SME Annual Meeting

Feb. 19 - 22, 2012, Seattle, WA

Preprint 12-034
EVALUATION OF CYANIDE PRODUCING MICROORGANISMS TO RECOVER GOLD FROM LOW-GRADE ORE
D. Shin, Korea Inst. of Geoscience and Mineral Resources, Daejeon, Republic of Korea
J. Jeong, Korea Inst. of Geoscience and Mineral Resources, Daejeon, Republic of Korea
B.D. Pandey, Nat. Metallurgical Lab. - Council of Scientific & Industrial Research (CSIR), Jamshedpur, India
J. Lee,, Korea Inst. of Geoscience and Mineral Resources, Daejeon, Republic of Korea

analyzed by X-Ray Diffraction. The analytical data were shown in Table

1. Some of the samples for the experimental tests were prepared by
grinding and sieving to <180 m.

INTRODUCTION
The gold leaching using cyanide from ore or waste has been
almost universally in use [1]. Recently, biological gold bioleaching using
cyanide producing bacteria, e.g. Chromobacterium violaceum,
Pseudomonas fluorescens, or P. aeruginosa, has been investigated as
environmental friendly and cost effective method. Among them, C.
violaceum has been reported as effective cyanide producing bacterium
on gold leaching [2, 3]. C. violaceum produces cyanide using glycine
as a direct precursor and subsequently detoxify the cyanide by cyanoalanine synthase [4]. However, until today only 0.026 16.9 mg/L
of cyanide production in YP medium has been reported in the
literatures by C. violaceum [4-6]. Comparing with adding cyanide
chemical, biogenic cyanide concentration is lower than the cyanide
concentration added in traditional cyanidation. Since cyanide also
forms complexes with a number of other elements such as iron, nickel,
zinc, silver, copper, and platinum, it is very important for gold recovery
to investigate the enhancement of biological cyanide production and
set optimal experimental condition.

Table 1. (a) Chemical and (b) mineralogical analysis of the ores.

(a)
R
S
H
*
AuCN
AuCN
ppm
0.629
1.389
6.184
AuFAA
ppm
1.149
1.343
21.201
**
AuFAA
AuCN/FA
%
55%
100%
29%
Ag
ppm
13.9
<2.0
176.6
ICP-260-X
Cu
ppm
60
120
136
* AuCN represents total cyanidable gold
** AuFAA represents total gold content analyzed by fire assay
(b)

Most of early studies on gold bioleaching by C. violaceum have

been performed to investigate gold leaching mechanism using gold
powder, and recently various resources such as ores, electronic scrap,
and printed circuit boards of waste electronics [7]. Few studies have
dealt with gold bioleaching from low-grade ore. In this research the
feasibility of gold bioleaching by C. violaceum from low-grade ores
containing gold was evaluated and fundamental study was performed
for further scale-up study. The aim of this study was to improve the
potential of gold bioleaching by C. violaceum from gold-containing ore;
in particular, enhancement of biological cyanide production.

MATERIALS AND METHODS

H

Bacterial strains and culture conditions

T
Chromobacterium violaceum DSM 30191 and Acidithiobacillus
T
ferrooxidans DSM 14882 were purchased from DSMZ (Braunschweig,
Germany) culture collection. Cyanide-producing bacterium, C.
violaceum was grown in yeast extract/peptone medium (YP medium;
peptone 10 g, yeast extract 5 g, distilled water 1 L) at 30C. For
cyanide production assay, 200 mL of YP medium were added in 250
mL Erlenmeyer flasks and adjusted pH 6.7, 8, 9, 9.5, 10, and 11 with 1
M NaOH. The glycine concentration in YP medium was changed in the
range of 0.5, 1, 2, 5, 10, and 20 g/L. Five-milliliter of pregrown C.
violaceum in YP medium was inoculated. The flasks were incubated
five days on a shaking incubator (220 rpm) at 30C. Ten milliliters of
samples were taken every day to analyze bacterial density and cyanide
concentration. Bacterial density was determined spectrophotometrically
by monitoring optical density at 600 nm.

Mineral
Quartz (SiO2)
Sanidine (K(Si3AlO8)
Pyrite (FeS2)
Muscovite (KAl2Si3AlO10(OH)2)
Magnetite (Fe3O4)
Gibbsite (Al(OH)3)
Hematite (Fe2O3)
Goethite (FeO(OH))
Kaolinite (Al2Si2O5(OH)4)
Rutile (TiO2)
Anatase (TiO2)
Magnetite (Fe3O4)
Quartz (SiO2)
Muscovite (KAl2Si3AlO10(OH)2)
Pyrite (FeS2)
Kaolinite (Al2Si2O5(OH)4)
Wollastonite (CaSiO3)

Shake flask culture

The gold bioleaching from the ores were determined in shake
flask culture with 20, 50, 100 g/L of pulp density. Five grams of the ores
were added into 250 mL of Erlenmeyer flask containing 200 mL of YP
medium and 5 mL of pregrown C. violaceum in YP medium was
inoculated. The flasks were incubated for five days on a shaking
incubator (220 rpm) at 30C. Ten milliliters of samples were taken every
day to analyze gold, copper, and silver concentration by atomic
absorption spectroscopy (AAS).
To enhance the bioleaching efficiency, four enhancement factors
were applied as follows: 1) pre-incubation, 2) grinding, 3) pH
adjustment, 4) pretreatment.

For pretreatment, A. ferrooxidans was maintained in the 9K

medium [8] which was filter-sterilized and adjusted to pH 1.8 with 0.1 N
H2SO4. The bacterial culture was cultivated on a shaking incubator with
220 rpm, at 30C.

1)

Ore characterization
The gold and silver content of ore samples was analyzed by fire
assay and copper was analyzed by inductively coupled plasma
emission spectrometry (ICP-ES). The mineralogy of the ores was

2)
3)

C. violaceum was inoculated into YP medium without glycine

and incubated overnight. Five grams of the ores and 5 g/L of
glycine were added in this pre-incubated culture and reacted
for five days.
The same procedure with 1) was used, except for using both
of ground (<180 m) and unground ores
The same procedure with 1) was used, except for adjusting

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pH to 11 when glycine was added.
The ores was incubated with A. ferrooxidans for 7 days
before incubating with C. violaceum. The rest experimental
procedure was the same with 1).

4)

2.0

1.6

Bacterial growth (OD600)

Analytical methods
Total cyanide concentration was analyzed by ion-selective
electrode (ISE) for cyanide (Orion ionplus combination cyanide
electrode; 9606BNWP, Thermo Scientific, USA) and titration against
standard AgNO3 solution. Gold, copper, silver content in leached liquor
was analyzed by atomic absorption analysis (AAnalyst 400, PerkinElmer, USA).
RESULTS AND DISCUSSIONS

Bacterial growth (OD600)

0.8
0.6

Incubation time (day)

(b)

40

glycine 0.5 g/L

glycine 1 g/L
glycine 2 g/L
glycine 5 g/L
glycine 10 g/L
glycine 20 g/L

30

20

10

1.4
1.2

0
0

1.0

Figure 2. (a) Cyanide production and (b) bacterial density of C.

violaceum with various glycine concentrations in YP medium with pH 9

0.6

Shake flask culture

To optimize sampling time and pulp density, the bioleaching of
gold, copper, and silver was determined in YP medium with glycine 5
g/L at pH 9 using H ore during five days and with 20, 50, 100 g/L of
pulp density. The results showed that copper was leached firstly and
gold was started to be leached after 2 days (Figure 3) due to the
0 3+
differences between their standard potential (E Au /Au: 1.52 V and
0 2+
E Cu /Cu: 0.34 V). The H ore was added at the same time with C.
violaceum inoculation.

0.2
0.0
0

Incubation time (day)

40
pH 6.7
pH 8
pH 9
pH 9.5

(b)
30

During the experimental period, leached metals were

accumulated continuously. From 20 to 100 g/L of pulp density was used
(Figure 4) since the gold concentration was too low to analyze in less
than 10 g/L of pulp density. The H ore was added after cultivating C.
violaceum overnight. With 20 g/L of pulp density, the maximum gold
bioleaching yield was observed (i.e., 22.489.39%) comparing with
higher pulp density. Throughout this study, 20 g/L of pulp density and
sampling at five days of incubation had been used. During the
experimental period, the pH was kept in the range of pH 8

20

10

The bioleaching yield of gold of the all ores was analyzed in batch
cultures and then for enhancing bioleaching efficiency various
enhancement factors were applied as follows: 1) pre-incubation, 2)
grinding, 3) pH adjustment, 4) pretreatment. In the case of batch
culture (i.e., the ores were added at the same time with C. violaceum
inoculation), the results showed very low bioleaching efficiency that
the bioleaching efficiencies of gold were 0%, 42.1965.36%, and
8.684.35% for R, S, and H, respectively (Figure 5 (a)). In the gold
leaching using cyanide, copper presents difficulties due to the lack of
selectivity of cyanide for gold over copper. For copper and silver,

0
0

Incubation time (day)

0.8

0.4

Cyanide concentration (mg/L)

1.0

0.0

Cyanide concentration (mg/L)

1.6

1.2

0.2

(a)

pH 6.7
pH 8
pH 9
pH 9.5

1.8

1.4

glycine 0.5 g/L

glycine 1 g/L
glycine 2 g/L
glycine 5 g/L
glycine 10 g/L
glycine 20 g/L

0.4

Cyanide production
Cyanide formation by C. violaceum was observed within five days
of incubation time with various pH (Figure 1 (a) and (b)) and glycine
concentrations (Figure 2 (a) and (b)). Characteristically, the peak value
of cyanide concentration was observed at two days of incubation and
the concentration decreased with time after that possibly due to
cyanide-utilizing enzyme activity. The bacterial growth reached lateexponential phase at one day and stationary phase after two days of
incubation. C. violaceum starts to produce cyanide from late
exponential phase and the cyanide-utilizing enzyme is induced after
the start of stationary phase [4]. The highest cyanide production (i.e.,
20 mg/L) was observed at pH 9 with glycine 5 g/L even though the
bacterial growth was higher at pH less than 9. It is because cyanide
exists primarily as CN rather than as HCN at pH above values 9.31.
Therefore, pH 9 and 5 g/L of glycine was used throughout this study.
2.0

(a)

1.8

Incubation time (day)

Figure 1. (a) Cyanide production and (b) bacterial density of C.

violaceum with various pH in YP medium containing glycine 5 g/L.

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Feb. 19 - 22, 2012, Seattle, WA
copper was leached from R, S, and H at 12.063.06%, 1.460.34%,
and 18.701.85%, respectively, and silver was leached from R and H
at 21.722.92% and 4.870.55%, respectively. Since copper may form
complex with cyanide than gold, the biogenic cyanide would be
consumed before reacting with gold.

The most effective pH for gold cyanidation has been reported at

pH 11 [9]. When adding glycine, pH of the C. violaceum culture was
adjusted to 11 and the H was added and incubated 5 days (Figure 7).
However, the gold bioleaching yield didnt change very much.
250

(a)

20
Au
Cu
Ag

200

Metal leached (%)

15

Metal leached (%)

Au
Cu
Ag

10

150

100

50

0
R

0
0

250

Incubation time (day)

(b)

Figure 3. Gold, copper, and silver bioleaching yield (% of total gold,

copper, and silver) by C. violaceum using H ore with 50 g/L of pulp
density.
Metal leached (%)

Au
Cu
Ag

30

Au
Cu
Ag

200

35

25

Metal leached (%)

20

150

100

50

15
0
Rosia Montana

10

Saramacca

Hollister

Figure 5. Gold, copper, and silver bioleaching yield (% of total gold,

copper, and silver) by C. violaceum with 20 g/L of pulp density at pH 9.
(a), Bioleaching assay in batch culture; (b) Bioleaching assay in preincubation culture.

0
20

50

100

Pulp density (g/L)

Figure 4. Gold, copper, and silver bioleaching yield (% of total gold,

copper, and silver) by C. violaceum using H ore with 20, 50, and 100
g/L of pulp density.

Au
Cu
Ag

200

Metal leached (%)

The methodology of pre-incubation was that C. violaceum was

cultivated in YP medium without glycine overnight and the ore samples
and glycine was added into the pre-incubation culture. As shown in
Figure 5 (b), the bioleaching efficiencies were enhanced to
4.3641.67%, 131.5573.93%, and 18.518.51% for R, S, and H,
respectively, as the effect of pre-incubation. The experimental error and
standard deviation was very high especially in S ore possibly due to
poor mixing and distribution of the ore samples. Since the bioleaching
efficiency was much enhanced by introducing the methodology of preincubation, pre-incubation had been used throughout this research.

150

100

50

By grinding, the bioleaching efficiency was enhanced as shown in

the Figure 6. Using ground sample (< 180 m), the bioleaching
efficiencies were enhanced to 60.9327.18% and 28.317.97% for R
and H, respectively. In the case of S, almost all of gold was already
leached out in pre-incubation so the leaching yield did not so increase.
Most of cyanidable gold were leached out in ground and pre-incubation
sample.

Figure 6. Gold, copper, and silver bioleaching yield (% of total gold,

copper, and silver) by C. violaceum with 20 g/L of pulp density at pH 9
using ground ore (<180 m).
If the gold particles are included in sulfide minerals such as pyrite,

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SME Annual Meeting

Feb. 19 - 22, 2012, Seattle, WA
the gold leaching agent (i.e., cyanide) cannot reach them [10, 11]. The
biooxidative pretreatment may be a useful tool to oxidize sulfide matrix
of the ore and liberate gold particle from the ore. Therefore, except for
S ore that most iron existed as ferric form, R and H ores were
incubated with A. ferrooxidans for 7 days and then filtrated, dried and
incubated again with C. violaceum. However, the bioleaching efficiency
was not enhanced after biooxidation (data not shown), it may be
because of short biooxidation time (i.e., 7 days). Overall, the
methodology of pre-incubation and grinding was the most effective to
enhance the bioleaching yield of gold. Further study is underway that
cyanide capturing system has been operated to concentrate cyanide
concentration and remove biofilm formation on the surface of ore. Also,
total cyanide produced by C. violaceum, the partitioned ratio of cyanide
between air and aqueous phase, and available cyanide for bioleaching
will be analyzed.
100

Faramarzi Mohammad A., Marion Stagars, Enrico Pensini, Walter

Krebs, and Helmut Brandl (2004). "Metal solubilization from metalcontaining solid materials by cyanogenic Chromobacterium
violaceum", Journal of Biotechnology, 113, pp. 321-326.

[4]

Rodgers Paul B. and Christopher J. Knowles (1978). "Cyanide

Production and Degradation During Growth of Chromobacterium
violaceum", Journal of General Microbiology, 108, pp. 261-267.

[5]

Kita Yoshito, Hiroshi Nishikawa, and Tadashi Takemoto (2006).

"Effects of cyanide and dissolved oxygen concentration on
biological Au recovery", Journal of Biotechnology, 124, pp. 545551.

[6]

Wissing Frode (1974). "Cyanide Formation from Oxidation of

Glycine by a Pseudomonas Species", Journal of Bacteriology,
117, pp. 1289-1294.

[7]

Brandl H., R. Bosshard, and M. Wegmann (2001). "Computermunching microbes: metal leaching from electronic scrap by
bacteria and fungi", Hydrometallurgy, 59, pp. 319-326.

[8]

Silverman M.P. and D.G. Lundgren (1959). "Studies on the

chemoautotrophic iron bacterium Ferrobacillus ferrooxidans: I. An
improved medium and a harvesting procedure for securing high
cell yields", Journal of Bacteriology, 77, pp. 642-647.

[9]

Rees K. L. and J. S. J. Van Deventer (1999). "The role of metalcyanide species in leaching gold from a copper concentrate",
Minerals Engineering, 12, pp. 877-892.

Au
Cu
Ag

80

Metal leached (%)

[3]

60

40

[10] Brown Edward J., Huan V. Luong, and Joan M. Forshaug (1982),
The Occurrence of Thiobacillus ferrooxidans and Arsenic in
Subarctic Streams Affected by Gold-Mine Drainage.. Vol. 35. 1982.

20

[11] Ubaldini S., F. Vegli, L. Toro, and C. Abbruzzese (1997).

"Biooxidation of arsenopyrite to improve gold cyanidation: study of
some parameters and comparison with grinding", International
Journal of Mineral Processing, 52, pp. 65-80.

0
pH (unadjusted)

pH (adjust to 11)

Figure 7. Gold, copper, and silver bioleaching yield (% of total gold,

copper, and silver) by C. violaceum using H ore with 20 g/L of pulp
density at pH 9 and 11 using ground ore (<180 m).
CONCLUSIONS
Cyanide producing bacterium C. violaceum was used to leach
gold from the ores. To enhance cyanide production by C. violaceum,
the growth parameters was investigated and pH 9 and 5 g/L of glycine
was the optimal condition. In shake flask culture, four enhancement
factors for gold bioleaching were introduced as follows: 1) preincubation, 2) grinding, 3) pH adjustment, 4) pretreatment. As applying
pre-incubation and grinding, the gold bioleaching yield by C. violaceum
reached the total cyanidable gold (Table 2). Further study is underway
that the amount total cyanide produced by C. violaceum will be
investigated by using NaOH trap to capture hydrogen cyanide. In
addition, the stoichiometric relationship will be analyzed between
produced cyanide and gold, silver, and copper.
Table 2. Comparison between total cyanidable gold and bioleaching
yield.

Cyanidable gold/total gold

%
Bioleached gold/total gold
%

55%

100%

29%

60.91
27.18%

89.35
38.87%

28.30
7.97%

REFERENCES
[1]

Chadwick B. M. and A. G. Sharpe (1966), "Transition Metal

Cyanides and Their Complexes in Advances in Inorganic
Chemistry, Emelus H. J. and Sharpe A. G., Editors Academic
Press. p. 83-176.

[2]

Brandl Helmut, Stefan Lehmann, Mohammad A. Faramarzi, and

Daniel Martinelli (2008). "Biomobilization of silver, gold, and
platinum from solid waste materials by HCN-forming
microorganisms", Hydrometallurgy, 94, pp. 14-17.

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