Resources, Conservation & Recycling 177 (2022) 105976

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Resources, Conservation & Recycling

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Simultaneous leaching of Cu, Al, and Ni from computer printed circuit

boards using Penicillium simplicissimum
Alireza Esmaeili 1, Mahdokht Arshadi 1, Professor of Chemical and Petroleum Engineering
Department Soheila Yaghmaei 1, *
1
Chemical and Petroleum Engineering Department, Sharif University of Technology, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: A short lifespan and increased consumption patterns make e-waste the world’s fastest-growing waste stream.
base metals Computers are one of the most significant parts of e-waste. Recycling of e-waste has been introduced as the main
e-waste solution to deal with environmental problems and to save natural mines. This research aims to investigate the
fungal leaching
bioleaching of Cu, Ni, and Al from computer printed circuit boards (CPCBs) using Penicillium simplicissimum. The
metabolites
mechanism
adaptation phase began at 1 g/l CPCBs powder with 107 spores and final pulp density was reached at 30 g/l. The
most effective parameters including pulp density, initial pH, and the sucrose concentration were optimized to
achieve maximum simultaneous leaching efficiency of Cu, Ni, and Al. The results showed the main mechanism of
Al and Ni leaching was acidolysis, while complexoysis was the main mechanism for leaching of Cu. The optimal
conditions of 60 g/l sucrose concentration, 16 g/l pulp density, and initial pH 6 led to leaching of 100% Cu, 70%
Ni, and 98% Al. Additionally, the initial CPCBs sample and the bioleaching residue were analyzed using XRD,
HPLC, SEM, and FTIR. The HPLC results confirmed that gluconic and oxalic acids were the main metabolites
produced by P. simplicissimum. The SEM micrograph revealed the effectiveness of the bioleaching process in
metal leaching. FTIR spectra validated conversion of the molecular structure to simpler materials, as well as the
presence of HIPS, PC, and PPO in CPCBs waste powder.

Introduction 15918625, estimated to reach 55199212 by 2022. 2678801 of electronic

equipment was discarded in 2013 in Iran and expected to reach
Discarded electrical and electronic instruments are regarded as 12567350 by 2022 (Mirgerami et al., 2018).
electronic waste (e-waste). A short lifespan and increased consumption E-waste contains different materials, including metals (such as toxic
patterns make e-waste the world’s fastest-growing waste stream. The metals, precious metals, and rare earth metals) and halogenated com­
annual generation of e-waste was 6.1 kg for every living person in 2016 pounds. It is categorized as hazardous waste and contains up to more
(Rienzie et al., 2019). United Nation University reported that only in than 1000 toxic substances (e.g. Pb, Cu, Co, and Cd) and resistant
2019, 53.6 million tons of electronic appliances were discarded, which is organic materials (e.g. dioxin, polychlorinated biphenyls, and bromi­
equal to 7.3 kg per inhabitant (Forti, 2020). nated flame retardants) that can threaten human society (Kiddee et al.,
It was reported 288000 tons of e-waste were generated in Iran in 2020). E-waste disposal in the landfill or incineration produce toxic
2012, which is corresponds to 5.37 kg per capita (Mirgerami et al., effect on the environment (Kumar et al., 2017). It is essential to develop
2018). However, this value is different for various cities in Iran. For the national e-waste policies and legislation worldwide to deal with
example, in Ahvaz, one of the economic centers of Iran, the total gen­ e-waste. 61 countries were covered by regulation, policy, or legislation
eration of e-waste was about 9952 tones and 9.95 kg per capita in 2011 in 2014, increased to 78 countries in 2019 (Forti et al., 2020). According
(Alavi et al., 2015). E-waste across Iran is estimated to be about 10 to the proposed general legislation from 2004 in Iran, all electronic
million tons annually, which was predicted to reach approximately importers or producer companies have to recycle their e-waste. Other­
361547 million tons by 2022 and 691225 million tons by 2050. The wise, 0.005% of their product value is assigned to a special fund for
number of electronic instruments purchased by Iranians in 2013 was e-waste recycling (Taghipour et al., 2012). E-waste is being recycled

* Corresponding Author. Tel: +98-21-66166430

E-mail addresses: [email protected] (M. Arshadi), [email protected] (P.C.P.E.D.S. Yaghmaei).

https://doi.org/10.1016/j.resconrec.2021.105976
Received 7 May 2021; Received in revised form 22 September 2021; Accepted 8 October 2021
Available online 23 October 2021
0921-3449/© 2021 Elsevier B.V. All rights reserved.
A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

legally using hydrometallurgy and pyrometallurgy methods by some three main mechanisms of fungal leaching, which occur through organic
companies (such as Yashilout and Pars Charkheshe Aisa) in Iran. The acids secreted by fungi. In acidolysis, the microbial secretion of organic
main problem for these companies is the segregation of e-waste that is acids leads to protonation of oxygen atoms covering the surface of the
non-industrially and unsystematically, leading to the failure of the large metals and results in changes of metal mobility. As Eq. (1) shows, in an
companies. Also, e-waste is recycled illegally under non-organized acidolysis reaction, water is generated from the reaction between pro­
conditions by the small home workshop with low-level technology tons and oxygen. As a result, the Ni is detached from the surface (Simate
that can threaten human health and the environment (Arshadi, 2019). et al., 2010).
Furthermore, e-waste contains precious metals like Au, and valuable
NiO + 2H + →Ni2+ + H2 O (1)
materials such as base metals (e.g. Cu) and plastics (Kiddee et al., 2020).
All types of E-wastes contain printed circuit boards (PCBs). Approxi­ In complexolysis, chelating agents or complexes are formed on the
mately 30% of the PCBs weight consists of metals such as, on average, metal surface and thus increase the metal mobility and their solubili­
16% Cu, 4% Sn, 3% Fe, 2% Ni, and 1% Zn (Lu and Xu, 2016). Recycling zation. For example, oxalic acid probably makes a complex with Al, Fe,
of e-waste reduces the word-wide demand for new metal sources and Mg or a complex of citric acid with Ca and Mg. According to Eq. (2),
(Kumar et al., 2017). Table 1 compares the precious metal content of the nickel citrate is produced through a complexolysis reaction (Aung,
different products (Arshadi et al., 2020b, Arshadi et al., 2018, Priya and 2004).
Hait, 2018). Computers are one of the most significant parts of e-waste.
The average lifespan of computers has reduced by 50% from 1992 to Ni2+ + C6 H8 O7 →Ni(C6 H5 O7 )− + 3H + (2)
2015 (Kiddee et al., 2020). In complexolysis, the solubilized metal ions in the acidolysis process
United Nation University reported that by 2016 in the United States, are stabilized. The environmental toxicity decreases for fungi due to the
50% of the people would use their own desktop computers. Annually, stability of metal ions. Due to Eq. 1, in the acidolysis mechanism, pro­
approximately 17 million computers are scrapped worldwide. It has tons are consumed, and pH decreases, while due to Eq. 2, in complex­
been reported that the gold content of computer printed circuit boards olysis reactions, protons are generated, and pH increases. The third
(CPCBs) is 17 times higher than that of gold mines (Yamane et al., 2011). mechanism is redoxolysis with oxidation and reduction processes lead­
The presence of precious metals in CPCBs, particularly Cu and Au, makes ing to an increase in metal mobility. This is an important function in
the recycling process economically justified. However, toxic metals chemolithoautotrophic bacteria leaching showing a non-significant role
make CPCBs hazardous waste (Castro and Martins, 2009). in the bioleaching of fungi (Wu and Ting, 2006).
Recycling of e-waste has been introduced as the main solution to deal Fungi can grow at high pHs compared to bacteria, making them more
with environmental problems and to save natural mines (Rienzie et al., efficient for leaching of alkaline waste (Arshadi et al., 2020a) like
2019). Traditional methods, such as pyrometallurgy and hydrometal­ e-waste (Arshadi et al., 2018). Fungi have a shorter lag phase and make a
lurgy, are expensive, need skilled labor, consume high energy, and chelating reaction with metals in ionic form by exerting organic acids
generate secondary pollutants. Biotechnology plays a crucial role in the (Wu and Ting, 2006). Fungi secreted various metabolites such as organic
future of metallurgy and the treatment of metals contained in solid acids, proteins, exopolysaccharides, and amino acids. Organic acids, by
waste (Nguyen et al., 2018). It is inexpensive and more environmentally supplying the necessary protons and ligands, play a key role in the
friendly for metal leaching efficiency from natural and artificial ore bioleaching process and contribute to the dissolution of metals (Saidan
compared to traditional methods (Rasoulnia et al., 2021). However, et al., 2012). The pH and metal ions existing in the solution affect the
there are some disadvantages to biotechnology. It is time-consuming concentration and type of produced organic acids (Wu and Ting, 2006).
(Valix, 2017), needing a large open area for treatment, inconsistent Penicillium and Aspergillus genera are the most efficient fungi utilized for
yield (microorganisms cannot grow uniformly) (Kumar and Yaashikaa, leaching activity (Das et al., 2019). Penicillium shows a higher leaching
2020), depending on atmospheric conditions (Asghari et al., 2013), need efficiency than A. niger (Brandl et al., 2001). It grows in the pH range of
to design the efficient processes. To solve these disadvantage points, 1.5-10. It can provide high concentration of organic acids like oxalate,
close cooperation between chemists, engineers, and microbiologists is gluconate, and citrate (Arshadi et al., 2019). Penicillium genera show
needed (Pollmann et al., 2016). more than 90% base metal leaching efficiency from solid waste (Amiri
Bioleaching, the main rout of biotechnology, mobilize metals from et al., 2011).
solid substance to aqueous solutions and catalyze this process by pro­ The reaction in the bioleaching process, between most important
duced microbial leaching agents (Rasoulnia et al., 2021). In the bio­ organic acids and metal are provided in Eq. 3-14 (Srivastava et al., 2020)
leaching process a wide range of microorganisms such as and Eq. 15-18 (Xia et al., 2018).
chemolithoautotrophic bacteria, heterotrophic bacteria, and fungi are Gluconic acid
utilized. Fungi and heterotrophic bacteria that secrete organic acids,
produce complex compounds, and change the oxidation state (Sethur­ C6 H12 O7 →C6 H11 O−7 + H + (pka = 3.86) (3)
ajan et al., 2018). Redoxolysis, complexolysis, and acidolysis are the

Table 1
Comparison of metal content of different type of e-waste
Metal ComputerPCBs Mobile TV Copy machine Fax Central Processing Laptops Washing Air
(%) phonePCBs PCBs PCBs machinePCBs Units machine Conditioner

Au 482 1270 91 1255 1039 2204 - - -

(ppm)
Pd 22 170 26 195 131 557 - - -
(ppm)
Cu (%) 14.5 21 14.44 21.29 21.03 30.51 23.11 3.85 5.2
Ag (%) 0.042 0.147 0.04 0.13 0.14 0.13 0.06 0.00 0.00
Sn (%) 0.46 2.354 3.61 2.51 3.04 1.80 - - -
Si (%) 8.22 9.425 2.155 9.34 8.82 9.56 - - -
Al (%) 6.051 5.793 4.14 5.06 6.14 5.10 0.52 0.52 0.42
Ni (%) 0.12 0.279 0.24 0.35 0.57 3.61 0.07 0.03 0.03
Zn (%) 0.31 0.93 2.06 0.93 1.26 0.27 1.18 0.48 0.42
Pb (%) 0.08 1.12 2.18 1.29 1.80 0.83 7.39 3.19 3.69

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A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

[ ] fermentation which the maximum amount of organic acids were pro­

n C6 H12 O−7 + M n+ →M[C6 H11 O7 ]n (Gluconic metallic complex) (4)
duced, the maximum leaching efficiency of Cu, Ni, and Zn were achieved
Oxalic acid 64%, 44%, and 99%, respectively (Faraji et al., 2018). In addition,
P. simplicissimum was utilized to bioleach some solid waste, such as power
C2 H2 O4 →C2 HO−4 + H + (pka1 = 1.25) (5)
plant residual ash (Rasoulnia and Mousavi, 2016b) and hydro-processing
catalysts (Anahid et al., 2011). Within the information collected, Brandl
C2 HO−4 →C2 O2−4 + H + (pka2 = 4.14) (6)
et al. (2001) have investigated the function of P. simplicissimum and
[ ] A. niger on the e-waste bioleaching. They concluded Sn and Cu was
n C2 HO−4 + M n+ →M[C2 HO4 ]n (Oxalic metallic complex) (7)
leached 60% and the leaching efficiency of Al, Ni, Pb, and Zn were 95%.
[ ] For pulp density more than 10 g/l the microbial growth was inhibited.
n C2 O2−4 + 2M n+ →M2 [C2 O4 ]n (Oxalic metallic complex) (8)
Also, the higher leaching efficiency of P. simplicissimum for mobilizing
Citric acid metals rather than A. niger was proved (Brandl et al., 2001). To the best of
our knowledge, no study was specifically examining the function of
C6 H8 O7 →C6 H7 O−7 + H + (pka1 = 3.09) (9) P. simplicissimum in the bioleaching process of e-waste.
Limited research has been conducted on the fungal leaching of e-
C6 H7 O1−7 →C6 H6 O2−7 + H + (pka2 = 4.75) (10) waste. Moreover, there are no data on the bioleaching of CPCBs using
P. simplicissimum, and no optimization has been carried out on the
C6 H6 O2−7 →C6 H5 O3−7 + H + (pka3 = 6.40) (11) bioleaching efficiency of e-waste using P. simplicissimum. In the present
[ ] study, P. simplicissimum was initially adapted to 30 g/l of CPCBs sample.
n C6 H7 O−7 + M n+ →M[C6 H7 O7 ]n (Citric metallic complex) (12) In the next step, the leaching of Cu, Ni, and Al was simultaneously
[ ] maximized using the central composite design (CCD) from RSM. The
n C6 H6 O2−7 + 2M n+ →M2 [C6 H6 O7 ]n (Citric metallic complex) (13) effects of critical parameters, including pulp density, initial pH, the
[ ] sucrose concentration, and their interaction were examined. Finally,
n C6 H5 O3−7 + 3M n+ →M3 [C6 H5 O7 ]n (Citric metallic complex) (14) the initial CPCBs waste and bioleaching residue were subject to phys­
Malic acid ical and chemical characterizations, such as Fourier transform infrared
spectrometer (FTIR), scanning electron microscopy (SEM), and high-
C4 H6 O5 →C4 H5 O−5 + H + (15) performance liquid chromatography (HPLC).

C4 H5 O−5 →C4 H4 O2−5 + H + (16) Materials and methods

[ ]
n C4 H5 O−5 + M n+ →M[C4 H5 O5 ]n (Malic metallic complex) (17) 2.1. Penicillium simplicissimum inoculum preparation
[ ]
n C4 O2−5 + 2M n+ →M2 [C4 O5 ]n (Malic metallic complex) (18) P. simplicissimum (BBRC20019) was obtained in lyophilized form
The parameters influencing the bioleaching process are processing from the Biochemical and Bioenvironmental Research Center (BBRC),
technique, type of waste, pulp density, particle size, microorganism, Tehran, Iran. It was grown on a 3.9% (w v− 1) potato dextrose agar (PDA)
Microbial population and tolerance, and physicochemical environment slants at 30 during a week. PDA is a general medium for fungi that
(including temperature, energy source concentration, surface tension, inhibit bacterial growth. The required spores for inoculation were pro­
nutrient, mass transfer of oxygen, pH). Acidity acts as the rate-limiting vided from washing the surface of slants. The number of spores was
step in the bioleaching environment (Rathna and Nakkeeran, 2020). counted using a microscope (Olympus, CH-B145-2, Japan) and a Neu­
Also, the diffusion of reactants and products would be the most limiting bauer chamber (HBG, Germany). Approximately 107 spores were inoc­
step (Picazo-Rodríguez et al., 2020). ulated in a Bosshard medium (containing 0.025 g/l MgSO4.7H2O, 0.025
Accordingly, a proper tool is required to optimize the fungal activity g/l KCl, 0.5g/l K2HPO4, 1.5 g/l NaNO3, 1.6 g/l yeast extract, and 100 g/l
for efficient bioleaching with high leaching efficiency of metals. To gain sucrose as the energy source which autoclaved at 121∘ C for 15 min)
the maximum metal leaching efficiency, some techniques have been under sterile conditions. It was incubated at 30∘ C and 130 rpm until a
suggested to optimize with statistical methods. A popular optimization sudden drop in pH was observed. A significant drop in pH shows the
technique is response surface methodology (RSM), optimizing biochem­ point that the maximum concentration of organic acids is produced. This
ical, bioenvironmental, and chemical parameters and their interaction in was used as the inoculum for the adaption period.
various processes. RSM was performed to design, model, and optimize P. simplicissimum was adapted to CPCBs powder, to increase micro­
different processes. Therefore, RSM leads to solving the optimization of bial resistance to the toxicity of the e-waste powder and production of
the bioleaching process to maximize the desirable metal leaching effi­ more organic acid (Bahaloo-Horeh et al., 2018). The serial passage was
ciency. RSM is the most suitable technique for fitting a quadratic model utilized with the steps of 1 g/l up to 10 g/l. Then, regarding the high
to optimize independent factors and introduce the interaction effect be­ resistance of fungi, each step was increased from 5 g/l up to 30 g/l. The
tween them (Montgomery, 2012). required time to reach a sudden drop in pH was 2-4 days for a pulp
There are several studies in the literature that have investigated the density less than 10 g/l. By continuing the adaptation period up to 30
function of bacteria, while only few studies have explored the leaching of g/l, the required time was increased to 7 days, being consistent with the
metals from waste PCBs using fungi. Some researchers have studied the results obtained by other researchers (Rasoulnia and Mousavi, 2016b).
bioleaching of e-waste using A. niger. Previously, the bioleaching of e- In this study, 250 ml Erlenmeyer flasks with a solution volume of 100 ml
waste using A. niger with one-step, two-step, and spent-medium bio­ were utilized for all experiments.
leaching methods was examined. Maximally, 97% of Cu and 74% of Ni
were recovered under optimal conditions (Arshadi et al., 2020a). Also, in 2.2. CPCBs powder and downstream steps
similar research, Bacci et al. (2020) studied the leaching of Cu and Zn
from PCBs using A. niger. In the presence of Fe3+ as an oxidant agent and Downstream processes are collection, transport, and steps following
25 g/l of pulp density, 60% of Cu and 40% of Zn were recovered after 21 manual dismantling, including mechanical processing and refining. The
days of fermentation (Becci et al., 2020). Another study examined the collection is the first step in the recycling chain for e-waste. Mechanical
bioleaching of Cu, Ni, and Zn from PCBs using A.niger. After 21 days of processes include crushing (shredding and milling), sizing (sieving and
screening, separation based on particle dimension), sorting (force

3
A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

separator, electric field, density, magnetic, and other physical charac­ gluconic acid, and citric acid were produced in the highest sucrose
teristics), recovery from dost, and selective treatment. Metals, synthetics, concentration, while malic acid was maximum in the lowest concen­
and glass in e-waste are considered for refining or conditioning to be sold tration. Due to this study, the inhibitory effect of sucrose occurred in the
as secondary raw materials or dispose of in a landfill (Ottiger et al., higher concentration of 150 g/l for oxalic acid, gluconic acid, and citric
2019). The downstream processes affect humans and animals by skin acid. The lower the sucrose concentration, the more malic acid was
contact, dust/soil ingestion, air inhalation, and dietary intake. The produced (Rasoulnia and Mousavi, 2016a). Accordingly, the sucrose
un-quantified downstream costs from informal recycling are borne by concentration was selected in the range of 60 g/l to 160 g/l.
workers and their families, the community, and the environment (Shaikh By adding CPCBs waste, the toxicity of the environment for fungal
et al., 2020). growth is increased, and the secreted metabolite is decreased (Wu and
CPCBs waste samples used in this research were provided from Pars Ting, 2006). However, to reduce the capital cost during bioleaching
Charkhesh Asia Company, Tehran, Iran, in dimensions of 2 cm. The process, a relatively high pulp density is needed. Due to the adaptation
sample particles were ground to less than 100 μm using a micronizer phase, which succeeded to 30 g/l, the pulp density range was selected
(Hertzog, Germany). X-ray fluorescence (XRF) spectroscopy (Spectro between 2 g/l to 30 g/l. The initial pH is one of the known important
Xepos, Germany) was conducted to identify the metal content of CPCBs parameters affecting the fungal activity and the produced metabolites
waste (Fig. 1). Among the metals containing in CPCBs waste, Cu, Ni, Ti, (Arshadi et al., 2019). The inhibitory effect of pH on P. simplicissimum
Fe, Al, Zn, and Sn (as the main base metals) are present with abundance growth was examined in the solid-state by other researchers. The largest
of 14.5%, 0.1%, 0.3%, 1.3%, 6.1%, 0.3%, and 0.5% (w/w), respectively. diameter of the colony and optimum growth rhythm of P. simplicissimum
Si is present about 8.2%. It has to be noted that Cd, U, Bi, Tl, Nd, Ga, V, occurred within a pH of 3-9. At the pH of 12, 2, 11, 10, the smallest
K, Cl, Th, Cr, Sr, As, Zr, and P were found in CPCBs metal content with a diameter of the colony was achieved (Bahaloo-Horeh, 2015). So, the
concentration of lower than 0.06 % (w/w) which have not been shown examined pH range was selected between of 3 to 9.
in Fig. 1. Due to Fig. 1, CPCBs contains 43.7% metals. Previously, the CCD design with n independent variables and m center points in­
characterization of personal computer PCBs was studied. Presence of cludes N experiments shown by Eq. (19) (Arshadi et al., 2020a). Conse­
45% metals, 27% polymers, and 28% ceramic was reported (Yamane quently, 18 experiments were designed with 4 center points shown in
et al., 2011). Table 2.
CPCBs waste phase was recognized using the X-ray diffraction (XRD)
N = 2n + 2n + m = 23 + 2(3) + 4 = 18 (19)
spectroscopy presented in Fig. 2. The results confirmed the presence of
the crystalline phase of CuO, CuBr, and SiO2. The highest pick belongs to
Cu, proving the high concentration of Cu in CPCBs waste. Al and Pb are • Response selection
present in their base form. Additionally, the sample contains a signifi­
cant amount of silicon dioxide. The order of economic value of base metals in CPCBs was reported as
follow by Arshadi et al. (2018): Cu> Ag> Si> Al> Sn> Ni> Zn> Pb>
Mn.
Statistical approach
Cu is the most economical base metal suggested to leach from
CPCBs. The International Copper Study Group (ICSG) has reported the
In this research, the most important bioleaching parameters for the
increasing worldwide demand for copper from 2011 to 2015 by more
metal leaching of e-waste were optimized by CCD from RSM. Three in­
than 17% (Awasthi et al., 2016). The economic values of Al and Ni are
dependent parameters, including A- initial pH (3-9), B- sucrose con­
more than Zn, Pb, and Mn in CPCBs waste (Arshadi et al., 2018).
centration (60-140 g/l), and C- pulp density (2-30 g/l) were selected for
Recycling of Al and Cu from e-waste provides 95% and 85% energy
the statistical analysis. Sucrose, as the energy source of heterotrophic
saving compared to natural ore, respectively (Cui and Forssberg, 2003).
fungi, is one of the most significant parameters affecting the secretion
Ag and Sn leaching is possible when cyanogen strains are used. So, the
and activity of organic acids. Determination of the optimal amount
most important base metals including Cu, Al, and Ni were chosen as the
of sucrose concentration improves the bioleaching efficiency and
responses. According to Fig. 1, a ton of CPCBs waste contains approxi­
economical aspect of the process. The Bosshard medium suggested 100
mately 145 kg, 10 kg, and 61 kg of Cu, Ni, and Al, respectively.
g/l sucrose as the energy source, while this amount is not optimal for all
produced metabolites. Rasoulnia and Mousavi optimized the organic
Bioleaching experiments
acid production by A. niger in a bubble column. Different concentration
of sucrose in the range of 50 g/l to 150 g/l was examined. Oxalic acid,
Two 250-ml Erlenmeyer flasks, including a flask containing a 100-ml
Bosshard medium with the desirable pH and a flask containing the
needed weight of CPCBs, were autoclaved at 121 ◦ C for 15 min, to
conduct each experiment designed. Under biological safety cabinet and
sterile conditions, the inoculum was added to the medium, and then it
was overflowed to the related CPCBs waste Erlenmeyer.
The Bosshard mediums were incubated at 30 ∘ Cand 130 rpm for a
week using a shaker incubator (Labcon 5082u, South Africa) to grow
fungi. The pH of the solution was controlled using sulfuric acid, sodium
hydroxide, and a pH meter (Milwaukee, Mi151, Romania). All chemicals
of analytical grade were obtained from Merck. The inoculum size was
constant and approximately 107 spores/ml for all experiments. Erlen­
meyer flasks were weighed every day. The weight loss is indicative of the
rate of evaporation. The data showed about 1 ml of the water in the
medium was vaporized in 24 h. 1 ml of distilled water was added to the
medium daily in sterile conditions. The experiments were conducted in
triplicate, and their mean was reported.

Fig. 1. The weight percentage (%) of base metal in CPCBs waste

4
A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

Fig. 2. The XRD of CPCBs waste

Table 2
Experimental plane based on CCD and response leaching efficiency
RunNumber A:pH B: Sucrose Cons. (g/l) C: Pulp density Al leaching efficiency (%) Ni leaching efficiency (%) Cu leaching efficiency (%)

1 4.5 80 9 84.6 95.3 84.6

2 7.5 80 9 87.3 98.7 89
3 4.5 120 9 80.1 94.1 84.3
4 7.5 120 9 80.3 84.8 78.9
5 4.5 80 23 76.4 70.2 10.1
6 7.5 80 23 66.3 78.5 69.2
7 4.5 120 23 74.7 87.9 67.9
8 7.5 120 23 73.5 85.9 73.5
9 3 100 16 76.5 85.7 80.4
10 9 100 16 74.4 84.6 78
11 6 60 16 73.2 84.3 76.6
12 6 140 16 67.8 79.9 69.5
13 6 100 2 99.9 99.7 100
14 6 100 30 72.8 70.8 7.51
15 6 100 16 81.3 90.6 83.1
16 6 100 16 76.3 87 80.8
17 6 100 16 77.9 88.2 81.1
18 6 100 16 83.8 98.3 87.3

Calculation of metal leaching efficiency mm × 4.6 mm, 5 µm, Germany) with an injection size of 20 µl. The
operating conditions were 30 ∘ C and 5.7 MPa. In this regard, 5 mM
To calculate metal leaching (Eq. 20), the metal absorbed by fungi and sulfuric acid with a flow rate of 0.5 ml/min was injected as the mobile
the metal ions in the medium were analyzed using inductively coupled phase. The calibration solutions (with minimum 4 points) were injected
plasma optical emission spectroscopy (ICP-OES, 730-ES, Varian). using the same procedure, and the peak area for the main effective
( ) metabolite in the bioleaching process (i.e., gluconic acid, oxalic acid,
(Cm + Cad ) × V
Lr (%) = × 100 (20) malic acid, and citric acid) at the selected wavelength was recovered.
P × Cr
FTIR analysis was conducted to identify the functional groups and
the chemical structure in CPCBs waste. A FTIR spectrophotometer
where Lr is the leaching efficiency of the responses (Cu, Ni, and Al). Cm
(Perkin Elmer, Spectrum Two, USA) in the wavelength between 450 and
and Cad denote the released metal ions in the medium and those absorbed
4000 cm− 1 was used for this purpose. CPCBs waste sample was mixed
by fungi, respectively. Additionally, V is the volume of the medium (for
with potassium bromide to form a pellet with a pressure of 5 tons.
all experiments, it is equal to 0.1 l). P is pulp density. Cr shows the metal
To examine the bioleaching process, the surface morphology of
concentration of the response contained in CPCBs waste.
CPCBs waste was examined using SEM (AIS-2100, 2005–2006, South
Korea). The e-waste sample was coated with gold using the sputtering
Analytical methods technique to enhance the conductivity. The coated CPCBs sample was
examined at an accelerating voltage of 20 kV.
XRD patterns were obtained using an X-ray diffractometer (EQUI
NOX 3000, Inel, France) equipped with CuKα radiation with a wave­ Results
length of 1.54056 angstroms. The operating conditions were adjusted at
40 kV/30 mA and a range of 2θ angle from 5◦ -118◦ with a scan speed of 3.1. Statistical analysis
0.04◦ /s. The concentration of secreted metabolites like organic acids
was estimated using HPLC (Sykam, Germany) equipped with a UV-VIS Fig. 3 presents the experiments designed by the software and
diode array detector at 210 nm and a Nucleodur C18ec column (250 the results of leaching efficiencies. Accordingly, RSM suggested three

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A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

quadratic models to predict the Cu, Ni, and Al leaching efficiency in Figs. 4(a), 4(c), and 4(e) present the contour plots of the pulp density
different amounts of independent variables presented in Table 3. As can versus the sucrose concentration at an initial pH of 4 for Cu, Al, and Ni,
be noted, these responses are significant owning to their p-values respectively. Fig. 4(b), Fig. 4(d), and Fig. 4(f) show the contour plots of
(0.0061 for Cu, 0.0006 for Al, and 0.0023 for Ni). The effect of the in­ the pulp density versus the initial pH at 80 g/l sucrose concentration for
dependent variables on the responses and the suitability of the suggested Cu, Al, and Ni, respectively.
equations are explained using the analysis of variance (ANOVA). In this All plots prove the maximum metal leaching efficiency was achieved
analysis, negative and positive coefficients show the antagonistic and in low pulp density regardless of the value of other parameters. This
synergistic effects on the leaching efficiency of the responses, respec­ figure confirms the negligible effect of the initial pH and the sucrose
tively. The high r-squared for Cu (0.8857), Al (0.9376), and Ni (0.9128) concentration compared to the pulp density, consistent with the co­
exhibited excellent confirmation of the suggested models with experi­ efficients reported in Table 3.
mental results. The normal plot of residuals and predicted versus actual The decrease in the metal leaching efficiency with increasing solid
data (Fig. A.1) for Cu, Al, and Ni confirms the accuracy of the models waste powder weight in the fungal leaching process has also been proved
(Arshadi et al., 2019). These responses are significant owning to their by Wu and Ting (Wu and Ting, 2006) and Nikfar (Nikfar et al., 2020). The
p-values (0.0061 for Cu, 0.0006 for Al, and 0.0023 for Ni). metals bioleaching from e-waste was investigated using A. niger. The
The effect of the independent variables on the responses and the results proved the high importance of pulp density on the metal leaching
suitability of the suggested equations are explained using the analysis of efficiency. Moreover, lower pulp density leads to higher efficiency (Wu
variance (ANOVA). and Ting, 2006). The bioleaching of Cr and Ni from an electroplating
sludge by A. niger was statistically examined by Nikfar et al. (2020). Pulp
density, temperature, and bioleaching duration were selected as the main
3.2. Pulp density parameters that affected the bioleaching process. CCD results exhibited
the higher importance of the pulp density compared to the temperature
The predicted models (Table 3) illustrate pulp density as the most and bioleaching duration (Nikfar et al., 2020).
influential and significant parameter affecting Cu, Ni, and Al. The Moreover, Amiri et al. (2011) examined the bioleaching of Ni, Mo,
negative coefficient of the pulp density in the suggested models shows and Al from the spent catalyst by P. simplicissimum. In the first step,
that the level of Cu, Ni, and Al leaching efficiency decreases by eleven parameters were analyzed for their importance on the metal
increasing the pulp density that can be attributed to the high metal leaching efficiency in the bioleaching process. The four most significant
toxicity and low dissolved oxygen (Wu and Ting, 2006). In addition, parameters, including yeast extract concentration, NaNO3 concentra­
based on ANOVA, the pulp density was the only significant independent tion, pulp density, and sucrose concentration, were selected and opti­
variable affecting the bioleaching of Cu, Ni, and Al. By adding the mized using CCD. The importance of the pulp density and the sucrose
concentration of CPCBs waste to the medium, the number of toxic metals concentration was almost the same, with a negative effect on Al, Ni, and
and consequently the environmental toxicity increase, leading to lower Mo. The screening proved the significance of the medium composition
fungal activity, lower metabolite production, and reducing bioleaching compared to the culture conditions for maximal metal leaching effi­
efficiency (Wu and Ting, 2006). ciency (Amiri et al., 2011), while in this study, the culture condition
Figs. 4(a)-4(f) illustrate the effect of the pulp density on Cu, Ni, and (pulp density) was known more important than the medium composi­
Al leaching efficiency. The contour plots represent the simultaneous tion (sucrose concentration). The reason may relate to the higher
interaction effects of independent parameters on a unique dependent toxicity of e-waste compared to the catalyst waste due to their metal
factor. They are used to establish the response values and selected pa­ composition. E-waste contains more than 50 metals, while the catalysts
rameters. In a contour plot, the response surface is viewed as a two- include only three metals. Due to the XRF analysis, a spent hydro­
dimensional plane where all points having same response are con­ cracking catalyst contains only 0.49% Na2O, 50.47% Al2O3, 33.44%
nected to produce contour lines. The contour lines of a model containing SiO2, 0.19% CaO, 1.13% Fe2O3, 4.05% NiO, and 10.24% MoO3 (Amiri
interaction effects will be curved and not straight. Contour and surface et al., 2011), while CPCBs contains about 40 metals (section 2.2.). By
plots help to illustrate the relationship between the two factors and the increasing the pulp density of e-waste, high densities of different metals
response (Antony, 2014).

Fig. 3. The results of leaching efficiencies versus run

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A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

Table 3
The results of the analysis of variance (ANOVA)
Response Response surface model Analysis of variance
Model R2 p-value Remarks

Cu (%) 82.38+3.69A+2.33B-18.82C-7.91AB+8.22AC+9.06BC-1.14 A2-2.68 B2-7.50 C2 Quadratic 0.8857 0.0061 significant

Al (%) 79.80-0.79A-1.05B-5.99C+0.81AB-1.78AC+2.13BC-1.11A2-2.34B2+1.62C2 Quadratic 0.9376 0.0006 significant
Ni (%) 91.31-0.1A+0.087B-6.76C-2.89AB+1.53AC+5.04BC-1.40A2-2.16B2-1.38C2 Quadratic 0.9128 0.0023 significant

inter to the solution that makes a highly toxic environment. So, the pulp Under the condition of the initial pH of 6, the sucrose concentration
density shows a highly significant effect on the bioleaching process of of 100 g/l, and the minimum possible pulp density of 2 g/l, the
e-waste; the culture condition is more important than the medium maximum leaching efficiency of Cu (100%) and Al (98.3%) occurred
composition. while Ni recovered about 70%.

3.4.1. Organic acids production

3.3. Optimal initial pH to gain maximum leaching efficiency
Hence, to identify the mechanism of the bioleaching process, it is
necessary to analyze produced organic acids. Fig. 5 presents the HPLC
The pH and metal of the environment significantly affect the con­
diagram under optimal conditions. According to Fig. 5 and the standard
centration and type of produced organic acids. The contour plots of pulp
curve of mentioned organic acids, approximately 712.45 ppm of oxalic
density versus initial pH at 80 g/l sucrose concentration for Cu, Al, and
acid and 10571.41 ppm of gluconic acid were produced by fungi. No
Ni have been drawn in Figs. 4(b), 4(d), and 4(f), respectively. Fig. 4(b)
citric acid and malic acid were identified. Similar to oxidation of
illustrates an increase in initial pH leads to an increase in the leaching
glucose, fungi metabolites are produced through metabolic mechanisms
efficiency of Cu. Also, ANOVA (Table 3) explains the coefficient of the
(Wu and Ting, 2006). According to the HPLC diagram, gluconic acid is
initial pH is positive for the Cu model. The main mechanism of Cu
the first and the main produced acid by P. simplicissimum. Similarly,
leaching is probably complexolysis due to the higher Cu leaching effi­
previous research indicated that gluconic acid was the main lixiviant
ciency with pH increasing. The contour plots in Fig. 4(d) and Fig. 4(f)
produced by fungi. Glucose oxidase enzymes at pH higher than 3.5
explain an increase in initial pH leads to a decrease in the leaching ef­
convert glucose into gluconic acid (Bahaloo-Horeh et al., 2018). Glu­
ficiency of Al and Ni. Similarly, Table 3 shows the coefficients of the
conic acid production by fungi is an enzymatic conversion. The main
initial pH for Al and Ni recoveries are negative. Accordingly, the prin­
enzyme is glucose oxidase in the mycelial cell wall. Glucose oxidase
cipal mechanism of Al and Ni leaching is acidolysis owing to their ten­
converts D-glucose to D-gluconolactone and hydrogen peroxide (Eq. 21).
dency to a more acidic environment. In more acidic conditions, Eq. (1)
Then, D-gluconolactone is hydrolyzed to gluconic acid by lactonase (Eq.
progresses, and in the presence of more OH− , Eq. (2) prevails.
22) and hydrogen peroxide is converted to water and oxygen by catalase
Other studies have also been reported an increase in Cu and a
(Eq. 23). This process needs a high oxygen concentration. As Eq. 24
decrease in Al and Ni fungal leaching efficiency, with increasing pH.
shows to generate 1 mol gluconic acid, 0.5 mol oxygen is consumed (Lu
Previously, Shah et al. (2020) studied the fungal leaching of Al from
et al., 2015).
bauxite ore using A. niger. The effect of pH was explained on Al disso­
lution. For all different tried methods including, direct one-step, direct Glucose Oxidase
β − D − Glucose ̅̅̅̅̅̅̅→ Glucono − δ − lactone + H2 O2 (21)
two-step, indirect bioleaching process, and controlled experiments, the
Al leaching efficiency was decreased with pH increasing (Shah et al., Glucono − δ − lactone ↔ Lactonase/Spontaneous Gluconic acid (22)
2020). The bioleaching of Cu and Ni using P. simplicissimum from
e-waste was optimized in non-conventional mediums. The effects of the
(23)
Catalase
H2 O2 ̅̅̅
̅→ 1/2O2 + H2 O
inoculums size, the initial pH, and the used medium were studied in the
mentioned research. The optimization experiments indicated that Glucose + 1/2O2 →Gluconic acid (24)
increasing the initial pH leads to an increase in Cu leaching efficiency
and a decrease in Ni leaching efficiency for cheese whey, molasses, and Fungi secrete oxalic acid through three pathways, including the
sugar mediums (Arshadi et al., 2019). cytoplasmic pathway, Kerb’s cycle, and the glyoxylate cycle. The cyto­
plasmic pathway is based on oxalate biosynthesis on glucose as an en­
ergy source. First, oxaloacetate is produced by fixation of carbon dioxide
3.4. Optimum condition onto pyruvate by the pyruvate carboxylase. In this cycle, cytosolic
oxaloacetate is hydrolyzed to oxalate and acetate by the Mn2+ depen­
The leaching efficiencies of Cu were obtained in the range of 7.51- dent oxaloacetate hydrolase. This pathway is the established route of
99.98%. The highest leaching efficiency was related to the initial pH of
oxalate biosynthesis on glucose as a carbon source Eq. 25-(26). In the
6, the sucrose concentration of 100 g/l, and the minimum possible pulp Kerbs cycle and the glyoxylate pathways, pyruvate is first oxidized into
density of 2 g/l. Meanwhile, the lowest one was attributed to the initial
acetyl-CoA (Eq. 27). The Kerbs cycle occurs in the mitochondria. Acetyl-
pH of 6, the sucrose concentration of 100 g/l, and the maximum possible CoA is converted to oxaloacetate and oxalic acid is secreted through the
pulp density of 30 g/l. The data emphasize the effect of the pulp density
cleavage of oxaloacetate by the oxaloacetate hydrolase (like cytoplasmic
on the leaching efficiency of Cu. The Al recoveries were in the range of pathway (Eq. 26)). Oxalic acid is secreted through the glyoxylate cycle,
66.26-98.30% for 18 experiments. The highest Al leaching efficiency
occurring in the glyoxysomes, when citrate is utilized as the carbon
gained was related to the initial pH of 6, the sucrose concentration of
source. In this cycle, acetyl-CoA is converted into glyoxylate by the
100 g/l, and the minimum possible pulp density of 2 g/l (like Cu), while
glyoxylate dehydrogenase then glyoxylate is converted to oxalate (Pal­
the lowest one was attributed to the initial pH of 7.5, the sucrose con­
mieri et al., 2019).
centration of 80 g/l, and the pulp density of 23 g/l. The Ni recoveries
were obtained in the range of 70.16-99.65% for all experiments. The pyruvate carboxylase
Pyruvate + ATP + HCO−3 ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅→ Oxaloaxetate + ADP + P
highest Ni leaching efficiency was related to the initial pH of 7.5, the
(25)
sucrose concentration of 80 g/l, and the minimum possible pulp density
of 9 g/l. The lowest one was attributed to the initial pH of 4.5, the su­
(26)
oxaloacetate hydrolase
Oxaloaxetate + H2 O ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅→ Oxalate + Acetate
crose concentration of 80 g/l, and the pulp density of 23 g/l.

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A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

Fig. 4. The contour plots of the pulp density versus: a) the sucrose concentration at an initial pH of 4 for Cu, b) the initial pH at 80 g/l sucrose concentration for Cu, c)
the sucrose concentration at an initial pH of 4 for Al, d) the initial pH at 80 g/l sucrose concentration for Al, e) the sucrose concentration at an initial pH of 4 for Ni, f)
the initial pH at 80 g/l sucrose concentration for Ni

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A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

Fig. 5. The HPLC diagram under optimum conditions

pyruvate dehydrogenase multienzyme complex 60 g/l, the pulp density of 16 g/l, and the initial pH of 9. The chemical and
Pyruvate + NAD+ + CoA − SH ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅→ NADH physical characteristics of the remained waste sample and the solution
+ CO2 + H + + Acetyl − CoA after the bioleaching process were analyzed to discuss bioleaching
(27) efficiency.
Table 4 presents the optimum condition, the predicted values from
Oxalic acid is one of the main metabolites excreted by fungi in an fitted models, and the results of the experiments conducted. RSM esti­
alkaline environment. The reason relates to the induction of the mated under the condition of 60 g/l sucrose concentration, 16 g/l pulp
oxaloacetate hydrolase enzyme through de novo synthesis (Gadd, density, and the initial pH of 9, the leaching efficiency of Cu, Al, and Ni
1999). Furthermore, in the presence of nickel, more oxalic acid is would be 100%, 67.7%, and 100%. In the confirmation test, Cu, Al, and
secreted (Santhiya and Ting, 2005). Consequently, according to the Ni were leached by 100%, 97.5%, and 70.2%, respectively. These values
alkalinity of CPCBs waste (Arshadi et al., 2018) and the presence of are in good agreement with the suggested leaching efficiency, proving
1200 ppm Ni (Fig. 1), the sensible concentration of oxalic acid was the accuracy of the proposed models. Higher concentrations of organic
produced by adding waste powder. In the presence of oxalic acid and acids exhibit a greater influence on the immobilization of metal through
insoluble oxalates, metal complexes and metal ions are immobilized, the surface complexation reactions occurring on the surface of the liquid
thereby decreasing the toxicity and increasing the tolerance level of phase and the powder particles (Bahaloo-Horeh et al., 2018). As
fungi. Eventually, the complexes formed between the metals of the mentioned in Subsection 3.3.1, only oxalic acid (712.45 ppm) and glu­
e-waste sample and oxalic acid promote fungi growth and the envi­ conic acid (10571.41 ppm) were secreted by fungi. The reactions for
ronment detoxification (Gadd, 1999). oxalic acid and gluconic acid with metals are presented in equations 3-8
Computer PCBs contain more than 14% copper and approximately (Introduction section).
0.12% Mn (Fig. 1). The presence of Cu, Zn, Fe, and Mn ions (the iso­ Any reaction with higher stability constant (k) will be more effi­
citrate dehydrogenase enzyme cofactors) strongly inhibits citric acid cient in metal removal. Log K for Cu complexes with gluconic acid and
accumulation. In the Krebs cycle, the isocitrate dehydrogenase enzyme oxalic acid is 18.29 and 6.3, respectively. Moreover, gluconic acid is
contributes to the conversion of the oxidative decarboxylation of iso­ the main lixiviant of copper leaching in the bioleaching process using
citrate to α-ketoglutarate. Therefore, in the presence of CPCCBs powder P. simplicissimum, and copper oxalate is insoluble in this process
and manganese ions, citric acid cannot accumulate, while citrate is (Gadd, 1999). Due to the high concentration of the produced gluconic
catalyzed to malate, fumarate, succinate, and so forth (Max et al., 2010). acid, the Cu leaching efficiency was 100%.
The unidentified peaks presented in Fig. 5 may be related to these The results of this study in the optimal conditions were compared
produced organic acids. Moreover, to secret citric acid, a lower pH is with other bioleaching studies using different microorganisms. Table 5
needed (Bahaloo-Horeh et al., 2018) thus no citric acid is identified on shows the used microorganisms and the yield of metals from PCBs or e-
the HPLC diagram. Additionally, due to Fig. 5, in the presence of man­ waste. Fungi and bacteria show a good Cu leaching efficiency. Ni
ganese ions, no sensible concentration of malic acid is accumulated leaching efficiency is more for P. simplicissimum. In this study utilizing
(Battat et al., 1991). P. simplicissimum, the base metal efficiency was improved.

3.4.2. Metal leaching efficiency

RSM estimated a set of conditions to receive the maximum simul­ Table 4
taneous Cu, Ni, and Al leaching efficiency. All conditions were exam­ The confirmation test under optimum conditions
ined. No condition was found that all the metals recover 100%, but there Metal leaching Ni Al Cu Sucrose Pulp Initial
was a suggestion that the pulp density could increase. By adopting the efficiency (%) (%) (%) conc. (g/ density pH
sucrose concentration of 60 g/l, pulp density of 16 g/l, and initial pH of l) (g/l)
9, the leaching efficiency of Cu, Al, and Ni were predicted 100%, 67.7%, Predicted 100 67.7 100 60 16 9
and 100%, respectively. The leaching efficiency of Al and Ni could not leaching
be 100% simultaneously. The reason is that Ni has low solubility in Al efficiency by
(0.01%-0.03%) (Medrano-Prieto et al., 2020). By increasing Ni ions, Al RSM
The results of 97.5 70.2 100
leaching efficiency decreases and vice versa. confirmation
Finally, to confirm the models, an experiment was run in the suggested test
optimum amounts of parameters, including the sucrose concentration of

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A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

Table 5 adaptation, column setup, solution flow, irrigation, pH, temperature,

The comparison of metal leaching efficiency in different bioleaching studies particle size, and mineralogical composition. For example, Ilyas et al.
from PCBs or e-waste (2010) studied the column bioleaching of e-waste. The column was 58
Microorganisms Metal leaching Reference cm in height and 13 cm in diameter with 10 kg of e-waste sample as the
efficiency feed and 50 ml/min irrigation flow rate. The consortium of bacteria was
Fungi adapted to 20 g/l of the e-waste sample. The bioleaching efficiency of
P. simplicissimum 100% Cu Present study 280 days for Zn, Al, Cu, and Ni were about 80%, 64%, 86%, and 74%,
98% Ni respectively (Ilyas et al., 2010).
70% Al
Accordingly, it is needed to conduct more studies about the indus­
Aspergillus niger 97% Cu (Arshadi et al.,
74% Ni 2020a) trialization of e-waste. Due to previous researches, it is probable to leach
Aspergillus niger 60% Cu (Becci et al., 2020) metals from several tones of e-waste on an industrial scale (heap or
40% Zn column bioleaching method) in more than 300 days.
Aspergillus niger 99% Zn (Faraji et al., 2018) After leaching, electrowinning was used to reach a pure metal from
44% Ni
64% Cu
e-waste in previous researches. Baniasadi et el. (2021) suggested closed-
Mixed Purpureocillium lilacinum and 56% Cu (Xia et al., 2018) loop recycling of Cu from e-waste involving the sequential application of
Aspergillus niger 15.7% Al bioleaching and electrowinning. After bioleaching, an electrowinning
20.5% Pb process was used to leaching efficiency copper from the bioleachate.
49.5% Zn 8.1% Sn
76% of copper was recovered with more than 99% purity (Baniasadi
Acidithiobacillus ferrooxidans 76% Cu (Baniasadi et al.,
2021) et al., 2021). Also, a cementation method was examined for metal
Aspergillus niger and 65% Cu 65% Sn (Brandl et al., leaching efficiency from e-waste resulting in 93% purity of copper
Penicillium simplicissimum More than 95% of 2001) (Sodha et al., 2020). In another study, an electrowinning process was
Al, Ni, Pb, Zn applied after bioleaching leading to copper leaching efficiency with
Bacteria
99.8% purity (Zhang et al., 2010).
Chromobacterium violaceum 34.5% Cu (Chi et al., 2011)
10% Au
Bacillus megaterium 36% Au (Arshadi and 3.5. Morphological characterization
13% Cu Mousavi, 2015)
Acidithiobacillus caldus 92% Cu (Akbari and
Fig. 6 depicts the surface morphologies of CPCBs powder before and
Sulfobacillus thermosulphidooxidans 67% Zn Ahmadi, 2019)
45% Al after the leaching process.
Acidithiobacillus ferrooxidans 90% Cu (Hong and Valix, The micrograph of the initial e-waste sample (Fig. 6(a) and Fig. 6(b)
2014) with the magnification of 50 µm and 5 µm, respectively) represents
Acidithiobacillus ferrooxidans 97% Cu (Wei et al., 2020) various particles, including chunks and rods with different textures,
Leptospirillum ferriphilum 99% Cu (Sodha et al.,
2020)
shapes, and sizes. The figure also represents the platform materials,
wire, and metal embedded in the e-waste sample powder. Piped-shaped
particles ranging from 22 µm to over 42 µm in Figs. 6(a) and 6(b) provide
Oxalic acid is the main organic acid in the bioleaching of Al and Ni the mechanical support to maintain metal layers that appeared after
(Santhiya and Ting, 2005). Log K for nickel oxalate and aluminum ox­ crushing (Rizki et al., 2019). The micrograph was taken from e-waste
alate complexes is 5.16 and 7.26, respectively. Lower Ni leaching effi­ powder particles with a diameter of approximately 35 µm. They are
ciency can be related to the formation of nickel oxalate, which has a low completely covered with particles smaller than 1 µm. Fig. 6(c) displays
solubility. Aluminum oxalate complexes of Al and oxalic acid are almost the micrograph of the leaching residue at a magnification of 50 µm. The
soluble (Sun and Qiu, 2012). The leaching efficiency for nickel (70.2%) mycelia of P. simplicissimum completely covered the surface of the res­
is approximately 30% lower than that for aluminum (97.5%) and copper idue. As Fig. 6(d) shows, the pores are rough, validating the good
(100%). Oxalate makes a bidentate complex with Al (III) easily. Al progress of the bioleaching process and the effectiveness of oxalic acid
(C2O4)(H2O)4+ and Al(C2O4)2(H2O)2– are two important forms of and gluconic acid on metal leaching. Owing to the destructive force of
aluminum oxalate. Aluminum oxalate reduces the Al toxicity. It affects mycelia which is approximately 10-20 µN/µm2, in the particles of CPCBs
the transfer and transformation of Al in the natural environment to a waste powder, pores are formed. By eroding the solid waste and for­
certain extent. Al (III) is taking into plant roots in the form of aluminum mation of pores, more active and fresh surfaces become available to
oxalate. Also, aluminum oxalate plays a significant role in Fe (III) up­ P. simplicissimum (Xiao et al., 2012). Furthermore, the leaching effi­
take. The significance of aluminum oxalate complexes to environmental ciency percentage of goal metals and leaching them into the liquid
chemistry is the increase of the level of dissolved aluminum resulting in medium demonstrates the effectiveness of the fungal activity and the
Al toxicity in the fishery, forestry, and agriculture (Jin et al., 2011). secreted metabolites.

• An industrial view of metal leaching from e-waste FTIR analysis

The present study was conducted in the Erlenmeyer. There are Fig. 7 presents the FTIR spectra in the range of 4000-500 cm− 1 of the
several types of industrial bioleaching, including, dump, heap, and initial CPCBs waste powder and the residue of bioleaching. FTIR peaks
column. The selected method depends on the solid waste, metal content, include the fingerprint region in the low wavenumber, triple bonds in
and geographical location of the setup. 2000-2500 cm− 1, double bonds in the 1500-2000 cm− 1, and single
The heap can be around 10 m tall and 100 m or more long and wide. bonds about 2500-4000 cm− 1 (Mohamed et al., 2017). According to this
For example, the Escondida Mine has constructed a sulfide copper ore finding and the peak wavelengths related to the FTIR spectra of poly­
heap contained 0.60% total Cu. This heap was designed to feed 14 ore mers defined in PCBs, the presence of polycarbonate (PC), poly­
strips at a steady state. Each strip was 125 m wide by 2000 m long. The phenylene oxide (PPO), and high-impact polystyrene (HIPS) is proved.
start of irrigation was about 1 month apart on each strip. Around 400 The functional groups of them are reported (Arnold et al., 2010, Reena
days of operation were considered as the leaching cycle for each strip et al., 2011):
(Jerez, 2017). HIPS: C-H aromatic at 3026-3032 cm− 1, C-H at 2863-2867 cm− 1 or
The column bioleaching of e-waste has been reported. It is influ­ 2923-2926 cm− 1, C⋅⋅− C aromatic at 1597-1602 cm− 1, benzene ring at
enced by several parameters, including process optimization, microbial 757-760 cm− 1 or 696-702 cm− 1

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A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

PC: C=O at 1734-1744 cm− 1, C⋅⋅− C aromatic at 1597-1602 cm− 1, C-O

ester/ether at 1028-1062 cm− 1, 1177-1187 cm− 1, or 986 cm− 1, C-H
aromatic at 3026-3032 cm− 1, C-H CH3 at 2923-2926 cm− 1 or 2863-2867
cm− 1, p-substitution at 839-843 cm− 1
PPO: C-H aromatic at 3026-3032 cm− 1, C-H bond of CH3 at 2923-
2926 cm− 1 or 2863-2867 cm− 1, C⋅⋅− C aromatic at 1597-1602 cm− 1, C-
O ester/ether at 1028-1062 cm− 1 or 986 cm− 1.
According to Fig. 7, the appeared peaks in both spectra occurred in a
similar wavelength. It proves the structure of the sample after bio­
leaching remains almost unchanged. But, the appeared peaks in the
initial CPCBs sample are wide and continue in a more wavelength range.
So, Fig. 7 proves the higher concentration of functional groups and
consequently higher amounts of polymers existed in the initial CPCBs
waste sample. After bioleaching, the concentration of all polymers re­
duces, emphasizing that the polymers are broken or bioleached then are
come to the solution.

3.7. Toxicity assessment

The main purpose of toxicity assessment is to protect human society

and environmental health by removing and reducing the toxic materials
from the waste. Several studies have been conducted in toxicity char­
acterization of the e-waste. E-waste contains organic and inorganic
substances, numerous additives, heavy metals, and brominated flame
retardants (BFRs), which cause numerous challenges for e-waste man­
agement, recovery, and recycling. Toxicity characteristic leaching pro­
cedure (TCLP), synthetic precipitation leaching procedure (SPLP), and
total threshold limit concentration (TTLC) tests are suggested to simu­
late material sitting inside a landfill due to the acidic conditions of the
landfills. The purpose is to determine the mobility of different materials,
such as heavy metals present in the leachate of the landfilled waste
(Rastegar et al., 2016).

• The recycling practices of e-waste were found to be more in informal

sectors. Improper handling of such materials leads to the uncon­
trolled release of toxic materials into the environment.
• E-waste is computed for 70% of heavy metals found in landfills. The
unscientific methods for metal recovery from e-waste have an
extremely high negative effect on the environment and human so­
ciety. Therefore, proper leachate management, removal of heavy
metals from leachate, and their proper burial should be illustrated in
future research.
• A study on toxicity evaluation of E-waste plastics on the potential
human health risks showed Hg contributed the major risk for car­
cinogens and non-cancer disease in the plastics. But the contribution
of Pb was also significant. In the case of eco-toxicity, Cr posed the
most significant risks in plastics. It is important to avoid the open
burning practices of e-waste plastics containing Hg, Pb, Cr, and Sb.
• The plastics of e-waste containing toxic substances such as BFRs are
suggested to manage by the Stockholm convention. This convention
guides the management of plastics of e-waste in an environment-
friendly manner and is not allowed to be reused or recovered
(Singh et al., 2020).

3.8. The practical applications of this work and future research prospects

In practice, e-waste contains many types of precious and base metals.

The results of this study help to conserve natural ores and the energy
needed to produce the new electronic instrument. In this way, heavy
metals are prevented from an environmental hazard. This study proved
Fig. 6. The surface morphology of CPCBs powder before the leaching process: all metals in the form of solid-state are oxidized using fungi in the bio­
a) 50 µm and b) 5 µm, and after the leaching process: c) 50 µm and d) 5 µm leaching technique. However, the required time for secreted organic acid,

11
A. Esmaeili et al. Resources, Conservation & Recycling 177 (2022) 105976

Fig. 7. The FTIR spectra of the initial CPCBs waste powder and the bioleached residue

recovery yield, and preparing sterile conditions for fungi culturing in the Acknowledgments
industry should be taken into account to ensure the cost-effective feature
of bioleaching. The development of mechanochemistry is probable in the Part of this study was financially supported by the deputy of research
near future; more research studies on applications of mechanochemistry and technology of Sharif University of Technology (Award Number QA:
to metal recovery are needed. Suitable milling instruments that can 970713). In addition, the authors are thankful to Iran National Science
produce sufficient particle size for industrial applications should be Foundation (Award Number: 99028031). The authors are grateful to
developed to reach high metal leaching efficiency. Due to the presence of Stat-Ease, Minneapolis, MN, USA, for the provision of the Design-Expert
sucrose as the energy source in Bosshard medium, future studies should 10 .0 .4 package.
be focused on introducing the optimum cheap carbon source to reduce
the cost of the bioleaching process. Finally, it is important to consider the Supplementary materials
toxicity assessment of the remained liquid and solid residue of bio­
leaching in future studies to have a safe landfilling. Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.resconrec.2021.105976.
Conclusions
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