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Indian Journal of Experimental Biology Vol. 40, March 2002, pp. 3 1 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 harnessed 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 mgIL) was observed with Aspergillus flavus (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 min­eral biotechnology has opened a new avenue for metal leaching from low-grade ores l •2• Microbial leaching processes have been considered economical, eco­friendly and technically viable alternatives for proc­essing of low-grade ores3• The microbe influenced extraction of metals from insoluble minerals is done through leaching by acidophili(; iron-oxidizing bacte­ria4. Chemolithotropic microorganisms like Thiobacil­Ius, Sulfolobus, Pseudomonas, Bacillus etc . , are among the microorganisms being used extensively in bioleaching studies4.8. Alternative to these extremo­philes, search is on for other acidophilic microorgan­isms. Some workers have indicated the possibility of metal dissolution by acidic metabolites of heterotro­phic fungi9. 12 . 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 alumina13 .

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.

Materials and Methods Substrate -Chalcopyrite, used in the present study

was collected from Chitradurga Copper Mine, Chi­tradurga, Karnataka. While the ore contains 1 .0 107%

*Correspondent author: E-mail: smgaddad@rediffmail .com

copper, the other major constituents are Si02 (68 .32%), Fe ( 10.55%) and S (3 .69%), Ni (4.5 ppm) and Co ( 1 .5 ppm) are present in trace concentrations.

Isolation offungi -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 purplel4• The spore suspension was prepared by growing fungi on Sabroud's Dex­trose Agar plates for 5 days at room temperature and spores harvested in 1 0ml sterile normal saline.

Bioleaching studies-All leaching experiments were conducted using modified Czepek Dox minimal mediuml5 supplemented with 1 % of ground chalcopy­rite ore ( 1 00 mesh). Hundred milliliters of the me­dium in 250 ml Erlenmeyer flask was inoculated with I ml of spore suspension (approximately 5 x 107

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 op­timum 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 concentra­tion (0.05-0.3%), pH (4.5-7.0), temperature ( lOD-40DC)

320 INDIAN J EXP BIOL, MARCH 2002

and different sugars (glucose, maltose, mannitol, su­crose 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 efficiency of different organic acids l 5, leaching ex­periments were conducted using 2% of different or­ganic 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 iso­lates, isolates were grown in Czepek Dox medium for 3 days. Fungal mycelium was separated by centrifugation (7000 rpm for 1 5 min) and the supernatant was qualitatively analysed for organic acidsl6

Analysis- Samples collected during leaching processes were centrifuged at 7,000 rpm for 1 5 min to remove biomass. The clear supernatant liquid was analysed for copper content by direct titration using Fast Sulphon Black F as indicator17•

Results and Discussion The elemental composition of the chalcopyrite ore

used in the present study indicates. that it is a low­grade ore, which is uneconomical for commercial ex­ploitation. 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 id�ntified by fol­lowing 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

178 1 DOF-O l 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 "

Penicillium sp.

A. niger

A. flavus

A. niger

A. flavus A. niger

Copper leached Maximum

(mgIL) 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 10 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 4 1 .67 14 53.57 08 62.50

16 65.47 1 2 1 7.86

08 53.57

14 62.50

14 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

.j �

RAO et at.: BIOLEACHING OF COPPER FROM CHALCOPYRITE ORE BY FUNGI 32 1

copper solubilised was found to be more in shake cul­tures than the static cultures in all the cases. It is 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 pres­ence 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.

flavus DSF-8, A niger DOF-8 and DOF- l and Rhizopus sp. (DOF-6), took 6- 10 days to leach about 66-78% copper. In comparison A niger (ATCC 1 7 8 1 ) was able to leach a maximum o f 69 mg/L (68.44%} of copper in 8 days (Table 1 ) . After recording the maxi­mum leaching, there was a slight decrease in the amount of copper in the leachate. This may be due to the adsorbence of -leached copper ions overtime onto the fungal hyphae19• Based on these preliminary re­sults only four isolates (A. flavus DSF-8, A. niger DOF-8, DOF- l and Rhizopus sp. DOF6) along with A. niger (ATCC 1 7 8 1 ) were used for further studies.

The dependency of copper leaching rate on initial pulp density is very important in case of bioleaching20,2 l . Figure 1 reveals that all the five isolates showed high percent of leaching at low pulp densities (0.5-2.0%). The strain Aflavus (DSF-8) was comparatively more efficient. 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 dioxide22• 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 oxygen22 and attrition problems with the mineral particles23.

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 microorgan­isms was found to increase with decrease in size of . I 2 1 23 Th

. f . I d . h . partiC es ' . e SIze 0 partlc es etermlOes t elr

surface area, which also affect the efficiency of leach­ing. 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 mm.

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. Aflavus (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 heterotro­phic microorganisms especially fungi 1 2. Its concentra­tion determines the quantity of acidic metabolites. The effect of different concentrations of glucose

Particle size (mm) (- - - - -) 0.05 0.1 0.15 0.2 0.25 0.3 0.5 0.6

100 r---t----t----t----t----+------li !

I : ' l: ; ; ;l;;"',,,,,: : : , , , ::;,,,�,.:!:;�,,.;::; : : : ' -:> U . . . . ...

. :::

: IL __ �_� __ t--_� ___

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).

0 1 781 . OOF-1 1I11 0SF-8 . OOF-8 Eil OOF-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.flavus (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 microor­ganisms and organic acid production. The effect of increasing phosphate concentrations on the bioI each­ing 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. flavus DSF-8 followed by A.niger DOF-l (74%) and A.niger A TCC 1781 (7 1 %). Further increase in phosphate concentration resulted in slight reduction of copper solubilization.

From Fig. 3(c), it is evident that the leaching effi­ciency was affected to some extent by the changes in

1 00 a

,....., 80 � e..... 't:I 60 Ql .c () C\I � 40 ,....., � C,) 20

0 0 .5 1 .5 2.5 3.5 4 .5

Glucose (%)

100 c

-- 80 :::R � 1J Q) 60 � 0 nI � -- 40 '5' () 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. flavus (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 interdependent25 • A temperature between 30° and 35°C was found to be optimum for copper bioleach­ing 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.

100

80

. (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 8 1 Malic acid 72

b

% of copper leached

38.688 83.328 68.440 7 1 .424 80.352 7 1 .424

'5' 40 ()

� � 1J Q)

� 0 nI � --'5' ()

20 �--�----�----�----�----�

0.05

1 00 80· 60 40 20

0 10

d

0. 1 0. 15 0.2 0.25 Phosphate as KH2P04 (mglL)

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 78 1 , - .& ­

DOF- l , - • - DSF-8, -0- DOF-8 and -/::.- 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-178 1 ) + + 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. flavus (DSF-8) was more adaptive than others as indi­cated by more than 65% Cu leaching even at 40°C. A.niger (ATCC 178 1 ) was able to leach 7 1 % and 56% of copper at 35°C and 40°C, respectively (Fig. 3d).

The leaching of metallic ores is facilitated by the release of organic acids as a result of growth and me­tabolism of microorganisms 12. 1 5.26. Chemical leaching studies using only organic acids (Table 2) showed that citric acid and lactic acid were the most efficient solu­bilisers 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 es­tablished, it appears that the acid metabolites released by fungi may be responsible for leachingl 5. Table 3 shows some of the organic acids produced by the fun­gal 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 I4.27. Thus it appears that production of organic acids, espe­cially citric acid and lactic acid, is responsible for the leaching of metallic ores. The production of organic acids was also confirmed by the drop in the pH during first half of the experiment.

In the bioleaching of metallic ores chemosynthetic bacteria have been extensively used4.28.32. Using Thio­bacillus ferrooxidans, Sakaguchi and Silver2 1 reported copper leaching up to 2 1 5 mg/L from chalcopyrite ore and Mehta and Murr33 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 ore23. Because of certain difficulties encountered in growing chemosyn­thetic bacteria, search is on for heterotrophic microor­ganisms. 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 incuba­tion. In a search for better fungal species for bioleach­ing of copper from its chalcopyrite ore, we are suc­cessful 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 ad­vantageous 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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