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Bacterial Cyanide Generation in the Presence of

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Journal of Chemical Engineering of Japan, Vol. 44, No. 10, pp. 692700, 2011 Research Paper

Bacterial Cyanide Generation in the Presence of Metal Ions (Na, Mg2,

Fe2, Pb2) and Gold Bioleaching from Waste PCBs
Chi Dac TRAN1,2, Jae-Chun LEE2, Banshi Dhar PANDEY2,3,
Jinki JEONG2, Kyoungkeun YOO4 and Trung Hai HUYNH5
1
University of Science and Technology (UST ), Daejeon 305-350, Korea
2
Korea Institute of Geoscience and Mineral Resources (KIGAM),
Daejeon 305-350, Korea
3
National Metallurgical Laboratory (NML), CSIR, Jamshedpur, India
4
Department of Energy and Resources Engineering,
Korea Maritime University, Busan, Korea
5
Institute for Environmental Science and Technology,
Hanoi University of Science and Technology, Hanoi, Vietnam

Keywords: C. violaceum, Cyanide Generation, Bioleaching, Printed Circuit Boards, Gold

As an alternative using cyanide chemicals for gold extraction, the application of a cyanogenic bacterium viz.
Chromobacterium violaceum (C. violaceum) in YP medium has been investigated. The catalytic roles of metal ions
such as Na, Mg2, Fe2, and Pb2, as well as the effect of Na2HPO4 nutrient addition on the cyanide generation
efficiency of the bacterium in this medium have been elucidated. While MgSO4 and FeSO4 added to the medium
were equally effective for cyanide generation, improved efficiency was obtained in the presence of Na2HPO4 and
Pb(NO3)2. In order to examine the effectiveness of C. violaceum cultured in YP medium for the generation of
cyanide ions, the dissolution of gold and copper from waste mobile phone printed circuit boards (PCBs), a good
source of gold and copper in alkaline conditions, was tested at 30C, for various pH values and metal ion con-
tents. Gold leaching was found to be 11% in 8 d at pH 11.0 in presence of 4.0  103 mol/L MgSO4, whereas; cop-
per recovery was high (11.4%) at pH 10.0. Addition of 1.0  102 mol/L Na2HPO4 and 3.0  106 mol/L Pb(NO3)2
to the YP medium increased copper leaching to 30.3% and 38.1%, respectively, at pH 10.0 in 8 d. However, this
effect was not observed for gold leaching.

ated metabolic activities. In this study, C. violaceum has

Introduction
Cyanidation is widely used in gold metallurgy to re-
cover gold from ores and concentrates, in spite of the fact
that cyanides are highly toxic. The impact of cyanidation
on the environment, especially on aquatic species and hu-
mans, is well known. In recent years, an alternate ap-
proach to the gold cyanidation process is being consid-
ered, in which the aim is to replace with a micro-organ-
ism, specifically cyanogenic bacteria that can produce
cyanide ions and dissolve gold. So far, several types of
cyanogenic bacteria have been identified and reported,
which include Pseudomonas aeruginosa, P. fluorescens,
Escherichia coli and Chromobacterium violaceum (C.
violaceum) (Moss and Ryall, 1981; Creczynski-Pasa et
al., 2004).
Among these bacteria, C. violaceum has been found
to be the most effective for the bio-dissolution of gold
from different materials because of its cyanide-associ-
Received on October 1, 2010; accepted on July 7, 2011
Correspondence concerning this article should be addressed to
J.-c. Lee (E-mail address: jclee@kigam.re.kr).
been cultured in YP medium containing glycine as one
of the components. It is a mesophilic, motile, Gram-
negative, and facultative anaerobe (Moss and Ryall,
1981). The importance attached to C. violaceum as a
cyanide-generating bacterial species stems from its char-
acteristic features of HCN synthase in comparison to
other cyanogenic bacteria. The versatility of energy me-
tabolism is better for C. violaceum than for bacteria
from any other genus because the former generally has
with six enzymes, whilst other bacteria lack one or more
of these enzymes (Creczynski-Pasa et al., 2004).
Metal cyanidation using a bacteria such as C. vio-
laceum is believed to follow an indirect leaching mecha-
nism involving two stages. First, glycine undergoes con-
version to HCN (leaching chemical) by the associated
enzymes under the metabolic activity of HCN synthase
(stage I) (Knowles and Bunch, 1986). This is followed
by reaction of the cyanide ions with the gold/copper sub-
strate, leading to dissolution of metal (stage II), as pre-
sented in Figure 1.
Actually, the amount of cyanide generated by C.
violaceum strongly depends on the medium in which it

692 Copyright 2011 The Society of Chemical Engineers, Japan


Fig. 1 Schematic of metal cyanidation using a microorganism
is grown. In previous investigations, media with differ-
ent organic componentspeptone 1% (w/v), L-glutamate
salts (Michaels and Corple, 1965); peptone 1% (wt/v),
2 mmol/L-glycine or 0.5 mmol/L-methionine (Rodgers
and Knowles, 1978); and several other organic compo-
nents (Collins et al., 1980) have been used. Besides or-
ganic components, some metal ions such as Na and
Mg2 added to the medium in low concentrations can
catalyze and enhance the cyanide generation capacity of
C. violaceum.
Addition of Na2HPO4 in small amounts is also
found to increase the amount of cyanide generated by
such bacteria. In earlier researches, the effect of addition
of these metal ions on the metabolic activity of bacteria
has been studied using media containing different or-
ganic components such as tryptone (10 g/L) and yeast
extract (5 g/L) (Faramarzi et al., 2004; Brandl et al.,
2008), or polypeptone (10 g/L) and yeast extract (2 g/L)
(Kita et al., 2005, 2008; Lawson et al., 1999). It is,
therefore, very difficult to compare the effect of different
metal ions on the cyanide generation capacity of C. vio-
laceum in a particular medium.
While the gold bioleaching process using a mi-
croorganism may be considered one of the most eco-
friendly options, the dissolution rate may not be as fast
as that in the case of direct addition of cyanide salts.
Earlier researches (Kita et al., 2005; Lawson et al.,
1999) have shown that the presence of some metal ions
can improve the bacterial cyanidation process. An en-
hanced rate of gold dissolution was observed with
cyanide solutions in presence of small amounts of
Pb(NO3)2, which activated the gold surface, leading to
higher reactivity and faster leaching (Sandenbergh and
Miller, 2001; May et al., 2005). However, the effect of
Pb2+ on the extent of cyanide generation by cyanogenic
micro-organisms has not been investigated.
For the application of cyanide-generating bacteria
to gold and copper leaching, major emphasis has been
placed on understanding the dissolution behavior of
metal powders (Brandl et al., 2008; Kita et al., 2006;
Smith and Hunt, 1985) in the presence of various mi-
crobes, and in a few cases the extraction of gold from
ores (Lawson et al., 1999). Recently, efforts were made
to treat secondary sources including waste printed circuit
boards PCBs, by using cyanobacteria (Faramarzi et al.,
2004; Brandl et al., 2008). In view of the fact that waste
mobile phone PCBs are a rich source of gold, besides
copper and other metals, the microbial recovery of gold
by cyanogenic bacteria may be worthwhile to pursue as a
part of the overall objective to develop a favorable recy-
cling technology for waste PCBs. The residue from bio-
leaching could then be treated by a suitable hydromet-
allurgical process to extract copper and other metals.
Thus, the effect of different metal ions including Pb2
and Na2HPO4 on cyanide generation by C. violaceum in
a medium with the predetermined organic components
has been investigated in this study. The potential use of
the biogenic cyanide produced from C. violaceum in the
presence of these metal ions to dissolve gold from waste
mobile phone PCBs has also been examined to establish
an alternative recycling process.
1. Experimental
1.1 Microorganism and waste PCBs
C. violaceum was supplied by Korea Collection for
Type Cultures (KCTC), Biological Resource Center
(BRC), Daejeon. Revival of C. violaceum in a medium
comprising 5 g/L polypeptone and 3 g/L beef extract in
100 mL solution was performed at pH 6.8 and 30C in
an incubator shaker at 150 rpm for 2 d. To check the cul-
ture purity, the revived culture was incubated on an agar
plate for 24 h at 37C. Viable suspended cells were
counted using the drop plate method with serial dilution
in an agar medium (beef extract 3.0 g/L, peptone 5 g/L,
agar 15 g/L). The plates containing pure cultures were
sealed with parafilm and stored under refrigerator. A
known volume of the culture medium containing viable
cells was inoculated aseptically for investigating the ef-
fect of different metal ions on the amount of cyanide
generated. In this research, a growth medium consisting
of yeast extract (5 g/L), polypeptone (10 g/L), and
glycine (5 g/L), designated as YP medium, was used.
Samples of waste mobile phone PCBs for the bioleach-
ing experiments were cut to 1 mm  1 mm size by using
scissors. Analysis showed a typical sample to consist of
around 34.5 wt% copper and 0.025 wt% gold. The effect
of different metal ions and Na2HPO4 on the cyanide pro-
duction of C. violaceum in the YP medium was studied.
Preliminary experiments were conducted by addition of
NaCl, MgSO4, and FeSO4 to the cultures, in order to de-
termine the metal ion concentrations at which the cell
population was not significantly affected. By adding
0.17 mol/L NaCl, 4.0  103 mol/L MgSO4, 1.8  103
mol/L FeSO4, and a mixture of these salts, besides vary-
ing the Pb(NO3)2 concentration in the range 3.0  106
to 6.0  105 mol/L, the cyanide concentration in the in-
oculated medium was measured. The concentration of
phosphate in the medium containing C. violaceum was
varied in the range 1.0  10-2 to 3.0  10-2 mol/L by
the addition of phosphate buffer. The metal content was
analyzed by atomic absorption spectrometry (AAS,
AAnalyst 400, Perkin Elmer Inc.).
693
1.2 Bioleaching
All experiments were carried out in a 250-mL ster-
ilized Erlenmeyer flask containing 200 mL of the YP
medium. A 1 mL aliquot of C. violaceum under log
phase culture conditions was added to the flask asepti-
cally. The desired amounts of phosphate buffer solution
and metal ions were then transferred to the flask. The pH
of the solution was adjusted by the addition of 2.0 mol/L
NaOH solution. The flask with the inoculated set was
placed in an incubator shaker maintained at 30C and in-
cubated for a specified period. The supernatant sample
was withdrawn every 24 h, and the cell count, pH, and
quantity of cyanide generated were determined. For each
inoculated set, a control experiment without metal ions
was also carried out. No air supply was maintained dur-
ing the experiments. The bacterial population was deter-
mined by counting the colonies, followed by cell count
on an agar plate incubated at 37C for 24 h, which was
correlated with the optical density. The morphological
features of C. violaceum were observed under a biologi-
cal microscope (BX 51, Olympus Corp.) and the cell
population was confirmed using a counting chamber.
In order to examine the bio-dissolution of gold and
copper from waste mobile phone PCBs, a known amount
of the sample (1 mm  1 mm in size) was placed in a
250-mL flask containing 200 mL of the medium inocu-
lated with a 2% active culture of C. violaceum. The
flasks were incubated at 30C and 15 g/L pulp density in
an orbital motion incubator with shaking at 150 rpm
while maintaining the pH in the range 8.01.0. The su-
pernatant was withdrawn at 24 h intervals to conduct a
bacterial cell count, and to measure the pH, dissolved
oxygen (DO), and cyanide concentration. After the bio-
leaching period was complete, the solution was filtered
through Whatman 42 filter paper. The obtained clear so-
lution was analyzed for gold and copper, and the per-
centage of metal leached was calculated (leaching effi-
ciency, %). The residue obtained in each experiment was
dissolved in aqua regia, and the metal in the leached and
aqua regia solutions was analyzed by AAS to calculate
the material balance. The concentration of cyanide gen-
erated by the bacteria during the process was determined
colorimetrically by UV/Vis spectrometry (UV-1601PC,
Shimadzu Corp.) at 580.5 nm using pyridine and barbi-
turic acid (Clescert et al., 1998). For this, 5 mL of the
sample supernatant was withdrawn from the experiment
flask, and the pH was adjusted to 11.0 with 4 mol/L
NaOH solution before filtration. To 10 mL of the clear
solution, 4 mL of NaH2PO4 (138 g/L) was added, and the
mixture was allowed to stand for 3 min; 2 mL of chlo-
ramine-T (10 g/L in distilled water) was added. After
5 min, 5 mL of the pyridinebarbituric acid mixture
(75 mL pyridine, 15 g barbituric acid in 1 L water) was
added to develop a stable violet color. The cyanide con-
centration was then analyzed colorimetrically at
580.5 nm against a standard solution. The DO value and
pH were determined at 24-h intervals by a DO meter
694
(HQ40d, Hach Co.) and a pH meter (720A, Orion
Research Inc.), respectively. Data given in the text are
the average values of duplicate sets of experiments, with
an accuracy of 3.0%.
2. Results and Discussion
2.1 Cyanide generation by C. violaceum
2.1.1 Bacterial cell population and cyanide generation
in the presence of metal ions The bacterial cell
population was determined in presence of different
metal ions at 30C and an initial pH of 7.4. The results
in Figure 2(a) show that the growth of C. violaceum
reached the log phase after 2 d and that the maximum
cell count was achieved in 5 d irrespective of the addi-
tion of metal ions. The decrease in the cell population
after 5 d of incubation could be attributed to the decrease
in cell multiplication once the optimum cell growth in
the medium was attained, owing to the short stationary
phase typically observed for cyano-bacteria. This also
reflects the decreasing metabolic activity of such mi-
crobes with time (Knowles and Bunch, 1986). When no
metal was present in the YP medium, the cell count
reached a maximum of 12.3  108 cells/mL, with very
little decrease in the bacterial population (11.3  108
cells/mL) until 7 d. Addition of 0.17 mol/L NaCl, 4.0 
103 mol/L MgSO4, and 1.8  103 mol/L FeSO4 sepa-
rately and the addition of a mixture of these salts to the
medium adversely affected the cell population. The cell
populations in the medium containing NaCl and the
mixture of metal salts were much lower than those in the
presence of MgSO4 and FeSO4. This could be attributed
to the toxicity and inhibition of bacterial growth caused
by the presence of Cl ions and the high concentration
of metal ions. While Fe2 was precipitated at pH 7.4 or
higher, adsorption of a number of bacterial cells led to
the observed decrease in the cell population in the pres-
ence of FeSO4.
In contrast to the effect of metal ion concentrations
on the cell population, the addition of a small amount of
metal ions can increase cyanide generation by C. vio-
laceum (Figure 2(b)). This is due to a catalytic effect en-
hancing the enzymatic process (Lawson et al., 1999).
As can be seen in the figure, Fe2 and Mg2 were
the most effective in enhancing cyanide generation,
yielding cyanide concentrations of 61 mg/L and
59 mg/L, respectively, in 5 d at an initial pH of 7.4. The
cyanide concentration obtained after the addition of
mixed metal ions was found to be approximately similar
(36 mg/L) to that observed in absence of these addi-
tives, after 7 d. Therefore, the YP medium with Mg2
was chosen for the subsequent experiments.
As air was not supplied to the medium, DO de-
creased quickly from 6.8 to 0.1 mg/L after the first day
of incubation (Figure 2 (c)). However, this drop in the
level of DO corresponded to an increase of the cell pop-
ulation as well as the cyanide concentration of the solu-
JOURNAL OF CHEMICAL ENGINEERING OF JAPAN

Fig. 2 Effect of metal ions on (a) cell population of C. vio-
laceum, (b) cyanide generation, and (c) consumption
of DO in the YP medium (temperature, 30C; initial
pH, 7.4)
tion for up to 5 d after which a decreasing trend was
observed. The consumption of dissolved oxygen is gen-
erally associated with the growth phase and bacterial
respiration (Kita et al., 2008).
The presence of various metal ions in the medium
had no effect on the oxygen consumption during the
bacterial growth. Thus, the influence of the dissolved
oxygen concentration on cyanide generation by C. vio-
laceum was not very significant after 24 h, and sustained
VOL. 44 NO. 10 2011
Fig. 3 Effect of Pb(NO3)2 on cyanide generation by C. vio-
laceum in the YP medium (4.0  103 mol/L MgSO4;
temperature, 30C; initial pH, 7.4). * YP media
with 4.0  103 mol/L MgSO4 and 1.0  102 mol/L
Na2HPO4
bacterial metabolism was confirmed to improve the
cyanide generation. During the growth of C. violaceum
in the YP medium that was not supplemented with metal
ions, the pH dropped quickly from 7.4 to 6.3 after one
day and thereafter increased to 8.1 in 6 d. The presence
of metal ions, particularly Mg2, or a mixture of salts re-
sulted in a similar trend with respect to the increase in
pH, which was found to be nearly 8.5 after 7 d. The de-
crease in the pH of the medium was attributed to the
acidification process for the conversion of organic com-
pounds, particularly glycine, into carboxylic acids such
as glyoxylic, cyanoformic, and oxamic acids. It was for
this reason that the quantity of cyanide generated in first
2 d was not high (Figure 2(b)). The increase in pH after
this point was accompanied by the increased generation
of cyanide via the conversion of intermediate products
such as carboxylic acids. Increased cyanide generation
occurred from the second day onwards and reached the
maximum after 5 d. A similar behavior has been ob-
served previously (Smith and Hunt, 1985).
In view of the improved dissolution of gold powder
by C. violaceum in presence of Pb2 (Sandenbergh and
Miller, 2001; May et al., 2005), the effect of Pb(NO3)2
on cyanide generation in the YP medium was also inves-
tigated. Because of the small difference in the cyanide
generation observed in the presence of Mg2 and Fe2,
4.0  103 mol/L of MgSO4 was added to the medium;
the results are presented in Figure 3.
It is apparent that the presence of Pb2 in the
medium affected the amount of cyanide produced by C.
violaceum. With 3.0  106 mol/L (1 mg/L) Pb(NO3)2
added to the YP medium, a higher quantity of cyanide
was generated (66 mg/L in 5 d and 59 mg/L in 7 d). A
Pb(NO3)2 concentration above 3.0  106 mol/L inhib-
ited the bacterial cyanide generation. As observed below,
the cyanide concentration was, however, found to be
695
Fig. 4 Cyanide generated by C. violaceum at varying phos-
phate concentrations in YP medium (4  103 mol/L
MgSO4; temperature, 30C; initial pH, 7.4)
slightly higher (69 mg/L in 5 d) with 1.0  102 mol/L
Na2HPO4 even in the absence of Pb(NO3)2. Incidentally,
the presence of a similar amount of Pb(NO3)2 has been
reported to improve gold recovery in the cyanidation
(chemical) process (Sandenbergh and Miller, 2001).
2.1.2 Effect of Na2HPO4 Phosphate has been re-
ported to increase the cyanogenesis process in C. vio-
laceum (Rodgers and Knowles, 1978; Lawson et al.,
1999). Its effect on cyanide generation in the YP
medium was investigated in presence of 4.0  103
mol/L MgSO4. As can be seen from Figure 4, the addi-
tion of phosphate buffer affected the efficiency of
cyanide generation. Cyanide generation was the highest
(69 mg/L in 5 d) when 1.0  102 mol/L of Na2HPO4
was added to the medium; a further increase in the
Na2HPO4 concentration decreased the cyanide concen-
tration in the solution, as reported in previous observa-
tion (Lawson et al., 1999).
The amount of cyanide generated (data not given in
Figure 4) was found to increase to 73 mg/L at pH 9.0 in
5 d. The concentration was found to be 63 mg/L and
58 mg/L at pH 10.0 and 11.0, respectively, after the same
culture period.
2.2 Bioleaching of gold and copper from waste mo-
bile phone PCBs by C. violaceum
Before examining the potential leaching of gold
from waste mobile phone PCBs by C. violaceum in a YP
medium containing 4.0  103 mol/L MgSO4, the effect
of pH on cell population and cyanide generation was in-
vestigated. The results presented in Figure 5(a) show
that the cell population decreased with an increase in the
pH of the medium. At pH 7.4, the cell population
reached a maximum (1.18  109 cells/mL) after 5 d of
culture, while at pH 11.0, the cell population decreased
to a lower value of 8.7  108 cells/mL. On the other
hand, the cyanide concentration at 5 d, presented in
Figure 5(b), increased from 59 mg/L to 68 mg/L when
the pH was raised from 7.4 to 9.0. The cyanide concen-
696
Fig. 5 Effect of pH on cell population of C. violaceum (a)
and cyanide generation (b) in the YP medium (4.0 
103 mol/L MgSO4; temperature, 30C)
tration dropped to 56 mg/L at pH 9.0 after 7 d under
these conditions. At pH 11.0, the cyanide concentration
was low and was found to be 50 mg/L after 7 d. In the
presence of the cyanide generated in the YP medium,
both gold and copper can dissolve, forming complexes
with the cyanide ions. Copper cyanide is produced in the
form of various complexes, depending on the pH and
other conditions, whereas gold mostly forms dicyanide
anions [Au(CN)2]. Since leaching of gold in the
cyanidation process critically depends on the solubiliza-
tion of copper in the sample, we have focused on the dis-
solution behavior of the latter along with that of in this
research. As can be seen below (reactions 14), the dis-
solution of gold and copper to form cyanide complexes
is thermodynamically favorable at 298 K in the presence
of oxygen (Alonso-Gonzales et al., 2010; Rees and
Vandeventer, 1999):
4 Au  8CN  O 2  2 H 2 O  4 Au ( CN )2  4 OH (1)
G298
0
392.2 kJ/mol
4 Cu  8CN  O 2  2 H 2 O  4 Cu ( CN )2  4 OH (2)
G298
0
322 kJ/mol
JOURNAL OF CHEMICAL ENGINEERING OF JAPAN

Fig. 6 Effect of pH on gold (a) and copper (b) leaching in
the presence of MgSO4 in YP medium (4.0  103
mol/L MgSO4; pulp density, 15 g/L; temperature,
30C)
Cu ( CN )2  CN  O 2  2 H 2 O  Cu ( CN )32  4 OH (3)
G298
0
228.6 kJ/mol
Cu ( CN )32  CN  O 2  2 H 2 O  Cu ( CN )4 3  4 OH (4)
G298
0
 163.3 kJ/mol
Copper cyanide complexes, particularly tetra-cyanate,
are more stable above pH 9.0 (Marsden and House,
1992) and tri-cyanate (stable in the pH range 7.012.0)
can dissolve gold when the cyanide concentration is high
(Vukcevic, 1996, 1997; Rees and Vandeventer, 1999).
With low concentrations of free cyanide, the Cu(CN)2
complex will be the most prevalent species, and it may
not oxidize gold.
2.2.1 Effect of pH on cyanidation/leaching of metals in
the presence of MgSO4 The effect of pH on the
leaching of gold and copper from waste mobile phone
PCBs was studied at 30C and 15 g/L pulp density in
presence of 4.0  103 mol/L MgSO4 in the YP medium;
the results are shown in Figure 6.
It is apparent that gold dissolution from the waste
PCBs increased from 7.8% (0.35 mg/L) to a maximum
VOL. 44 NO. 10 2011
value of 11% (0.46 mg/L) when the pH was increased
from 8.0 to 11.0 (Figure 6 (a)). The trend was observed
in the leaching of copper was found to be similar to that
in the case of gold. The copper leaching efficiency was
found to increase with pH, but a maximum of 11.4%
(878 mg/L) was achieved at pH 10.0, and this value de-
creased marginally to 10.2% (815 mg/L) when the pH
was further raised to 11.0 after 8 d of leaching (Figure
6(b)). Though, cyanide generation was higher (68 mg/L
in 5 d and 56 mg/L in 7 d) at pH 9.0 (Figure 5(b)), high
gold and copper leaching was observed at pH 11.0 and
10.0, respectively. This may be attributed to the require-
ment of a higher pH to form stable metal cyanide com-
plexes under the present experimental conditions
(Marsden and House, 1992). At pH 11.0, the cyanide
concentration was analyzed to be 54 mg/L after 5 d and
50 mg/L after 78 d when gold leaching was the maxi-
mum (11%).
2.2.2 Effect of pH on cyanidation/leaching of metals in
the presence of Na2HPO4 The effect of the addition
of 1.0  102 mol/L Na2HPO4 to the YP medium on
metal dissolution was investigated for varying pH values
in the range 8.011.0; the results are presented in Figure
7.
In the presence of Na2HPO4, the gold and copper
dissolution trend was similar to that observed in the
presence of Mg2+ up to a pH of 9.0. Gold leaching was
not significantly different in the pH range 8.011.0, with
a maximum dissolution of 9.2% gold at pH 11.0 (Figure
7(a)). Similarly, copper recovery was low within the pH
range 8.09.0. However, copper recovery was found to
increase substantially to 30.3% and 29.2% at higher pH
values of 10.0 and 11.0 (Figure 7(b)), respectively. Thus,
it is apparent that because the amount of cyanide gener-
ated (Figure 4) in the presence of Na2HPO4 is higher
than that in the presence of MgSO4 (Figure 5(b)), copper
leaching is more efficient than gold dissolution from
PCBs under the above mentioned conditions. Therefore,
pH is a critical factor affecting copper recovery, besides
cyanide concentration.
2.2.3 Effect of Pb(NO3)2 on leaching/cyanidation of
metals The results obtained for the addition of
3.0  106 mol/L Pb(NO3)2 to the YP medium in the ab-
sence of Na2HPO4 and MgSO4 are presented in Figure 8
for gold and copper bioleaching at pH 11.0 and 10.0, re-
spectively. To compare the effect of other additives, the
dissolution patterns of gold and copper in the presence
of Na2HPO4 and MgSO4 are also shown for the same
conditions. On the basis of the optimum pH derived
from Figures 6 and 7, the leach recovery of gold and
copper is also briefly mentioned (plots not presented) for
pH 10.0 and 11.0, respectively, in the presence of a small
amount of Pb(NO3)2. Copper bioleaching at pH 10.0 was
found to be higher (38.1%) than that at pH 11.0 (29.3%,
data not shown) in the presence of a small amount of
Pb(NO3)2. However, higher gold bioleaching (10.1%)
was observed at pH 11.0 (Figure 8(a)) than at pH 10.0
697

Fig. 7 Effect of pH on gold (a) and copper (b) leaching in
the presence of Na2HPO4 in YP medium (1.0  102
mol/L Na2HPO4; pulp density, 15 g/L; temperature,
30C)
(9.4%, data not shown). Figure 8(b) also shows that cop-
per bioleaching can be increased by 3 to 4 times when
MgSO4 is substituted by Na2HPO4 and Pb(NO3)2 in the
YP medium. As can be seen, copper bioleaching was
11.4% (878 mg/L) with MgSO4 which increased to
30.3% (2,156 mg/L) and 38.1% (2,668 mg/L) in the
presence of small amounts of Na2HPO4 and Pb(NO3)2,
respectively. In contrast, gold leaching in the presence of
MgSO4 at pH 11.0 (11%) was only marginally higher
than that in the presence of Na2HPO4 (9.2%) and
Pb(NO3)2 (10.1%) (Figure 8(a)).
From these results, it can be said that the addition
of a low concentration of Na2HPO4 and Pb(NO3)2 en-
hances copper leaching because of the generation of
a higher quantity of cyanide, (typically 69 mg/L and
66 mg/L, respectively) at pH 7.4, as compared to that in
the presence of MgSO4 (59 mg/L cyanide at pH 7.4).
Similarly, at higher pH values of 10.0 and 11.0, the
concentration of cyanide generated in the presence of
Na2HPO4 was clearly higher (63 mg/L and 58 mg/L in
5 d) than that in the presence of MgSO4 (57 mg/L and
54 mg/L cyanide), thus favoring copper bio-dissolution.
698
Fig. 8 Effect of different metal ions on the bioleaching of
gold at pH 11.0 (a) and copper at pH 10.0 (b) in the
YP medium (pulp density, 15 g/L; temperature, 30C)
The concentration of copper obtained in the pres-
ence of Pb(II) is higher than that obtained in the pres-
ence of Na2HPO4, probably because of the activation of
the copper surface by Pb(II), which increases the reactiv-
ity of the surface toward cyanide. The higher solubility
of copper over gold may also be related to the electro-
chemical behavior of the two metals, as gold is nobler
0 0
(EAu 3
/Au: 1.52 V) than copper (ECu2/Cu: 0.34 V), and
therefore, copper may dissolve preferentially while con-
suming bio-genic cyanide. Secondly, gold leaching will
be facilitated in the presence of a high concentration of
cyanide (Marsden and House, 1992), and the low level
of bio-generated CN in the present case clearly favors
the copper leaching at the expense of gold. As men-
tioned above, the Cu(CN)2 complex available because
of the low cyanide concentration and DO in this study
will have very weak ability to dissolve gold.
In Figure 9, the variation in the redox potential
during the leaching of waste PCBs shows a low value of
potential (about 2 mV at 6 d) at pH 11.0 (Figure 9(b)),
favoring gold leaching over leaching at a low pH of 10.0
and below. The leaching behaviors of gold and copper at
pH 9.0 and below were different from those at pH 10.0
and 11.0. This may be attributed to the stability of the
JOURNAL OF CHEMICAL ENGINEERING OF JAPAN

Fig. 9 Variation of the potential during bioleaching of cop-
per at pH 10.0 (a) and gold at pH 11.0 (b) from waste
mobile phone PCBs with C. violaceum in the YP
medium (pulp density, 15 g/L; temperature, 30C)
iron hexacyanide complex [Fe(CN)64] up to pH 9.0,
which may decompose at higher pH and thus contribute
to metal dissolution (Rees and Vandeventer, 1999).
Our recent studies (data not included) involving the
addition of 0.004%(v/v) hydrogen peroxide to supple-
ment the DO level during the leaching of waste mobile
phone PCBs by C. violaceum show that copper recovery
and gold recovery can be improved by 810% and 23%
over those obtained in this study, depending on the metal
ions present.
In view of the above results, it appears that gold
bioleaching is adversely affected in the presence of a
high copper concentration in the PCBs, and thus, it is
necessary to reduce the copper content of the sample.
The low leaching of gold may eventually affect the ex-
ploitation of the results. In order to make the present
process/technology suitable for complete recycling of
waste mobile phone PCBs, the gold recovery process
must be improved. Precious metals, particularly gold,
are mostly present as bonding wires and coated thin
films in PCBs.
As observed in our earlier research (Korea Institute
VOL. 44 NO. 10 2011
of Geoscience and Mineral Resources, 2010), by the
physical beneficiation of waste PCBs, such as grinding
and gravity separation, gold can be enriched (about
8085 wt%) in the separated non-metallic portion con-
taining a low quantities of metals. Therefore, it would be
desirable to reduce the copper content by physical bene-
ficiation and study the bioleaching of gold from the
gold-enriched non-metallic component using C. vio-
laceum. The major metallic portion enriched with copper
and other metals can be treated by a suitable hydrometal-
lurgical method to recycle the waste mobile phone PCBs
completely.
The main aspects of this research are as follows.
The amounts of biogenic cyanide produced by C. vio-
laceum in the presence of various metal ions in a partic-
ular medium (YP medium) were compared. The cyanide
generated was used in gold leaching from waste mobile
phone PCBs in order to recycle it, while understanding
the effect of high copper content in the sample on the
bio-dissolution of gold.
Conclusion
Cyanide generation by cyanogenic bacteria
Chromobacterium violaceum in YP medium can be en-
hanced by adding a low amount of metal ions to the cul-
ture medium. The addition of MgSO4 and FeSO4 to the
medium was found to be equally effective for cyanide
generation by the bacteria, and the presence of Na2HPO4
and Pb(NO3)2 enhanced cyanide generation further. The
dissolved oxygen concentration did not affect cyanide
generation by C. violaceum. The results of bioleaching
of valuable metals from waste mobile phone PCBs
showed that the maximum amount of gold could be
leached out (11% in 8 d) at pH 11.0 in the presence of
4.0  103 mol/L MgSO4 in the YP medium; however,
the copper recovery was high (11.4%) at pH 10.0.
When 1.0  102 mol/L Na2HPO4 and 3.0  106 mol/L
Pb(NO3)2 were added to the YP medium, copper recov-
ery was found to increase almost three fold (30.3%) and
four fold (38.1%) at pH 10.0, respectively, in 8 d. The
presence of phosphate and Pb(NO3)2 in the medium fa-
vored copper bio-dissolution, but it was not effective for
gold leaching. The low concentration of cyanide gener-
ated by the metabolic activity of C. violaceum, and dis-
solved oxygen, favored copper bioleaching at the ex-
pense of gold from the PCBs. The results demonstrate
the possibility of recovering gold and copper using C.
violaceum.
Acknowledgements
This study was supported by the Korea Institute of Energy
Technology Evaluation and Planning (KETEP) under the project en-
titled Development of New Technology for the Recycling of Rare
Metals from Urbane Ore. One of the authors (Chi Dac Tran) is thank-
ful to the Korea Institute of Geoscience and Mineral Resources
(KIGAM) for providing the fellowship. Dr. B.D. Pandey is also thank-
ful to the Korean Federation of Science and Technology Societies for
699
 the Brain Pool Scientist award.
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