Hydrometallurgy 105 (2010) 8995

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Hydrometallurgy
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t

Leaching of copper, nickel and cobalt from Indian Ocean manganese nodules by
Aspergillus niger
K.D. Mehta, Chitrangada Das, B.D. Pandey
MEF Div., National Metallurgical Laboratory, CSIR, Jamshedpur-831007, India

a r t i c l e

i n f o

Article history:
Received 29 June 2010
Received in revised form 10 August 2010
Accepted 10 August 2010
Available online 13 August 2010
Keywords:
Polymetallic manganese nodules
Bioleaching
Aspergillus niger
Fungus
Copper
Nickel
Cobalt

a b s t r a c t
The leaching of copper, nickel and cobalt from polymetallic manganese nodules from the Indian Ocean was
investigated using a fungus Aspergillus niger. Parameters such as initial pH, pulp density, particle size and
duration of leaching were optimized for the bio-recovery of metals. At an initial pH of 4.5, 35 C temperature
and 5% (w/v) pulp density, about 97% Cu, 98% Ni, 86% Co, 91% Mn and 36% Fe were dissolved in 30 days time
using adapted fungus as against only 4.9% Cu, 8.2% Ni, 27% Co, 6.3% Mn and 7.1% Fe solubilized in control
experiment. The results indicate that A. niger released organic acids such as oxalic and citric acids which in
turn reduced the host metal oxides/hydroxides to their lower valence states, and thus dissolving the base
metals following the indirect mechanism. A comparison of results obtained with chemical leaching of sea
nodules using citric and oxalic acids and bio-leaching using A. niger show that the leaching of metals was
more effective in presence of the fungus. The appearance of some lower oxide phases of manganese and iron
in the leach residue identied by XRD phase analyses may account for unlocking of the host lattice leading to
release and dissolution of metals during leaching.
2010 Elsevier B.V. All rights reserved.

1. Introduction
In view of gradual exploitation of land based mineral resources
and the lack of mineral resources in some countries, efforts are
increasingly being made to explore alternate resources. Among the
marine mineral resources, the polymetallic nodules from the deep
ocean are of special interest as they represent an important renewable
source of several valuable metals. The nodules are generated slowly
on the sea bed and comprise mainly of manganese and iron oxides/
hydroxides together with metals such as copper, nickel, cobalt and
zinc (Monhemius, 1980). As these transition metals are often present
in the lattice of manganese and iron phases rather than in separate
identiable mineral phases within the nodules, reductive dissolution
of the host oxides/hydroxides is a prerequisite to release and dissolve
the base metals (Cronan and Tooms, 1967).
Many researchers have studied the chemical dissolution of metals
in mineral acids/ammonia with/without pretreatment of ocean
nodules. Mention may be made of a few of these viz., glucose
ammonia leaching (Das et al., 1986), reductionroastammonia
leaching (Jana et al., 1999; Hoover et al., 1975), ammonia sulfur
dioxidepressure leaching (Anand et al., 1988; Mittal and Sen, 2003),
and sulfuric acid leaching at elevated temperature (Han and
Fuerstenau, 1975) for the recovery of valuable metals. These processes

Corresponding author. Mineral Resources Research Division, KIGAM, 92, Gwahangno, Yuseong-gu, Daejeon 305-350, South Korea.
E-mail addresses: bdpnml@gmail.com, bd_pandey@yahoo.co.uk (B.D. Pandey).
0304-386X/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.hydromet.2010.08.002

consume high energy and are often associated with excessive

corrosion and create adverse impact on environment.
With the reported biogenesis of sea nodules (Ehrlich, 1968) and its
subsequent reduction by bacteria, the investigation of the mechanism
of bioleaching (Ehrlich, 1997) and the microbial recovery of valuable
metals (Ehrlich 2001) from ocean nodules has gained importance.
With regard to the bioprocessing of low grade oxide ores, there have
been various reports on the dissolution of metals from Indian ocean
nodules by Acidithiobacillus ferrooxidans (Mehta et al., 2002, 2003)
and a marine isolate (Mukherjee et al., 2003a,b). Also, on the leaching
of manganese from manganese ore by Penicillium citrinum (Acharya
et al., 2001) and nickel from lateritic nickel ore by a heterotrophic
microorganism (Sukla and Panchnadikar, 1993).
Metal dissolution with the help of a fungus has been studied
using A. niger to leach copper and zinc from an oxidized ore (Dave
and Natarajan, 1981). Whilst the treatment of a domestic low
grade limonitic/hematitic laterite ore by Aspergillus sp. and
Penicillium sp. were studied by Agatzini and Tzeferis (1997),
where low metal recoveries (5070%) were obtained even after
acid wash of the fungal biomass. Likewise, A. niger and Penicillium
sp. were used for bioleaching of zinc and nickel from silicates viz.,
calamine and garnierite (Castro et al., 2000). Mulligan and Kamali
(1998) also reported bio-dissolution of copper and other metals
embodied in low grade mining residues using A. niger. In recent
years, Mukherjee et al. (2003a,b, 2004) investigated the role of a
marine isolateBacillus M1 to extract cobalt from ocean nodules at
neutral pH while examining the role of pH and redox potential in
metal bio-dissolution.

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K.D. Mehta et al. / Hydrometallurgy 105 (2010) 8995

Aspergillus niger is known to secrete certain organic acids as a part

of its metabolic activity (Guebel et al., 2001) which can act as a
lixiviant by following a combination of reductive acid leaching and
complexation to dissolve some metals from the oxide ores (Mulligan
et al., 2004; Anjum et al., 2010). It is also reported that the release of
carboxylic acids is adversely affected in presence of metals like
manganese (Aghaie et al., 2009; Mulligan et al., 2004). In view of this,
it was considered worthwhile to investigate the effectiveness of A.
niger in the bioleaching of polymetallic sea nodules containing high
amounts of manganese with the aim of extracting associated metals
such as copper, nickel and cobalt.
The microbial leaching of manganese with fungi usually occurs
indirectly by non-enzymatic reduction process (Abbruzzese et al.,
1990). A. niger releases reductive acids (oxalic and citric) which in
turn converts Fe(III) to Fe(II) and MnO2 to Mn(II) (Stone, 1987). The
mechanism of the dissolution process can be represented by
reactions 1 and 2. Oxalic acid for example released by A. niger rst
reacts with manganese dioxide and goethite while destroying the host
lattice of oxide/hydroxide. The metals associated with these two are
thus released and get dissolved at lower pH, besides forming soluble
metal complexes (Mulligan et al., 2004; Anjum et al., 2010). Some of
the metals are in turn adsorbed on fungal cell walls and are easily
desorbed again by acid wash of the biomass.

2CO2 2H2 O

2CO2 4H2 O

MnO2 HOOCCOOH 2H Mn

2FeOOH COOH2 4H 2Fe

2. Materials and methods

2.1. Nodule analysis and characterization
Indian Ocean nodules in the air dried form were provided by
National Institute of Oceanography, Goa. Using XRD, the major phases
in the sea nodules were found to be todorokite, birnesite, lithiophorite
and silica, whereas, maghemite, goethite, and alumina were the minor
phases. The sample of nodules contained 0.89% Cu, 0.99% Ni, 0.12% Co,
6.4% Fe, 18.3% Mn, 0.09% Zn, 0.04% Mo, 0.7% C, 16% SiO2 and 8% Al2O3.
The nodules were crushed, ground and passed through a sieve of
300 m. A representative sample was prepared by coning and
quartering method and metals were analysed by acid digestion and
atomic absorption spectrometry (AAS). A known amount of the
nodule powder was also passed through different sieves to get
different fractions for bioleaching experiments. Chemical analyses of
the sieve fractions are given in Table 1.
All the chemicals used were of Anala R grade.
2.2. Microorganism and medium
Fungus Aspergillus niger (MTCC 281) was obtained in the
lyophilised form from Microbial Culture Collection Centre, IMTECH
Chandigarh, India. The A. niger culture was revived at 37 C using

Table 1
Composition of different size fractions of sea nodules.

Czapek Dox medium (sucrose 30 g/L, sodium nitrate 3 g/L, dipotassium phosphate 1 g/L, magnesium sulfate 0.5 g/L, potassium chloride
0.5 g/L, ferrous sulfate 0.01 g/L). The revived culture was streaked on
sterile agar plates to check the purity. The plates containing pure
culture were saved in a refrigerator sealed with paralm at 4 C.
2.3. Growth and adaptation of microorganism
Since metal toxicity is a well known phenomenon to fungal culture
(Hughes and Poole, 1989) it was necessary to adapt A. niger to the higher
metal ion concentration. Initially, A. niger was inoculated into 100 mL
Czapek Dox medium at 35 C in presence of 0.5 g/L Cu2+ and Ni2+ each,
and 0.05 g/L Co2+ and incubated for 96 h at 120 rpm with a count in
spores formed. The culture was then progressively adapted to higher
concentrations of Cu2+ and Ni2+ (2.5 g/L each), and Co2+ (0.25 g/L) in
the nutrient broth while incubating under the same condition (35 C,
120 rpm and 96 h). The growth was monitored by counting the spores
formed. Fungus thus became tolerant to 5 g/L (Cu +Ni) and 0.25 g/L Co
which was assumed to be the concentration of metal ions in the leaching
medium when higher than 10% (w/v) pulp density (PD) was used.
Despite their acclimatization to synthetic divalent metal salt medium
the fungus could hardly grow in medium containing ocean nodules
because of the adverse effects like cell trauma, reduction in oxygen mass
transfer rate and presence of non-substrate solid particles (Bailey and
Hansford, 1993). The adaptation of the culture on the nodules was done
by progressively increasing the pulp density from 0.55% (w/v).
Repeated sub-culturing was done and stored at 4 C (2 108 spores/
mL) for further use.
The stored culture was grown in 5% w/v PD at 3 months interval to
maintain its activity; and its purity was also checked on sterile agar
plate. The amount of carboxylic acid generated from A. niger at
different sucrose levels in Czapek Dox medium was estimated (Castro
et al., 2000). The acid generated in an inoculated set during the
bioleaching of metals from ocean nodule at a pulp density of 5% (w/v)
was also analyzed. The supernatant sample drawn from the
inoculated set was centrifuged at 8000 g for 10 min and was ltered
using a 0.45 m microlter. The method described by Kiel et al (1981)
was employed, in which the solution was analyzed for carboxylic
acids by titration with standard sodium hydroxide solution. In this
method, citric acid was measured by titration with 0.1 N NaOH
solution with phenolphthalein as an indicator after precipitation of
oxalic acid by CaCl2.
2.4. Bioleaching experiments
Bioleaching experiments were carried out in sterile Erlenmeyer
asks taking 25 g sea nodules of known size at the desired
temperature in an orbital motion incubator under shaking condition
(120 rpm). Adapted fungal suspension (5% v/v) was inoculated in
nutrient broth in presence of the nodules. All the experiments were
done in duplicate and the average values (with variation within 2%)
were determined to see the effect of pH, pulp density and particle size
of the nodules on the aqueous recovery of metals. Progress of
bioleaching of metals was studied by collecting the samples at specic
intervals and analysing the metal content by AAS. The redox potential
(Eh) against saturated calomel electrode (SCE) was measured at
5 days interval and expressed as such; a stable redox potential value
was the indication of the end of bioleaching process.
2.5. Acid wash of residue

Particle size
(m)

Fraction
retained,
%

Cumulative
fraction retained,
%

Composition (%)
Cu

Ni

Co

Mn

Fe

300150
150100
10075
b 75

27.33
36.12
14.97
21.58

27.33
63.45
78.42
100

0.884
0.856
0.847
0.849

0.989
0.99
0.850
0.981

0.1035
0.105
0.105
0.107

18.30
19.20
19.73
18.81

7.2
7.7
7.98
8.13

During the bioleaching process, sometimes manganese is reported

to precipitate. Also, manganese ion adsorbs on MnO2 at pH N5
(Marshall, 1979) and limits further leaching (Buys et al., 1986). As
manganese dioxide has negligible solubility in dilute sulfuric acid at
ambient temperature, the adsorbed manganese(II) ion can be

K.D. Mehta et al. / Hydrometallurgy 105 (2010) 8995

3. Results and discussion

3.1. Indirect and chemical leaching
Considering the release of a rich variety of carboxylic acids by
Aspergillus group of fungi in the aqueous system, particularly those of
citric and oxalic acids (Guebel and Darias, 2001), the use of these acids
on leaching of metals from the ocean nodules was investigated. A.
niger was grown in varying sugar concentrations for 15 days and
oxalic and citric acid were estimated (Castro et al., 2000) in the
supernatant.
As shown in Table 2 the values of all analyses for citric and oxalic
acids were found to be in the range 05 g/L. The pH prole for 15 days
old A. niger culture show that it typically dropped from an initial value
of pH 6.5 to 3.1 at a sucrose concentration of 15% although, pH
decreased to 4.4 in 5 days with the secretion of 2 g/L oxalic acid and
1.8 g/L citric acid. With 3% sucrose in the media the decrease in pH
was not appreciable till 10 days. However, pH fell down to 4.1 in
15 days time because of secretion of 3.0 g/L oxalic acid and 2.8 g/L
citric acid. The growth of A. niger may be correlated with the amount
of carboxylic acid secreted and the concentration of sucrose added in
the media. The growth was very high in 5 days time with 15% of
sucrose as compared to 15 days time required with 3% sucrose level.
The lower concentration of carboxylic acids (Table 2) detected in
solution after 10 days may be the result of manganese present in the
nodules that might have affected their secretion. However, some
amount of organic acids oxidized by MnO2 and consumed during the
leaching process may not be ruled out.
Since the maximum amount of any organic acid secreted by A.
niger was ~5 g/L in 15 days at 15% sugar concentration, chemical

Table 3
Chemical leaching by organic acids and bioleaching by A. niger. Time8 days, temp
35 C, PD5%(w/v), initial pH6.5.
Condition

Recovery (%)

Oxalic: 1 g/L
3 g/L
5 g/L
Citric: 1 g/L
3 g/L
5 g/L
3 g/L citric + 3 g/L oxalic acid
Metabolite (15 d old culture)
Bioleaching

Cu

Ni

Co

8.2
11.6
26.1
9.9
11.4
28.1
30
21
34

6.4
13.6
22
12.5
18.8
27
26
24
24

9.6
10.6
18
9
11.4
17.8
17.9
8.8
11

leaching experiments were carried out with the reaction mixture

containing 15 g/L carboxylic acid to compare the results with the
fungal bioleaching. The results given in Table 3 show that chemical
leaching of nickel using citric acid (5 g/L) is more effective (27%) than
that (22%) with oxalic acid in 8 days at 35 C with initial pH of 6.5. This
may be attributed to the low solubility of nickel oxalate (Mulligan
et al., 2004). However, with a combination of both acids (3 g/L each),
the nickel chemical leaching is nearly same (26%) as that observed
with citric acid due to the formation of nickel citrate complex as
observed by Tzeferis et al. (1994). On the other hand, the leaching rate
of metals from the nodules shown in Fig. 1 suggests that oxalic acid
(5 g/L) is more effective over 6 days of leaching for copper and cobalt.
After 8 days, the amount of metal extraction decreased in chemical
leaching with oxalic acid probably due to some precipitation of metals
already dissolved. When a lower concentration of citric acid and oxalic
acid was used, metal extraction was found to be much lower (Table 3).
In the case of leaching using metabolite of A. niger (15 days old
culture with 3% sucrose) containing 3.0 g/L oxalic acid and 2.8 g/L

35

Cu

Ni

Co

30

Recovery, %

redissolved in sulfuric acid (Imai, 1978). Once the manganese ion

comes in leach liquor it indicates the release of other entrapped metal
ions viz., Cu2+, Ni2+ and Co2+. So by washing the leach residue with
dilute H2SO4 the adsorbed metal ions on residue as well as on biomass
could be recovered.
The leach residue after ltration was washed with distilled water
and dried in oven at 110 C. To the half portion of the residue was
added dilute H2SO4 (pH: 1) and stirred for 2 h followed by
centrifugation at 10,000 rpm. The solution was analyzed for metal
ions by AAS. The other half of the residue was kept at 200 C
temperature in an oven for 24 h for the biomass to be burnt. The burnt
mass was washed thoroughly with ethanol, dried and washed with
above acid, and metal content was analyzed by AAS. By comparing
both data, metal adsorbed on biomass was determined. Unless stated
otherwise, the total metal recovery given in the text is the sum of acid
desorbed metal and soluble metal extraction yield. In several cases the
residues were also analyzed to check the mass balance.

91

25
20
15
10
5

a. Citric acid

0
0

10

Days
Table 2
Organic acid released by A. niger and corresponding pH prole in 15 days at varying
sucrose concentration. Initial pH: 6.5, temperature: 35 C.
Sucrose conc.
(%)
5

10

15

15

3
5
10
15
3
5
10
15
3
5
10
15
3( with nodules5% PD )

Final
pH

Oxalic acid
(g/L)

(g/L)

6.4
5.8
5.9
4.4
6.2
5.8
5.2
4.1
4.1
4.0
3.9
3.1
4.6

0.5
0.5
1.4
2
0.6
0.5
2.1
3.1
3.0
3.5
3.8
4.9
2.8

0
0.1
0.8
1.8
0.2
1.2
1.8
2.3
2.8
3.3
3.1
3.8
2.4

Cu

Ni

Co

25

Citric acid

Recovery, %

Days

30

20
15
10
5

b. Oxalic acid

0
0

10

Days
Fig. 1. Metal recovery in chemical leaching by oxalic and citric acid in 10 days. Acid
conc.: 5 g/L, temperature: 35 C, PD: 5% w/v.

92

K.D. Mehta et al. / Hydrometallurgy 105 (2010) 8995

b
pH 3
pH4
pH5

60

pH3.5
pH4.5
pH 4.5 (C)

pH3.5
pH4.5
pH 4.5 (C)

60

Metal recovery (%)

50

Metal recovery (%)

pH 3
pH4
pH5

70

40
Cu
30
20
10

50
40
30
20

Ni

10

0
0

10

15

20

25

30

10

15

Days

25

30

c
pH 3
pH4
pH5

70

pH3.5
pH4.5
pH 4.5 (C)

pH 3
pH4
pH5

80

pH3.5
pH4.5
pH 4.5 (C)

70

Metal recovery (%)

80

Metal recovery (%)

20

Days

60
50
40
30
Co

20
10

60
50
40
30
Mn

20
10

0
0

10

15

20

25

30

Days

10

15

20

25

30

Days

Fig. 2. (ad): Metal recovery (without acid wash) at varying pH in 30 days time during bioleaching at 35 C and 5% PD.

Fig. 2(ad). Acid wash was performed only after completion of the
bioleaching experiment and not at the intermediate stage. High metal
recoveries of 97% Cu, 98% Ni, 86% Co, 91% Mn and 36% Fe were
obtained at initial pH 4.5, whereas cobalt and iron recovery was
higher at initial pH 4.0 (96 and 66% respectively) after acid wash. The
analysis of the solution during leaching at initial pH 4.5 shows the
presence of increased amounts of oxalic acid (3.2 g/L in 15 days),
besides citric acid (2.3 g/L) which improved the dissolution of metals

3.2. Direct bioleaching

350

If the effectiveness of metal dissolution in presence of fungus and

the synthetic carboxylic acid (indirect process) is considered after
15 days, one can nd that copper extraction is maximum (34%) in the
case of fungal leaching, again with low cobalt extraction (11%), which
is comparable to indirect leaching. But, as discussed below, in fungal
leaching the extraction process goes on for almost 2530 days, when
high metal extraction (N90%) can be achieved; whilst in case of the
indirect process, the dissolution of metals reaches to a stationary level
(data not shown) soon after 10 days of leaching.

300

250

200

150

3.2.1. Effect of initial pH

The effect of initial pH on the biodissolution of metals was
investigated at 35 C and 5% (w/v) PD using 5% (v/v) suspension of
adapted A. niger culture in 250 mL leaching medium. Recoveries of
metals at different pH in 30 days without acid wash are shown in

Eh

400

pH

citric acid (pH 4.1), a similar trend in metal dissolution as that of any
organic acid was observed although with a low cobalt recovery
(8.8%). The low cobalt leaching with the metabolite and also with the
organic acids is related to its association with iron(III) phase in the
nodules (Han and Fuerstenau, 1975) which is dissolved to a much
lesser extent. Iron(III) from the nodules could be mostly reduced by
oxalic acid while forming a complex. Also, the dissolution of iron(III)
from the nodules may be affected by citric acid which forms Fe(II)
citrate and Fe(III) citrate complexes which are stable and dissolve
slowly (Tzeferis et al., 1994).

100
Eh

50

pH

0
0

10

20

0
30

Days
Fig. 3. pH and redox potential prole during bioleaching by Aspergillus niger in 30 days
at 35 C and initial pH of 6.8.

K.D. Mehta et al. / Hydrometallurgy 105 (2010) 8995

Metal recovery (%)

Table 5
Recovery of metals by A. niger in 30 days at 35 C and 5% (w/v) PD.

Cu

100

Ni

Metals

Co

80

Recovery (%) at initial pH of 4.5

Leaching

Mn
Fe

60

93

Cu
Ni
Co
Mn
Fe

40

51
46
51
67
14

Acid wash

Total

Ore

Biomass

34
15
28
11
10

12
17
7
13
12

97
98
86
91
36

20
0
0

10

15

20

PD (%)
Fig. 4. Metal recovery (with acid wash) in 21 days at varying PD during bioleaching by
A. niger at initial pH of 4.5 and 35 C.

from the nodules. Still higher amounts of oxalic acid were found in
solution at initial pH 4.0 which may account for higher iron and cobalt
leaching. Decreased recovery at pH 5 in all the cases is attributed to
lower solubility and higher precipitation of metal ions.
It is pertinent to note that during the leaching process, the pH
variation shows opposite tendencies, i.e., higher pH in the indirect
process with synthetic organic acid and lower pH in the presence of
fungus. Considering nearly equal amount of organic acids were
present in both cases, it can be concluded that the pH increase with
synthetic acid is due to proton consumption while converting the
oxides/hydroxides phases of nodules into soluble metal salts. Whilst
the pH decrease in the fungal leaching process is due to more protons
produced extracellularly that exceeds the demands for this reaction.
The fungal leaching process also has to be viewed in the context of
both redox and chemical reactions. Initially the pH of the medium was
high with a low redox potential value of 140 mV (Fig. 3) i.e., high

Table 4
Metal recovery at varying particle size of nodules in 30 days at 35 C, 5% PD and 4.5 pH
(initial).
Size fraction
(m)/condition

Days

(300150)

7
26
14
45
21
58
30
57

41

97
7
29
14
53
21
48
30
61

36

97
7
29
14
73
21
70
30
64

33

97
7
29
14
67
21
66
30
71

25

96
Control leaching
30
15

Acid wash
Total
(150100)

Acid wash
Total
(10075)

Acid wash
Total
b 75

Acid wash
Total
(300150)

Biorecovery (%)
Cu

Ni

Co

Mn

24
52
67
78
20
98
26
57
65
74
19
93
37
32
82
74
23
97
43
89
73
79
20
99

24
27
29
27
11
38
20
35
38
41
13
55
34
45
26
38
14
52
62
87
59
79
18
97

28
66
67
82
11
93
27
68
62
71
10
81
37
58
65
69
12
81
48
96
77
86
12
98

32

0.7

Fe
3
3

3
13
15
2.3
3
2

16
16
3
5
2

15
15
8
3
4
2
16
18
0.9

Fe(II) concentration. The ferrous ion reduced MnO2 to Mn (II) and was
oxidized to Fe(III) which hydrolysed thus the redox potential
increased with time and pH decreased. After 25 days of experiment,
the Eh became stable at 380 mV(SCE) indicating that the biological
activity reached to a saturation level. The fall in pH as the leaching
proceeded may also be attributed to the secretion of organic acids
such as oxalic and citric by the fungus as discussed earlier. To meet the
continuous proton demand, the presence of A. niger is preferable
because organic acids are continuously generated, whereas in the
indirect process at some point the leaching terminates without
completion due to the limited supply of protons.

3.2.2. Effect of pulp density and particle size

Fig. 4 depicts dissolution of metals with A. niger at varying pulp
density in the range 320 (w/v) %. An increase in pulp density (PD)
resulted in a decrease in metal dissolution. At 5% (w/v) PD about 79.5%
Mn was recovered along with 89.2% Cu, 79.5% Ni, 63% Co and 36% Fe
after acid wash after 21 days at initial pH 4.5 and 35 C. These values
drastically came down at 20% w/v PD under the same experimental
conditions. Besides the limitation of the microbial growth, the amount
of carboxylic acid secreted at higher pulp density may not match the
acid requirement for reduction and metal leaching thereby yielding
lower extraction. From these results 5% w/v pulp density was
considered optimum for the recovery of all the metals of importance.
In the sterile control experiment recovery was much less; e.g. at 5% w/
v PD after 21 days the leach recovery was 10% Cu, 7.8% Ni, 32% Co and
6.3% Mn.
To nd out the inuence of different particle size of the sea
nodules, bioleaching experiments were carried out at initial pH 4.5,
35 C and 5% w/v PD using A. niger along with a set of control
experiments. Data on metal recovery vs. size fraction of the sea
nodules are shown in Table 4. The composition of the different size
fractions given in Table 1 is not very different except a little low value
of nickel (0.85%) in 10075 m size. So total recovery as seen in
Table 4 are very similar, excepting that of cobalt, which shows
increasing trend with the neness of the particles. The total biorecovery after acid wash was found to be 97% Cu, 98% Ni, 38% Co, 93%
Mn and 15% Fe for the coarser size of the nodules (300150 m) as
against recovery of 15% Cu, 32% Ni, 9% Co, 0.7% Mn and 0.9% Fe under
the sterile conditions in 30 days. However, cobalt recovery was as
high as 97% from the particles of b75 m size.

Table 6
XRD phase identication of ocean nodules before and after bioleaching at 35 C in
30 days.
Sample
Ocean nodules

Phases

Major: todorokite, birnesite, lithiophorite, silica

Minor: maghemite, goethite, alumina
Bioleach residue pH 3.5 Major: birnesite, lithiophorite, silica, hydronium jarosite,
Minor: MnO, Mn2O3, braunite, maghemite, alumina
pH 4.5 Major: silica, birnesite, lithiphorite and Fe(OH)3
Minor: goethite, alumina

94

K.D. Mehta et al. / Hydrometallurgy 105 (2010) 8995

the attack of host lattice by the combination of reduction and acid

leaching and release of metals entrapped in them. A change in surface
morphology of the leach residue further reected the bioleaching of
metals. As can be seen from the SEM images (Fig. 5), the surface of the
leach residue after acid wash (Fig. 5b) was observed to be highly
corroded as compared to the morphology exhibited by the unreacted
sea nodules (Fig. 5a).

4. Conclusions

It may be concluded that the dissolution of metals from the Indian

Ocean nodules by A. niger is an indirect process proceeding through
the secretion of oxalic and citric acids which in turn reduce the host
lattice of manganese(IV) and iron(III) oxides thereby releasing the
valuable metals particularly copper, nickel and cobalt. The bioleaching
of the metals has been found to be more effective than the chemical
leaching by carboxylic acids or by fungal metabolites. The overall
leaching of about 97% Cu, 98% Ni, 86% Co, 91% Mn and 36% Fe was
achieved after acid wash of the residue in presence of A. niger under
the optimum conditions after 30 days at initial pH of 4.5, 35 C and 5%
pulp density using a particle size b300 m. The low values of redox
potential (380 mV) observed during the course of leaching and the
XRD phase analysis of the leach residue showed the presence of lower
valence manganese and iron phases further conrmed the usefulness
of the fungus to dissolve the metals effectively with the help of
secreted carboxylic acids.
Acknowledgments
The authors are thankful to the Department of Science and
Technology, New Delhi for nancial support and the Director, NML,
Jamshedpur, India for giving permission to publish the paper. One of
the authors (C. Das) is thankful to the Council of Scientic & Industrial
Research for the award of Research Associateship.
References

Fig. 5. SEM images of sea nodules (a) and the leach residue (b) by leaching with A. niger
at initial pH of 4.5 and 35 C.

3.2.3. Effect of acid washing of biomass and residue

After each batch of bioleaching, the residue along with fungal
biomass was washed with dilute sulfuric acid to evaluate the acid
desorbed manganese and other metals. The total recovery of metals
rose by 2160% as shown in Table 5, a part of which was precipitated
on residue and about 713% of metal was adsorbed on biomass. The
change in colour of the mycelial ball observed after acid wash and easy
recovery of most of the adsorbed metals indicated that the metal ions
were adsorbed and accumulated mainly on the surface of fungal
hyphae as was observed earlier by Ghiorse and Ehrlich (1992). This
was further substantiated by insignicant recovery of metals during
acid wash of leach residue from control experiments at pH 4.5.
3.3. Residue phase analysis
The phases identied by XRD analyses of leach residue by A. niger
at pH 3.5 and pH 4.5 and also the nodules are presented in Table 6.
Apart from the manganese phases such as birnesite, lithiophorite and
silica, formation of hydronium jarosite at pH 3.5 and iron-hydroxide at
pH 4.5 as major phases was the interesting feature. Besides, some
amount of MnO, Mn2O3, braunite and maghemite were also formed
as minor phases in leach residue at pH 3.5 whilst goethite was found
in the residue at pH 4.5. Formation of phases comprising of mixed and
lower oxidation states of manganese and iron clearly demonstrates

Abbruzzese, C., Duarte, M.Y., Paponetti, B., Toro, L., 1990. Biological and chemical
processing of low grade manganese ore. Miner. Eng. 3 (34), 307318.
Acharya, C., Kar, R.N., Sukla, L.B., 2001. Microbial extraction of manganese from low
grade manganese ore. Trans. Indian Inst. Met. 54 (3), 99103.
Agatzini, S., Tzeferis, P., 1997. Bioleaching of nickelcobalt oxide ores. Aus IMM Proc. 1,
915.
Aghaie, E., Pazouki, M., Hosseini, M.R., Ranjbar, M., Ghavipanjeh, F., 2009. Response
surface methodology (RSM) analysis of organic acid production for kaolin
beneciation by Aspergillus niger. Chem. Eng. J. 147, 245251.
Anand, S., Das, S.C., Das, R.P., 1988. Leaching of manganese nodules at elevated
temperature and pressure in presence of oxygen. Hydrometallurgy 21, 4155.
Anjum, F., Bhatti, H.N., Ghaur, M.A., 2010. Enhanced bioleaching of metals from black
shale using ultrasonics. Hydrometallurgy 100 122-12.
Bailey, A.D., Hansford, D.S., 1993. Factors affecting biooxidation of sulphide minerals at
high concentrations of solids. Biotechnol. Bioeng. 42 (10), 11641174.
Buys, H.M., Chan, S.M., Chun, U.H., Cho, D.W., Davis, P., Mackay, B., Madwick, J.,
Pannowitz, D., Poi, G., Varga, R.A., 1986. Acid removal of manganese ion adsorbed
on ore surface during microbial manganese dioxide leaching. Proc. Aus. I.M.M. 291,
7173.
Castro, I.M., Fietto, J.L.R., Vieira, R.X., Tropia, M.J.M., Campos, L.M.M., Paniago, E.B.,
Brandao, R.L., 2000. Bioleaching of zinc and and nickel from silicates using
Aspergillus niger cultures. Hydrometallurgy 57, 3949.
Cronan, D.S., Tooms, J.S., 1967. Geochemistry of Mn-nodules from N. W. Indian Ocean.
Deep Sea Res. 15, 215223.
Das, R.P., Anand, S., Jena, P.K., 1986. Leaching of manganese nodules in ammoniacal
medium using glucose as reductant. Hydrometallurgy 16, 335344.
Dave, S.R., Natarajan, K.A., 1981. Bioleaching of copper and zinc from oxidised ores by
fungi. Hydrometallurgy 7, 235242.
Ehrlich, H.L., 1968. Bacteriology of manganese nodules. Appl. Microbiol. 16 (2),
197202.
Ehrlich, H.L., 1997. Microbes and metals. Appl. Microbiol. Biotechnol. 48, 687692.
Ehrlich, H.L., 2001. Past, present and future of biohydrometallurgy. Hydrometallurgy
59, 127134.
Ghiorse, W.C., Ehrlich, H.L., 1992. Microbial Biomineralization of Iron and Manganese. In:
Skinner, H.C.W., Fitzpatrick, R.W. (Eds.), Biomineralization process of iron and
manganese in modern and ancient environments: Catena supplement 21, pp. 7599.
Catena, Verlag, Crelingen, Germany.

K.D. Mehta et al. / Hydrometallurgy 105 (2010) 8995

Guebel, D.V., Darias, N.V.T., 2001. Optimisation of citric acid production by A. niger
through a metabolite ux balance model. Process. Biotechnol. 4 (1), 111.
Han, K.N., Fuerstenau, D.W., 1975. Acid leaching of ocean oor manganese at elevated
temperature. Int. J. Miner. Process. 2, 163171.
Hoover, M., Han, K.N., Fuerstenau, D.W., 1975. Segregation roasting of Cu, Ni, Co from
deep sea manganese nodules. Int. J. Miner. Process. 2, 173185.
Hughes, M.N., Poole, R.K., 1989. Metals and Microorganisms. Chapman and Hall,
London. 412 pp.
Imai, K., 1978. On the Mechanism of Bacterial Leaching. In: Murr, L., Torma, A.E.,
Brierleyi, J.A. (Eds.), Metallurgical Applications of Bacterial Leaching and Related
Microbiological Phenomena. Academic Press, New York, pp. 276295.
Jana, R.K., Pandey, B.D., Premchand, 1999. Ammoniacal leaching of roast reduced deep
sea manganese nodules. Hydrometallurgy 53, 4556.
Kiel, H., Guvrin, R., Henis, Y., 1981. Citric acid fermentation by Aspergillus niger on low
suger concentrations and cotton waste. Appl. Environ. Microbiol. 42 (1), 14.
Marshall, K.C., 1979. In: Trudinger, P.A., Swaine (Eds.), Biogeochemistry of manganese
minerals. : Biogeochemical Cycling of Mineral Forming Elements. Elsevier,
Amsterdam, pp. 253292. Chapter 5.
Mehta, K.D., Pandey, B.D., Mankhand, T.R., 2002. Biochemical recovery of valuable
metals from Indian Ocean Nodules in presence of pyrite and sulphur. Trans. Indian
Inst. Met. 55 (3), 7180.
Mehta, K.D., Pandey, B.D., Mankhand, T.R., 2003. Studies on kinetics of bio-dissolution of
metals from Indian Ocean nodules. Miner. Eng. 16, 523527.

95

Mittal, N.K., Sen, P.K., 2003. India's rst medium scale demonstration plant for treating
polymetallic nodules. Miner. Eng. 16, 865868.
Monhemius, A.J., 1980. In: Burkin, A.R. (Ed.), The extractive metallurgy of deep sea
nodules: Topics in Non-ferrous Extractive Metallurgy. Blackwell SCI, London,
pp. 4269.
Mukherjee, A., Raichur, A.M., Modak, J.M., Natarajan, K.A., 2003a. Bioprocessing of Indian
Ocean nodules using marine isolateeffect of organics. Miner. Eng. 16, 651657.
Mukherjee, A., Raichur, A.M., Modak, J.M., Natarajan, K.A., 2003b. Solubilisation of
cobalt from ocean nodules at neutral pHa novel bioprocess. J. Ind. Microbiol.
Biotechnol. 30 (10), 606612.
Mukherjee, A., Raichur, A.M., Modak, J.M., Natarajan, K.A., 2004. Exploring process
options to enhance metal dissolution in bioleaching of Indian Ocean nodules. J.
Chem. Technol. Biotechnol. 79 (5), 512517.
Mulligan, C.N., Kamali, M., 1998. Bioleaching of copper and other metals from low grade
oxidised ores by Aspergillus niger. J. Chem. Technol. Biotechnol. 78, 447450.
Mulligan, C.N., Kamali, M., Gibbs, B.F., 2004. Bioleaching of heavy metals from a lowgrade mining ore using Aspergillus niger. J. Hazard. Mater. 110, 7784.
Stone, A.T., 1987. Microbial metabolites and the reductive dissolution of manganese
oxides: oxalate and pyruvate. Geochim. Cosmochim. Acta 51, 919925.
Sukla, L.B., Panchnadikar, V., 1993. Bioleaching of lateritic nickel ore using a
heterotrophic microorganism. Hydrometallurgy 32, 373379.
Tzeferis, P.G., Agatzini, S., Nerantzis, E.T., 1994. Mineral leaching of non-sulphide nickel
ores using heterotrophic micro-organisms. Lett. Appl. Microbiol. 18, 209213.