The European Journal of Mineral Processing and Environmental Protection

Vol.2, No.3, 1303-0868, 2002, pp. 197-204

Bioleaching of Low Grade Manganese Ore with Penicillium citrinum

C. Acharya*, R.N. Kar and L.B. Sukla

Dept. of Biomineral & Biotechnology, Regional Research Laboratory,

(Council of Scientific & Industrial Research), Bhubaneswar-751 013, India

Received 1 April 2002 accepted 4 October 2002

ABSTRACT

A native microorganism, Penicillium citrinum was isolated from the top soil of a manganese mine. Based
on its efficiency for manganese solubilisation, it was utilized for the leaching of a low-grade manganese ore.
The effects of various parameters such as pulp density, particle size, sucrose concentration, inoculum size
and bioleaching duration on manganese ore were studied. The optimised conditions for maximum
solubilization of the manganese ore (64.58% Mn) were : a particle size of 45µm; a pulp density of 2% (w/v);
a sucrose concentration of 10% (w/v); an inoculum dosage of 10% (v/v); and a 30 day duration. © 2002
SDU. All rights reserved.

Keywords: Manganese ore; Penicillium citrinum; Leaching

1. INTRODUCTION

India has been recognized as one of the leading manganese producing countries in the
world. Considering the environmental problems related to conventional processes, both
pyrometallurgical and hydrometallurgical, its recovery through bioleaching utilizing different
kinds of microorganisms has been studied extensively by several workers (Baglin et al., 1992;
Gupta and Ehrlich, 1989; Srimekanond et al., 1992). Leaching of manganese by heterotrophic
microorgaisms is caused by metabolites such as organic acids (Baglin et al., 1992). The
proposed mechanisms for the bioleaching of manganese diooxide include both direct or indirect
(Ehrlich, 1976; Toro et al., 1988). In the former case, the microbes are said to be capable of
utilising the MnO2 as a final acceptor of electrons in the respiratory chain of their metabolism
instead of oxygen (Ehrlich, 1987). In the latter case, the reductive process is associated with the
formation of reductive compounds(citric acid) resulting from their metabolism (Toro et al.,
1988).
The following communication provides the preliminary evidence of the mechanism for the
dissolution of manganese from a manganese ore using a native strain of Penicillium citrinum.
Studies on isolation, identification, and leaching characteristics of this fungus are described in
this paper.

2. MATERIALS AND METHODS

2.1. Substrate

Manganese ore was obtained from Joda East manganese mines of Tata Iron & Steel
Company Ltd. (TISCO) in Keonjhar District in Orissa. The ore used in this study was of low grade,
the manganese content ranging from 22-29% (Table 1). The XRD analysis of the ore indicated
the presence of pyrolusite (MnO2), chalcophanite (ZnMn3O7.3H2O), hematite (Fe2O3) and
goethite (FeOOH). For leaching experiments, the bulk ore was ground and sieved to get

* Corresponding author. E-mail: [email protected]

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different size fractions. After acid digestion, the elemental analysis of ore was carried out using
a Perkin Elmer-3100 Atomic Absorption Spectrophotometer.

Table 1
Chemical analysis of manganese ore
Average Particle Element (%)
size (microns) Mn Fe Zn Cd Pb Cu Ca Mg
2812 29.08 34.92 0.01 0.00 0.00 0.01 0.00 0.00
1831 24.54 26.24 0.00 0.00 0.00 0.01 0.01 0.00
500 22.96 23.18 0.02 0.00 0.00 0.01 0.00 0.00
112.5 25.72 24.94 0.02 0.00 0.00 0.01 0.02 0.01
60 23.99 22.55 0.00 0.00 0.03 0.01 0.01 0.01
45 23.04 22.06 0.48 0.05 1.07 0.02 0.02 0.01

2.2. Microorganism

2.2.1.Isolation

The microorganisms were isolated from the top soil of a manganese mine. A total of eight
isolates was obtained and those isolates were used for selection of the best species for
manganese solubilisation. Colony growth of fungal and bacterial species were observed using
agar plates. Individual colonies, which grew well on solid media were selected on the basis of
colour, size, shape and dominance.

2.2.2. Screening

The colonies were purified by single colony streaking and then each organism was
restreaked on solid media impregnated with sterile, sub-micron, ball-milled manganese ore. The
species which removed suspended ore particles and produced halos around colonies growing
on agar plates were sub-cultured for taxonomic identification (Burgstaller and Schinner, 1993).
Submerged leaching was also carried out using all of the eight isolates at 2% (w/v) pulp
density at an initial pH of 6.5 with a 500 microns particle size and 10% (v/v) inoculum dosage
for a period of 10 days. The mineral salt media used for leaching contained the following
constituents in 1 liter of distilled water, 3g NH4NO3, 1g KH2PO4, 0.5g MgSO4.7H2O, 100g
Sucrose. After the preliminary screening, a fungal culture was selected on the basis of
maximum manganese solubilisation from the ore.

2.2.3. Identification

The fungal culture was identified by the Institute of Microbial Technology, Chandigarh, India.
This fungus was further used for bioleaching of manganese ore.

2.3. Bioleaching studies

Leaching experiments were carried out in 250ml Erlenmeyer flasks containing 90ml salt
medium and 2-10% (v/v) (106spores/ml) inoculum, 2-10% (w/v) sucrose at 2-10% (w/v) pulp
density of ore and particle sizes of manganese ore ranging from 2812 microns to 45 microns
for 90 days. The flasks were incubated at room temperature (32oC) while shaking at 140rpm.
Experiments were performed in duplicate. At the termination of each set of experiment, the
flasks were sterilised. The contents of the flasks were filtered. The final pH was measured. The
biomass and the ore which remained on the filter paper were thoroughly washed with distilled
water and then with dilute sulphuric acid. The washings were added to the filtrate. The overall
manganese extractions thus stated here is the sum of acid desorbed and soluble

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manganese at the termination of each set of experiment. The soluble/acid desorbed

concentrations of manganese/iron were calculated as (Vegliò et al., 1997):

C  Ci
Manganese (or iron) extraction yield (%) = x 100 (1)
Co
or recovery

where, C = total manganese/iron recovered after 30 days treatment (%),

Co = initial manganese / iron content in the leaching system (%),
Ci = Mn2+ due to inoculum (%), (Here, Ci was taken as zero)

3.RESULTS AND DISCUSSION

3.1. Influence of particle size

Various size fractions ranging from 2812 microns to 45 microns were used for the study.
The microbial leaching of manganese ore with heterotrophic microorganisms like fungi
generally follows an indirect mechanism (Abbruzzese et al.,1990; Toro et al., 1993) where
manganese solubilization is due to reduction.
Table 2 shows that the total manganese extraction yield is highest (64.58%) for the finest
particle size of 45 microns due to the accessibility of the ore particles to the fungus. Soluble
manganese in the culture fluids ranged from 4.13% to 19.88% for the different granulometric
size ranges.

Table 2
Influence of particle size on bioleaching of manganese ore
Conditions: Pulp density: 2% (w/v) Inoculum: 10% (v/v) Temperature: 32oC Shaking: 140rpm
Duration: 30 days pH initial: 6.5 Sucrose: 10% (w/v)
Average Final pH Soluble manganese Acid desorbed Total extraction (%)
Particle size and iron extraction manganese and iron
(microns) yield (%) extraction yield (%)
Mn Fe Mn Fe Mn Fe
2812 5.95 4.13 0.08 8.08 2.90 12.21 2.98
1831 5.85 4.81 0.08 9.78 3.88 14.59 3.96
500 5.76 13.72 0.09 19.25 6.69 32.97 6.78
112.5 5.70 15.01 1.28 41.99 9.02 57.0 10.3
60 5.62 16.80 0.26 42.93 10.02 59.73 10.28
45 5.60 19.88 0.14 44.70 12.24 64.58 12.38

There is not a strong pH decrease as can be seen from Table 2. Similar observations were
made by Paponetti et al. (1989) while leaching MnO2 using Aspergillus niger. They proposed
the indirect mechanism of dissolution where organic acids produced during the process (oxalic
and citric acids) were responsible for reduction and solubilization of the manganese ore.
Penicillium sp. is also said to produce citric and oxalic acids (Silverman and Munoz, 1970;
Tzeferis, 1994). There seems to be greater evidence for the indirect mechanism and hence, it
was assumed that dissolution of manganese ore in our case with P. citrinum also followed
indirect reaction mechanism. During the bioleaching process, manganese may precipitate. Also,
manganese ions adsorbs on the surface of the MnO2 ore particles at pH>5 (Marshall, 1979)
which may limit further leaching (Buys et al., 1986). As manganese oxide has negligible
solubility in dilute sulphuric acid at ambient temperature (Imai, 1978), the adsorbed manganous
ions were re-dissolved in the dilute sulphuric acid wash. Therefore the total Mn2+ extacted is
the sum of acid desorbed manganese extraction yeild and soluble manganese extraction yeild
at the end of each batch of experiment.

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The iron dissolution was also highest for the finest particle size of 45 microns corresponding
to the peak in manganese extraction. The maximum concentration of soluble manganese in the
culture fluids (19.88%) at the 45 microns size was not as high as expected. It may be due to the
influence of the mineralogy of the manganese ore. There is a trend towards slower microbial
leach rates on oxides with more ordered or crystalline structures like pyrolusite. XRD data of the
manganese ore in our investigation indicated the presence of more pyrolusite than other
minerals like cryptomelane, rhodochrosite, calcite, etc.Manganese oxides containing relatively
more pyrolusite have been shown to be less susceptible to microbial attack than those with less
(Srimekanond et al., 1992).

3.2. Influence of pulp density

A series of experiments were carried out with different pulp density suspensions ranging
from 2 to 10%(w/v) manganese ore and the data is summarized in Table 3. A constant particle
size of 500 microns was used in the experiments. The highest dissolved or solubilized
manganese concentration (13.8%) was achieved with a 2% (w/v) pulp density at 32oC (Table
3). There was no appreciable change in pH which again indicates a non-enzymatic reduction of
manganese by Penicillium citrinum. Higher Mn2+ extraction yields are achieved with lower pulp
densities. Similar observations were made by Vegliò et al., 1997. The total manganese recovery
or manganese extraction yield after 30 days treatment was evaluated in a similar fashion to
previous tests, adding the soluble (Mn2+) and the precipitated manganese after acid treatment
of the solid leached residue.
The maximum total recovery of 33% was achieved for the 2% (w/v) pulp density.Iron
extraction was also maximized at this pulp density, 6.78%. As the pulp density increased, the
manganese extraction yields decreased.This may be due to a decrease in the availability of
oxygen or nutrients for the fungus. MnO2- reduction in an acid solution under anoxic conditions
represented by the following reaction:

MnO2 + 2H+  Mn2+ + 1/2O2 + H2O (2)

is not very likely due to the high energy of activation (Ehrlich, 1996).

Table 3
Influence of pulp density on bioleaching of manganese ore using P. citrinum
Conditions: Sucrose: 10% (w/v) Inoculum: 10% (v/v) Temperature: 32oC
Shaking: 140rpm Duration: 30 days pH initial: 6.5
Particle size: 500 microns
Pulp density Final pH Soluble manganese Acid desorbed Total extraction (%)
(% w/v) and iron extraction manganese and iron
yield (%) extraction yield (%)
Mn Fe Mn Fe Mn Fe
2 5.76 13.72 0.09 19.25 6.69 32.97 6.78
4 5.80 12.54 0.08 17.51 4.66 30.05 4.74
6 5.82 7.40 0.04 16.77 4.40 24.17 4.44
8 5.85 7.06 0.02 15.81 4.28 22.87 4.30
10 5.95 4.75 0.35 12.63 3.54 17.38 3.89

3.3. Influence of sucrose concentration

The experiments conducted to observe the influence of sucrose concentration on the

bioleaching of manganese ore indicated that there is a direct relationship between manganese
extraction and the total amount of sucrose present in the media. Heterotrophs like Penicillium
sp. were found to solubilize MnO2 by producing citric and oxalic acids in a culture medium
containing sucrose (Silverman and Munoz, 1970). This type of media is commonly utilized in
microaerobial monitored conditions. It was previously mentioned that strong evidence exists for

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the reduction of MnO2 by fungi through the indirect mechanism or non-enzymatically

(Paponetti et al., 1989). Fungi produce metabolic products like oxalic or citric acids by utilising
sucrose. These acids are strong enough reductants for MnO2. Since the electron transport
mechanism in fungi, which are eukaryotic organisms is located in mitochondrial membranes
and not in plasma membrane, as in procaryotic cells, fungi cannot be expected to reduce MnO2
enzymatically (Ehrlich, 1978), except possibly by extracellular enzymes. The overall indirect
reaction utilizing sucrose can be expressed as follows :

25 MnO2 + C12H22O11 + 50H+  25 Mn2+ + 12CO2 + 36H2O (3)

Mn2+ + CO2 + H2O  MnCO3 + 2H+ (4)

Sucrose consumption was greater in the presence rather than in absence of manganese
(Toro et al., 1991). The growth of the microorganism began with a short lag phase which was
followed by an exponential growth phase when manganese was extracted from ore, hence
utilising the sucrose.
In these investigations, it was observed that a maximum extraction of manganese of 32.97%
was obtained with 10% sucrose with a particle size of 500 microns (Table 4). The manganese
extraction yields were increased as the sucrose concentration in the culture media increased.
Similar observations were made by (Vegliò et al., 1995). Correspondingly, iron extraction was
also peak (6.99%) with a 10% sucrose concentration. In the control (media without sucrose)
there was a low recovery of Mn2+ (6.98%) and iron (0.9%). In the absence of energy substrate
like sucrose Penicillium citrinum could not produce organic acids to reduce the MnO2 and
hence, a low extraction was observed.

Table 4
Influence of sucrose concentration on the bioleaching of manganese ore using P. Citrinum
Conditions: Pulp density: 2% (w/v) Inoculum: 10% (v/v) Temperature : 32oC
Particle size: 500 microns Shaking: 140rpm Incubation: 30 days
pH initial: 6.5
Sucrose Final pH Soluble manganese Acid desorbed Total extraction (%)
concentration and iron extraction manganese and iron
(% w/v) yield (%) extraction yield (%)
Mn Fe Mn Fe Mn Fe
2 5.90 10.83 0.09 12.63 5.82 23.46 5.91
4 5.85 11.59 0.09 12.84 6.04 24.43 6.13
6 5.83 12.68 0.17 15.03 6.13 27.71 6.30
8 5.80 13.66 0.13 16.6 6.47 30.26 6.60
10 5.76 13.72 0.30 19.25 6.69 32.97 6.99
Control 6.35 6.42 0.04 0.56 0.86 6.98 0.90

3.4. Influence of inoculum size

It was observed from these studies that the product formation (manganese reduction) was
related closely to the microbial inoculum or growth (product associated to the growth). The
greater the inoculum concentration, the greater the production of organic acids which will
eventually lead to manganese reduction. A dosage of 10% (v/v) inoculum gave the maximum
total manganese extraction of 32.97% as shown in Figure 1. One of the major drawbacks with
submerged cultivation technique employing fungus is that the leaching material is adsorbed to
fungal mycelium (Burgstaller and Schinner, 1993). As stated above, the growth of the fungus
was enhanced in presence of the manganese ore as manganese is an essential element for
growth and differentiation of the fungus (Auling, 1989). Greater than 10% (v/v) inoculum was
not employed for this investigation as it would have caused clumping of the mycelia and the
adsorption of the leached manganese to the mycelia. The control with no fungal spore inoculum
under the similar experimental conditions produced a manganese extraction of only 1.18% and
only after acid washing.The soluble manganese extracted into the culture media was only
0.003%, the final pH (6.45) of the culture media also reflects the lack of extraction. In general,

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the manganese reduction is enhaced as the inoculum concentration increases to the point
where mycelia clumping and adsorption becomes problematic.

35

Total Mn extraction (%)

30
25
20
15
10
5
0
0 2 4 6 8 10
Inoculum (%v/v)

Figure 1. Influence of inoculum size on Manganese extraction

3.5. Influence of time period

The effect of leach duration on the extraction of manganese with P. citrinum was studied.A
maximum period of 90 days was employed using a finer particle size of 112.5 microns. The
studies were conducted with an initial pH of 6.5 at 32oC. It appears that the manganese was
extracted from the ore by indirect mechanism or non-enzymatically by the fungus P. citrinum.
This is supported by a decrease in the pH of the leach liquor.The pH decreased to 5.25 in 30
days giving a total extraction of 58% [16.1% of soluble manganese and 41.9% of acid washed
manganese in the leach system (Figure 2)]. There was a net acid consumption at the time of
maximum Mn2+ reduction. In the acidic medium (organic acids released by the fungus), the iron
was also reduced. A total recovery of 10.30% iron was achieved within a period of 30 days.
There was an increasing trend of total manganese extraction up to 45 days (68.3%). After 45
days, there was a decrease in the total manganese extraction (59.48%) as well as iron (6.01%).
It has been suggested that the manganese which is leached out gets accumulated on the
surface of some structure of the fungal hyphae (Ghiorse and Ehrlich, 1992). It was observed that
the leaching was growth independent and most likely the result of microaerobic fermentation
where the reductive process was associated with the production of reductive compounds
(Madgwick, 1993). The reduction in the recovery of manganese after 45 days may be due to :

1. the accumulation of leached material (manganese) on the surface of fungal hyphae

(Ghiorse and Ehrlich, 1992).
2. the production of volatile metabolites instead of organic acids and decay of the enzymes
of the fungus following its death phase (Harrison and Pirt, 1967) as a result of which there
is a slight rise in pH of the media.

30
25
Mn recovery (%)

20
15
%Mn recovery (in-situ
10 leaching)
5 %Mn recovery (culture
filtrate leaching)
0
0 10 20 30 40 50 60 70 80 90
Time (days)

Figure 2. %Mn recovery in 1) in-situ leaching (soluble manganese) 2) culture filtrate leaching

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The experiments were terminated at the end of 90 days following a reduction in the
manganese extraction yield.

4. CONCLUSIONS

The fungus Penicillium citrinum, a native organism from the top soil of a manganese ore
mine, was found suitable for the leaching of a low grade manganese ore. A maximum
manganese extraction of 64.58% was achieved with a particle size of 45 microns, a pulp
density of 2% (w/v), a sucrose concentration of 10%(w/v), an inoculum dosage of 10% (v/v)
and a duration of 30 days.

ACKNOWLEDGEMENT

The authors are thankful to Prof. V.N. Misra, Director, Regional Research Laboratory for his
permission to publish the paper.

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