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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: [email protected] .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)


    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 idntified 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


    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-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



    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



    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 or