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Indian Joural mExperimental Biology

Vol. 40, March 2002, pp. 31 9-324
Bioleaching of copper from chalcopyrite ore by fungi
D Venkateshwara Rao, Channappa T Shivannavar & Subhaschandra M Gaddad*
Department of Studies and Research in Microbiology, Gulbarga University, Gulbarga 585 1 06, India
Received 28 March 2001; revised 12 December 2001
Microorganisms have been geologically active in mineral formation, mineral diagenesis and sedimentation via direct
action of their enzymes or indirectly through chemical action of their metabolic products. This property of microorganisms
is being haessed during the recent years for extraction of metals from their ores, especially from low-grade ores. In the
present study bioleaching of copper from its low-grade chalcopyrite ore using 26 isolates of acidophilic fungi is reported.
Most of these fungal strains belonged to the genera Aspergillus, Penicillium and Rhizopus. The leaching experiments were
conducted in Czepek Dox minimal medium containing 1 % ( 1 00 mesh) ore with shaking at room temperature for 20 days.
Out of these, 4 isolates exhibited significant bioleaching activities. Maximum leaching of copper (78 mgI) was observed
with Aspergillus favus (DSF-8) and Aspergillus niger (DOF- I ). Nutritional and environmental conditions for optimum
bioleaching were standardized. Present study indicates the usefulness of acidophilic fungi in bioleaching of copper from its
low-grade ores.
The potential and ability of microorganisms in min
eral biotechnology has opened a new avenue for metal
leaching from low-grade ores
l

2
Microbial leaching
processes have been considered economical, eco
friendly and technically viable alteratives for proc
essing of low-grade ores
3
The microbe influenced
extraction of metals from insoluble minerals is done
through leaching by acidophili( iron-oxidizing bacte
ria
4
. Chemolithotropic microorganisms like Thiobacil
Ius, Sulolobus, Pseudomonas, Bacillus etc. , are
among the microorganisms being used extensively in
bioleaching studies
4
.
8
. Alterative to these extremo
philes, search is on for other acidophilic microorgan
isms. Some workers have indicated the possibility of
metal dissolution by acidic metabolites of heterotro
phic fungi
9
.
1 2
. The presence of such fungi along with
yeast:, algae and protozoa in natural leach solutions
has been reported. Several moulds isolated from mine
water samples and strains belonging to the genus
Cladosporium were found to solubilise alumina
1 3
.
In the present study, the usefulness of acidophi l ic
fungi for extraction of copper from the chalcopyrite
ore were investigated and some of the bioleaching
parameters were standardized.
Materals and Methods
SubstItC~Chalcopyrite, used in the present study
was collected from Chitradurga Copper Mine, Chi
tradurga, Karataka. While the ore contains 1 .01 07%
*Correspondent author: E-mail: smgaddad@rediffmail .com
copper, the other major constituents are Si0
2
(68. 32%), Fe ( 1 0. 55%) and S (3. 69%), Ni (4.5 ppm)
and Co ( 1 . 5 ppm) are present in trace concentrations.
Isolation offngi ~Twenty six fungal strains were
isolated from freshly collected dumped chalcopyrite
ore, soil collected from Chitradurga Copper Mine and
spoiled fruits and foods (like tomato, pickles, etc) by
using Sabroud' s Dextrose Agar and Czepek Dox
Agar. The fungal isolates were preliminarily screened
for leaching on the basis of their acidophilic activities
by using acid indicator medium (AIM) containing
0.04% of bromocresol purple
l4
The spore suspension
was prepared by growing fungi on Sabroud's Dex
trose Agar plates for 5 days at room temperature and
spores harvested in 1 0ml steril e normal saline.
Bioleaching studies-All leaching experiments
were conducted using modifed Czepek Dox minimal
medium
l 5
supplemented with 1 % of ground chalcopy
rite ore ( 1 00 mesh). Hundred milliliters of the me
dium in 250 ml Erlenmeyer flask was inoculated with
I ml of spore suspension (approximately 5 x 10
7
spores/ml). Control flask was not inoculated. All the
flasks were incubated at room temperature (28D-35DC)
on a rotary shaker (200 rpm) for 20 days.
Standardization of leaching conditions-For op
timum bioleaching various leaching conditions were
standardized. Effect of shaking, ore concentration
(0. 5-5%), ore particle size (0.05-0.6 mm diameter),
glucose concentration (0. 5-5%), phosphate concentra
tion (0.05-0.3%), pH (4.5-7.0), temperature ( l OD-40DC)
320
INDIAN J EXP BIOL, MARCH 2002
and different sugars (glucose, maltose, mannitol, su
crose and lactose at 1 %) on the bioleaching of copper
by the five fungal isolates was evaluated by using the
standard leaching conditions except for the variable
parameter.
Chemical leaching -In order to find out leaching
effciency of different organic acids
l 5
, leaching ex
periments were conducted using 2% of different or
ganic acids for 3 days at room temperature under
shaking conditions. After 3 days of incubation, the
samples were collected for metal analysis. In order to
find out the organic acid production by the fungal iso
lates, isolates were grown in Czepek Dox medium for
3 days. Fungal mycelium was separated by
centrifugation (7000 rpm for 1 5 min) and the
superatant was qualitatively analysed for organic
al6
AnalysisSamples collected during leaching
processes were centrifuged at 7,000 rpm for 1 5 min to
remove biomass. The clear superatant liquid was
analysed for copper content by direct titration using
Fast Sulphon Black F as indicator
1 7

Results and Discussion
The elemental composition of the chalcopyrite ore
used in the present study indicates. that it is a low
grade ore, which is uneconomical for commercial ex
ploitation. The XRD-analysis of the ore has revealed
the presence of covellite, melnicovite, spholirite,
quartz, goethite and magnetite. The 26 fungal strains
isolated from various sources were idntified by fol
lowing the standard mycological procedures
1 8
.
The maximum amount of copper leached by the
different isolates is shown in Table 1 . Amount of
Table I -Amount of copper leached (maximum) by the different isolates of fungi
Organism
1 781
DOF-Ol
DOF-02
DOF-03
DOF-04
DOF-05
DOF-06
DOF-07
DOF-08
DOF-09
DOF- I O
DOF- I I
DOF- 1 2
DOF- 1 3
DSF-OI
DSF-02
DSF-03
DSF-04
DSF-05
DSF-06
DSF-07
DSF-08
DSF-09
DSF- I O
DSF- l 1
DSF- 1 2
DSF- 1 3
Control
Isolate/ Strain No.
(ATCC) Aspergillus niger
Rhizopus sp.
A. niger
Y
Penicillium sp.
A. niger
A. flavus
A. niger
A. favus
A. niger
Copper leached
Maximum
(mgI)
69
75
60
57
69
48
66
57
75
5 1
54
42
54
63
66
1 8
54
63
57
66
48
78
54
54
63
63
60
09
Time taken % of Cu
(Days) leached
08 68.44
1 0 74.40
1 6 59. 52
08 56.54
1 6 62.50
14 47.62
06 65.47
1 6 56.54
08 74.40
08 50.60
1 8 53.57
14 41 .67
1 4 53. 57
08 62.50
1 6 65.47
1 2 1 7.86
08 53.57
14 62.50
1 4 56.54
1 6 65.47
1 8 47.62
08 77.38
1 0 53. 57
1 6 53. 57
1 6 62.50
1 2 62.50
08 59. 52
20 08.93
.

RAO et at.: BIOLEACHING OF COPPER FROM CHALCOPYRITE ORE BY FUNGI
321
copper solubilised was found to be more in shake cul
tures than the static cultures in all the cases. It i s well
known that, due to continuous supply of oxygen in the
liquid medium, the hyphal growth will be enhanced
and consequently more solubilisation of copper from
chalcopyrite is achieved. In the control flask without
any inoculation a maximum of only 9 mg/L (8. 93%)
of copper leaching was observed.
All the fungal isolates showed growth in the pres
ence of chalcopyrite but the amount of copper leached
versus time taken for maximum leaching varied. It
was observed that more efficient strains such as A.
favus DSF-8, A niger DOF-8 and DOF- l and
Rhizopus sp. (DOF-6), took 6- 1 0 days to leach about
66-78% copper. In comparison A niger (ATCC 1 781 )
was able to leach a maximum of 69 mg/L (68.44%} of
copper in 8 days (Table 1 ) . After recording the maxi
mum leaching, there was a slight decrease in the
amount of copper in the leachate. This may be due to
the adsorbence of -eached copper ions overtime onto
the fungal hyphae
1 9
Based on these preliminary re
sults only four isolates (A. favus DSF-8, A. niger
DOF-8, DOF- l and Rhizopus sp. DOF6) along with
A. niger (ATCC 1 781 ) were used for further studies.
The dependency of copper leaching rate on initial
pulp density is very important in case of bioleaching
20
,
2l
.
Figure 1 reveals that all the five isolates showed high
percent of leaching at low pulp densities (0.5-2.0%).
The strain Afavus (DSF-8) was comparatively more
efcient. The intensity of mineral oxidation depends
on the pulp density of ground ore for optimum water
permeability and aeration and supply and dissolution
of oxygen and carbon dioxide
22
Several studies have
pointed out the possible causes of this limitation as
the growth of toxic products in the medium and the
non-availability of carbon dioxide and oxygen
22
and
attrition problems with the mineral particles
23
.
Figure 1 reveals that the particle size of the ore had
a profound effect on the leaching efficiency. The rate
of leaching of metals from their ores by microorgan
isms was found to increase with decrease in size of
.
I
2
1 2
3
Th
.
f
.
I d
.
h
.
partiC es ' . e SIze O partlc es etermlOes t elr
surface area, which also affect the efficiency of leach
ing. Significant leaching of copper from chalcopyrite
was achieved upto 0. 1 5 mm, with further increase in
the particle size there was a gradual decrease in the
copper leaching and only 30-40% leaching was
achieved at 0.6 U.
Sugars are the best source of organic carbon and
energy. The influence of different sugars (glucose,
maltose, mannitol, sucrose and lactose) on the
bioleaching activity of various fungi was tested
(Fig. 2). It is evident that glucose is the best substrate
followed by sucrose, mannitol and maltose. Lactose
was a poor inducer of bioleaching activities. Afavus
(DSF-8) was again very versatile with respect to its
organic carbon requirements exhibiting equal
(77 . 38%) bioleaching activities with glucose and
mannitol and slightly reduced activities (7 1 .42%, and
65. 47%) with sucrose and maltose respectively. A
niger (DOF-8) was the organism better suited for
utilization of lactose for bioleaching activities.
Glucose is considered to be the best organic carbon
source for the growth and metabolism of heterotro
phic microorganisms especially fungi
1 2
. Its concentra
tion determines the quantity of acidic metabolites.
The effect of different concentrations of glucose
Paricle size (mm) (" - " " ")
0.05 0.1 0.15 0.2 0.25 0.3 0.5 0.6
100

!
I
: ! 1._ __

:
U

:..
I L , , , ,
0.5
Pulp density (%) ( * )
Fig. 1 -Effect of pulp density and ore particle size on copper
leaching from chalcopyrite by fungi (-.- 1 78 1 , - .- OOF- I , - .
OSF-8, -o-OOF-8 and -- OOF-6).
01 781 .OOF-1 10SF-8 .OOF-8 EOOF-6
Glucose Maltose Mannitol Sucrose Lactose
Fig. 2 -Effect of different sugars on copper leaching from
chalcopyrite ore by fungi.
322
. INDIAN J EXP BIOL, MARCH 2002
(0.5-5.0%) on Cu (II) leaching was investigated
(Fig. 3a). One percent glucose supported the optimum
leaching by all the five isolates, with A.favus (DSF-8)
showing maximum activity. The growth of the fungal
isolates as observed by biomass was almost the same
in concentrations from 1 % to 4%. Further increase in
glucose concentration exhibited very little effect on
leaching of copper.
Though phosphate is one of the important nutrients,
higher concentrations affect the growth of microor
ganisms and organic acid production. The effect of
increasing phosphate concentrations on the bioI each
ing of copper from chalcopyrite is shown in Figure
3(b). A phosphate concentration of 0. 1 5% was found
to be optimum for leaching of copper. The maximum
leaching of about 77% was observed with A. favus
DSF-8 followed by A. niger DOF- l (74%) and A. niger
A TCC 1 781 (71 %). Further increase in phosphate
concentration resulted in slight reduction of copper
solubilization.
From Fig. 3(c), it is evident that the leaching ef
ciency was affected to some extent by the changes in
1 UU
a
~
dU

'
U Q
.
(
C

4U
.

C
ZU
U
U. 1 . Z. . 4.
Gl ucose (%)
10
C
~
8

1
Q

0
n

40
'
L
20
0
.
4.5 5 5. 5 6 6. 5 7
pH

the pH of the medium. A pH of 5. 6 was found to be

optimum for all the strains. A. favus (DSF-8) was
more versatile with respect to pH as it depicted copper
leaching over a range of pH (5. 3-6. 5). The optimum
pH values for oxidation of different minerals by fungi
have been reported to vary slightly
24
.
The effect of temperature and pH of the medium on
microbial growth and mineralization are reported to
be interdependent
25
A temperature between 30 and
35C was found to be optimum for copper bioleach
ing by all the isolates. However, a reduction was ob-
Table 2 -Leaching of copper from chalcopyrite by organic acids
Sl. No.
1 .
2.
3.
4.
5.
6.
1 0
8
.
(at 2%)
Organic acid mgIL copper
leached
in 72 hr
Oxalic acid 39
Citric acid 84
Aceti'c acid 69
Tartaric acid 72
Lactic acid 81
Malic acid 72
b
% of copper
leached
38.688
83.328
68.440
7 1 .424
80.352
7 1 .424
' 40
(

1
Q

0
n

'
(
20 -
0.05
1 00
80
60
40
20
0
1 0
d
0. 1 0. 1 5 0.2 0.25
Phosphate as KH2P04 (mgL)
20 30
Temperature ("C)
0. 3
40
Fig. 3-Effect of dextrose, phosphate (as KH2P04), pH and temperature on copper leaching from chalcopyrite by fungi (-.- 1 781 , &
DOF- l , - - DSF-8, -0- DOF-8 and -0- DOF-6).
. \
RAO et al.: BIOLEACHING OF COPPER FROM CHALCOPYRITE ORE BY FUNGI
323
Table 3 -0rganic acids produced by the fungal isolates
Organism Acetic Oxalic
acid acid
A. niger (ATCC- 1 781 ) + +
A. niger (DOF- l ) +
A. flavus (DSF-8) + +
A. niger (DOF-8) + +
Rhizopus sp. (DOF-6) + +
served both below 30 and at 40 C temperature. A.
favus (DSF-8) was more adaptive than others as indi
cated by more than 65% Cu leaching even at 40C.
A. niger (ATCC 1781 ) was able to leach 7 1 % and 56%
of copper at 35C and 40C, respectively (Fig. 3d).
The leaching of metallic ores is facilitated by the
release of organic acids as a result of growth and me
tabolism of microorganisms
1
2. 1 5.26
. Chemical leaching
studies using only organic acids (Table 2) showed that
citric acid and lactic acid were the most effcient solu
bilisers effecting about 83% and 80% copper
solubilization respectively in 3 days. Tartaric acid,
malic acid and acetic acid on the other hand, were
moderate and oxalic acid showed very poor activity.
Though the mechanism has not been thoroughly es
tablished, it appears that the acid metabolites released
by fungi may be responsible for leaching
l
5
. Table 3
shows some of the organic acids produced by the fun
gal isolates. While citric acid, lactic acid and oxalic
acids were produced by all the fungal isolates, acetic
acid and malic acids were not produced by DOF- l ,
and none produced tartaric acid. Production of citric
acid by fungi is a well established phenomenon
I 4
.
27
.
Thus it appears that production of organic acids, espe
cially citric acid and lactic acid, is responsible for the
leaching of metallic ores. The production of organic
acids was also confrmed by the drop in the pH during
frst half of the experiment.
In the bioleaching of metallic ores chemosynthetic
bacteria have been extensively used
4
.
28
.
32
. Using Thio
bacillus ferrooxidans, Sakaguchi and Silver
2 1
reported
copper leaching up to 21 5 mg/ from chalcopyrite ore
and Mehta and Murr
33
reported up to 55% dissolution
of copper from ore. A mixed culture of moderately
thermophilic microorganisms was used to leach up to
60% of copper from chalcopyrite ore
23
. Because of
certain difculties encountered in growing chemosyn
thetic bacteria, search is on for heterotrophic microor
ganisms. Sukla et ai.
12

33
have shown that the fungus
A. niger was capable of leaching 92% nickel and 34%
cobalt from lateritic nickel ore. Khan et ai.
1
5
reported
Tartaric Citric Lactic Malic
acid acid acid acid
+ + +
+ +
+ + +
+ + +
+ +
85% of copper leaching by Aspergillus species from
ferromanganese sea nodules in seven days of incuba
tion. In a search for better fungal species for bioleach
ing of copper from its chalcopyrite ore, we are suc
cessful in isolating certain fungal isolates capable of
leaching up to 77% of copper. The versatility of these
fungi especially A.flavus (DSF-8), with respect to
sugar utilization, temperature and pH prove to be ad
vantageous for leaching copper from its chalcopyrite
ore.
Acknowledgement
The authors are thankful to Dr. Ilangovan, Manager
(Metallurgy), Chitradurga Gold Unit, Chitradurga for
providing ore samples and necessary information.
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