Bioresource Technology 102 (2011) 1567–1573

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Bioleaching of tungsten-rich spent hydrocracking catalyst using

Penicillium simplicissimum
F. Amiri a, S. Yaghmaei a,⇑, S.M. Mousavi b,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
Department of Chemical Engineering, Lappeenranta University of Technology, Lappeenranta, Finland

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

Article history: Adaptation of Penicillium simplicissimum with different heavy metals present in a spent hydrocracking
Received 6 June 2010 catalyst, as well as one-step, two-step, and spent medium bioleaching of the spent catalyst by the
Received in revised form 18 August 2010 adapted fungus, was examined in batch cultures. Adaptation experiments with the single metal ions
Accepted 20 August 2010
Ni, Mo, Fe, and W showed that the fungus could tolerate up to 1500 mg/L Ni, 8000 mg/L Mo, 3000 mg/
Available online 21 September 2010
L Fe, and 8000 mg/L W. In the presence of multi-metals, the fungus was able to tolerate up to 300 mg/
L Ni, 200 mg/L Mo, 150 mg/L Fe and 2500 mg/L W. A total of 3% (w/v) spent catalyst generally gave
Keywords:
the maximum extraction yields in the two-step bioleaching process (100% of W, 100% of Fe, 92.7% of
Spent catalyst
Bioleaching
Mo, 66.43% of Ni, and 25% of Al). The main lixiviant in the bioleaching was shown to be gluconic acid.
Tungsten The red pigment produced by the fungus could also possibly act as an agent in Al leaching.
Penicillium simplicissimum Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Large quantities of catalysts are used in the refining industry for
purifying and upgrading various petroleum streams and residues
(Marafi and Stanislaus, 2003). The quantity of spent hydroprocess-
ing catalysts that is discarded as solid waste has increased remark-
ably in recent years due to a steady increase in the processing of
heavier feedstock in the petroleum refining industries (Marafi
and Stanislaus, 2008). Due to their toxic nature, spent hydropro-
cessing catalysts have been branded as hazardous wastes, and
refiners are experiencing pressure from environmental authorities
to handle them safely (Marafi and Stanislaus, 2003, 2008; Mishra
et al., 2007, 2008; Aung and Ting, 2005; Santhiya and Ting, 2005,
2006).
In Iran, spent catalysts, after a few cycles of regeneration and re-
use, are managed via: (i) selling spent catalysts containing precious
metals such as platinum and rhenium (spent reforming catalysts)
to foreign companies for recovery, (ii) using spent FCC catalysts
in cement production, and (iii) landfilling for ultimate disposal
(substantially hydroprocessing catalysts).
Spent hydroprocessing catalysts contain various hazardous com-
ponents, such as W, Mo, Ni, V, Co, Al, and some organic contaminants
⇑ Corresponding author. Tel.: +98 21 66166430.
⇑⇑ Corresponding author. Address: Department of Chemical Engineering, Tarbiat
Modares University, Jalal al Ahmad High Way, Tehran, Iran. Tel.: +98 21 82884917;
fax: +98 21 82884931.
E-mail addresses: yaghmaei@sharif.edu (S. Yaghmaei), mousavi_m@modares.
ac.ir (S.M. Mousavi).
(Marafi and Stanislaus, 2008; Santhiya and Ting, 2006). Due to strict
environmental regulations and economical conditions, proper
recovery of valuable metals from the catalysts has become essential
(Marafi and Stanislaus, 2003, 2008; Mishra et al., 2007, 2008; Aung
and Ting, 2005; Santhiya and Ting, 2005, 2006). The bioleaching of
industrial waste materials, such as municipal solid waste, incinera-
tor fly ash, spent catalysts, and electronic computer scraps, has been
considered more economical and environmentally friendly than the
traditional metal extraction methods that have high energy costs
and produce environmental pollution (Mishra et al., 2007, 2008;
Aung and Ting, 2005; Santhiya and Ting, 2005, 2006).
Microorganisms have been shown to possess an ability to sur-
vive by adaptation or mutation to high concentrations of toxic hea-
vy metals (Liu et al., 2008; Santhiya and Ting, 2006; Le et al., 2006;
Tang et al., 2006; Valix and Loon, 2003). Some researchers have
adapted such an approach in order to obtain heavy metals-tolerant
fungus strains, including Penicillium funiculosum, Aspergillus
foetidus and Penicillium simplicissimum, for the bioleaching of nickel
laterite ores (Le et al., 2006; Tang et al., 2006; Valix and Loon,
2003). The adaptation of Aspergillus niger that was exposed to the
single metal ions Ni, Mo, and Al and a mixture of these metal ions
was examined in order to increase the tolerance of this fungus to a
spent refinery processing catalyst (Santhiya and Ting, 2006).
Comparative studies of one-step and two-step bioleaching, as
well as bioleaching by spent microbial culture, were reported by
different researchers, and the feasibility of the latter was demon-
strated. For industrial application, bioleaching by spent culture fil-
trate is believed to be appropriate for increasing leaching efficiency
(Mishra et al., 2008; Aung and Ting, 2005; Wu and Ting, 2006).

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.08.087
1568 F. Amiri et al. / Bioresource Technology 102 (2011) 1567–1573

There is no data available on the fungal leaching of tungsten Table 1

from spent catalysts. In this investigation, the fungus P. simpliciss- Elemental composition of spent hydrocracking catalyst.

imum was initially adapted to W, Mo, Fe, and Ni singly (at 500– Element Digestion (mg/kg) XRF (mg/kg)
8000 mg/L) and to a mixture of these metals (as present in the Ag 40 nt
spent catalyst), and the adaptation was then continued with tung- Si nt 109,700
sten-rich spent hydrocracking catalyst (0.5–5 g/100 mL). In the Al 210,000 129,700
next stage, bioleaching of the spent catalyst using one-step and Fe 6000 14,400
Ca <100 1900
two-step processes, as well as leaching by the spent medium at Na 950 100
various pulp densities, was investigated. The physical and chemical Mg 70 100
characterization of the spent catalyst was initially carried out be- K 130 400
fore the spent catalyst was bioleached using Penicillium simpliciss- Ti nt 450
Mn 30 10
imum and its spent medium. The different constituents of the spent
Co <20 <10
medium were analyzed in the absence and presence of the spent Cr 100 120
catalyst at various pulp densities. Cu 45 <10
Ni 8000 14,100
Pb 90 <10
2. Methods V 25 500
W 77,000 268,800
2.1. Source and description of spent catalyst Zn 130 <10
Mo 6800 15,500
Hg <50 nt
Spent hydrocracking catalyst (Criterion HC-102 W/Ni/Al2O3/SiO2) Ga <20 nt
was provided by the National Iranian Oil Refining & Distribution
Company (NIORDC), and this material was the basis of all samples nt, not tested.

used in the experiments. The spent catalyst was extrudate tri-lobe

with a black covering. The spent catalyst was pre-treated by heat-
ing in a furnace at 600 °C for 4 h, during which decoking resulted
in turning the spent catalyst color to yellow. The decoked spent
catalyst was gently dry-ground and sieved. All spent catalysts used
in this study were of a particle size less than 150 lm.
2.2. Microorganism and chemicals
Penicillium simplicissimum BBRC-20019 provided by the Bio-
chemical and Bioenvironmental Research Center (BBRC) in Sharif
University of Technology (Iran), was used in this study. All chemi-
cals were analytical grade reagents (AR) and all aqueous solutions
were prepared using distilled water.
2.3. Characterization of spent refinery hydroprocessing catalyst
2.3.1. Catalyst composition
The elemental composition of the spent catalyst was determined
using chemical analysis and fluorescence (XRF) (Philips Magix Pro)
(Table 1). X-ray diffraction (XRD) (PHILIPS Xpert Pro) was also used
to determine the crystalline phases of the spent catalyst. XRD anal-
ysis showed that lithium aluminum tungsten oxide (LiAlW2O4),
nickel molybdenum oxide (NiMoO4), tungsten oxide (WO3), and sil-
icon oxide (SiO2) are the major components in the spent catalyst.
2.3.1.1. Chemical analysis. Partial chemical composition was deter-
mined by a homemade method; 0.02 to 0.05 g of sample was di-
gested for 2 h using 10 mL of concentrated HNO3 and 10 mL of
concentrated HF and then heated for about 2 h up to 120 °C. The
sample was almost completely soluble. The digestate was cooled, fil-
tered and brought to a volume of 100 mL using deionized water and
subjected to chemical analysis using an inductively coupled plasma-
atomic emission spectrometer (ICP-AES) (Varian LIBERTY – RL).
2.4. Acclimatization of Penicillium simplicissimum
2.4.1. Solid media preparation and acclimatization of fungus with
metal ions
Adaptation was conducted through a series of repeated sub-cul-
turing of the fungi exposed to heavy metals including W, Mo, Ni,
and Fe in plate tests singly as well as together. Penicillium simpli-
cissimum was grown in a Czapek yeast extract agar media that con-
sisted of 1 L distilled water, 1.0 g/L K2HPO4, 3.0 g/L NaNO3, 0.5 g/L
MgSO47H2O, 0.5 g/L KCl, 0.01 g/L FeSO47H2O, 30.0 g/L sucrose,
5.0 g/L yeast extract, 15.0 g/L agar, and trace elements. The concen-
tration of the single heavy metals (W, Mo, Fe, and Ni) in the culture
medium was increased from 500 to 8000 mg/L in steps of 500 mg/
L. In the W: Mo: Ni: Fe adaptation, various concentrations of the
metals to simulate the full leaching of spent catalyst at different
pulp densities (0.5–3% w/v) were calculated from the digestion
data in Table 1 and used in experiments. At each step, the desired
metal concentrations (Na2WO42H2O, FeSO47H2O, NiSO46H2O,
and Na2MoO42H2O) were added to the CYEA growth media prior
to sterilization of the solution at 121 °C for 15 min. After steriliza-
tion, the medium was allowed to cool down to around 60 °C and
poured into a 12-cm diameter petri dish for culture growth. The
plates were inoculated by placing a 3 mm circular extract of the
growing fungus onto the plates. The plates were incubated at
30 °C to establish their growth. A culture in the absence of the me-
tal elements was also conducted as a control.
The growth was monitored by measuring and averaging the
spread of the culture from the point of inoculation or the center
of the colony to the end of the longest and smallest hyphae.
The tolerance index, an indication of the organismal response to
metal stress, was calculated from the growth of the strain
exposed to the metals divided by the growth in the control plate.
The growth of the fungal strain was mapped according to proce-
dures outlined by Valix et al. (2001). Fungi subjected to abiotic
factors responded with a growth pattern characterized by five
stages: (a) lag phase which occurs at the beginning of the inocu-
lation, where very little growth occurs; (b) rapid growth rate, but
the absolute growth is often suppressed relative to the control;
(c) decline in the growth rate; (d) leveling of the tolerance index
indicating that the rate of growth of the fungi with metals and
the control are similar; and finally (e) the second growth phase
in which the absolute growth rate often exceeds that of the
control (a tolerance index greater than 1). Strains demonstrating
growth at phase ‘‘e’’ were sampled and further exposed to higher
metal concentrations.
2.4.2. Acclimatization of fungus with spent catalyst
This stage is necessary because some other elements, such as
Mn, Ti, Cr, Pb, Hg, and Cl that exist in spent catalysts, may have
inhibitory effects on fungus growth. Also, adaptation in the
 F. Amiri et al. / Bioresource Technology 102 (2011) 1567–1573
bioleaching medium before major experiments is supposed to be
helpful in increasing the efficiency of bioleaching.
The acclimatized fungus from the final stage of the adaptation
with the mixture of metal ions was cultured on 3.9% (w/v) PDA
(potato dextrose agar) slant. In order to obtain a sufficient number
of spores, the culture was incubated at 30 °C for five days. The ma-
ture conidia were then washed off of the surface of the PDA med-
ium using sterilized physiologic saline (9 g/L NaCl). The number of
spores was counted using a Neubauer counting chamber and
adjusted using sterilized physiologic saline to approximately 107
spores/mL. Two milliliters of the spore suspension was added to a
500 mL Erlenmeyer flask containing 0.5% (w/v) of the spent catalyst
and 100 mL of sucrose medium with the following composition: su-
crose (100 g/L), NaNO3 (1.5 g/L), KH2PO4 (0.5 g/L), MgSO47H2O
(0.025 g/L), KCl (0.025 g/L), and yeast extract (1.6 g/L). The flasks were
agitated in an incubator with orbital shaking at 120 rpm and at 30 °C.
Acclimatization of the fungus with the spent catalyst was conducted
by increasing the spent catalyst concentration from 0.5% (w/v) until
no fungal growth occurred in steps of 0.5% (w/v). A 10% (v/v) inocu-
lation was carried out in each step, which was obtained from the pre-
vious culture.
2.5. Different methods of bioleaching
In order to obtain sufficient numbers of spores for the bioleach-
ing experiments, the acclimatized fungus from the final stage of
adaptation with the spent catalyst was cultured as described in
Section 2.4.2. A sterile experimental set-up was achieved by auto-
claving at 121 °C for 15 min prior to inoculation. Bioleaching was
performed in 500 mL Erlenmeyer flasks with 100 mL of sucrose
medium containing the spent catalyst at various pulp densities
(1, 2, 3, 4, and 5% w/v). All of the experiments were carried out
in an orbital shaking incubator at 30 ± 1 °C and 120 rpm. Three dif-
ferent methods of bioleaching were carried out: (i) the fungus was
incubated together with the medium and the spent catalyst (one-
step bioleaching), (ii) the fungus was first cultured in sucrose med-
ium without spent catalyst for four days, and after a sudden reduc-
tion in pH (the beginning of organic acid production), the sterilized
catalyst was added (two-step bioleaching), and (iii) the fungus was
first cultured in sucrose medium for 14 days. The suspensions were
then filtered through MN 640d and 0.2 lm (Millipore) filter paper
to obtain the cell-free spent medium, and the filtrate that con-
tained bio-produced metabolites was used for the leaching of spent
sterilized catalyst added to the filtrate (spent medium leaching).
The control experiments were conducted using fresh sucrose
medium.
2.6. Analytical methods
After the desired bioleaching time, the culture from each flask
was filtered, and the filtrate was analyzed for organic acids (i.e., cit-
ric, oxalic, and gluconic acids) using high performance liquid chro-
matography (HPLC) (WATERS, model: ALLINACE 2695), with a
variable wavelength detector (VWD) at 210 nm for the organic
acids and a refractive index detector (RID) for the sugars.
The filtrate was also analyzed for 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 calibration.
Additionally, the pH of the leached liquor was measured using a
digital pH meter (Metrohm, model: 827 pH lab). The residue (i.e.,
the fungal biomass with the bioleached catalyst) obtained from
the filter paper was carefully transferred to a pre-weighed evapo-
rating dish and was dried at 80 °C for 24 h. The dried residue was
ashed at 500 °C for 4 h to determine the dry weight of the biomass.
1569
In order to extract and measure the biomass-accumulated and
associated metals, the method described by Santhiya and Ting
(2006) was used.
A scanning electron microscope was used to observe the mor-
phology of the catalysts. The spent and bioleached catalysts were
used for the SEM examination. The spent and bioleached catalysts
were mounted with silver paste on aluminum stubs, then coated
with 300–400 Å Au in a sputtering unit and finally examined in a
scanning microscope. Image analysis was conducted under an
accelerating voltage of 10 kV and under high vacuum.
3. Results and discussion
3.1. Composition analysis of spent hydrocracking catalyst
Elemental composition of the spent hydrocracking catalyst, as
analyzed using chemical digestion and XRF, is shown in Table 1.
The most abundant elements (>50,000 mg/kg) include Al, W, and
Si. Most of the minor constituents (1000–50,000 mg/kg) were hea-
vy 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
(<1000 mg/kg).
3.2. Effect of heavy metal concentration on Penicillium simplicissimum
tolerance development
The effect of sub-culturing Penicillium simplicissimum in increas-
ing concentrations of W, Mo, Ni, and Fe is shown in Table 2. After
developing tolerance at 500 mg/L, Penicillium simplicissimum was
shown to grow more vigorously, particularly at higher concentra-
tions of heavy metals. As seen in Table 2, increasing the metal con-
centration resulted in increasing of the tolerance index at phase d
(the similar growth phase) and enhanced growth rate at phase e as
well as decreasing of the lag phase in phase a. There is an exception
about Ni, with which the Penicillium simplicissimum lag phase in-
creased from one to three days with increasing metal concentra-
tions. The stimulating effect of the higher metal concentration on
the tolerance development of the fungus is unclear, although it ap-
pears to be related to their initial adaptive behaviors (growth rate
at phase b and death rate at phase c). According to Table 2, a higher
growth rate at phase b and a lower retarded rate at phase c have a
positive effect on the overall tolerance achieved at phase d. This
was confirmed from previous studies by Valix et al. (2001) and
Tang et al. (2006). There is no growth with Ni and Fe concentra-
tions higher than 1500 and 3000 mg/L, respectively. This pattern
shows the great toxic effect of high concentrations of Ni and Fe.
Heavy metals are able to exert harmful effects due to their strong
coordinating capabilities. These toxic effects include blocking of
biologically important functional groups and the denaturation of
enzymes (Valix and Loon, 2003). According to Table 2, the order
of the toxicity of the metals is as follows:
Ni > Fe > W > Mo
Le et al. (2006) reported that the presence of multi-metals im-
posed a greater toxicity to the fungi growth than single metals.
Tang et al. (2006) also found that the growth behavior of Aspergillus
foetidus adapted to tolerate Ni, Co, Fe, Mg, and Mn concentrations
up to 1000 mg/L becomes sluggish when exposed to a 1095 mg/L
multi-metals mixture in which the single heavy metal concentra-
tions are much less than 1000 mg/L. Yang et al. (2009) also con-
firmed the synergistic toxicity of multi-metals to Aspergillus niger
trained for the bioleaching of fly ash. Therefore, it was necessary
to train Penicillium simplicissimum to tolerate multi-metals. The
growth pattern of the Penicillium simplicissimum strain in the pres-
ence of various multi-metal concentrations is shown in Fig. 1. The
1570 F. Amiri et al. / Bioresource Technology 102 (2011) 1567–1573

Table 2
Effect of heavy metal concentration on the growth characteristics of Penicillium simplicissimum.

Metal concentration Penicillium simplicissimum

(mg/L)
Phase a Phase b Phase c Phase d Phase e
lag days rapid growth rate retarded growth rate similar growth enhanced growth rate
Nickel
500 1 0.16 0.02 0.07 0.09
1000 2 0.23 0.01 0.16 0.2
1500 3 0.57 0.01 0.4 0.5
Molybdenum
500 1 0.54 0.01 0.78 0.5
1000 1 1.08 0.16 0.81 0.8
2000 1 1.86 0.08 0.85 0.9
3000 1 1.96 0.05 0.95 1
5000 1 2 0.02 1 1.2
Iron
500 2 0.135 0.01 0.15 0.11
1000 2 0.29 0.03 0.16 0.15
2000 1 0.3 0.02 0.28 0.18
3000 1 0.62 0.02 0.47 0.5
Tungsten
500 1 0.41 0.11 0.6 0.15
1000 1 0.51 0.07 0.63 0.3
2000 1 1.2 0.09 0.63 0.5
3000 1 1.6 0.09 0.8 0.7
5000 1 1.85 0.1 0.88 1
8000 1 2.34 0.14 0.95 1.2

results indicated that Penicillium simplicissimum tolerated multi-
metals present in up to in 3% (w/v) in the spent catalyst. As shown
in Fig. 1, raising the multi-metal concentration resulted in increas-
ing the lag time from one to three days and a reduction of the
phase b growth rate (from 0.94 to 0.05), which consequently re-
sulted in the tolerance index decreasing at phase d from 0.78 to
0.1. The tolerance index at phase d under multi-metal stresses
(0.1–0.78) was below that of adapted fungi in the presence of sin-
gle metals. This implies that multi-metals act in synergy to impose
a higher toxic response to the fungi growth behavior in comparison
The produced morphology of Penicillium simplicissimum in the
submerged culture was dispersed small pellets. Pelleted suspen-
sions of fungal cells are generally not viscous and usually only
deviate from Newtonian behavior at high biomass concentrations
(Gibbs et al., 2000). Fig. 2 shows the changes in pH, biomass,
a 25 6.5
Biomass Dry weight (g/L)

20 6
with single metals alone.

15 5.5
3.3. Penicillium simplicissimum pure culture investigation

pH
10 5
Prior to the bioleaching experiments, pure cultures of adapted
Penicillium simplicissimum were incubated under identical biole-
aching conditions and investigated over 30 days in order to deter- 5 4.5
mine the optimum time for the addition of catalyst in two-step
bioleaching and filtration of the culture in the spent medium 0 4
bioleaching. 0 10 20 30 40
Time (day)
1.2 Dry weight (g/L) pH

1 b 120 160
Gloconic acid con. (mg/L)
Suger Concentration (g/L)
Tolerance Index

140
0.8 100
120
0.6 80 100
60 80
0.4
60
40
0.2 40
20 20
0
0 2 4 6 8 10 12 14 0 0
Time (day) 0 10 20 30 40
0.50% 1%
Time (day)
2% 3% Fructose Glucose Sucrose Gluconic acid

Fig. 1. Tolerance index of Penicillium simplicissimum exposed to different multi- Fig. 2. (a) Concentration of biomass and pH changes, and (b) concentration of
metal concentrations simulating heavy metals present in different spent catalyst organic acids and sugars in pure culture of Penicillium simplicissimum, during
pulp densities (0.5–3% w/v). 30 days of incubation.
 F. Amiri et al. / Bioresource Technology 102 (2011) 1567–1573 1571

sugars, and organic acid concentrations during 30 days of incuba- a 6.5

tion. Sucrose was substantially hydrolyzed to glucose and fructose
through the action of invertase (Ruijter et al., 2002) within three 6
days of incubation (Fig. 2b), during which the pH increased from
5.94 to 6.23 (Fig. 2a). At about the fourth day, the sporulating 5.5
mycelia developed a green pigment. Following the reduction in

pH
glucose concentration and the increase in gluconic acid concentra-
5
tion on the fourth day (Fig. 2b), the pH decreased from 6.23 to 5.8.
The decrease in glucose concentration was accompanied by an in-
4.5
crease in fungal biomass; at the tenth day, the biomass and glu-
conic acid concentration reached their highest values at 20.39 g/L
4
and 1379 mg/L, respectively, and the pH reached its lowest value 0 10 20 30
at 4.8; otherwise, red water-soluble pigment was recognizable in time (day)
the medium that intensified day after day, along with the fructose 1% 2% 3% 4% 5%
consumption. At about the 14th day, the red color was completely
obvious in the medium. These red pigments are polyketide com- b 6.5

pounds that are the product of the polyketide pathway, the major
6
route for the formation of secondary metabolites in filamentous
fungi (Jiang et al., 2005). Following the red pigment production, 5.5
the biomass dry weight decreased. This may be due to the toxicity

pH
5
of secondary metabolites accumulating in the medium. The red
pigments produced are mainly in the cell-bound state, and the in- 4.5
tra-concentration of pigments is higher than that secreted into the
medium (Jiang et al., 2005). Therefore, the increase of the red pig- 4
ment concentration in the fungus death phase from the 20th to
3.5
30th day is probably due to biomass lysis, which causes the release
0 10 20 30
of the intracellular red pigment into the medium; meanwhile, the time (day)
increase of the pH from 5 to 6 confirmed the alkaline buffering nat- 1% 2% 3% 4% 5%
ure of these metabolites, as stated by Bhardwaj and Shukla (2007).
The increase in acid and biomass concentration and the com- c 5.1
plete hydrolysis of sucrose at the fourth day of incubation indi- 5
cated that Penicillium simplicissimum was in the active growth 4.9
phase. Thus, the spent catalyst was added to the culture for biole- 4.8
aching after four days of incubation (under the two-step process). 4.7
pH

After 14 days of incubation marked the end of the active growth 4.6
phase, the spent medium was obtained by filtering the culture. 4.5
4.4
3.4. Comparison of different methods of bioleaching 4.3
4.2
3.4.1. pH changes during bioleaching 4.1
0 5 10 15
There are different factors that affect pH, such as the produced
time (day)
metabolites and the amounts of anions and cations in the medium
1% 2% 3% 4% 5%
(ionic strength). Fig. 3 shows the changes in pH during (a) one-step
and (b) two-step bioleaching by Penicillium simplicissimum as well Fig. 3. Change in pH during different methods of bioleaching: (a) one-step, (b) two-
as (c) spent medium leaching by Penicillium simplicissimum metab- step, and (c) spent medium.
olites. In one-step bioleaching (Fig. 3a), the initial pH values of the
crease occurred after 12 days of incubation. The pH decreased to
suspension with 1%, 2%, 3%, 4%, and 5% (w/v) spent catalyst, which
5.48, 4.94, 4.53, 4.11, and 3.87 at pulp densities of 1, 2, 3, 4, and
were, respectively, 4.82, 4.65, 4.44, 4.25, and 4.25, suddenly in-
5% (w/v), respectively, after 30 days of incubation.
creased to values of 6.27, 5.83, 5.33, 5.03, and 4.97 after 2, 3, 4.5,
The pH of the spent medium (Fig. 3c), which reached 5.13 after
6, and 10 days of incubation, respectively. As described in Sec-
14 days of incubation, decreased with the addition of spent cata-
tion 3.3, the initial pH increase occurs simultaneously with sucrose
lyst to 5.02, 4.82, 4.62, 4.41, and 4.31 at pulp densities of 1, 2, 3,
hydrolysis, which implies the beginning of fungal growth; there-
4, and 5% (w/v), respectively. The changes in the pH of the spent
fore, the pH results of one-step bioleaching show higher lag times
medium during 15 days of leaching are slight compared to those
with increasing pulp density. The sudden decrease in pH to 4.25,
in one-step and two-step bioleaching. This is because there was
4.26, 4.41, 4.6, and 4.72 at pulp densities of 1, 2, 3, 4, and 5% (w/
no producing agent in the medium and the metabolites concentra-
v), respectively, after 4.5, 5.5, 7, 7, and 15 days, could be due to
tions were constant. At pulp densities of 1%, 2%, and 3% (w/v), the
the initiation of organic acid production (Brandl et al., 2001; Ruijter
pH of the spent medium decreased to 4.53, 4.31, and 4.25, respec-
et al., 2002). Increases in the pH after 15 days of incubation at pulp
tively, after five days. At about 4% and 5% (w/v), there was a pH in-
densities of 1, 2, 3, and 4% (w/v) and after 20 days at a pulp density
crease to 4.46 after two days before a pH decrease to 4.2 at the 5th
of 5% (w/v) occurred simultaneously by red pigment production.
day. The pH remained constant for about six days, from the 9th to
In the two-step bioleaching process (Fig. 3b), by decreasing in
15th of leaching.
the pH to 5.8 after four days, the spent catalyst added to the med-
ium. The spent catalyst introduced to the medium caused de-
creases in the pH to values of 5.65, 5.5, 5.35, 5.2, and 5.14 at 3.4.2. Metal ion recovery using different bioleaching methods
pulp densities of 1, 2, 3, 4, and 5% (w/v), respectively. Pulp density Fig. 4a–e shows the recovery yield of various metal ions under
increases resulted in greater pH changes. At 1% (w/v), a gradual in- different conditions and pulp densities. As is seen in this figure,
1572 F. Amiri et al. / Bioresource Technology 102 (2011) 1567–1573

a 100 b
90 100
80 90
% Metal Recovery

% Metal Recovery
70 80
70
60
60
50
50
40
40
30 30
20 20
10 10
0 0
W Al Fe Mo Ni W Al Fe Mo Ni
One-step bioleaching Two-step bioleaching One-step bioleaching Two-step bioleaching
Spent-medium bioleaching Fresh-medium leaching Spent-medium bioleaching Fresh-medium leaching

c 100 d 100
90
90
% Metal Recovery

80

% Metal Recovery
80
70 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
W Al Fe Mo Ni W Al Fe Mo Ni
One-step bioleaching Two-step bioleaching One-step bioleaching Two-step bioleaching
Spent-medium bioleaching Fresh-medium leaching Spent-medium bioleaching Fresh-medium leaching

e 100
90
% Metal Recovery

80
70
60
50
40
30
20
10
0
W Al Fe Mo Ni
One-step bioleaching Two-step bioleaching
Spent-medium bioleaching Fresh-medium leaching

Fig. 4. Recovery yields of various metals from the spent catalyst under different pulp densities and leaching conditions: (a) 1% (w/v), (b) 2% (w/v), (c) 3% (w/v), (d) 4% (w/v),
and (e) 5% (w/v).

the optimum pulp densities in one-step, two-step, and spent med- for W and Mo, except for the 3% (w/v) pulp density, belonged to
ium bioleaching are 1% (w/v), 3% (w/v), and 1% (w/v), respectively. spent medium leaching with a leaching efficiency of 65.13–
In two-step bioleaching at a 3% (w/v) pulp density, the highest me- 93.45% W and 60.77–83% Mo.
tal ion recoveries were achieved. In this condition, Penicillium sim- The recovery yield in descending order for the two-step and
plicissimum leached virtually all W and Fe, while the recovery spent medium bioleaching are shown below, respectively, and
yields of Al, Mo, and Ni were 25%, 92.7%, and 66.4%, respectively. are found to be slightly different.
In one-step and spent medium bioleaching, an increase in pulp
Fe > Ni  W  Mo > Al ð1Þ
density led to a decrease in the recovery yield, as observed by
Wu and Ting (2006). The decrease in metal recovery was higher W  Fe  Mo > Ni > Al ð2Þ
in the case of the one-step bioleaching, which is likely to be due In control experiments (using fresh medium), the recovery
to the inhibition of fungal growth at higher concentrations of toxic yields of Al and Ni were found to be negligible under all pulp den-
metals as well as a decrease in the initial pH of the spent catalyst sities. Depending on the pulp density, the fresh medium effected
suspension with an increase in the pulp density. Spent medium an extraction of 8–13.4% W, 6.5–11.9% Mo, and 14.5–19.5% Fe.
leaching consistently resulted in a higher metal leaching efficiency The behavior of Fe leaching was different from that of W and
than that of the one-step bioleaching for all metals except for Al. Mo; by increasing the pulp density and consequently decreasing
This is possibly due to the higher red pigment concentration in the pH (from 4.82 to 4.2), the extraction yield of Fe decreased in
one-step bioleaching compared with spent medium leaching. This good agreement with Wu and Ting (2006), while W and Mo extrac-
compound has a hemiquinonoid structure and resembles alumi- tion increased.
num complexants (Rezza et al., 2001); therefore, this compound The SEM photomicrograph of the spent catalyst revealed the
could possibly act as a leaching agent. Two-step bioleaching had rock shape of the ground spent and bioleached catalysts, with con-
the highest efficiencies for Fe and Ni at all pulp densities, with a siderable variation in particle size. In the case of the bioleached
leaching efficiency of approximately 69.84–100% Fe and 50– catalyst, however, a more homogeneous distribution of the dis-
70.2% Ni. In the two-step bioleaching at a 5% pulp density, the crete particles with no fine particles was seen. The absence of fine
bioleaching efficiency for W and Mo decreased significantly to particles and deposits in the bioleached catalyst is possibly due to
13.8% and 19%, respectively. Meanwhile, the highest efficiencies dilution under the effect of bioleaching.
 F. Amiri et al. / Bioresource Technology 102 (2011) 1567–1573
Table 3
Organic acid concentration in different methods of bioleaching.
Organic acids (mg/L) One-stepa
Spent catalyst pulp density (%w/v)
1% 2% 3% 4%
Oxalic acid <1 32 4 54
Citric acid 791 197 <1 <1
Gluconic acid <1 <1 235 660
a
After 30 days of incubation in the presence of spent catalyst.
b
After 14 days of incubation in the absence of spent catalyst.
3.4.3. Effect of spent catalyst on the production of organic acid
Metal leaching by heterotrophic microorganisms generally in-
volves an indirect process with the microbial production of organic
acids, amino acids, and other metabolites. These metabolites dis-
solve metals by displacement of metal ions from the solid matrix
by hydrogen ions or by the formation of soluble metal complexes
and chelates (Rezza et al., 2001). It was noted that bio-produced
organic acids were the most important leaching agent in the biole-
aching process using fungi (Yang et al., 2009). The organic acids se-
creted by the fungus Penicillium simplicissimum were analyzed in
the absence and presence of spent catalyst and are listed in Table 3.
As it is obvious, the main leaching agent in the bioleaching was
gluconic acid. The amount of organic acids produced depends on
many factors, including the buffering capacity of the medium,
the carbon source, the ratio of nitrogen and phosphate in medium,
and the experimental conditions for the fungal growth (Gadd and
Sayer, 2000). According to Table 3, in the absence of the spent cat-
alyst, the fungus secreted 1350 mg/L after 14 days of incubation;
neither citric acid nor oxalic acid was produced. Pulp densities of
1% and 2% (w/v) in the one-step and a pulp density of 1% (w/v)
in the two-step bioleaching induced secretion of citric acid, while
no gluconic acid was detected in these conditions. The lack of citric
acid production at the higher pulp density is presumably due to
increasing concentrations of metal ions such as Mn and Fe. Mn
strongly inhibits citric acid accumulation by stimulating the en-
zymes in the TCA cycle (Ruijter et al., 2002). The oxalic acid con-
centrations are negligible in the one-step and two-step
bioleaching processes compared to gluconic and citric acid, and
they remained relatively independent of the concentration of the
spent catalyst. It is noteworthy that a significant increase in the
concentration of gluconic acid occurred with an increase in
the spent catalyst pulp density in one-step bioleaching. The highest
gluconic acid concentration belongs to the 3% (w/v) pulp density
(the optimum pulp density) in two-step bioleaching. Because the
highest metal recovery also occurred in this condition, it can be
concluded that gluconic acid has a major role in spent catalyst
bioleaching by Penicillium simplicissimum. Reduction in gluconic
acid concentrations at higher pulp densities resulted in decreasing
in metal extraction yield, as described in Section 3.4.2.
4. Conclusion
The adaptation of Penicillium simplicissimum to the metal ions
showed an increase in the tolerance index of the fungi with
increasing concentrations of the metals, and the cumulative action
of multi-metals enhanced the toxic effect. The main agent in biole-
aching was gluconic acid; red pigments could also be responsible
for Al leaching. The spent medium leaching showed effective re-
sults in heavy metal leaching, especially for W and Mo. But the
most favorable results for all of the metals were obtained using
two-step bioleaching at the optimum pulp density (3% w/v). These
1573
Two-stepa Spent mediumb
Spent catalyst pulp density (%w/v)
5% 1% 2% 3% 4% 5%
36 <1 <1 <1 18 10 <1
<1 498 <1 <1 32 <1 <1
1192 <1 234 1381 885 116 1350
results suggest that optimized two-step bioleaching can be an
alternative to conventional treatment methods.
Acknowledgements
The authors thank the National Iranian Oil Refining & Distribu-
tion Company (NIORDC) for their financial support (contract No.
88-1098). Special thanks to Eng. Zahra Ghobadinejad for her kind
cooperation in the microbial work.
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