectrophotometrically using 5- sulfosalicylic acid. During the study after
finding the optimum range of one factor other factors were studied at that range. The
sequence of the studied factors was pH, iron concentration, temperature, ammonium
concentration and magnesium concentration respectively.
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The results from this section of study are divided to five parts as bellow:
3.3.1. EFFECT OF pH
Acidity of the environment controls the bacterial activity within a system. The H+ ion is
in fact vital for acidophilic microorganisms since bacteria utilize it as a proton source for
the reduction of O2. The effect of pH on growth and activity of bacterium was studied in
the range of 1.4 to 2. As, pH 1.2 was the lowest pH which the bacterial cell could tolerate
we did not study the effect of pH below 1.4. The results are presented in Table 3.3.
The effect of acidity (pH 1.4 - 2.0) on the growth of bacterium and oxidation of iron (Fig.
3.5 & 3.6) showed that increase in the acidity (pH 1.4) led to the significant increase in the
growth and efficiency of bacterium and increase in the pH up to 2 has an adverse effect on
the growth and efficiency of bacterium for the conversion of ferrous to ferric. The main
focus of this experiment was to establish that the optimum pH required for the growth (1.4)
and efficiency of conversion (1.6) of bacteria are not the same. As can be seen from the
Fig.3.5 and 3.6 the optimum pH required for growth of bacterium is different from the pH
which was required for maximum oxidation of ferrous iron and at pH 1.6 the amount of
oxidation of iron was higher than at pH 1.4, although the cells number was higher at pH
1.4. The biooxidation rate of 0.183, 1.163 and 0.146 g/l h was obtained for pH 1.6, 1.4 and
1.8 respectively.
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66.36.66.97.27.57.88.18.48.7
99.3
0 5 10 15 20 25 30 35 40 45 50 55 60Time (h)
Log
of c
ell N
o.pH 1.4 pH 1.6 pH 1.8 pH 2
Fig. 3.5. Effect of different pH on growth of At. ferrooxidans D F1. The cell
numbers were converted to Logarithm with bases of 10 to get the better and correct
view of the bacterial growth. The initial cell number was 5X106. During the experiment
the initial pH was kept constant by adding 10 N H2SO4
Table 3.3 Bacterial cell number (Log10) during the growth at different pH
Time (h) pH 1.4 pH 1.6 pH 1.8 pH 2
0 6.6284973 6.627409 6.6396558 6.639656 5 6.6310979 6.642191 6.6655608 6.648464
10 6.6467996 6.720599 6.7055723 6.68201 15 6.8134796 6.718835 6.8657197 6.719892 20 6.9054726 6.882994 6.9288734 6.863447 25 7.3702925 7.251144 7.1846298 6.94814 30 7.6897003 7.610523 7.3019519 7.18099 35 7.9744008 7.892449 7.5797828 7.544595 40 8.1655475 8.046403 7.8334585 7.667949 45 8.4757328 8.294137 7.9575151 7.649293 50 8.7468648 8.448759 8.1652376 7.770068 55 8.8860523 8.52134 8.1465894 7.783812 60 8.8986206 8.528301 8.079227 7.778874
RESULTS 104
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00.5
11.5
22.5
33.5
44.5
55.5
6
0 5 10 15 20 25 30 35 40 45 50 55Time (h)
Fe3+
con
cent
ratio
n (g
/l)pH 1.4 pH 1.6 pH 1.8 pH 2
Fig. 3.6. The effect of different pH on the efficiency of bacterium D.F.1 for
oxidizing ferrous iron to ferric. As can be seen initially we have more oxidation of
iron at pH 1.4 and 1.8 but finally the maximum oxidation of iron (over 90%) was taken
place at pH 1.6. The initial concentration of Fe(II) was 6 g/l.
Table 3.4 Fe3+ concentration at different pH during the growth of the isolate.
Time (h) pH 1.4 pH 1.6 pH 1.8 pH 2
0 0.25 0.28 0.23 0.25 5 0.303 0.35 0.345 0.34
14 0.633 0.68 0.678 0.75 17 1.216 1.431 0.766 0.8 21 2.057 2.482 0.968 1.1 24 2.607 3.183 1.164 1.38 29 3.672 4.06 2.04 1.53 33 4.428 5.039 2.866 2.22 38 4.637 5.2459 3.756 2.53 44 4.604 5.288 3.929 2.61 48 4.869 5.197 3.82 2.76 52 4.977 5.169 3.8 2.95
RESULTS 105
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3.3.2. EFFECT OF IRON CONCENTRATION
At. ferrooxidans is a chemolithotrophic bacterium and they use inorganic compounds
(iron or sulfur) as an energy source, in the 9-K medium also ferrous sulfate is the sole
source of energy. Hence finding the best range of iron concentration on which bacterial
cell has the maximum growth and activity is necessary. We have also studied effect of
different concentrations of ferrous iron on growth and activity of the isolate.
Within the optimal pH range we have studied the effect of four different concentrations
of iron on growth and activity of bacterium. It has been observed that at high concentration
of Fe2+ there is a prolong lag phase (24-28h) but at a low concentration of Fe2+ the lag
phase was reduced to 10-15 hours which shows the extent of the lag phase depends on the
initial concentration of ferrous iron (Fig 3.7.). In the case of activity of bacteria,
concentration of Fe3+ in solutions was measured in the time interval of 2 hours. While there
was very little conversion in the early stages of exponential phase the rate was quite high
as the exponential phase progresses. Unlike the lag phase the specific growth rate of
bacterium was significantly higher at high concentration of iron as compared with lower
concentrations.
RESULTS 106
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6
6.5
7
7.5
8
8.5
9
9.5
0 10 20 30 40 50 6Time (h)
Log
of c
ell N
o.
0
2 g/l Fe(II) 4 g/l Fe(II) 6 g/l Fe(II) 8 g/l Fe(II)
Fig 3.7. Effect of different concentrations of initial iron on growth of bacterium
DF1. Initial pH of medium was 1.4 with incubation at 35oC and 180 rpm in orbital shaker.
Table 3.5. Bacterial cell number (Log10) at different concentrations of Fe2+
Time (h) 2 g/l Fe2+ 4 g/l Fe2+ 6 g/l Fe2+ 8 g/l Fe2+
0 6.6571058 6.6087077 6.6484635 6.6396557 5 6.7131505 6.6102972 6.6102974 6.6379575
10 6.7824418 6.6289319 6.6467996 6.7265361 15 7.0257506 6.8567648 6.6820078 6.7587789 20 7.3217123 7.0497384 6.84782 6.8033876 25 7.4634906 7.404948 7.1891556 6.9827854 30 7.8238957 7.7286209 7.46156 7.2539217 35 7.8967947 8.0061113 7.8405824 7.5258537 40 8.0038266 8.4316434 8.1348213 7.747226 45 8.1146532 8.7426128 8.4609615 7.9789024 50 8.1776834 8.9995699 8.7468648 8.2931313 55 8.2926172 9.0871566 8.8971998 8.5327971 60 8.2967014 9.1346108 8.8931009 8.535151
RESULTS 107
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Initial Fe2+ concentration of 4 g/l led to the maximum biooxidation rate (0.24 g/l h)
and it showed shorter lag phase and better specific growth rate in comparison with 6 and 8
g/l initial Fe2+ (Fig. 3.8). The analysis of growth carve of bacterium also indicted that at
this concentration (4 g/l) the number of free bacterial cells in solution was maximum and
there was better growth. So it can be concluded that 4 g/l of initial Fe2+ is the best iron
concentration for growth and activity of our bacterium and unlike pH effect, the required
concentration of iron for best growth and activity is same.
0102030405060708090
100
2 4 6 8Intial Fe(II) concentration (g/l)
Fe(I
I) o
xida
tion
(%)
0.10.120.140.160.180.20.220.240.26
Bio
oxid
atio
n ra
te (g
/l h)
Fe(II) coxidation (%) Biooxidation rate
Fig. 3.8. Effect of initial iron concentration on efficiency of bacterium DF1 for
conversion of Fe (II) to Fe (III). The experiment was performed at pH 1.6, with
incubation in orbital shaker with 35 oC and 180 rpm speed. The pH of medium was
maintained constant by adding 10 N sulfuric acid or 10 N NaOH.
RESULTS 108
CHAPTER THREE
3.3.4. EFFECT OF TEMPERATURE
Optimum activity of each type of bacteria takes place in a relatively well-defined range
of temperature at which these microorganisms grow most efficiently. This indicates the
temperature dependence of bioleaching processes. In the previous experiment we could
find out the temperature range for growth of the isolate (10-40 oC) but here we have
studied the effect of different temperature on growth of bacterium to find out the optimal
temperature of growth.
We have studied the effect of temperature in the range of 25 to 40 0C over the optimal
pH and iron concentration range. As Fig 3.9 shows the bioleaching efficiency and growth
of bacteria tended to increase with increasing the temperature but optimum temperature for
growth was 35 0C. Decreases in the oxidation activity of the bacterium at temperature
beyond the optimum may be attributed to the likely denaturation of proteins involved in
oxidation system of bacteria. The increase in bacterial growth and efficiency is with the
same rate while the temperature is increasing
RESULTS 109
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0.10.120.140.160.18
0.20.220.240.260.28
25 30 35 40Temperature (oC)
Bio
oxid
atio
n ra
te (g
/l h)
0
0.05
0.1
0.15
0.2
0.25
0.3
Gro
wth
rat
e (1
/h)
specific growth rate biooxidation rate
Fig. 3.9. Effect of temperature on growth and activity of bacterium DF1. The iron
concentration was 4 g/l with pH 1.4 and 1.6 for study effect on growth and activity
respectively.
3.3.4. EFFECT OF AMMONIUM SULFATE CONCENTRATION
A culture medium is mixtures of chemical compounds, which provide all the elements
required for cell mass production and sufficient energy for biosynthesis and maintenance.
A typical nutrient solution is mainly composed of nitrogen introduced as an ammonium
salt, phosphorus as a potassium salt of phosphoric acid, magnesium as magnesium sulfate
and other salts such as calcium nitrate or calcium chloride are sometimes added. In the
culture medium of Thiobacillus (9K) after iron, (NH4)2SO4 is the main part of medium and
having higher concentration in compare to other elements and also like iron this element is
RESULTS 110
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also is the only source of nitrogen in the medium. Hence, we have studied the effect of
different ammonium sulfate concentrations on growth and activity of bacterium.
The results are shown in Fig 3.10. As can be seen from the results increase in the
concentration of nitrogen led to increase the cell density and maximum growth was
observed at 3 g/l (NH4)2SO4. The reason that the oxidation activity of bacterium was
decreased at 4g/l (NH4)2SO4 than 3g/l is the possible precipitation of phosphate, potassium
and ammonium as jarosites due to higher concentration of these salts in the medium. This
is also one of the major detractions to 9-K liquid medium (Deveci et al., 2003).
3
3.5
4
4.5
5
5.5
6
1 2 3 4(NH4)2SO4 concentration (g/l)
Fe+3
coc
entr
atio
n (g
/l)
6
6.7
7.4
8.1
8.8
9.5
Log
of c
ell N
o.
Fe(III) Concertration Cell density
Fig. 3.10. Effect of different (NH4)2SO4 concentrations on growth and activity of
bacterium D.F.1. Initial Fe(II) concentration was 4 g/l and culture was incubated at 35 oC
and 180 rpm. The pH of the medium was 1.4 and 1.6 respectively for study growth and
activity
RESULTS 111
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3.3.5. EFFECT OF Mg2+ CONCENTRATION
The effect of Mg2+ concentration at 20, 40, 60 and 80 mg/l, on the efficiency of the
biooxidation process has been studied (Fig. 3.11). The results indicated that the
concentration higher than 40-mg/l doses not has any effect on the biooxidation of ferrous
sulfate. Though Mg2+ ions are essential for the biooxidation process the dose of Mg2+
required for an efficient oxidation process is as low as 40 mg/l.
50
60
70
80
90
100
20 40 60 80Mg2+ concentration (mg/l)
Fe(I
I) o
xida
tion
(%)
0
0.05
0.1
0.15
0.2
0.25
0.3
Gro
wth
rat
e (1
/h)
Iron oxidation Growth rate
Fig.3.11. Effect of Mg2+ concentration of the growth and activity of bacterium D.F.1
RESULTS 112
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3.4. ZINC AND LEAD EXTRACTION AT LABORATORY SCALE
As this bacterium was isolated from zinc and lead complex mine the ability of
bacterium for extraction of zinc and lead was evaluated at laboratory scale in flasks. Hence
we have performed the series of experiments to standardize the bioleaching process. For
this reason the ability of the isolate for bioleaching of zinc and lead was studied at two
different conditions including basic condition of 9-k medium and optimum conditions
which we were obtained before. In the second experiment the effect of different
concentration of Fe2+ on the extraction efficiency of zinc and lead was evaluated.
3.4.1 ZINC AND LEAD EXTRACTION AT BASIC AND OPTIMUM CONDITIONS
Results on zinc and lead extraction at basic and optimum conditions are presented in
Fig. 3.12. Results showed (Fig 3-12.) that at the basic condition of 9-K medium the isolate
can only extract 65 % and 16 % of zinc and lead respectively whereas at optimum
conditions there was more than 85% extraction of zinc and 22% lead. The required time for
complete extraction at basic conditions was about 17 days and the prolonged lag phase was
observed but at optimum conditions the lag phase was reduced and required incubation
time for complete oxidation was 14 days. These results were expectable since we have
observed very less growth rate and consequently oxidation efficiency of bacteria at basic
condition of 9-k medium in the previous experiment (optimization process).
RESULTS 113
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0102030405060708090
100
0 2 4 6 8 10 12 14 16 18 20Time (days)
Ext
ract
ed m
etal
(%)
Zn extracted at Control Pb extracted at ControlZn extracted at Optimum Pb extracted at Optimum
Fig 3.12. The amount of zinc and lead extraction at basic condition and optimum
conditions (4 g/l Fe2+, 3 g/l (NH4)2SO4, pH 1.6, 20 mg/l Mg2+ and 35 oC) of 9K medium
by DF1 strain.
3.4.2 EFFECT OF IRON CONCENTRATIONS ON EFFICIENCY OF
CONVERSIONS
In this experiment at optimal condition (pH, temperature, ammonium concentration and
magnesium concentration) the effect of different concentrations of initial ferrous iron on
bioleaching capability of the isolate for extraction of zinc and lead from the ore sample
was investigated. The iron concentrations were 2, 4, 6 and 8 g/l Fe2+. Table 3.6.(A, B) and
Figs 3-13 (A, B) illustrate the efficiency of extraction of zinc from the ore sample by the
isolate at different concentrations of iron. Results indicated that maximum zinc extraction
RESULTS 114
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was at 4 g/l of Fe2+ (over 86%) and lead extraction has reached to 22 %. Hence we can
conclude that in the optimal condition the efficiency of bacterium for bioleaching of zinc
and lead tended to increase 30 to 38 % for zinc and lead respectively. After this
concentration, we had maximum extraction of zinc at 6, 2 and 8 g/l of Fe2+ respectively.
Table. 3.6. The amount of zinc (A) and lead (B) extraction (%) at different
concentrations of initial Fe2+ concentration during 16 days.
Time (day) 2 g/l Fe2+ 4 g/l Fe2+ 6 g/l Fe2+ 8 g/l Fe2+
0 0 0 0 0 2 6 4 3 1 4 10 10 6 5 6 12 21 18 11 8 15 35 31 18
10 22 48 40 26 12 30 69 52 30 14 38 82 63 33 16 42 86 67 35
(A)
Time (h) 2 g/l Fe2+ 4 g/l Fe2+ 6 g/l Fe2+ 8 g/l Fe2+
0 0 0 0 0 2 2 2 2 1 4 6 5 5 4 6 9 10 9 6 8 12 13 12 8
10 14.5 16.5 14 11 12 17 19.5 17 13.5 14 18 21 19 15 16 19 22 20 16
(B)
RESULTS 115
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08
16243240485664728088
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Time (days)
Zin
c ex
trac
tion
(%)
2 g/l Fe(II) 4 g/l Fe(II) 6 g/l Fe(II) 8 g/l Fe(II)
(A)
0
3
6
912
15
18
21
24
0 2 4 6 8 10 12 14 16Time (days)
Lea
d ex
trac
tion
(%)
2 g/l Fe(II) 4 g/l Fe(II) 6 g/l Fe(II) 8 g/l Fe(II)
(B)
Fig. 3.13. The effect of different iron concentrations on bacterial efficiency for
extraction of zinc (A) and lead (B).
RESULTS 116
CHAPTER THREE
These results can be compared with the optimization process results where we had
maximum efficiency of bacteria in the concentration of 4 g/l Fe2+ that ultimately it also
concluded maximum Zn extraction. These results also showed that extraction of zinc is
with the same rate of growth and iron oxidation rate of bacterium as in the first 5 days of
experiment we had more extraction at lower concentrations of iron but in the next 5 days it
shifted to higher concentrations. But with regard to extraction of lead in all concentration
of Fe2+ there was same amount of extraction (20-25%) with same rate except in case of 8
g/l Fe2+. The reason of less extraction of zinc and lead at higher concentration of Fe2+ (6
and 8 g/l) could be the presence of high concentration of iron in the mine sample (more
than 24 %) which has leached out from the sample while bioleaching process progresses.
This leached iron together with high concentration of initial iron (8 g/l) might have toxic
effect on bacterial growth.
RESULTS 117
CHAPTER THREE
3.5 ZINC AND LEAD EXTRACTION IN COLUMN BIOREACTOR
After studying the bioleaching ability of our isolate at laboratory scale, we were
examined this ability at large scale. Hence, the bioleaching of zinc and lead was studied in
column bioreactor. On trial and error basis we have designed bioreactor. Reactor design
was based on a glass column with inlet for air and outlet for effluent at the bottom. The
bioreactor size was 50 cm in diameter and 3 m length. Total operation volume of
bioreactor was about 450 l and around 700 kg of the ore was transferred into it. Air was
supplied from bottom and fresh medium from the top with the use of peristaltic pump with
the rate of 9 l/(m2.h) to 24 l/(m2.h). At every 30 cm of bioreactor a sampling port and
temperature indicator were provided. During the experiment factors like pH, temperature
and etc were continually monitored. Extraction of zinc and lead were monitored in the
period of 100 days. Every 24 hours the column was sampled by removing a 1-mL aliquot
of the leach solution form all the sampling ports, which was then used for analysis of
metals (Zn, Fe and Pb) and for monitoring pH. The pH was adjusted using 10 N H2SO4
when it deviated towards neutrality from the initial preset values. As a control, for
measuring the amount of chemical oxidation 5 ml of 0.5% (v/v) formaline in ethanol was
added to the 9-k medium. Free bacteria in solution were counted by direct counting and
soluble zinc and lead in the leached solutions were measured by an atomic absorption
spectrophotometer. Fig 3.14 shows the schematic design of the column bioreactor used in
this study and Fig 3.15 A and B are showing the pictures of column bioreactor.
RESULTS 118
CHAPTER THREE
In this column also like the laboratory scale we have studied two different
experiments. The first experiment was the study of zinc and lead extraction at basic and
optimum condition and in the second one we were studied the effect of different pH and
Fe2+ concentrations on bioleaching of zinc and lead.
Fig. 3.14. The schematic design of column bioreactor.
RESULTS 119
CHAPTER THREE
Fig. 3.15.A. Pictures of column bioreactor
RESULTS 120
CHAPTER THREE
Fig.3.15. B. Pictures of column bioreactor. The left side is the temperature analyzer
machine and the right is the close up photo of temperature sensor and sampling port.
RESULTS 121
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3.5.1 STUDY THE EXTRACTION OF ZINC AND LEAD AT BASIC
AND OPTIMUM CONDITIONS
In this experiment the extraction of zinc and lead, once studied by using the bacterial
culture with the basic composition of 9k medium and then the bioleaching of these metals
were studied at optimal conditions of bacterial activity. The results indicated (Fig.3.16 and
Table 3.7) that at the optimum conditions we have more than 69% extraction of zinc in
about 87 days and after this period there was a slight increase in zinc extraction, but at the
basic conditions (9-K medium) only 54 % extraction obtained in 97 days. In case of lead
recovery also at optimum conditions, lead was leached more than 19% during the complete
run (100 days) whereas at basic condition in the same period only 12% of lead was
extracted. These results confirmed our laboratory scale results where also we had more
than 35 % increase in bioleaching ability of bacterium at optimal conditions.
RESULTS 122
CHAPTER THREE
01020304050607080
0 10 20 30 40 50 60 70 80 90 100Time (Day)
Met
als
extr
acte
d (%
)Zn at basic Zn at optimumPb at basic Pb at optimum
Fig. 3.16. Comparison between basic and optimum conditions for extraction of zinc
and lead in the column bioreactor in period of 100 days.
Table. 3.7. The amount of zinc and lead extraction at basic and optimum condition
Time (Days) Zinc extraction
at basic (%)
Zinc extraction
at Optimum (%)
Lead extraction
at Basic (%)
Lead extraction
at Optimum (%)
0 0 0 0 0 10 5 7 1 2 20 10 16 1 3 30 20 28 1 4 40 25 40 2 6 50 32 51 3 8 60 38 58 4 10 70 45 64 6 12 80 50 69 7 14 90 52 70 9 16 97 54 71 11 18
100 54.5 71 11.5 19
RESULTS 123
CHAPTER THREE
3.5.2 STUDY THE EFFECT OF pH AND Fe2+ CONCENTRATION ON
BIOLEACHING OF ZINC AND LEAD
In the second experiment for better comparison between laboratory scale and large scale
results two factors (pH and Fe2+) were selected among those five factors which were
studied in laboratory scale, for optimizing the bioleaching process in column. The reason
of selecting these two factors was that these two factors were having maximum effect on
bacterial growth and activity.
3.5.2.1. EFFECT OF INITIAL pH
The effect of the initial pH on the column feed for the zinc and lead leaching was tested
in the range from 1.4 to 2 in a medium containing 4 g/l Fe2+ and 3 g/l ammonium sulfate.
Fig.3.17 indicates that the best pH for sphalerite dissolution in the column was 1.6 at
which the maximum bioleaching of zinc was 71 %. Zinc extraction in experiments carried
out at higher pH than 1.6 was lower. Unlike laboratory scale (process optimization)
experiment that at pH 1.4 we had maximum cell number and best growth rate in this
experiment there was a reduction in the cell population in solution with pH 1.4. In case of
lead extraction also the similar results obtained with maximum of about 20% extraction at
pH 1.6. Less extraction of lead in comparison with zinc extraction was because of the toxic
RESULTS 124
CHAPTER THREE
effect of lead on bacterial growth, while the bioleaching process progresses and ultimately
the concentration of soluble lead rises in the solution.
0
10
20
30
40
50
60
70
80
0 10 20 30 40 50 60 70 80 90 100Time (Day)
Zin
c ex
trac
ted
(%)
pH 1.4 pH 1.6 pH 1.8 pH 2
Fig. 3.17. Effect of different pH on zinc extraction in column bioreactor. At 4 g/l Fe2+,
3 g/l (NH4)2SO4 and 35 oC
3.5.2.2 EFFECT OF INITIAL Fe(II) CONCENTRATION
Fig. 3.18 shows zinc recovery by our isolate in the presence of different concentrations
of initial Fe(II) in 9-K medium. The studied concentrations was 2, 4, 6 and 8 g/l. Results
showed that at 2 g/l Fe(II) the extraction of zinc and lead was at the lowest. This was the
expected results because due to lack of sufficient substrate for bacterial growth and
RESULTS 125
CHAPTER THREE
consequently of oxidant (ferric iron) for the sulfide oxidation bacterial cell has less growth
and activity.
Among the higher concentrations of ferrous iron the concentration of 4 g/l Fe2+ causes
the maximum extraction of zinc and lead. At higher concentrations more than 4 g/l of iron
the efficiency of metals extraction was decreased. The feasible reason of this decreasing, is
the same that we observed at laboratory scale experiments, where the high concentrations
of initial iron combine with leached iron was preventing the bacterial growth.
As we said before, high concentration of initial iron in the medium could increase the
duration of lag phase of bacterial growth. Similar results were also observed in these
experiments. We have observed less oxidation at early stages and this is because of
prolonged lag phase of bacterial growth at high concentration of ferrous ion, since the
oxidation occurs by a chemical mechanism and the rate of bacterial oxidation of ferrous
ion is low in the initial stage of experiments. As can be seen from Fig. 3.18 and Table 3.8
in the first 35 days, the zinc extraction obtained in the experiment with 6 and 8 g/l Fe(II)
was around 18% whereas in experiment with 4 g/l Fe(II) was 29%.
RESULTS 126
CHAPTER THREE
01020304050607080
0 10 20 30 40 50 60 70 80 90 100
Time (Day)
Zin
c ex
trac
ted
(%)
2 g/l Fe(II) 4 g/l Fe(II) 6 g/l Fe(II) 8 g/l Fe(II) Control
Fig.3.18. Effect of different concentrations of initial Fe2+ on extraction of zinc. pH of
the medium was 1.6 and with 3 g/l (NH4)2SO4 at 35 oC.
Table 3.8. The amount of zinc extraction (%) at different concentration of Fe2+
Time (h) 2 g/l Fe2+ 4 g/l Fe2+ 6 g/l Fe2+ 8 g/l Fe2+ Control
0 0 0 0 0 0 10 8 7 3 2 1 20 14 16 8 6 2 30 20 25 12 9 4 40 23 42 20 17 6 50 25 50 32 22 9 60 28 58 37 30 11 70 30 64 50 40 12 80 33 69 57 43 13 90 34 70 58 44 14
100 35 71 59 45 15
RESULTS 127
CHAPTER THREE
In comparison between the metals extraction in the medium without microorganisms
and medium containing bacterial suspension; zinc extraction was low in the absence of
inoculum of microorganisms and only 15% extraction of zinc was observed in control
condition.
During the study of each factor the cell density was measured by direct counting the
bacterial cell under the microscope and we were compared the relationship between cell
density and zinc extraction rate. As presence in Fig. 3.19, zinc extraction rate increased
with the same rate of increase in the bacterial cell density and maximum cell density
(9.36E+8) and zinc extraction rate (0.975 Kg/l day) were recorded at 4 g/l Fe2+ and 1.6 pH.
1.00E+05
2.00E+08
4.00E+08
6.00E+08
8.00E+08
1.00E+09
1.20E+09
Cel
l den
sity
(cel
l/ml)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
zinc
ext
actio
n ra
te (k
g/l d
ay)
Cell No. Bioox rate
C e ll N o . 7 .0 2 E+0 8 9 .6 3 E+0 8 5 .8 0 E+0 8 4 .8 0 E+0 8 3 .9 8 E+0 8 9 .6 3 E+0 8 7 .0 2 E+0 8 5 .0 3 E+0 8
B io o x ra te 0 .12 8 0 .16 5 0 .11 0 .0 8 8 0 .0 7 9 8 0 .16 5 0 .13 7 0 .10 2
pH 1.4 pH 1.6 pH 1.8 pH 22 g / l
F e (II)4 g / l
F e (II)6 g / l
F e (II)8 g / l
F e (II)
Fig 3.17. The relationship between bacterial growth and rate of zinc extraction in
column bioreactor. The cell number is the direct count of bacterial cell after 100 days.
RESULTS 128
CHAPTER THREE
3.6 HEAVY METAL TOXICITY AND PROTEIN ANALYSIS
At. ferrooxidans is resistance to several toxic metals. However, strain specific
difference in the level of tolerance have been reported for At. ferrooxidans isolates from
various mine sites (Garcia and Silva, 1991; Leduce et al.,1997). Among different metals
At. ferrooxidans shows an unusual resistance to some metals, such as zinc, nickel, cobalt
and copper (Tuovinen et al., 1971; Hutchins et al., 1986; Garcia and Silva 1991), unlike
most heterotrophic bacteria. However some metals (e.g. mercury and silver) are very toxic
to the bacteria even at low concentration (Hoffman and Hendrix, 1976; Mahapatra and
Mishra, 1984; Tuovinen et al., 1985).
Most of these metals are present in high concentration at mines environment due to
this, bioextration of metals from low-grade sulfide ores can only be effective if the
bacterium is resistant to the metal recovered as well as to other in the environment (Leduce
et al. 1997). Hence it would be interesting to isolate bacterium having high resistance to
such metals and try to increase its resistance by slowly exposing it to higher concentration
of certain metal that it can resist. These improved bacteria can be good source to be
applying in different mines for extracting different metals. According to Modak &
Ntarajan (1995), it is necessary to develop At. ferrooxidans strains which are more tolerant
to high concentrations of metal and temperature fluctuations which will improve
bioleaching.
RESULTS 129
CHAPTER THREE
In the present work, the tolerant of At. ferrooxidas D.F.1 to nine different heavy metals
(e.g. zinc, lead, arsenate, nickel, mercury, manganese, cobalt, copper, and chromium) was
investigated by measuring growth and iron oxidation capacity of isolates when exposed to
heavy metals.
3.6.1 DETERMINATION OF MINIMUM INHIBITORY
CONCENTRATION (MIC)
The MIC of the nine metals, i.e., Zn, Pb, Cu, Co, Ni, As, Hg, Mn and Cr was
determined by macrodilution method. The metal salts used for the determination of MIC
were lead nitrate, cobalt chloride, zinc sulphate, copper sulphate, sodium arsenite, sodium
arsenate, nickel chloride, manganese sulphate, mercury chloride and chromium oxide. Iron
oxidation was determined as an indicator for growth and activity of bacteria by
colorimetric method.
The MIC of these metals revealed that this is the highly resistant bacterium. It is clear
from the results presented in table 3.9, this isolate is having the ability to tolerate Zn and
Mn toxicity as high as 700 mM. After these two metals it is having higher tolerance to
nickel and cobalt with 150 and 80 mM respectively (Table.3.9). These results could be
expected; because the mine where this bacterium was isolated from, contains different
concentrations of most of these metals specially zinc and lead. So the organisms from this
mine must be adapted to high concentrations of such metals. Although the resistance of
RESULTS 130
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bacterium to other metals was less than 100 mM but in comparison to other reports, this
much resistance also is high, especially in case of lead and chromium (10mM). Thus we
can conclude that this strain is one of the most resistant strains of At. ferrooxidans to
different heavy metals.
Table 3.9. The MIC of nine different heavy metals during the growth of strain D.F.1
Metals Zn Mn Ni Co Cu As Cr Pb Hg
MIC
(mM)
650 700 150 80 50 25 10 10 0.005
3.6.2. GROWTH PROFILE IN RESPONSE OF METALS
The growth profile of the organism was studied in 9-K medium in present of two
different concentrations of these heavy metals. The iron oxidation (Fe3+ concentration) was
recorded spectrophotometerecally. The organisms’ growth response was different in all
metal salts when compared with control as shown in Fig.3.20 (A - D). There is a
significant raise in the lag phase of bacterial growth while growing in presence of metals.
At the control condition 45 h of incubation was necessary for complete oxidation of
ferrous iron by bacterium whereas in the presence of 650 mM Zn and 10 mM Pb oxidation
RESULTS 131
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of ferrous iron was completed in 160 h. Similar results were also obtained for manganese,
nickel and copper that more than 140 h was required for oxidation of ferrous iron. In the
case of other heavy metals 70 – 90 h incubation time was necessary for completion of
growth. The DF1 strain had a lag phase of 40 to 50 h in the presence of different heavy
metals as compared to 15-20 h in the control. However in the medium containing
chromium less lag phase obtained than the control. Hence we have tested the effect of
chromium oxide on iron oxidation in the medium without inoculum. As can be seen from
Fig.3 19 in the medium without inoculum also we observed some amount of oxidation of
iron as 35-40 % of iron was oxidized after 30 h of incubation but the remaining 65 % of
oxidation was microbially. Hence, we can conclude that in the medium containing this salt
due to oxidation activity of chromium oxide the rate of iron oxidation increased.
Table. 3.10. The amount of Fe(III) concentration (g/l) during the growth of bacterial
strain DF1 at different concentrations of heavy metals. (A) zinc and lead, (B) arsenate,
mercury and copper (C) chromium and cobalt, (D); manganese and nickel
(A)
Time (h) Control 400 mM Zn 650 mM Zn 5 mM Pb 10 mM Pb 0 0.144 0.18 0.25 0.375 0.382 19 0.399 0.269 0.247 0.319 0.233 25 0.836 0.6 0.245 0.756 0.256 43 3.58 0.954 0.282 1.302 0.432 50 3.65 1.274 0.262 1.543 0.547 69 3.944 3.869 0.622 4.096 0.805 91 3.732 3.62 0.819 3.977 0.807 125 1.408 1.334 150 2.581 2.531 170 3 3
RESULTS 132
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(B):
Time (h) Control 25mM Cu 50mM Cu 15mM As 25mM As 0.005mM
Hg 0 0.144 0.2 0.25 0.28 0.125 0.06 19 0.399 0.399 0.312 0.331 0.155 0.062 25 0.836 0.401 0.42 0.35 0.25 0.15 43 3.58 0.437 0.447 0.365 0.355 0.2 50 3.65 0.53 0.52 0.45 0.42 0.25 69 3.944 0.661 0.739 0.848 0.658 0.38 91 3.732 2.105 1.408 1.206 0.925 0.72 125 2.7 2.2 1.6 1.65 1 150 3.2 2.8 2.3 2.02 1.6 170 3.3 3 2.5 2.35 1.9
(C)
Time (h) Cotrol 5mM Cr 10 mM Cr 40mM Co 80 mM Co0 0.144 0.76 1.44 0.25 0.3 19 0.399 1.081 2.1 0.697 0.495 25 0.836 2.05 2.68 1.2 0.9 43 3.58 3.55 3.68 1.617 1.35 50 3.65 3.52 3.4 2 1.8 69 3.944 3.552 4.2 2.358 2.2 91 3.732 3.312 3.524 3 2.9
(D)
Time (h) Control 500mm Mn 700mM Mn 150mM Ni 100mM Ni 0 0.144 0.22 0.256 0.195 0.28 19 0.399 0.271 0.375 0.375 0.331 25 0.836 0.408 0.38 0.4 0.8 43 3.58 1.75 0.373 0.783 1.502 50 3.65 2 0.8 1 2 69 3.944 2.358 1.461 1.639 2.634 91 3.732 2.8 2.72 1.819 2.864 125 3 3.2 2.5 3.3 150 3
RESULTS 133
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0
0.7
1.4
2.1
2.8
3.5
4.2
4.9
0 20 40 60 80 100 120 140 160 180Time (h)
Fe3+
con
cent
ratio
n (g
/l)control 400 mM Zn 650 mM Zn 5 mM Pb 10 mM Pb
(A)
0
1
2
3
4
5
0 10 20 30 40 50 60 70 80 90 100Time (h)
Fe3+
con
cent
ratio
n (g
/l)
Control 5 mM Cr 10 mM Cr40 mM Co 80 mM Co
(B)
RESULTS 134
CHAPTER THREE
00.5
11.5
22.5
33.5
44.5
0 20 40 60 80 100 120 140 160Time (h)
Fe3+
con
cent
ratio
n (g
/l)Control 25 mM Cu 50 mM Cu15 mM As 25 mM As 0.005 mM Hg
(C)
00.5
11.5
22.5
33.5
44.5
0 20 40 60 80 100 120 140 160Time (h)
Fe3+
con
cent
ratio
n (g
/l)
Control 500 mM Mn 700 mM Mn150 mM Ni 75 mM Ni
(D)
Fig.3.20 A-D. Growth profile of DF.1 strain in response to different
concentrations of different heavy metals. (A) zinc and lead, (B) arsenate, mercury
and copper(C) chromium and cobalt, (D); manganese and nickel
RESULTS 135
CHAPTER THREE
0
1
2
3
4
5
0 10 20 30 40 50 60 70 80 90Time (h)
Fe3+
con
cent
ratio
n (g
/l)
With Inoculum Without Inoculum
Fig. 3.21 The amount of iron oxidation in the medium with inoculum and without
inoculum. To measure the amount of chemical oxidation. 10 mM Cr was used in both the
media.
RESULTS 136
CHAPTER THREE
3.6.3 PROTEOMIC APPROACH TO FIND OUT THE MECHANISM
OF RESISTANCE TO ZINC AND LEAD
Proteomics provide direct information of the dynamic protein expression in tissue or
whole cells, giving us a global analysis. One important aspect of proteomics is to
characterize proteins differentially expressed by dissimilar cell types or cells imposed to
different environmental conditions. Two-dimensional polyacrylamide gel electrophoresis
(2D PAGE) in combination with mass spectrometry is currently the most widely used
technology for comparative bacterial proteomics analysis (Gygi et al., 2000). As described
in the material and methods, intracellular proteins were extracted from the cells exposed to
zinc and lead as well as from the cells grown under control conditions. These proteins were
then resolved on two dimensional electrophoresis using glass tube gels as well as
immobilized pH gradient (IPG) strips.
The reasons of selecting these metals was that, this bacterium was isolated from the
zinc and lead mine and it is going to be used for extraction of these metals. It is s also
showing high resistance to these metals. Hence in this section it was proposed to study the
possible mechanism of resistance of bacterium to these metals. For this propose the
bacterial cell were exposed to 500 mM and 5 mM zinc and lead respectively. The proteins
of bacterium were extracted in all three conditions; control, with zinc and with lead
Hence, Isoelectric focusing (IEF) was performed using both 7 cm IPG strips and tube gel
of pH range 3-10 using the BioRad IEF cell. After IEF, proteins were separated in the
second dimension by using 12 % SDS-PAGE gel. Differentially expressed proteins (over
RESULTS 137
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expressed) were detected visually and treated as separate spots. The 2D electrophoresis
was repeated three times to confirm the differentially expressed proteins. Spots were
excised and digested with proteolytic enzymes. Peptide mass fingerprints were created and
analyzed with MADLI-TOF. Proteins were identified by different bioinformatics
softwares. MALDI mass spectra were recorded in the mass range of 800-4000 Da. For
protein identification two search engines, MASCOT and ProFound, were used for database
interrogation. A protein was considered as identified when the same ID was found as first
hit in both ProFound and MASCOT searches. The probability-based score of either 50 in
MASCOT or 1.5 in Profound was taken as acceptable.
There are certain spots which are specific to each of the growth conditions. Some of the
proteins are showing over expression or under expression when the cells were exposed to
metals. These differences are marked in the gels with arrow as shown in the Fig. 3.22. The
2_D profile of the bacterium showed some differential expression of protein in the
presence of lead and zinc when compared with control.
The results of 2D PAGE have indicated that under the influence of metals, there was a
differential regulation of proteins to cope-up with the metal toxicity. More than 13 proteins
have been differentially expressed. In presence of lead there were four protein spots, which
were differentially expressed in lead treated cells when compared to the control. In
presence of zinc also there were seven protein spots, which were differentially expressed in
zinc treated cell. Certain spots were present in control which were absent or under
expressed in zinc and lead treated cells. From the above results it indicates that certain
proteins were differentially expressed in presence of zinc and lead treated cells, which
RESULTS 138
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were not present in control. It can be speculated that these proteins have some role to play
in the tolerance of metals. Some proteins also were found over expressed in both the
metals treated cell which suggested it can be some common proteins for tolerance to both
the metals.
The mostly over expressed protein was the protein with 30-40 KDa molecular weight
and with pI 4-5 which were over expressed in presence of both the metals (lead and zinc)
but the amount of over expressions was higher in case of lead treated cells (spot No. 1, Fig.
3.22. This protein was identified as major Outer Membrane Protein of At. ferrooxidans
(OMP40) with significant ProFound score (2.25) (Table. 3.11). It seems that this protein
has the significant role in resistance to metals toxicity as it was over expressed in response
to both the metals and it was the highest over expressed protein in both the metals in
comparison with other over expressed proteins.
The second most over expressed protein, had 60 KD molecular weight with pI of 6-7
which were over expressed in presence of zinc and lead (Spot 4., Fig.3.22) but the amount
of over expressions was higher in case of zinc treated cells. This protein was showed
highest significant ProFound score (1.64) to Putative DNA Restriction Methylase
(Salmonella typhi) (Table 3.11). These enzyme protect the cell from exogenous DNA,
most species have DNA modification methylase but the actual role of this enzyme in metal
resistance is still unclear and there is no report for that.
RESULTS 139
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Fig. 3.22 2D PAGE diagram showing the differential expression of intracellular
proteins in bacterial grown in the presence of metals. Spot nos. 1, 2, and 3 are over
expressed in lead and zinc treated samples. Spot nos. 4, 5 and 6 are over expressed only
in zinc treated cells. Spot nos. 7, 8, 9, 10 are down regulated in metals treated cells
when compared with control. Spot nos. 11, 12, 13 only down regulated in lead treated
cells.
RESULTS 140
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RESULTS 141
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In the presence of lead we had two more over expressed proteins (Spots No.2 and 3) but
most of other proteins was down regulated or completely disappeared in presence of lead
in compare to the control. Those over expressed proteins were shown less ProFound score
but one of them was very basic protein (pI 9) which was shown more similarity to Holo-
synthase Protein (Spot No 2). This protein also with less intensity in comparison with lead
was over expressed in presence of zinc. This enzyme belongs to the family of transferees,
specifically those transferring non-standard substituted phosphate groups.
In contrast to lead the number of over expressed proteins in the presence of zinc was
higher in comparison with the control and lead. Most of them were in the pI range of 5 -7
with different molecular weights (Spots No 3, 4, 12). Among these protein spots one of
them was the protein with molecular weight of around 60 KD (Spot No 3) which showed
more similarity to Chapronin 60 kDa subunit with 0.9 ProFound score (Table 3.11).
Another over expressed spot (Spot No 12) in presence of zinc was similar to Hypothetical
protein of Pseudomonas syringae with top score of 62 in Mascot search (Table 3.11). All
this proteins are described and the possible roles of them in metals resistance are discussed
in discussion section.
As compared to control five proteins have been found to down regulated or completed
in strain exposed to zinc and lead. One of these proteins (Spot No 11) was identified as
CBBL (Ribulose bisphosphate carboxylase large subunite) of At. ferrooxidans with the
high Profound score of 2.25 and Mascot score 115 (Table 3.11). This is an enzyme that
RESULTS 142
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plays a role in Calvin cycle to catalyze the first major step of carbon fixation. The results
also showed that the level of enzyme decreased in presence of heavy metals (zinc and
lead). RuBisCo is very important in term of biological impact and it’s very vital and
important in chemolitotrophic bacteria for carbon fixation. There are other 2-3 proteins
which have been down regulated in metal treated cells have similar molecular weigh in the
pI range of 5-6. One of these proteins (Spot No. 8) showed more similarity to Hypothetical
protein SO-408 of Shewanella oneidensis with the marching of 11 of 35 peptides. Other
(Spot No 10) is Putative glutamines with 9 out of 54 peptides matching (Table 3.11).
RESULTS 143
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Table 3.11 MALDI-TOF analysis of 2D spots using ProFound and MASCOT search
engines
Spot No 1 4 11 2
Protein Major Outer
Membrane
Protein of At.
ferrooxidans
Putative DNA
restriction
methylase
(Salmonella typhi)
Riboluse
bisphosphate
carboxylase
Holo-synthase
(Syntrophicus
aciditrophicus)
NCBI
Accession No
Gi|4138616|e
mb|CAA1010
7.1|
Gi|10957232|ref|NP_
058256.1|
gi|4836660|gb|AA
D30508.1|
Q2LYJ7_SYNAS
MW (kDa)
Exp/pred
35 / 42.23
55 / 63.35
60 / 53
55 / 50
PI exp/pred 4.5 / 4.9 6 / 5.7 6 / 5.8 9.5 / 9.7
No. of peaks
matched
10 / 28 13 / 72 18 / 69 4 / 15
ProFound /
MASCOT
score
2.25/ - 1.64/ - 2.25/ 115 -/ 50
Level of
expression
Over
expressed in
lead and zinc
Over expressed in
zinc and lead
Down regulated in
lead and zinc
Over expressed in
lead and zinc
Function Porin, role in
metal
resistance
Protecting cells
from external DNA
Carbon fixation transferring non-
standard
substituted
phosphate groups
RESULTS 144
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Mass Spectra of Spot Number One
RESULTS 145