Journal of Hazardous Materials 264 (2014) 203–210
Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
Bioleaching of metals from printed circuit boards supported with
surfactant-producing bacteria
Ewa Karwowska a,1 , Dorota Andrzejewska-Morzuch a , Maria Łebkowska a ,
Agnieszka Tabernacka a,∗ , Małgorzata Wojtkowska b , Alicja Telepko b ,
Agnieszka Konarzewska b
a
b
Warsaw University of Technology, Faculty of Environmental Engineering, Biology Division, Nowowiejska 20, 00-653 Warsaw, Poland
Warsaw University of Technology, Faculty of Environmental Engineering, Nowowiejska 20, 00-653 Warsaw, Poland
h i g h l i g h t s
• Bioleaching of metals from printed circuit boards by BSAC-producing bacteria was estimated.
• Aeration increased the release of all metals in medium with sulphur and biosurfactant.
• Increase in Cu, Pb, Ni and Cr removal rate was observed at 37 ◦ C in acidic medium.
a r t i c l e
i n f o
Article history:
Received 27 June 2013
Received in revised form 7 November 2013
Accepted 9 November 2013
Available online 16 November 2013
Keywords:
Heavy metal
Bioleaching
Printed circuit boards
Biosurfactant producing bacteria
a b s t r a c t
This study has evaluated the possibility of bioleaching zinc, copper, lead, nickel, cadmium and chromium
from printed circuit boards by applying a culture of sulphur-oxidising bacteria and a mixed culture of
biosurfactant-producing bacteria and sulphur-oxidising bacteria. It was revealed that zinc was removed
effectively both in a traditional solution acidified by a way of microbial oxidation of sulphur and when
using a microbial culture containing sulphur-oxidising and biosurfactant-producing bacteria. The average
process efficiency was 48% for Zn dissolution. Cadmium removal was similar in both media, with a highest
metal release of 93%. For nickel and copper, a better effect was obtained in the acidic medium, with a process effectiveness of 48.5% and 53%, respectively. Chromium was the only metal that was removed more
effectively in the bioleaching medium containing both sulphur-oxidising and biosurfactant-producing
bacteria. Lead was removed from the printed circuit boards with very low effectiveness (below 0.5%).
Aerating the culture medium with compressed air increased the release of all metals in the medium with
sulphur and biosurfactant, and of Ni, Cu, Zn and Cr in the acidic medium. Increasing the temperature
of the medium (to 37 ◦ C) had a more significant impact in the acidic environment than in the neutral
environment.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Technological innovation and modern business marketing strategy results in the development of electronic products that are
cheaper, better and more readily available than older versions.
As a consequence, much electronic equipment becomes outdated
and redundant quicker than ever and is consigned to waste [1].
Electronic scrap is now the fastest growing waste category [2].
According to UN Environment Programme estimates, about 20–50
∗ Corresponding author. Tel.: +48 22 234 5943; fax: +48 22 621 2979.
E-mail addresses: ewa.karwowska@is.pw.edu.pl (E. Karwowska),
agnieszka.tabernacka@is.pw.edu.pl, atabernacka@life.pl (A. Tabernacka).
1
Tel.: +48 22 234 5944; fax: +48 22 621 2979.
0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jhazmat.2013.11.018
million Mg of electric and electronic scrap is generated yearly, and
the volume of electronic waste is increasing three times faster than
other types of municipal waste [3]. In the UK 50,000 tonnes of
printed circuit board (PCB) scrap is currently generated every year,
with only 15% subjected to any form of recycling [4].
PCBs are integral elements of many electronic systems, including computers and other consumer electronics as well as devices
used in military and medical applications. They constitute about
3% of the total weight of electronic scrap. Their main components
are non-conducting substrates or laminates, conductive circuits
printed on or inside the substrate and mounted components (chips,
connectors, capacitors) [1].
Printed circuit boards are composed of polymers, ceramics and
metals. About 28–30% of the content is metal, with 10–20% copper,
1–5% lead, 1–3% nickel, and 0.3–0.4% precious metals like silver,
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E. Karwowska et al. / Journal of Hazardous Materials 264 (2014) 203–210
platinum and gold [5]. Many other elements (Ga, In, Ti, Si, Ge, As,
Sb, Se and Te) may be found in chips, with An, Pb and Cd in solder
joints, and Ga, Si, Se and Ge in semiconductors, and tantalum in
capacitors [1]. Other materials in PCBs are plastics (19%), bromine
(4%), glass and ceramics (49%), isocyanates, phosgene, and acrylic
and phenolic resins [5]. Ceramics present in PCBs include silica,
alumina, alkaline Earth oxides, mica and barium titanate [1].
In the US and the EU the main way of treating PCB scrap is by
incineration or by landfill (and more than 70% is disposed without recycling) [1,2]. For example, in 1997, 3.2 million tonnes of US
electronic waste was landfilled [1]. This poses a problem due to the
toxic properties of both the metallic and non-metallic PCB components [1]. The toxicity of PCB waste is connected with the presence
of brominated flame retardants (BFR), PVC plastic and heavy metals
– metals with elemental densities above 7 g/cm3 . During landfilling, pollutant-containing leachate may contaminate groundwater
and soil. On the other hand, incineration of waste PCBs may result
in the release of potentially hazardous byproducts into the atmosphere, such as dioxins, furans, polybrominated organic pollutants
and polycyclic aromatic hydrocarbons, as a result of burning BFR,
epoxy resins and plastics [3].
Legislation, including the Waste from Electrical and Electronic
Equipment (WEEE) directive, necessitates the increasing recovery
and recycling of materials found in scrap electronic components
[4]. It has been claimed that the purity level of precious metals in
waste PCBs is more than 10 times higher than that of rich-content
minerals, making them a potentially economically viable mineral
resource [3]. However, the recycling of electronic scrap is rather difficult because of its complexity and the heterogeneity of electronic
components [5].
Scrap PCBs are classified into three categories, according to the
precious metal content: H (high), M (medium) and L (low) grade
scrap. For scrap PCBs classified as low grade, the traditional smelting technique appears to be uneconomic. The approximate value
of metals found in medium grade PCB scrap is £0.07/kg for silver,
£0.13/kg for copper, £1.63/kg for gold and £0.8/kg for palladium.
The total value for all metals is £2.74/kg [4].
The traditional techniques for electronic waste treatment – both
pyrometallurgical and hydrometallurgical – often require great
energy consumption, are high cost and low efficiency, and cause
serious secondary pollution [5,6]. Pyrometallurgy, based on incineration, smelting in a plasma arc furnace or blast furnace, drossing,
sintering, melting and high temperature reactions in a gas phase,
is used for the recovery of non-ferrous metals as well as precious metals from waste PCBs [3]. Hydrometallurgical processing
of PCB waste usually includes both chemical leaching and electrochemical processing [1]. This involves a series of acid or caustic
(cyanide, halide, thiourea, thiosulphate) leachings of the waste. The
obtained solutions are subjected to precipitation of impurities, solvent extraction, adsorption and ion-exchange in order to purify and
concentrate the metals [3,7].
Some other technological approaches have been used in order to
recover valuable components from electronic waste. One method
is an application of a mixture of nitric acid and hydrochloric acid,
although a large amount of waste gases and acid solutions are produced in this process. In the US, a method based on solvolysis
has been developed. It allows recovery of both metals and plastic materials (such as epoxides) and, additionally, the extraction
of halogens and brominated hydrocarbon derivatives [4]. A dissolution of PCB scrap in sulphuric/nitric acid leachants followed by
electrolytic copper recovery is used commercially in the US [1]. In
Germany, a mechanical process involving shredding, granulation,
magnetic separation, classification and electrostatic separation has
been commercialised by Fuba [4]. A consortium based at Imperial
College, London, has developed the use of non-selective leachants
to dissolve the metal content of PCB scrap. This consortium’s
Table 1
The concentration of metals in the electronic waste.
Metal
Cd
Cr
Cu
Ni
Pb
Zn
Concentration (mg/kg)
1.9
72.4
41,237.3
13,244.7
14,129.9
12,471.4
method involves chemical leaching with electrogenerated chlorine in an acidic aqueous solution of high chloride ion activity.
As a result, a multi-metal leach electrolyte containing all of the
available metal content is obtained. Additionally, metal recovery
via electrolytic membrane cells with discrete metal separation has
been demonstrated [4]. Kim et al. [8] applied a similar method
and reported that the leaching rate of the metals increased with
an increase in density, temperature and time in reactor and that
the dissolution kinetics of copper with electro-generated chlorine
followed an empirical logarithmic law controlled by surface layer
diffusion.
In some areas (though mainly in the mining industry to
date), hydrometallurgical processes are being replaced with biohydrometallurgical (bioleaching) techniques [6]. The advantages of
biological methods are low operating costs, reduced environment
pollution, minimisation of the volume of end-products and highly
efficient effluent detoxification [3].
Bioleaching technology offers many advantages over conventional methods, including its relative simplicity, less exacting
operational requirements, low energy input, reduced need for
skilled labour, and environmental friendliness. However, it requires
a longer period of operation compared to other methods, such as
chemical leaching [3,9]. For these reasons, techniques facilitating
and improving the bioleaching process should be of interest. Additionally, there is a need for research on seeking the available source
of active bioleaching biomass [3,10].
There are some suggestions in the literature that the addition of
surfactants, complexing agents or other additives may positively
influence bioleaching with mesophilic bacteria [7,11]. It has been
observed that the addition of citric acid increases copper solubility
in the bioleaching solution from 37 wt% to 80 wt% [7].
This research has evaluated the possibility of achieving heavy
metal bioleaching from printed circuit boards by applying, respectively, a culture of sulphur-oxidising bacteria and a mixed culture
of biosurfactant producing bacteria and sulphur-oxidising bacteria.
The impact of selected process parameters, such as temperature
and aeration, on bioleaching effectiveness has also been estimated.
This is the first step to metal recycling.
2. Materials and methods
2.1. Waste characteristics
The printed circuit boards were obtained in crumbled (fined)
form. The grain fractions of the crumbled printed circuit boards
used in the experiment were as follows: d < 0.063 mm – 2.8%,
0.063 mm – 1.6%, 0.01 mm – 22.0%, 0.25 mm – 22.6%, 0.5 mm – 4.0%,
1 mm – 9.6%, 2 mm – 37.4%. The relative humidity of the waste was
0.84%. Table 1 shows the concentration of metals in the waste.
2.2. Bioleaching media
The experiments were carried out using two bioleaching media.
The M-I medium was a mixture of municipal activated sludge
and municipal wastewater with a solids concentration of about
3–4 g dry weight/L. Indigenous sulphur-oxidising bacteria were
E. Karwowska et al. / Journal of Hazardous Materials 264 (2014) 203–210
enriched in the sludge with the addition of 1% powdered sulphur
and “grown” for 4 weeks to lower the pH to 2–3. The concentration
of bacteria Acidithiobacillus sp. in M-I was about 106 CFU/mL. The
medium was labelled as “culture with 1% sulphur”.
The M-II medium was prepared as a (1:1) mixture of M-I
medium with a culture of bacteria capable of producing biological
surface-active compounds (BSAC). Surfactin producing strains of
Bacillus subtilis PCM 2021 and Bacillus cereus PCM 2019 were cultivated in medium containing 6 g/L of Na2 HPO4 , 3 g/L of KH2 PO4 ,
0.5 g/L of NaCl, 0.25 mg/L of yeast extract, starch and peptone, and
2 mL/L of plant oil. The final solution, with about 15 mg/L of BSAC,
was produced after 10 days of incubation in a shaker (120 rpm) at
room temperature. The concentration of biosurfactant-producing
bacteria in M-II was 1.5 × 109 CFU/mL. The M-II medium was
labelled as “culture with 1% sulphur and biosurfactant”.
2.3. Experimental procedure
The first part of the experiment compared the bioleaching effectiveness of the two bioleaching media. A fixed amount of waste
(10 g) was placed in flasks with 150 mL of bioleaching medium – MI and M-II, respectively. The flasks were put into a shaker (120 rpm)
to keep the content in homogenous form at room temperature.
Cultures were carried out in non-sterile conditions for 25 days.
The control flasks contained waste in 150 mL of distilled water to
compare the results to the natural washing process. The metal concentrations in these solutions were determined after 2, 7, 14 and
25 days.
The aim of the second experiment was to evaluate the influence
of chosen process parameters on heavy metal removal from electronic waste. The bioleaching process was carried out for 14 days
in three experimental variants:
- aerated by shaking (100 rpm) at 22 ◦ C
- aerated with compressed air at 22 ◦ C
- aerated by shaking (100 rpm) at 37 ◦ C
The waste and bioleaching medium amounts were similar to
those used in the first part of the experiment. The metal concentrations were analysed after 2, 7 and 14 days of the experiment.
In order to evaluate the total metabolic activity of microorganisms, the ATP concentration in the bioleaching cultures was
determined. The sulphur-oxidising activity was evaluated by determining the sulphate ion concentration.
2.4. Control analyses
The metal concentrations were analysed with a flame atomic
absorption (AAS Thermo-Jarrel Ash SH-1150) spectrophotometer.
The metal removal efficiency was evaluated as the ratio between
the amount of metal in solution and the initial total metal content
in a sludge. The standard error, standard deviation and variation
coefficient were calculated.
Biosurfactant concentration in media was detected using cobalt
thiocyanate active substances assay (CTAS assay).
The pH was determined according to Polish Standard PN-90/C04540/01.
The adenosine-triphosphate (ATP) content was evaluated by
a luminometric method, using a HY-LiTE® luminometer (Merck),
after 7 and 14 days of the experiment. The total ATP concentration
was calculated based on the relative light unit (RLU) number, where
1 g/mL ATP causes the emission of 1036 RLU.
The sulphate ion concentrations in the bioleaching media were
determined according to Polish Standard PN-ISO 9280:2002.
The detection and differentiation of sulphur-oxidising bacteria
were assessed both by a polymerase chain reaction (PCR) based
205
technique and fluorescent in situ hybridisation (FISH). Bacteria
belonging to Acidithiobacillus ferrooxidans (previously Thiobacillus
ferrooxidans), Acidithiobacillus thiooxidans (previously Thiobacillus
thiooxidans), Thiobacillus denitrificans and Thiobacillus thioparus
species were examined. The design of the PCR primers was based
on 16S rRNA genes sequences, applying the AlignX program and
in accordance with literature data. The most suitable sequences
were identified using the AmplifiX program. DNA starters were
synthesised at the Institute of Biochemistry and Biophysics, Polish
Academy of Sciences. The appropriate FISH probes (EUB-338, EUB-II
338, EUB-III 338 and ATT023) were supplied by biomers.net GmbH.
3. Results and discussion
Fig. 1 presents the results of the heavy metal bioleaching in the
two different bioleaching cultures and Tables 2–4 set out the results
of the statistical calculations.
Zinc was removed effectively in both bioleaching media,
although the metal was released more intensively in the strongly
acidic M-I (1% sulphur) medium, achieving a maximum value after
15 days. The zinc bioleaching efficiency in the medium with sulphur and biosurfactant (of pH about 7) needed 25 days to reach the
same level, with an average Zn removal of about 6000 mg/kg (48%).
Copper was bioleached more effectively in the M-I medium,
with an average effectiveness after 15 days of about 20,000 mg/kg
(48.5%). From day 15 to day 25, the rate of copper removal in the MI medium decreased. By the end of the experiment (day 25), it was
about 25%, similar to the effectiveness of the M-II medium (29%).
The effectiveness of lead removal from printed circuit boards
was very low (below 0.5%), with the M-I medium being slightly
more effective than the M-II medium.
In the case of cadmium, the bioleaching effect was similar in
both media, with a temporary increase in Cd release (average
effectiveness of about 93%) at the beginning of the experiment (a
phenomenon also seen in the control sample).
Nickel was removed effectively from the waste in the culture
with 1% sulphur medium (M-I) but not in the biosurfactantcontaining culture. The average Ni removal after 25 days was about
53%.
Only chromium was removed more effectively in the bioleaching medium containing both sulphur-oxidising and biosurfactantproducing bacteria (M-II), with the highest effectiveness on day 2
of experiment and an average Cr removal of 23%.
Fig. 2 presents the maximum effectiveness of bioleaching for
particular heavy metals obtained during the experiment. The highest percentages of metal to be released from the printed circuit
boards were obtained for copper (>90%) and cadmium (100%). In
contrast, only trace amounts of lead were removed from the waste.
An assessment of pH changes in the examined bioleaching
media (Fig. 3) confirmed that the metal bioleaching in M-I and
M-II media occurred in acidic conditions and in a neutral/slightly
alkaline environment, respectively.
The bioleaching cultures were examined in order to determine
the occurrence of bacteria capable of sulphur oxidation in bioleaching media. A PCR-based technique revealed the presence of A.
thiooxidans in both the M-I and M-II media, which was confirmed by
a FISH technique (Photos 1 and 2). A. ferrooxidans, T. denitrificans and
T. thioparus were not detected. The final concentration of sulphuroxidising bacteria in media M-I and M-II was 103 CFU/mL. The total
concentration of heterotrophic bacteria including biosurfactantproducing bacteria was 107 CFU/mL in medium M-II.
The results of the experiment concerning the influence of
temperature and aeration mode on the bioleaching effectiveness
revealed that aeration of the bioleaching medium stimulated the
release of Ni, Cu, Zn and Cr but not Cd and Pb in the M-I medium
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E. Karwowska et al. / Journal of Hazardous Materials 264 (2014) 203–210
7000
25000
6000
Copper removal [mg/kg]
Zinc removal [mg/kg]
20000
5000
4000
3000
2000
15000
10000
5000
1000
0
0
0
5
10
15
20
25
30
0
5
10
15
days
a)
culture with 1% sulphur
20
25
30
days
culture with 1% sulphur and biosurfactant
b)
control
culture with 1% sulphur
100
culture with 1% sulphur and biosurfactant
control
6
90
5
Cdmium removal [mg/kg]
Lead removal [mg/kg]
80
70
60
50
40
30
20
4
3
2
1
10
0
0
0
5
10
15
20
25
30
0
5
10
15
days
c)
culture with 1% sulphur and biosurfactant
d)
control
culture with 1% sulphur
8000
18
7000
16
Chromium removal [mg/kg]
Nickel removal [mg/kg]
culture with 1% sulphur
6000
5000
4000
3000
2000
1000
25
30
culture with 1% sulphur and biosurfactant
control
14
12
10
8
6
4
2
0
0
0
5
10
15
20
25
0
30
5
10
15
culture with 1% sulphur
20
25
30
days
days
e)
20
days
culture with 1% sulphur and biosurfactant
control
f)
culture with 1% sulphur
culture with 1% sulphur and biosurfactant
control
Fig. 1. Average bioleaching from electronic waste: (a) zinc, (b) copper, (c) lead, (d) cadmium, (e) nickel, (f) chromium.
Table 2
Statistical data of heavy metal removal from electronic waste using the bioleaching medium M-I containing sulphur-oxidising bacteria.
Time
Type of the metal
Zn
Cu
Pb
Cd
Ni
Cr
Day 2
Mean value (mg/kg) and standard error
Standard deviation
Variation coefficient
3568.95 ± 35.18
58.81
1.65
16,426.12 ± 87.68
146.56
0.89
66.78 ± 3.48
5.81
8.7
5.01 ± 0.0
0
0
5804.94 ± 68.08
113.8
1.96
7.52 ± 0.58
0.96
12.83
Day 7
Mean value (mg/kg) and standard error
Standard deviation
Variation coefficient
2626.91 ± 1481.39
2476
94.25
14,295.2 ± 7326.2
12,245.0
85.66
24.22 ± 6.34
10.61
43.8
1.19 ± 0.33
0.56
47.26
6173.99 ± 33.46
55.92
0.91
9.19 ± 1.73
2.89
31.49
Day 14
Mean value (mg/kg) and standard error
Standard deviation
Variation coefficient
5998.64 ± 1836.74
3069.93
51.18
19,754.43 ± 13,096.0
21,888.7
110.8
86.01 ± 12.11
20.24
23.54
1.67 ± 0.0
0
0
6193.2 ± 36.34
60.74
0.98
6.68 ± 0.0
0
0
Day 25
Mean value (mg/kg) and standard error
Standard deviation
Variation coefficient
5975.26 ± 1064.89
1779.88
29.79
10,417.46 ± 3869.61
6467.69
62.09
70.98 ± 1.73
2.89
4.08
1.67 ± 0.0
0
0
7039.89 ± 556.67
930.43
13.22
1.67 ± 0.0
0
0
E. Karwowska et al. / Journal of Hazardous Materials 264 (2014) 203–210
207
Table 3
Statistical data of heavy metal removal from electronic waste using the bioleaching medium M-II containing both sulphur-oxidising bacteria and BSAC-producing bacteria.
Time
Type of the metal
Zn
Cu
Pb
Cd
Ni
Cr
Day 2
Mean value (mg/kg) and standard error
Standard deviation
Variation coefficient
4025.52 ± 1599.84
2673.98
66.43
3138.91 ± 1073.59
1794.4
57.17
42.45 ± 14.82
24.77
58.36
5.59 ± 1.31
2.19
39.21
86.75 ± 48.56
81.16
93.55
16.84 ± 10.16
16.98
10,082
Day 7
Mean value (mg/kg) and standard error
Standard deviation
Variation coefficient
4336.1 ± 1797.99
3005.16
69.31
12,457.42 ± 4190.44
7003.92
56.22
46.61 ± 12.54
20.96
44.98
1.53 ± 0.17
0.29
18.66
149.93 ± 44.74
74.78
49.88
12.34 ± 2.77
4.64
37.6
Day 14
Mean value (mg/kg) and standard error
Standard deviation
Variation coefficient
2277.55 ± 1535.39
2566.56
112.88
4979.14 ± 3322.76
5553.67
111.54
52.26 ± 20.33
33.98
65.04
1.43 ± 0.29
0.49
33.98
97.23 ± 44.83
74.93
77.07
11.12 ± 2.04
3.41
30.66
Day 25
Mean value (mg/kg) and standard error
Standard deviation
Variation coefficient
6087.53 ± 4188.3
7000.34
115.0
11,512.31 ± 11,369.16
19,950.12
173.29
29.44 ± 16.74
27.98
95.06
1.48 ± 0.23
0.39
26.06
116.39 ± 39.42
65.89
56.61
9.19 ± 0.41
0.68
7.42
100
80
70
60
culture with 1% sulphur
50
culture with 1% sulphur and
biosurfactant
40
30
20
10
0
Zn
Cu
Pb
Cd
Ni
Cr
Fig. 2. The maximum obtained values of heavy metal removal from printed circuit boards.
9
8
7
6
5
pH
maximum metal removal [%]
90
4
3
2
1
0
0
5
10
15
20
25
days
culture with 1% sulphur
culture with 1% sulphur and biosurfactant
Fig. 3. pH of the bioleaching media during the experiment.
control (water)
30
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E. Karwowska et al. / Journal of Hazardous Materials 264 (2014) 203–210
Table 5
Sulphate ion concentration in various experimental conditions.
Photo 1. Acidithiobacillus thiooxidans in bioleaching medium M-I.
Photo 2. Acidithiobacillus thiooxidans in bioleaching medium M-II.
(1% sulphur culture), while in the M-II medium (1% sulphur and biosurfactant culture) all the metals were bioleached more effectively
in the process that involved aeration with compressed air.
At a temperature of 37 ◦ C, an increase in Cu, Pb, Ni and Cr removal
in the M-I medium was observed, compared with the removal rate
at 22 ◦ C. In the M-II medium, a similar effect was only observed for
nickel and chromium. For other metals, a bioleaching temperature
of 22 ◦ C was favourable at pH 7.
For the M-II medium, the metal removal assessment was accompanied by some additional tests, including ATP content assessment
and sulphate ion concentration.
The amount of adenosine-triphosphate (ATP) in the bioleaching media was determined at the start and after 7 and 14 days
of the bioleaching experiment. The initial ATP concentration in
the 1% sulphur medium was 202.7 mg/L, while in the mixed
Table 4
ATP concentration in M-II bioleaching medium containing surfactin produced by
Bacillus subtilis PCM 2021 and Bacillus cereus PCM 2019 in various experimental
conditions.
Experimental variant
Control (distilled water)
Aerated by shaking (100 rpm) at 22 ◦ C
Aerated with compressed air at 22 ◦ C
Aerated by shaking (100 rpm) at 37 ◦ C
ATP concentration (mg/L)
7 days
14 days
36.49
851.35
1121.62
310.81
13.38
1405.41
1891.89
1351.35
Time (days)
Experimental variant
Sulphate ion
concentration (mg/L)
0
14
14
14
14
–
Control (distilled water)
Aerated by shaking (100 rpm) at 22 ◦ C
Aerated with compressed air at 22 ◦ C
Aerated by shaking (100 rpm) at 37 ◦ C
1039
100
1820
2030
1950
medium containing both sulphur-oxidising and biosurfactantproducing bacteria it was 1858.1 mg/L. Towards the beginning of
the bioleaching process (after 7 days), a significant decrease in ATP
concentration was observed, but after 14 days the concentration
increased almost to the initial value, especially in case of the sample
aerated with compressed air (Table 4), which proves the metabolic
activity of microorganisms during the metal removal process.
The microbial activity in sulphur oxidation was proved by the
sulphate ion assessment (Table 5), which was accomplished at
the beginning and after 14 days of the bioleaching process. It
revealed active sulphuric acid production by A. thiooxidans bacteria
in the bioleaching medium containing both sulphur-oxidising and
biosurfactant-producing bacteria.
The highest values for both ATP concentration and sulphate
ion concentration were observed in the sample aerated with compressed air at room temperature (Tables 4 and 5). The differences
in microbial activity between variants aerated by shaking at temperature 22 ◦ C and 37 ◦ C, respectively, were not significant.
Previous research has suggested that the use of bioleaching for
the recovery of metals from waste could be an economical alternative to traditional PCB treatment processes [5]. The bioleaching
process can be applied to the extraction of metals from solid waste,
such as fly ash, galvanic sludge, spent batteries and electronic
scrap as well as different types of spent catalysts [9,12–17]. The
proposed biological agents useful in metal recovery are sulphuric
acid formed by sulphur-oxidising microorganisms, Fe(III) ions produced by iron-oxidising bacteria or some organic acids produced
in bacterial or fungal metabolism resulting in organic acidolysis,
complexation and chelation [2].
The microorganisms most widely used in bioleaching processes
are acidophilic sulphur-oxidising and iron-oxidising bacteria,
mainly A. ferrooxidans and A. thiooxidans [6,18]. A. ferrooxidans has
been used as model microbe in bioleaching systems [19–22].
The important environmental conditions for the active growth
of bacteria of genus Acidithiobacillus are acidic pH values, high redox
potential (maintained by aeration), and the availability of substrate
(sulphur). A. thiooxidans use reduced forms of inorganic sulphur
but not ferrous iron as energy sources. The bacteria are highly acidophilic (pH 0.5–5.5, optimum pH 2–3.5), and may decrease the pH
in the medium to between 1.5 and 1 and even lower [18].
During metal bioleaching from contaminated sediments, A.
thiooxidans was found to be more effective bacteria than A. ferrooxidans, because it oxidised sulphur rapidly and gave sufficient cell
concentrations in the bioleaching medium [18]. A. thiooxidans can
survive the harsh conditions in a concentrated solution of metals,
with the bioleaching supported by the mechanism facilitating the
attachment of cells to the surface of sulphur or sulphidic particles,
involving effects of electric charges, surface irregularity, cell membrane characteristics and the excretion of extracellular polymeric
substances (EPS). In the case of zinc and cadmium, specific metalbinding proteins are produced by the bacteria [23]. Therefore, the
bioleaching cultures applied in this research were enriched with
A. thiooxidans in presence of 1% sulphur and the presence of these
bacteria in the bioleaching cultures was confirmed using both PCR
and FISH techniques.
E. Karwowska et al. / Journal of Hazardous Materials 264 (2014) 203–210
It was reported that bioleaching systems are usually characterised by low species diversity, even if they are inoculated with
a multi-species bacterial community. Goebel and Stackebrand [24]
identified bacteria from genera Leptospirillium, Acidiphilium, Sulfobacillus, and Acidithiobacillus in a bioleaching system. He et al.
[22], using the denaturing gradient gel electrophoresis (DGGE), PCR
and sequencing over time, confirmed the domination of bacteria
belonging to genera Acidithiobacillus and Leptospirillum in bioleaching of Ni–Cu sulphide.
In this research the low diversity of acidophilic bacteria in
the bioleaching cultures was confirmed – only A. thiooxidans was
present in the media while A. ferrooxidans, T. denitrificans and T.
thioparus were not detected. The activated sludge served as a source
of bioleaching microflora.
Data pertaining to the application of the biotechnological processes for the extraction of metals from the electronic waste
material is still scanty [5]. A few studies have been undertaken
on the biohydrometallurgical removal of metals from electronic
scrap. For example, Faramarzi et al. [25] applied cyanogenic bacteria Chromobacterium violaceum and demonstrated that gold can be
microbially solubilised from printed circuit boards. Brandl et al.
[13] used the mixed culture of A. ferrooxidans and A. thiooxidans
for metal removal from the dust collected from shredding processes of electronic scrap. Moderately thermophilic bacteria were
used for column bioleaching of metals from printed circuit boards
[26]. Wang et al. [2] applied A. ferrooxidans and A. thiooxidans
strains isolated from an acidic mine drainage and acclimated in
the presence of electronic waste to solubilise copper, lead and
zinc from printed wire boards. They revealed that the bioleaching
effectiveness increased with a decrease in the sieve fraction of the
sample and in the waste concentration. For copper, the solubilisation results after 9 days, at a scrap concentration of 7.8 g/L and a
sieve fraction of 0.5–1.0 mm, were 99.0%, 74.9% and 99.9% for the
pure culture of A. ferrooxidans, the pure culture of A. thiooxidans and
a mixture of the strains, respectively. For a sieve fraction <0.35 mm,
the process effectiveness exceeded 88.9% for copper, lead and zinc
in all the tested cultures [2].
The waste applied in this research consisted mainly of two
fractions: 0.1–0.25 mm and 2 mm. It probably influenced the process effectiveness, although even in the presence of larger sample
grains the highest process efficiencies reached >90% for copper and
100% for cadmium. The waste concentration was 6.7%, higher than
that applied by Amiri et al. [9] for the effective metal recovery
(14.2–100%) from spent catalysts by the mould Aspergillus niger
producing gluconic acid (1% and 3%), and comparable with that
applied for bacterial bioleaching [2].
The physico-chemical properties of the electronic scrap may
influence its bioleaching behaviour. The non-metallic components
of the electronic scrap may contribute towards its alkalinity [5],
which results in an increase of the pH of the bioleaching media
[2,13,18,26]. Therefore, techniques to recover metals from scrap
with higher pH values should be of interest. Lan et al. [11] observed
that that the addition of surfactant o-phenylenediamine (OPD) in
low concentrations positively influenced the bio-oxidation of elemental sulphur by mixed cultures of A. ferrooxidans, A. thiooxidans
and L. ferrooxidans [11]. The positive effect of OPD on zinc dissolution rates during oxidative leaching of sphalerite was revealed by
Owusu et al. [27]. The application of microbiological surfactants –
effective, biodegradable and less toxic than synthetic surfactants –
may also support the bioleaching process [28]. In this research the
bioleaching activity of sulphur-oxidising bacteria was supported
with the addition of biosurfactants produced by B. cereus and B.
subtilis strains.
The experiment carried out in this research allowed a comparison between metal bioleaching in a traditional solution acidified
by a way of microbial oxidation of sulphur with a bioleaching
209
process using a microbial culture containing sulphur-oxidising
and biosurfactant-producing bacteria. It was revealed that the use
of biosurfactant-producing bacteria is statistically significant to
the bioleaching process for copper, lead, nickel and chromium.
Zinc was removed effectively in both bioleaching media, but
faster in the acidic environment. The final average process efficacy was 48%. Cadmium removal was similar in both the pH 2–3
and pH 6–8 media, with a highest metal release of about 93%.
For nickel and copper, a better effect was obtained in the acidic
medium, with a process effectiveness of 48.5% and 53%, respectively. Chromium was the only metal that was removed more
effectively in the bioleaching medium containing both sulphuroxidising and biosurfactant-producing bacteria, which had a pH of
about 6–8. Lead was removed from the printed circuit boards with
very low effectiveness (below 0.5%).
The process effectiveness varied depending on the type of metal.
In this research, the highest metal removal rates were observed
for copper and cadmium, while lead was bioleached only in trace
amounts. Some difficulties in lead removal from the scrap might be
related to forming slightly soluble salts [18], a finding confirmed in
literature by the speciation analysis of precipitates [7]. Xin et al.
[6] also revealed that bioleaching mechanisms applied to spent
batteries vary with different metals, species and energy source
types.
Many factors may affect the bioleaching process. Yang et al.
[29,30] revealed that the copper leaching from electronic scrap
using A. ferrooxidans was influenced by the process variables, such
as Fe(III) concentration, quantity of stock culture added, and pH.
They obtained high leaching rates of copper in the presence of
6.66 g/L of Fe3+ , 100% addition quantities of stock culture, and a pH
of 1.5, confirming the feasibility of copper bioleaching from printed
circuit boards. It was stated that for A. ferrooxidans in a sulphide
mineral medium, particulate matter content above 16% was the
main growth-limiting factor due to a decrease in the rates of oxygen transfer and removal of bacterial metabolic products [23]. The
limiting impact of oxygen deficit on metal release efficiency has
been shown [31]. Bioleaching may also be limited by the temperature of the process. Stimulation of acid production by bacteria, and
the specific growth rate and microbial activity, in higher temperatures has been observed [32].
The results of the experiment concerning the influence of
temperature and aeration mode on the bioleaching effectiveness
revealed the different susceptibility of metals to the bioleaching
process in both tested cultures in applied process conditions. The
aeration of the culture medium with compressed air increased the
release of all metals in the medium with sulphur and biosurfactant,
and of nickel, copper, zinc and chromium in the acidic medium. The
influence of a higher temperature (of 37 ◦ C) was more significant
in the acidic environment (four metals release stimulated) than in
the neutral environment (only nickel and chromium). The sample
aeration influenced positively both ATP content and sulphuric acid
production, with no temperature effect.
4. Conclusions
The experiments carried out in this study revealed that although
an acidic bioleaching solution is the most effective in metal
bioleaching from electronic scrap, some metals may be effectively released also in neutral pH using mixed consortium of
biosurfactant-producing bacteria and sulphur-oxidising bacteria,
which may be useful due to alkalinity of the waste. Metals with high
bioleaching potential in pH 6–8 are copper, cadmium and zinc. The
bioleaching process is influenced by the type of aeration and the
temperature of the process, depending on the bioleaching culture
and the type of metal.
210
E. Karwowska et al. / Journal of Hazardous Materials 264 (2014) 203–210
Acknowledgement
The research work was financed by Ministry of Science and
Higher Education in a project no. O680/B/PO1/2009.
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