Hydrometallurgy 109 (2011) 65–71

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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

Recovery of metals from spent refinery hydrocracking catalyst using adapted

Aspergillus niger
F. Amiri a, S. Yaghmaei a,⁎, S.M. Mousavi b,⁎⁎, S. Sheibani c
a
Department of Chemical and Petroleum Engineering, Center of Excellence, Development and Strategic Plants for Bioprocess Technology, Sharif University of Technology, Tehran, Iran
b
Biotechnology Group, Department of Chemical Engineering, Tarbiat Modares University, Tehran, Iran
c
R & T Management Department, National Iranian Oil Refining and Distribution Company, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: This study was designed to compare one-step, two-step and spent medium bioleaching of spent catalyst by
Received 9 February 2011 adapted Aspergillus niger in batch cultures. Aspergillus niger, which was adapted to heavy metal ions, Ni, Mo,
Received in revised form 17 April 2011 Fe, and W, was grown in medium containing up to 5% (w/v) of spent catalyst. The main lixiviant in bioleaching
Accepted 20 May 2011
was gluconic acid, which was produced at all pulp densities in the one-step bioleaching process. Gluconic acid
Available online 27 May 2011
was also produced in the two-step bioleaching process when the spent catalyst was present at pulp densities
Keywords:
greater than 1% (w/v). In the spent medium leaching, however, the primary agent was citric acid. The pulp
Spent catalyst density of the spent catalyst was varied, and this resulted in different amounts of solubilized metals. A total of
Tungsten 3% (w/v) spent catalyst generally produced maximum extraction yields in the one-step bioleaching process;
Bioleaching the amounts of leached metals were 100% of W, 77.8% of Fe, 90.9% of Mo, 65.8% of Ni, and 14.2% of Al. The
Aspergillus niger highest concentration of gluconic acid in this condition demonstrated that it was the primary bioleaching
agent. Compared with chemical leaching at 1% pulp density, the fungus was more efficient at the leaching of
W, Al, Mo, and Fe and equally efficient in the extraction of Ni.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Rapid urbanization, industrialization and various technological
innovations have created an increased need to dispose of industrial
solid wastes, such as spent catalysts, fly ash, spent batteries and
electronic scraps. Heavy metal pollution resulting from industrial
solid waste disposal is a major environmental problem. These heavy
metals are required by all life forms, but only in limited amounts. At
high concentrations, heavy metal ions react to form toxic compounds
in cells. Another major problem with metals is their indefinite
persistence in the food chain (Gupta et al., 2000; Spain, 2003;
Gavrilesea, 2004). Despite the environmental problems they cause,
heavy metals are valuable and have many applications in industry.
Hydroprocessing catalysts are used extensively in the petroleum
refining industry to produce clean fuels from petroleum distillates and
residues. These catalysts usually consist of molybdenum (Mo) or
tungsten (W) supported on an alumina or silica-alumina carrier with
promoters, such as cobalt (Co) or nickel (Ni). The catalysts enhance the
cracking and removal of undesirable impurities, such as sulfur, nitrogen
and metals (V and Ni) present in the feed (Marafi and Stanislaus, 2003).
Worldwide, the quantity of spent hydroprocessing catalysts generated is
⁎ Corresponding author. Tel.: + 98 2166166430.
⁎⁎ Corresponding author. Tel.: + 98 21 82884917; fax: + 98 21 82884931.
E-mail addresses: yaghmaei@sharif.edu (S. Yaghmaei), mousavi_m@modares.ac.ir
(S.M. Mousavi).
in the range of 150,000–170,000 tons year− 1. This will increase in the
coming years because of the increasing demand for clean fuels. In view of
the environmental and economic benefits, increasing attention has been
paid to the development of processes to recover metals and other
valuable materials from the spent catalysts (Marafi and Stanislaus, 2008).
Bioleaching is a novel approach to recover metals from various
solids. Bioleaching relies on the ability of microorganisms (bacteria and
fungi) to transform solid compounds. This transformation occurs
through the production of organic or inorganic acids, which results in
soluble and extractable elements that can be recovered (Aung and Ting,
2005; Santhiya and Ting, 2005, Santhiya and Ting, 2006; Mishra et al.,
2007, Mishra et al., 2008). Bioleaching has been used in the mining of
metals, such as copper, uranium and gold (Tzeferis, 1994; Mulligan and
Cloutier, 2000; Mulligan et al., 2004; Le et al., 2006). The process can also
be applied to the extraction of metals from solid wastes, which may be
considered ‘artificial ores’. These include bio-dissolution of metals from
fly ash, spent batteries and electronic scraps, as well as different types of
spent catalysts (Brandl et al., 2001; Aung and Ting, 2005; Santhiya and
Ting, 2005, Santhiya and Ting, 2006; Wu and Ting, 2006; Mishra et al.,
2007, Mishra et al., 2008; Yang et al., 2009). The most active leaching
fungi that have been isolated and used are from the genera Penicillium or
Aspergillus. In recent studies, heavy metals in solid waste have been
demonstrated to be extractable by fungus through four main mecha-
nisms within the bioleaching process: acidolysis, complexolysis,
redoxolysis and bioaccumulation (Aung and Ting, 2005; Santhiya and
Ting, 2005, Santhiya and Ting, 2006; Wu and Ting, 2006; Yang et al.,

0304-386X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.hydromet.2011.05.008
66 F. Amiri et al. / Hydrometallurgy 109 (2011) 65–71

2009). Metal leaching by heterotrophic microorganisms generally Magix Pro) (Table 1). X-ray diffraction (XRD) (Philips, model: Xpert
involves an indirect process with microbial production of organic Pro) was also used to determine the crystalline phases of the spent
acids, amino acids and other metabolites. These metabolites dissolve catalyst (Fig. 1).
metals from minerals either by replacing metal ions in the solid matrix
with hydrogen ions or by forming soluble metal complexes and chelates 2.3.1.1. Chemical analysis. Partial chemical composition was deter-
(Rezza and Salinas, 2001). mined using a homemade method. Briefly, 0.02 to 0.05 g of sample
Comparisons of one-step and two-step bioleaching, as well as was digested for 2 h in 10 ml of concentrated HNO3 and 10 ml of
bioleaching by spent microbial culture, have been reported by several concentrated HF, and the sample was then heated to 120 °C for
researchers, and the feasibility of the latter has been demonstrated. For approximately 2 h. The sample was almost completely soluble in the
industrial applications, bioleaching by spent culture is believed to be digest solution. The digestate was cooled, filtered and brought up to a
desirable to increase leaching efficiency ( Aung and Ting, 2005; Wu and total volume of 100 ml using deionized water. The sample was then
Ting, 2006; Mishra et al., 2008). There are few reports on bioleaching of subjected to chemical analysis using an inductively coupled plasma
Mo, Ni, Al, and V from spent catalyst by Aspergillus niger. However, there optical emission spectrometer ICP-AES (Varian, model: LIBERTY - RL).
have been no reports on bioleaching of tungsten by this fungal strain. In
this work, we are the first to report on the fungal leaching of tungsten
2.4. Acclimatization of fungus with spent catalyst
from spent hydrocracking catalyst by our native Aspergillus niger strain.
The aim of this work was to compare metal recovery from
The fungus from the final stage of adaptation with a mixture of
tungsten-rich spent hydrocracking catalyst using different methods of
metal ions was cultured in a potato dextrose agar (PDA, 3.9% (w/v))
bioleaching by an adapted Aspergillus niger strain. Furthermore, we
slant. In order to obtain a sufficient number of spores, the culture was
investigated the role of produced organic acids in various bioleaching
incubated at 30 °C for 5 days. The mature conidia were then washed
methods as the main agents of metal extraction. To reach this aim,
from the surface of the PDA medium using sterilized physiologic
first the fungus Aspergillus niger was adopted to spent catalyst. This
saline (9 g/L NaCl). The number of spores was counted using a
strain had been initially adapted to W, Mo, Fe and Ni (data not shown)
Neubauer counting chamber and was adjusted using sterilized
in a procedure described by Amiri et al. (2011). This process was
physiologic saline to a final concentration of approximately
continued using tungsten-rich spent hydrocracking catalyst; pulp
10 7 spores/mL. Two milliliters of spore suspension was added to a
densities were increased until no more fungal growth was observed.
500 mL Erlenmeyer flasks containing 0.5% (w/v) of spent catalyst and
In the next stage, bioleaching of spent catalyst using one-step (the
100 mL of sucrose medium with the following composition: sucrose
fungus and the spent catalyst were introduced to the medium
(100 g/L), NaNO3 (1.5 g/L), KH2PO4 (0.5 g/L), MgSO4·7H2O (0.025 g/L),
simultaneously) and two-step process (the fungus was added first
KCl (0.025 g/L) and yeast extract (1.6 g/L). The flasks were agitated in
and the spent catalyst was added when biometabolite production
an incubator with orbital shaking at 120 rpm and 30 °C. Acclimatiza-
commenced) as well as leaching by spent medium (produced
tion of fungus to spent catalyst was achieved by increasing the
biometabolites by the fungus after a specified time) were investigated
concentration of spent catalyst, in steps of 0.5% (w/v), from 0.5% (w/v)
at various pulp densities. Finally, we investigated the role of organic
to a concentration at which no fungal growth occurred. For each step, a
acids in the leaching of W, Mo, Fe, Ni, and Al. Chemical leaching
10% (v/v) inoculation was performed using a sample obtained from
experiment was carried out using commercial organic acids at the
the previous step. Finally, following the final stage of adaptation to the
same concentrations as were measured in the spent medium. Physical
spent catalyst, the fungus was maintained in PDA (3.9% (w/v)) slant.
and chemical characterization of the spent catalyst was performed
prior to bioleaching using Aspergillus niger and its spent medium. The
various constituents of the spent medium were analyzed in the 2.5. Different methods of bioleaching
absence and presence of spent catalyst at various pulp densities.
In order to obtain a sufficient number of spores for the bioleaching
2. Materials and methods experiments, the acclimatized fungus from the final stage of

2.1. Source and description of spent catalyst

Table 1
Spent hydrocracking catalyst (Criterion HC-102 W/Ni/Al2O3/SiO2) Elemental composition of spent hydrocracking catalyst.

was provided by National Iranian Oil Refining & Distribution Company Element Digestion (mg/kg) XRF (mg/kg)
(NIORDC), and this material was used in all experiments. The spent Ag 40 nt
catalyst was extrudate trilobe with black covering. The spent catalyst Si nt 109,700
was pretreated by heating it in a furnace at 600 °C for 4 h, and this Al 210,000 129,700
decoking resulted in the spent catalyst turning yellow in color. The Fe 6000 14,400
Ca b 100 1900
decoked spent catalyst was gently dry ground and sieved. All spent
Na 950 100
catalysts used in this study were of particle size less than 150 μm. Mg 70 100
K 130 400
2.2. Microorganism Ti nt 450
Mn 30 10
Co b 20 b10
The microorganism used in this study was Aspergillus niger BBRC- Cr 100 120
20018. This microorganism was provided by the Biochemical and Cu 45 b10
Bioenvironmental Research Center (BBRC), Sharif University of Ni 8000 14,100
Technology (SUT), Tehran, Iran. Pb 90 b10
V 25 500
W 77,000 268,800
2.3. Characterization of spent refinery hydroprocessing catalyst Zn 130 b10
Mo 6800 15,500
2.3.1. Catalyst composition Hg b 50 nt
The elemental composition of the spent catalyst was determined Ga b 20 nt

using chemical analysis and X-ray fluorescence (XRF) (Philips, model: nt: not tested.
 F. Amiri et al. / Hydrometallurgy 109 (2011) 65–71
Fig. 1. X-ray diffraction of decoked spent hydro-cracking catalyst.
adaptation to spent catalyst was cultured as described in Section 2.4.
Sterile experimental set-up was achieved by autoclaving at 121 °C
for 15 min prior to inoculation. Bioleaching was performed in
500 ml Erlenmeyer flasks containing 100 ml of sucrose medium
and the spent catalyst at various pulp densities (1, 2, 3, 4 and 5%
(w/v)). All experiments were conducted in an orbital shaking
incubator at 30 ± 1 °C and 120 rpm. Three different methods of
bioleaching were investigated. The first method was one-step
bioleaching, in which the fungus was incubated together with the
medium and spent catalyst. The second method was two-step
bioleaching, in which the fungus was first cultured in sucrose
medium without the spent catalyst for 2 days and then, following
a decrease in pH (beginning of organic acid production), the
sterilized catalyst was added. The third method was spent medium
leaching, which was performed by culturing the fungus in sucrose
medium for 10 days. In this method, the suspensions were then
filtered through MN 640 d and 0.2 μm (Millipore) filter paper to
obtain cell free spent medium. Finally, the filtrate that contained
the fungal metabolites was used for the leaching of sterilized spent
catalyst that was added to the filtrate. Control experiments were
conducted using fresh sucrose medium.
2.6. Chemical leaching
Because there are other biometabolites besides organic acids in the
bioleaching medium, such as amino acids which involve in metal
extraction, the purpose to perform the chemical leaching was to
investigate the importance of the measured organic acids as agents in
the bioleaching of W, Mo, Fe, Ni, and Al.
Chemical leaching of the heavy metals was conducted in a solution
containing commercial organic acids at the same concentrations as
those produced by A. niger in the absence of the spent catalyst.
2.7. Analytical methods
After the desired bioleaching time, the culture from each flask was
filtered, and the filtrate was analyzed for organic acid content (i.e.,
citric, oxalic and gluconic acids). Analysis was performed using high
performance liquid chromatography (HPLC) (Waters, model: ALLI-
NACE 2695), with a variable wavelength detector (VWD) at 210 nm to
detect the organic acids and a refractive index detector (RID) to detect
the sugars.
The filtrate was also analyzed to determine the concentration of
various metal ions. Metal ions were analyzed using an ICP-AES
(Varian, model: LIBERTY - RL). Multi-element standards (Merck) were
used for the calibration.
67
Additionally, the pH of the leached liquor was measured using a
digital pH meter (Metrohm, model: 827 pH lab).
The fungal biomass obtained from the filter paper was carefully
transferred to a preweighed evaporating dish and dried at 80 °C for
24 h to determine the dry weight of the biomass.
Extraction and measurement of the accumulated and associated
metals were performed as described by Santhiya and Ting (2006).
Briefly, at 80 °C, the dried residue, consisting of residual catalyst and
biomass, was placed in a 250 mL beaker in an orbital shaker at room
temperature for 2 h in 50 mL of 5% HNO3. The solution was then
heated gently for 30 min at 80 °C and filtered through an MN 640 d
filter paper. Finally, the solution was brought up to a total volume of
100 mL with distilled water and analyzed for metal ions using an ICP-
AES (Varian, model: LIBERTY - RL). Similarly, blank experiments were
conducted using the same spent catalyst concentration to determine
the dissolution of metals from the catalyst by 5% HNO3.
The scanning electron microscope (SEM) was used to observe the
morphology of the catalysts. The spent and bioleached catalysts were
subjected to SEM examination. Samples were mounted with silver
paste on aluminum stubs and then coated with 300–400 Å Au in a
sputtering unit. Finally, samples were scanned under an accelerating
voltage of 10 kV and high vacuum.
3. Results and discussion
3.1. The spent catalyst compositions
Elemental composition of spent hydro-cracking catalyst was
determined using chemical digestion and XRF (Table 1). The most
abundant elements (N50,000 mg/kg) were Al, W and Si. Most of the
minor constituents (1000–50,000 mg/kg) were heavy metals that
included Ni, Mo, and Fe. Other heavy metals, such as V, Ti, Cr, Mn, Co,
Cu, Pb, Hg and Zn were found in trace amounts (b1000 mg/kg).
Crystalline phases in solid samples were identified by X-ray
diffraction analysis (Fig. 1). The X-ray diffractogram showed four
crystalline phases: lithium aluminum tungsten oxide (LiAlW2O4),
silicon oxide (SiO2), tungsten oxide (WO3), and nickel molybdenum
oxide (NiMoO4).
3.2. Adaptation of Aspergillus niger with the spent catalyst
This stage was necessary because there are some elements, such as
Mn, Ti, Cr, Pb and Hg (Table 1), as well as Cl (data not shown), in the
spent catalysts that may have inhibitory effects on fungus growth.
Additionally, adaptation in the bioleaching medium before major
experiments is helpful in increasing the efficiency of bioleaching.
The adaptation was continued up to a pulp density of 5% (w/v).
More than this concentration, no more fungal growth was observed.
Accordingly, bioleaching experiments were conducted at pulp
densities up to 5% (w/v).
3.3. Mechanism of organic acid production and investigation of
Aspergillus niger pure culture
The production of organic acids, as the main agents in the
bioleaching of metals, from sucrose involves a large number of
enzymatic steps that occur in two different membrane-bound cellular
compartments: the cytosol and the mitochondrion. Sucrose is
substantially hydrolyzed to glucose and fructose through the action
of invertase. Glucose is taken into the cell and converted to the three-
carbon acid, pyruvate, via the glycolytic pathway in the cytosol. One
molecule of pyruvate is decarboxylated through the formation of
acetyl-CoA by the mitochondrial pyruvate dehydrogenase complex,
and another is carboxylated to oxaloacetate in the cytosol by pyruvate
carboxylase. Oxaloacetate must be transported into the mitochondri-
on (via malate) and condensed with acetyl-CoA to form citrate
68 F. Amiri et al. / Hydrometallurgy 109 (2011) 65–71

(Grewal and Kalra, 1995). Oxalate biosynthesis is exclusively due to decreased to 2.88. This decrease in pH caused the gluconic acid
the action of oxaloacetase, which catalyzes the hydrolysis of production to cease and the citric acid production to increase.
oxaloacetate to oxalate and acetate. Gluconic acid is produced
extracellularly. In a two-step reaction, glucose in the medium is
3.4. Influence of pulp density on organic acid production
oxidized to gluconic acid through the action of glucose oxidase
(Ruijter et al., 2002).
The fungal bioleaching processes, mainly acidolysis and complex-
Prior to the bioleaching experiments, pure cultures of adapted
olysis, are mediated through the secondary metabolites produced by
Aspergillus niger were incubated under conditions identical to
the fungus. Thus, it is important to investigate these metabolites to
bioleaching. The cultures were investigated over the course of
better understand the bioleaching mechanisms. The organic acids
30 days to determine the optimum time for addition of catalyst in
secreted by the fungus A. niger were analyzed in the absence and
two-step bioleaching and filtration of the culture for spent medium
presence of the spent catalyst and are listed in Table 2.
bioleaching. The increase in acid and biomass concentration and the
As shown in Table 2, the main agent in one-step and two-step
substantial hydrolysis of sucrose on the second day of incubation
bioleaching was gluconic acid. Gluconic acid was produced at all pulp
(Fig. 2) indicated that Aspergillus niger had entered the active growth
densities in one-step bioleaching and at pulp densities of more than
phase; the same result was also reported by Aung and Ting (2005).
1% (w/v) in two-step bioleaching. In spent medium leaching,
Thus, the spent catalyst was added to the culture for bioleaching after
however, the main lixiviant was citric acid. The concentration of
2 days of incubation (under the two-step process). We found that
gluconic acid produced during fungal leaching (i.e., in the presence of
10 days of incubation marked the end of the active growth phase. At
spent catalyst) was higher than in the absence of the spent catalyst.
this point, the citric acid concentration reached its maximum
The concentration of oxalic acid was found to be very low compared to
concentration of 691 mg/L. Accordingly, the spent medium was
gluconic and citric acid; this was also reported by Wu and Ting (2006).
obtained by filtering the culture after this period. This result is
The results show that the presence of spent catalyst induces gluconic
different from that reported by Aung and Ting (2005), in which
acid production by A. niger. The lack of citric acid production in the
14 days of incubation marked the end of the active growth phase.
presence of spent catalyst is presumably due to the presence of metal
Within the first 2 days of incubation, the main organic acid
ions, such as Mn and Fe, in the spent catalyst (Grewal and Kalra,
produced was gluconic acid; after 5 days of incubation, citric acid was
1995). Among these metal ions, Mn is reported to strongly inhibit
the main product (Fig. 2b). It has been reported that glucose oxidase is
citric acid accumulation by stimulating the enzymes in the TCA cycle
most efficient at a pH of 4.5–6.5 and is inactivated at pH levels below 3
(Burgstaller and Schinner, 1993; Wu and Ting, 2006).
(Ruijter et al., 2002; Wu and Ting, 2006). However, citric acid
fermentation by A. niger commences when pH levels decrease to
below 3 and continues through pH 2 (Grewal and Kalra, 1995). 3.5. Influence of pulp density on metal recovery and fungal growth
Therefore, when incubation began at pH 5.94, gluconic acid
production was induced, but after the 5th day of incubation, the pH Pulp density was an important variable for the outcome of the
various bioleaching methods investigated, especially one-step and
spent medium bioleaching. The optimum pulp density for spent
catalyst bioleaching occurred at 3% (w/v) for the one-step process and
a 35 7
at 1% (w/v) for the spent medium leaching. In the two-step
bioleaching method, the effect of pulp density on metal recovery
Fungus dry weight (g/L)

30 6 was not significant. The optimal pulp density in the one-step

25 5 bioleaching method was greater than reported in previous studies
(Aung and Ting, 2005; Wu and Ting, 2006; Amiri et al., 2011). There
20 4
was a direct relationship between the percentage of metal recovery
pH

15 3 and the fungal growth (Fig. 3a and b). Higher fungal growth led to
10 2 higher bio-metabolite production and, thus, resulted in higher metal
5 1
recovery. At pulp densities higher than the optimum in the one-step
process, the metal leaching yield decreased (Fig. 3a). This decrease
0 0
0 5 10 15 20 25 30 was due to the high toxic metal concentration in the medium (Xu and
Time (day) Ting (2004)) as well as the decrease in the initial pH of the spent
catalyst suspensions, which may inhibit fungal growth and organic
Fungus dry weight pH
acid production. As mentioned above, the change in metal recovery as
a function of pulp density was not significant in the two-step
b 120
Organic acid concentration (mg/L)

800 bioleaching. This is may be due to the considerable increase in

Sugar concentration (g/L)

700
100
600
80
500
60 400 Table 2
Organic acid concentrations resulting from different methods of bioleaching.
300
40
200 Organic acids One-Step(a) Two-Step(a) Spent
20 (mg/L) medium(b)
100 Spent catalyst pulp density Spent catalyst pulp density
(%w/v) (%w/v)
0 0
0 5 10 15 20 25 30 1 2 3 4 5 1 2 3 4 5
Time (day) Oxalic acid b1 15 19 13 13 b 1 2 1 b1 16 2
Fructose Glucose Sucrose Citric acid Gluconic acid Citric acid b1 b1 b 1 b 1 b 1 22 b1 b1 b1 b1 691
Gluconic acid 111 584 808 539 609 b 1 26 57 162 956 61
(a)
Fig. 2. (a) Concentration of biomass and pH changes, and (b) concentration of organic After 30 days of incubation in the presence of spent catalyst.
(b)
acids and sugars in pure culture of Aspergillus niger during 30 days of incubation. After 10 days of incubation in the absence of spent catalyst.
 F. Amiri et al. / Hydrometallurgy 109 (2011) 65–71 69

a 100 30

Fungus dry weight (g/L)

Metal recovery (%)

80 25

60 20

40 15

20 10

0 5
1% 2% 3% 4% 5%
Pulp density
W Al Fe Mo Ni Fungus dry weight

b 80 28

70

Fungus dry weight (g/L)

Metal recovery (%)

60 26

50

40 24

30

20 22

0 20
1% 2% 3% 4% 5%
Pulp density
W Al Fe Mo Ni Fungus dry weight

c 100
Metal recovery (%)

80

60

40 Fig. 4. SEM photomicrograph of spent and bioleached catalysts: (a) spent catalyst
(5000× magnification), and (b) bioleached catalyst (5000× magnification).
20

0
1% 2% 3% 4% 5% 3.6. Comparison of bioleaching and chemical leaching methods
Pulp density
W Al Fe Mo Ni
As shown in Fig. 5, the most favorable results for all metals were
obtained in the one-step bioleaching at a pulp density of 3% (w/v)
Fig. 3. Metal recovery at different pulp densities in the a) one-step method, b) two-step
method, and c) spent medium method.

100
90
80
gluconic acid secretion that occurred when the spent catalyst pulp
Metal recovery (%)

70
density was increased (Table 2).
In the case of spent medium leaching, the decrease in leaching 60
yield with an increase in pulp density was likely due to the constant 50
metabolite concentration at all pulp densities (Fig. 3c). This result was 40
consistent with that reported by Aung and Ting (2005). With the
30
exception of the case of 3% (w/v) pulp density in the one-step
bioleaching process, the highest efficiencies for W and Mo were 20
obtained in the spent medium leaching process. The leaching 10
efficiencies were 70.1–93.6% for W and 68.0–85.0% for Mo. Very 0
similar results were achieved in our previous study by Penicillium W Al Fe Mo Ni
simplicissimum (Amiri et al., 2011). One-step bioleaching Two-step bioleaching
The SEM photomicrograph of the spent (Fig. 4a) and bioleached Spent-medium bioleaching Chemical leaching
catalyst (Fig. 4b) revealed an absence of fine particles and deposits on
the surface of the bioleached catalyst. This may be due to dilution Fig. 5. Metal recovery at optimum pulp density in different bioleaching and leaching
under the effect of bioleaching. methods.
70 F. Amiri et al. / Hydrometallurgy 109 (2011) 65–71

which the results were 100% of W, 77.8% of Fe, 90.9% of Mo, 65.8% of a
Ni, and 14.2% of Al. 4.8
Higher Al recovery was achieved in our previous study using 4.6
Penicillium simplicissimum (7.6–25.1%) compared to the present study
4.4
(5.71–14.23%). This reduced recovery may have been due to the
4.2

pH
production of red pigment by Penicillium simplicissimum. This
compound has a hemiquinonoid structure and resembles aluminum 4
complexants (Rezza and Salinas, 2001; Amiri et al., 2011).
3.8
At optimum pulp density, the recovery yields for one-step, two-
step and spent medium bioleaching, respectively, are shown below. 3.6
The yields for the one-step and spent medium bioleaching methods 3.4
were quite similar but were very different from the two-step 0 5 10 15 20 25 30
bioleaching method. Time (day)
pH 1% pH 3% pH 5%
WNMoNFeNNiNAl ð1Þ

b 6.5
FeNNiNW≈MoNAl ð2Þ 6
5.5
WNMoNFeNNiNAl: ð3Þ 5

pH
In control experiments (data not shown) using fresh medium 4.5
(sucrose medium), the recovery yield of Al and Ni was negligible at all
4
pulp densities. Depending on the pulp density, the fresh medium
effected an extraction of 8.0–13.4% W, 6.5–11.9% Mo, and 14.5–19.5% Fe. 3.5
The behavior of Fe leaching was different from that of W and Mo. We
3
observed that an increase in the pulp density (from 1 to 5% (w/v)) and a 0 5 10 15 20 25 30
corresponding decrease in pH (from 4.82 to 4.20) led to a reduction of the Time (day)
extraction yield of Fe and an increase in the extraction yield of W and Mo.
pH 1% pH 3% pH 5%
These results for Fe were consistent with those of Wu and Ting (2006).
Fig. 5 shows the results of chemical leaching of 1% (w/v) spent
catalyst using a mixture of organic acids at the same concentration as c
those produced by A. niger for spent medium leaching (691 mg/L citric 3.4
acid, 61 mg/L gluconic acid and 2 mg/L oxalic acid). It was found that
similar extraction yield was achieved for Ni in both spent medium and 3.2
chemical leaching. This result demonstrates that citric acid is the main
agents responsible for Ni leaching in spent medium. In the case of
pH

other metals, spent medium leaching resulted in significantly higher 3

yields than chemical leaching; 93.6% W, 12.5 Al, 75.1% Fe, and 85.0%
Mo were extracted in spent medium leaching, and only 28.2% W, 2.8%
Al, 15.3% Fe, and 45.4% Mo were extracted using chemical leaching.
This difference shows that metabolites other than the measured
organic acids must be involved in metal leaching.
3.7. Influence of pulp density on pH changes
Fig. 6 shows the changes in pH during bioleaching by A. niger at
various pulp densities (1, 3 and 5% (w/v)) in (a) one-step, (2) two-
step, and (c) spent medium processes. There are many factors that
affect pH, such as the kind and concentration of produced metabolites
and the amounts of anions and cations in the medium (ionic strength).
In one-step bioleaching (Fig. 6a), the initial pH (4.82) of the suspension
with 1% (w/v) spent catalyst gradually decreased to pH 3.54 after
20 days of incubation, and thereafter it remained relatively constant
for up to about 30 days. At 3% and 5% (w/v), pH increased after 5 days
to 4.75 and 4.58, respectively. This increase in pH at 5% (w/v)
continued for up to 10 days of incubation. The initial pH increase
occurred simultaneously with initiation of sucrose hydrolysis, which
implies the beginning of fungal growth. The pH increase for 1% (w/v)
likely occurred within the 5 days of incubation. Thus, the pH results
from one-step bioleaching showed higher lag times with increasing
pulp density. The sudden decrease in pH to 4.08 and 4.12 at pulp
densities of 3 and 5% (w/v), respectively, after 5 and 10 days could be
due to the initiation of organic acid production (Brandl et al., 2001;
Ruijter et al., 2002). In the two-step bioleaching process (Fig. 6b), the
2.8
2.6
0 5 10 15 20
Time (day)
pH 1% pH 3% pH 5%
Fig. 6. Change in pH during different methods of bioleaching: (a) one-step, (b) two-
step, and (c) spent medium.
pH decreased to 3.92 after two days, and the spent catalyst was then
added to the medium. The introduction of the spent catalyst to the
medium caused a further decrease in the pH to values of 3.6, 3.52, and
3.4 at pulp densities of 1, 3, and 5% (w/v), respectively. The rate of pH
reduction decreased with increasing spent catalyst content. This result
occurred due to the higher buffering capacity of suspension with
higher solid content; such solutions required more time and acid to
lower the pH. Similar results have been reported by Chen and Lin
(2010). In the one-step and the two-step bioleaching process, the pH
of the medium remained above 3 (Fig. 6a, b). Either the pH of the
bioleaching medium or the presence of Fe and Mn may have caused the
negligible amount of citric acid production (Table 2). The pH of the
spent medium (Fig. 6c), which reached a value of 3.1 after 10 days of
incubation, decreased with the addition of spent catalyst to 3, 2.95, and
 F. Amiri et al. / Hydrometallurgy 109 (2011) 65–71
2.92 at pulp densities of 1, 3, and 5% (w/v), respectively. Following a
gradual increase in pH after 11 days of leaching, the pH remained
constant up to 20 days. The changes in the pH of the spent medium
during leaching were minimal compared to those in the one-step and
two-step bioleaching; Wu and Ting (2006) have reported a similar
observation. This lack of change was the due to the fact that there was
no producing agent in the medium, and the metabolites concentra-
tions were constant.
4. Conclusion
This work has shown that heavy metals from spent industrial
catalyst may be mobilized by different methods of bioleaching with
Aspergillus niger. It is the first time that data on tungsten bioleaching
by Aspergillus niger strain have been presented. The presence of spent
catalyst induced gluconic acid production, while in its absence, citric
acid was mainly produced. Spent medium leaching was effective for
heavy metal leaching and especially for W and Mo. However, one-step
bioleaching at a pulp density of 3% (w/v) produced the highest metal
recovery. Under these optimal conditions, the following amounts of
metals were leached: 100% of W, 77.8% of Fe, 90.9% of Mo, 65.8% of
Ni, and 14.2% of Al. Chemical leaching of 1% (w/v) spent catalyst was
performed using a mixture of organic acids in the same concentra-
tion as those produced by A. niger for spent medium leaching. This
chemical leaching resulted in lower extraction yields for W, Mo, Fe,
and Al and the same yield for Ni. These results indicate the importance
of citric acid in Ni recovery and the involvement of biometabolites
other than the measured organic acids in the extraction of W, Mo, Fe,
and Al. Achieving the complete bioleaching of W by A. niger and, in
our previous work, by Penicillium simplicissimum, shows the high
potential of fungal leaching in recovery of this heavy metal.
Although bioleaching technology offers many advantages over
other conventional methods due to its relative simplicity, mild
operating condition requirements, low energy input, reduced skilled
labor requirements, and environmental friendliness, it requires a
longer period of operation compared to other methods, such as
chemical leaching. These results suggest that optimizing the bioleach-
ing method using Aspergillus niger could facilitate the creation of an
alternative to conventional waste treatment methods. Such a method
could be used to obtain higher efficiencies and rates of metal recovery,
while minimizing process costs by using cheaper substrate sources.
Acknowledgments
The authors thank the National Iranian Oil Refining & Distribution
Company (NIORDC) for financial support (contract No. 88-1098). We
give special thanks to Eng. Zahra Ghabadinejad for helpful discussion.
71
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