International Journal of Chemical Engineering Research

ISSN 0975 – 6442 Volume 1, Number 2 (2009), pp. 143–154

© Research India Publications
http://www.ripublication.com/ijcher.htm

Isolation and Characterization Two Novel Indigenous

Acidophilic Species Involved Copper Extraction
(Bioleaching) from the Aliabad Copper Mine in Iran

Jamshid Raheb1,*, Ali Azimi2, Mohammad Hadi Ardabili2, Bahram Nasernejad3,

Mahmood Arabnezhad2 and Mohammad Javad Hajipour1
1
National Institute for genetic engineering and biotechnology, Tehran, Iran,
2
National Iranian Copper industries Company, Tehran, Iran,
3
Amirkabir University, Tehran, Iran
*
Corresponding author E-mail: jam@nigeb.ac.ir

Abstract

We successfully isolated two novel gram-negative aerobic chemolithotrophic

sulfur-oxidizing bacterium, designated strain NICI1 and strain NICI2, from
mine ores growing under acidophilic conditions. Strain NICI2 oxidized
elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions and
cause to reduce pH. Strain NICI1 was able to oxidize the Fe++ to Fe+++. The
rate of oxidation of elemental iron and sulfur from NICI1and NICI2 were
higher than those of Thiobacillus ferrooxidans DSM583 and Acidithiobacillus
thiooxidans DSM622 as standard strains respedtively. Further isolation 16S-
23S rRNA intergenic spacer gene sequence analysis revealed that newly
isolated strains NICI1 and NICI2 were affiliated with members of the genus
Acidithiobacillus. These results demonstrated that NICI1 was
Acidithiobacillus ferrooxidans and NICI2 was Acidithiobacillus thiooxidans
(Genbank accession number of 16S-23S intergenic spacer rRNA gene
fragment of NICI1 and NICI2 are AM404268 and AM404265 respectively ).
NICI1 and NICI2 were capable to extract the copper. Therefore the amount of
copper extraction of these bacteria were measured in shaking flasks and
showed that the efficiency of copper extraction in these bacteria were higher
than standard strains. According to results obtained from this study, the newly
indigenous NICI1 and NICI2 can be a promising candidate for industrial
application in copper extraction (Bioleaching).

Keyword: Acidithiobacillus ferrooxidans DSM583, Acidithiobacillus

thiooxidans DSM622, Bioleaching, chemolithotrophic, 16S-23S rRNA.
144 Jamshid Raheb et al

Introduction
Bioleaching, a general term refers to the conversion of an insoluble metal (e.g. CuS)
into a soluble form (usually CuSO4) by biological oxidation and by applying microbes
[1, 2, 3]. Metals for which this technique is mainly employed include copper, cobalt,
nickel, zinc and uranium. For recovery of gold and silver the activity of leaching
bacteria is applied only to remove interfering metal sulfides from ores bearing the
precious metals prior to cyanidation treatment [3]. The sulfur cycle, driven by sulfate
reduction and sulfide oxidation, is an important cycle of minerals in various sulfur and
sulfide-rich environments and it is closely linked with other cycles such as carbon,
oxygen, and nitrogen. For instance, up to 50% of organic matter can be mineralized by
sulfate reduction in marine sediments [4, 5]. The use of microbes in bioleaching
process has some distinct advantage over traditional physicochemical methods. This
process is more environmental friendly, less consuming energy and useful for the
low-grade ores. And so, it is increasingly being used because of its economical
advantages [3, 6, 7]. Bioleaching is carried out by astonishingly diverse groups of
bacteria. Today, at least 11 putative prokaryote divisions can be related to this
phenomenon [8]. The most common microorganisms; belong to the genera
Thiobacillus and Leptosprillium which are mesophile, acidophile and
chemolithoautotrophe. They obtain energy from oxidation of either ferrous ion to
ferric or reduced sulfur compounds to sulfuric acid [9, 10]. The first step in
development of industrial bioleaching process in a metal mine is to investigate of its
bacterial population. One of the major obstacles encountered in studying the ecology
of these organisms is the difficulty involved in isolating, identifying and enumerating
individual species and strains from an environment which contains a plethora of
strains with similar metabolic requirements [11]. Yates and his colleagues were the
first to suggest the use of genetic probes to identify different species of bioleaching
bacteria commonly isolated from biomining operation [12]. In PCR-mediated
detection of bioleaching bacteria, the DNA obtained from samples after laboratory
cultures was analyzed by PCR of the spacer regions between 16s-23s intergenic
spacer [13], 16s rDNA [14], or 23s rDNA [13]. In this study the composition of
bacterial populations in Aliabad copper mine was isolated and investigated by
electron microscopy. This analysis consisted of the characterization of the spacer
regions between the 16s and 23s genes in the bacterial rRNA genetic loci after PCR
amplification. The size of the spacer region amplified from DNAs obtained from
samples, were compared with the sizes of that obtained from culture of the
bioleaching bacterial reference (Acidothiobacillus Ferrooxidans DSMZ 583) and the
16s-23s intergenic spacer regions sequences in database. Further identification of the
locale species was achieved by sequencing of the PCR product. In the next step the
elemental iron oxidation rate of NICI1 and Acidothiobacillus Ferrooxidans DSMZ
583 were compared. Then the sulfur oxidation rate of NICI2 measured using
elemental sulfur and the pH analysis. Afterward the capability of these two strain
(NICI1, NICI2) in copper extraction (Bioleachig) were evaluated and compared with
standard strains (Acidithiobacillus ferrooxidans DSM583 and Acidithiobacillus
thiooxidans DSM622).
Isolation and Characterization Two Novel Indigenous 145

Material and Methods

Strains. The strains used in this study were Acidothiobacillus ferrooxidans DSM 583,
NICI1, NICI2 and Acidithiobacillus thiooxidans DSM622.
Sample collection. Ore samples were collected from various damp sites in Iranian
mine (Aliabad) in November 2008, using polypropylene flasks. Samples maintained
at room temperature.

Cultures
Acidothiobacillus ferrooxidans DSM 583 and NICI1 cultured in 9k medium contained
(per liter): KH2PO4 (0.4 gr), CaCl2.2H2O (0.2gr), MgSO4 . 7H2O (0.4
gr),(NH4)2SO4(0.4 gr),FeSO4 . 7H2O (33.3gr), PH: 1.5-2 at 30ºC and 180 rpm. Strain
NICI2 and Acidithiobacillus thiooxidans DSM622 cultured in 35 medium contained
(per liter): NH4Cl (0.1g), KH2PO4 (0.3g), MgCl2.6H2O (0.1g), CaCl2. 2H2O (0.14g),
Sulfur (10g), pH=3 at 30ºC and 180 rpm. Ore samples treated with H2SO4 (0.5 N) for
three weeks, then, crushed into small particles (0.2-0.5 mm in diameter). 5 gram of
the sample incubated in 200ml of 9k and 35 medium. Cultures medium were
incubated for 1 to 8 weeks until growth was observed microscopically. In cultures the
growth of Acidithiobacillus ferrooxidans was accompanied by a characteristics ferric
precipitation and orange coloring of medium and the growth of Acidithiobacillus
thiooxidans was confirmed by pH changes.

DNA extraction
200ml from the cultured media at the end of exponential phase was filtered with
through a Millipore filter (pore size, 0.22µm), and washed twice with sterile distilled
water. The pellet resuspended in 100µlit TE (0.01M Tris,0.001M EDTA[PH:7.5]) and
DNA extracted by DNA extraction kit (Cinnagene Co. Cat. No. DN8115C).

PCR
The spacer region between 16s and 23s genes was amplified by PCR using 1.5ng of
DNA, G1 (GAAGTCGTAACAAGG) and L1 (CAAGGCATCCACCCGT)
primers.G1 oligonucleotide is located about 30 to 40 nucleotide upstream from the
spacer boundary and L1 oligonucleotide is located about 20 bases downstream from
the spacer boundary [15]. PCR was performed according to Pizzaro et al., including
25 cycles at 15s at 94ºC, 7 min at 55ºC and 30s at 72ºC and the one additional cycle
was run at 72ºC for 10 min. electrophoresis was performed on 1% agarose gel with
TAE buffer and the DNA was visualize by staining the gel with ethidium bromide.

Scanning electron microscopy

Bacterial samples were fixed on specimen stubs, gold coated by auto spotter coater
(BioRad E5200) and examined by a SEM (Cambridje S-360) at 20kv.

Sequencing
The nucleotide sequence of the PCR bands was directly determined by automated
sequencing 3700ABI (Gene fanavaran, Macrogene Seoul,Korea).
146 Jamshid Raheb et al

Iron analysis
The concentration of ferrous ions in solution was determined by titration with
potassium dichromate by using sodium diphenylamine sulfonate as the indicator [7].

pH analysis
NICI2 bacterium converted S0 to SO2-4 and caused pH changed. The range of pH
changes were measured by pHmeter for 8 days.

Evaluation of copper extraction using NICI1 and NICI2 bacteria

In this method four flask which contained 6g of copper ore sample powdered and 200
ml of 0.9K medium contained (per liter): KH2PO4 (0.4 gr), CaCl2.2H2O (0.2gr),
MgSO4 . 7H2O (0.4 gr), (NH4)2SO4 (0.4 gr), FeSO4. 7H2O (3.33gr), PH: 1.5-2 were
propagated. Afterward NICI1 bacterium added into the flask number 1, NICI2
bacterium added into the flask number 2, NICI1 and NICI 2 bacteria added into the
flask number 3 and flask number 4 used as negative control. These samples were
incubated at 30ºC and 180 rpm. Then the amount of soluble copper was measured by
atomic absorption.

Results
Enrichment, isolation and identification of strains NICI1 and NICI2.
For enrichment of strains NICI1, ores samples were gently washed, acidified,
homogenized, and incubated under oxic conditions in 9K medium. Iron oxidation
observed after 3 days incubation. The culture produced constantly in 9K medium, and
concomitant cell growth was confirmed. The use of elemental sulfur as an electron
donor was effective for recovering the NICI1 population. After 10 days culture in 35
medium, there was no cell growth of NICI1 as expected. However the NICI2 cell
growth was observed after several transfers in fresh 35 medium which is confirmed
by Light Microscopy. At this point, we examined the purity of the enrichment cultures
by 16S-23S rRNA-based molecular approaches (i.e., PCR amplification
andsequensing). PCR amplification with the primer set specific for NICI1 clearly
showed the presence of strain NICI1 in the cultures (Fig. 2). The partial sequence
(about 500 bp) of the PCR-amplified 16S-23S intergenic spacer rRNA gene fragment
was approximately identical to the sequence of NICI1 obtained previously, and PCR
amplification with the designed primer for NICI2 clearly showed the presence of
strain NICI2 in the cultures (Fig. 2). The partial sequence (about 400 bp) of the PCR-
amplified 16S-23S intergenic spacer rRNA gene fragment was approximately
identical to the sequence of NICI2 obtained previously. The accession numbers of
16S-23S intergenic spacer rRNA gene fragment from NICI1 and NICI2 are
AM404268 and AM404265 respectively (GenBank). Furthermore, two novel strains
of thiobacillus were isolated from Aliabad mine copper ores using 9k medium. Since
thiobacillus strains are lithoautotrophe, they are not easily culturable in solid medium.
The samples were prepared and examined for light and scanning electron microscope
(Fig1). The primary examination of SEM as a microscopic result and applying the
Isolation and Characterization Two Novel Indigenous 147

stringent condition of culture medium (pH: 1.5-2) demonstrated that the strain is
Acidithiobacillus species.

Iron oxidation of NICI1

Analyses of the growths rate of NICI1 were applied using the Fe+++ production which
was compared with Acidothiobacillus ferrooxidans DSM583 and showed that the rate
of iron oxidation in NICI1 was higher than Acidothiobacillus ferrooxidans DSM583.
According to Figure 3, the amount of iron oxidation of NICI1 were 64% and 95%
after 3 and 5 days respectively. The amount of iron oxidation of Acidothiobacillus
ferrooxidans DSM583 were 32% and 82% after 3 and 5 days respectively.

Sulfur oxidation of NICI2

The rate of sulfur oxidation from NICI2 in 35 medium evaluated measuring the pH
changes. According to Figure 4, the sulfur oxidation activity of NICI2 increased after
3 days and caused to reduce pH of 35 medium from 3 to 1.7 after 5 days. In contrast
the pH of the 35 medium which has no NICI2 bacterium (Negative control) increased
after 5 days (Fig. 4).

Copper extraction using NICI1 and NICI2

The copper extraction of NICI1 was measured in 0.9K medium and showed that the
amount of copper extraction from NICI1 was higher than that of Acidothiobacillus
ferrooxidans DSM583 up to 6 fold (Fig. 5). The copper extraction of NICI2 was
measured in 0.9K medium and compared with Acidothiobacillus thiooxidans
DSM622, this comparison demonstrated that the amount of copper extraction from
NICI2 was higher than that of Acidothiobacillus thiooxidans DSM622 (Fig. 6). After
the comparison of copper extraction in NICI1 and NICI2, it is demonstrated that the
efficiency of copper extraction in NICI1 was higher than that of NICI2 (Fig. 7).
Finally the copper extraction activity of mixture of both strains NICI1 and NICI2 in
0.9K medium were compared with that of NICI1, NICI2, Acidothiobacillus
thiooxidans DSM622, Acidothiobacillus ferrooxidans DSM583 and control media
(0.9K medium) separately. The data obtained here showed that the efficiency of
copper extraction of mix culture (NICI1 and NICI2) was higher than that of other
cultures (Fig. 8).
148 Jamshid Raheb et al

1a 1b
Figure 1: Light (b) and scanning electron microscopy photograph (a) (1000x and
10000x) of NICI1 bacterium.

1 2 3 4

500
400

Figure 2: PCR amplification product of spacer regions between the 16S and 23S
RNA genes of DNA from cultured sample. Lane 1 is NICI2, lane 2 is NICI1, lane 3 is
Acidothiobacillus Ferrooxidans DSM583 and lane 4 is Molecolar weight
marker100bp (Roche).
Isolation and Characterization Two Novel Indigenous 149

100
90
80
70
Fe +++ (%)

60
50
40
30
20
10
0
0 1 2 3 4 5 6
Time (day)

9k DSM583 NICI1
Figure 3: The chart of Fe+++ production rate in 9k medium. Square is
Acidothiobacillus Ferrooxidans DSM 583, triangle is NICI1 and diamond is 9K
medium which has no bacteria.

3.5
3
2.5
2
pH

1.5
1
0.5
0
0 2 4 6 8 10
Time(day)

35 NICI2

Figure 4: The chart of pH changes. Square is 35 medium contain NICI2 and diamond
is 35 medium which has no bacteria.
150 Jamshid Raheb et al

40
35
30
25
Cu(ppm)

20
15
10
5
0
0 10 20 30 40
Time(day)

0.9k DSM583 NICI1

Figure 5: Comparison of copper extraction from NICI1 and Acidothiobacillus

Ferrooxidans DSM583. Square is Acidothiobacillus Ferrooxidans DSM583, triangle
is NICI1 and diamond is 0.9K medium which has no bacteria.

30

25

20
Cu(ppm)

15

10

0
0 10 20 30 40
Time(day)

0.9k DSM622 NICI2

Figure 6: Comparison of copper extraction from NICI2 and Acidothiobacillus

thiooxidans DSM622. Square is Acidothiobacillus thiooxidans DSM622, triangle is
NICI2 and diamond is 0.9K medium which has no bacteria.
Isolation and Characterization Two Novel Indigenous 151

40
35
30
25
Cu(ppm)

20
15
10
5
0
0 10 20 30 40
Time(day)

0.9k NICI2 NICI1

Figure 7: Comparison of copper extraction from NICI1 and NICI2. Square is NICI2,
triangle is NICI1 and diamond is 0.9K medium which has no bacteria.
50

45

40

35

30
Cu(ppm)

25

20

15

10

0
0 5 10 15 20 25 30 35 40
Time(day)

0.9k NICI2 NICI1 Mix(NICI1+NICI2) DSM583 DSM622

Figure 8: Comparison of copper extraction from mix culture, NICI1, NICI2,

Acidothiobacillus Ferrooxidans DSM583 and Acidothiobacillus thiooxidans
DSM622. Square is NICI2, triangle is NICI1, multiple is mix culture (NICI1 +
NICI2), stare is Acidothiobacillus Ferrooxidans DSM583, circle is Acidothiobacillus
thiooxidans DSM622 and diamond is 0.9K medium which has no bacteria.
152 Jamshid Raheb et al

Discussion
The most microorganisms require suitable environmental factors for optimal growth
conditions. To isolate a specific strain of bacteria, from its habitat, it is necessary to
bring together all sufficient chemical and physical environmental factors in a selective
medium [16]. In this investigation variety of medium with the required factors were
used to isolate an important native microorganism acid mine drainage and bioleaching
environments Acidithiobacillus ferrooxidans from Iranian Aliabad ores. A modified
9K medium was specific in identification and isolation of this strain. The optimal
growth environmental factors for most bacteria arrange in specific range; however
there are broad variety range of environmental factors in a microorganism as a natural
habitat flora due to their structure and chemical combination of ores. The most
common type of bacteria grows well in a natural temperature between 25ºC and 40ºC,
while the range for a natural flora is quiet variable [17]. Although the optimal
temperature seems to be 30 ºC, but it must be modified in pilot plan particularly in
natural habitat conditions. The optimal pH for the most bacteria to grow best is
located between 6.5 and 2.5 [18] since the bacteria isolated from Aliabad ore are
acidophilic grow in pH range much lower than above range. In addition due to
interfere of organic acid with some media ingredients, the external buffer may needed
to maintain the correct pH. In addition to above primary isolation based on
environmental factors, PCR amplification of the 16s-23s intergenic spacer region by
using the highly conserved flanking sequence was applied. Sequence of 16s-23s
spacer regions was identical to the other bacteria of the same group using nucleotide
blast software [19]. The rRNA genes PCR have been extensively characterized by the
investigators such as Moriera and his colleagues (Moriera and Amils 1996). PCR
amplification of the 16s, 23s (Moriera and Amils 1996) and 5s region were used to
bacterial identification, by investigators. On the basis of the results obtained, PCR
amplification of 16s-23s spacer region demonstrated that the significant promise as a
tool for the identification of strains belonging to a wider range of bacteria. In this
study for the first time we isolated and identified the newly indigenous NICI1 and
NICI2 bacteria from Aliabad ore in Iran which were involve in copper extraction.
Strains NICI1 is capable to oxidize Fe++ to Fe+++ and follow to this reaction leads to
oxidizing of the copper of minerals. NICI2 is capable to oxidize the sulfur of minerals
to SO42- which cause to converting the insoluble oxidized copper to soluble CuSO4
compounds. The oxidation of sulfur leads to reduction of the pH in media provided
the optimum growth condition for NICI1 and NICI2 bacteria. The efficiency of
copper extraction of NICI1 and NICI2 were higher than that of Acidothiobacillus
ferrooxidans DSM583 and Acidothiobacillus thiooxidans DSM622 respectively. In
addition, the copper extraction activity of NICI2 was higher than that of
Acidithiobacillus ferrooxidans DSM583, however, Acidithiobacillus ferrooxidans is
known as the best bacteria in the bioleaching processes. The data obtained here
showed that the efficiency of copper extraction for both these newly indigenous
NICI1 and NICI2 bacteria were higer than that of standard strains. Furthermore, these
analyses demonstrated that mix culture of NICI1 and NICI2 extracted the highest
amount of copper from the ore. According to the results obtained here, two newly
Isolation and Characterization Two Novel Indigenous 153

indigenous strains NICI1 and NICI2 could be a promising candidate for industrial
application in bioleaching processes.

Acknowledgements
This work was funded by the National Iranian Copper industries Company.
Borgne, S.L., and Quintero, R., 2003, "Biotechnological process for the refining of
petroleum," Fuel. Process. Techno., 81, pp. 155-169.

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