Journal of Biotechnology 113 (2004) 321–326

Metal solubilization from metal-containing solid materials by

cyanogenic Chromobacterium violaceum
Mohammad A. Faramarzia,1 , Marion Stagarsa , Enrico Pensinib ,
Walter Krebsb , Helmut Brandla,∗
a Institute of Environmental Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
b Department of Chemistry and Biological Chemistry, Zurich University of Applied Sciences,

Technikumstrasse 9, CH-8401 Winterthur, Switzerland

Received 1 September 2003; received in revised form 11 March 2004; accepted 19 March 2004

Abstract

Different cyanogenic bacterial strains (Chromobacterium violaceum, Pseudomonas fluorescens, Bacillus megaterium) were
cultivated under cyanide-forming conditions in the presence of metal-containing solids such as nickel powder or electronic scrap.
All microorganisms were able to form water-soluble metal cyanides, however, with different efficiencies. C. violaceum was able
to mobilize nickel as tetracyanonickelate [Ni(CN)4 2− ] from fine-grained nickel powder. Gold was microbially solubilized as
dicyanaoaurate [Au(CN)2 − ] from electronic waste. Additionally, cyanide-complexed copper was detected during biological
treatment of shredded printed circuit boards scrap. Regarding the formation of tetracyanonickelate, C. violaceum was more
effective than P. fluorescens or B. megaterium. Besides a few previous reports on gold solubilization from gold-containing ores
or native gold by C. violaceum, the findings demonstrate for the first time the microbial mobilization of metals other than gold
from solid materials and represent a novel type of microbial metal mobilization based on the ability of certain microbes to form
HCN. The results might have the potential for industrial applications (biorecovery, bioremediation) regarding the treatment of
metal-containing solids since metal cyanides can easily be separated by chromatographic means and be recovered by sorption
onto activated carbon.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Cyanogens; Hydrocyanic acid; Metal cyanides; Bioleaching; Chromobacterium violaceum; Pseudomonas fluorescens; Bacillus
megaterium

∗ Corresponding author. Tel.: +41-1-635-61-25; fax: +41-1-635-57-11.

E-mail address: hbrandl@uwinst.unizh.ch (H. Brandl).

1 Present address: Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences,

P.O. Box 14155-6451, Tehran 14174, Iran.

0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2004.03.031
322 M.A. Faramarzi et al. / Journal of Biotechnology 113 (2004) 321–326
1. Introduction
Certain microorganisms are able to mobilize met-
als from solid materials (minerals, ores, wastes) by the
formation of organic or inorganic acids (protons), by
oxidation and reduction reactions; and by the excretion
of complexing agents (Bosecker, 1997; Brandl, 2001b).
Sulfuric acid is the main inorganic acid found in leach-
ing environments (Rawlings, 2002; Sand et al., 2001).
It is formed by sulfur-oxidizing microorganisms such
as Acidithiobacillus species. A series of organic acids
are formed by bacterial (as well as fungal) metabolism
resulting in organic acidolysis, complex and chelate
formation (Brandl, 2001a; Burgstaller and Schinner,
1993). These microbial activities can be applied in the
industry for the recovery of metals from solid materials
(Ehrlich, 2002). The technology has successfully found
practical application in copper and gold mining (termed
“bioleaching” or “biomining”) where low-grade ores
are biologically treated to obtain metal values which
are not accessible by conventional (mechanical or ther-
mal) treatments (Agate, 1996).
Hydrocyanic acid (HCN) is formed by a vari-
ety of bacteria, e.g. Chromobacterium violaceum,
Pseudomonas fluorescens, P. aeruginosa, and fungi,
e.g. Marasmius oreades, Clitocybe sp., Polysporus
sp., many of them belonging to the soil microflora
(Knowles and Bunch, 1986). Although cyanide for-
mation by microorganisms is known for many years
(Clawson and Young, 1913; Greshoff, 1909), quanti-
tative data on the ability of HCN formation of many
species are still missing. Mainly fungi are poorly in-
vestigated and mainly qualitative data are available for
most of the strains.
Cyanide is formed as secondary metabolite. It is as-
sumed that its formation has an advantage for the organ-
ism by inhibiting competing microorganisms (Blumer
and Haas, 2000). Glycine is a precursor of cyanide
which is formed by a oxidative decarboxylation. In
certain ecosystems (e.g. soils, especially the plant
rhizosphere) cyanogenic microorganisms represent a
large part (upto 50%) of the soil microbial commu-
nity (Kremer and Souissi, 2001). Typically, cyanide
is formed during growth only during a short time pe-
riod (early stationary phase) (Knowles, 1976). Signif-
icant cyanide formation can be obtained only in cer-
tain growth media under specific conditions (Castric,
1981; Michaels and Corpe, 1965). Iron is known to
stimulate cyanide formation by gram-negative bacte-
ria (Kleid et al., 1995). Magnesium stimulates the for-
mation by gram-positive bacteria, copper and zinc the
formation by fungi.
Cyanide occurs in solution as free cyanide which
includes the cyanide anion (CN− ) and the non-
dissociated hydrocyanic acid (HCN). At physiological
pH, cyanide is present mainly as HCN due to its pKa
value of 9.3 and is, therefore, volatile. In the presence of
salts this value decreases to approximately 8.3 and the
volatility is reduced (Fagan, 1998). Generally, cyanide
can interact with a series of metals. It is known that
nearly all transitions metals (except lanthanides and ac-
tinides) form well-defined cyanides complexes which
show often a very good water solubility and a very
high chemical stability (Barnes et al., 2000; Chadwick
and Sharpe, 1966). However, until today a combina-
tion of this chemical knowledge with microbiologi-
cal principles (biological HCN formation) regarding
metal solubilization from metal-containing solids and
the formation of water-soluble cyanide complexes has
been considered very marginally. Only very few reports
can be found in the literature describing the biological
solublization of gold by C. violaceum and microbe-
mediated formation of gold cyanide (Campbell et al.,
2001; Lawson et al., 1999; Smith and Hunt, 1985).
The objective of this study was to evaluate the poten-
tial of cyanogenic microorganisms to solubilize met-
als from metal-containing solids, in particular (i) the
biological treatment of solid metal-containing materi-
als using cyanogenic bacteria; (ii) the comparison of
different cyanogenic strains regarding their ability to
form metal cyanide complexes; (iii) the optimization of
metal cyanide complex formation by varying glycine
and solid material concentration; and (iv) the detection
of water-soluble metal cyanide complexes in bacterial
culture media by HPLC.
2. Materials and methods
C. violaceum was obtained from the German Col-
lection of Microorganisms and Cell Cultures (DSMZ
strain 30191), P. fluorescens from the Swiss Federal In-
stitute of Technology (ETH Zurich), and B. megaterium
from our own strain collection. Cells were routinely
cultured in a complex medium containing (in g/l) l-
glutamate (4.4); KH2 PO4 (1.4); Na2 HPO4 ·7H2 O (2.1);
 M.A. Faramarzi et al. / Journal of Biotechnology 113 (2004) 321–326 323

MgSO4 ·7H2 O (0.2); FeCl3 ·6H2 O (0.005); glycine

(0.75) and l-methionine (1.5) under sterile conditions
in baffled Erlenmeyer flasks and incubated at 30 ◦ C
on a rotary shaker at 150 rpm. Alternatively, organ-
isms (C. violaceum, B. megaterium, P. fluorescens)
were grown in Luria-Bertani Broth containing tryp-
tone (10.0), yeast extract (5.0), sodium chloride (10.0).
Glycine and metal-containing solids were additionally
supplemented in different amounts according to the
experimental setup. Long-term storage of the organ-
isms was carried out in 15% glycerol at −80 ◦ C. Bac-
terial growth was monitored by determining the ab-
sorbance at 600, 450 or 660 nm, (for C. violaceum,
P. fluorescens, and B. megaterium, respectively). The
pH was recorded additionally. For metal mobilization
experiments, different amounts (upto 10 g/l) of solid Fig. 1. Growth as well as formation of free cyanide and tetracyano-
nickelate by C. violaceum in LB medium supplemented with glycine
materials (e.g. nickel powder, electronic scrap) were
(0.75 g/l ) and powdered elemental nickel (1 g/l): (䊉) OD at 600 nm;
added to the medium. (
) pH; () free cyanide; () tetracyanonickelate.
Free cyanide was analyzed colorimetrically at
605 nm using the pyridine barbituric acid colorimet- ture increased during incubation from 7.2 to approx-
ric method (Anonymous, 1981). Analyses of metal imately 9.
complexed cyanides were performed by reversed The effect of increasing amounts of nickel pow-
phase high pressure liquid chromatography (rP-HPLC) der was investigated (Fig. 2). The presence of nickel
(Hilton and Haddad, 1986). Metal-complexed cyanides in the growth media (upto 4 g/l) influenced growth
were separated at 40 ◦ C on a hydrophobic C-18 column. (measured as optical density) of C. violaceum only
The eluent consisted of 60 mM tetrabutylammonium slightly. However, at concentrations higher than 4 g/l
hydroxide (TBAOH); 150 mM ortho-phosphoric acid;
25% acetonitrile; 2.34 mM sodium perchlorate·H2 O.
Flow rate was adjusted to 1 ml/min. Metal cyanides
were measured by UV detection at 230 nm (Fe, Cu,
Ag, Au, and Pt) and 267 nm (Ni).

3. Results and discussion

Approximately 4 mM of free cyanide was detected

during growth of C. violaceum (Fig. 1). Generally,
cyanide concentration always decreased after reaching
a maximum. This is probably due to cyanide consump-
tion by cyanide-consuming (cyanicidic) compounds or
to cyanide degradation. Only very small cyanide con-
centrations were measured in the gas phase of closed
vessels. C. violaceum was able to mobilize nickel as
tetracyanonickelate from fine-grained nickel powder
Fig. 2. Growth as well as formation of free cyanide and tetracyano-
(Fig. 1). Cyanide-complexed nickel was already de-
nickelate by C. violaceum in LB medium supplemented with glycine
tected 4 h after inoculation (data not shown). Approx- (0.75 g/l) as function of different amounts of powdered elemental
imately 9% of the initial nickel powder added was nickel added. Measurements were taken after an incubation of 48 h:
recovered as tetracyanonickelate. The pH of the cul- (䊉) OD at 600 nm; () free cyanide; () tetracyanonickelate.
324 M.A. Faramarzi et al. / Journal of Biotechnology 113 (2004) 321–326
growth was drastically reduced. The reasons might be
either toxic effects and/or mechanical stress due to in-
creased pulp densities. Pulp density effects have al-
ready been observed during biological treatment of
pyrite by Acidithiobacillus species (Baldi et al., 1992).
Quartz particles at densities of 80 g/l almost com-
pletely inhibited the biological oxidation of covellite
by Acidithiobacillus (formerly Thiobacillus) ferrooxi-
dans (Curutchet et al., 1990). Regarding the formation
of tetracyanonickelate, the optimal concentrations of
solid nickel are between 0.5 and 1 g/l.
Since hydrocyanic acid is directly produced from
glycine (Wissing, 1968), optimal glycine concentra-
tions were determined for the cultivation of C. vio-
laceum in the presence of 1 g nickel powder per liter
(Fig. 3). These are in the range of 8–10 g/l and resulted
in the highest cyanide as well as tetracyanonickelate
concentrations. The concentration of tetracyanonick-
elate determined corresponded to a mobilization of
43.5% of the nickel initially added. Increased glycine
concentrations (>10 g/l) led to reduced growth as well
as reduced cyanide and tetracyanonickelate formation.
When comparing different microbial strains known
to be cyanogenic, C. violaceum proved to be the most
efficient under the same conditions regarding the for-
mation of tetracyanonickelate (Fig. 4). All three or-
Fig. 3. Growth as well as formation of free cyanide and tetracyano-
nickelate by C. violaceum in LB medium supplemented with pow-
dered elemental nickel (1 g/l) as function of different amounts of
glycine added. Measurements were taken after an incubation of 48 h:
(䊉) OD at 600 nm; () free cyanide; () tetracyanonickelate.
Fig. 4. Formation of tetracyanonickelate from powdered elemental
nickel (1 g/l added to the growth medium) by different microbial
strains.
ganisms can be isolated from soil. From an ecological
point of view microbially formed cyanide might act in
soil environments − besides controlling agent to in-
hibit competitors or predators (Gallagher and Manoil,
2001) − as lixiviant for metal compounds which can be
subsequently been taken up by plants and microorgan-
isms. Until today, there are no reports on this hypoth-
esis although certain plant rhizospheres (e.g. Solanum
tuberculosum or Euphorbia sp.) are known of harbor-
ing cyanogenic microorganisms (Kremer and Souissi,
2001).
Residues from the mechanical recycling of used
electronic equipment (e.g. computers) represent a
highly complex metal-containing matrix (Brandl et al.,
2001). These solid wastes often contain metals with
a high economic value. Particularly gold is of special
interest and can occur in concentrations of 20 mg/kg
shredded printed circuit boards (Oh et al., 2003). Gold-
containing pieces (5 mm × 10 mm) of printed circuit
boards were used for growth experiments. These were
obtained by manually cutting printed circuit boards
followed by manual sorting. Each piece contained ap-
proximately 10 mg of gold. Using C. violaceum, it was
demonstrated that gold can be microbially solubilized
from printed circuit boards (Fig. 5). The maximum di-
cyanoaurate [Au(CN)2 − ] measured corresponds to a
14.9% dissolution of the initially gold added.
In conclusion, the results represent a novel type of
microbial metal mobilization based on the ability of
 M.A. Faramarzi et al. / Journal of Biotechnology 113 (2004) 321–326
Fig. 5. Growth and dicyanoaurate formation by C. violaceum in LB
medium supplemented with glycine (0.75 g/l) and shredded pieces
of printed circuit boards originating from electronic scrap: (䊉) OD
at 600 nm; (
) dicyanoaurate.
certain microbes to form HCN. The findings might
have the potential for a microbially based industrial ap-
plication regarding the treatment of metal-containing
solids or the biological remediation of metal-polluted
soils since metal cyanides can be separated by chro-
matographic means and easily be recovered by sorp-
tion onto activated carbon. However, cyanogenic mi-
crobes have to be comprehensively evaluated to fully
exploit the potential to form water-soluble metal com-
plexes from solid metal-containing solids. Growth and
cyanide formation have to be optimized as well as
metal mobilizing efficiencies. In particular, the pres-
ence of cyanide-consuming compounds in the growth
medium originating from the solids treated has to be
addressed.
In addition, the formation of HCN under anoxic con-
ditions (Campbell et al., 2001; Mascher et al., 2003) as
well as the regulation of HCN formation by signaling
compounds such as homoserine lactones (Martinelli et
al., 2002) can be considered when attempting to in-
crease the HCN content in the growth media.
Acknowledgements
Pseudomonas fluorescens was kindly provided by
Dr. Monika Maurhofer (Institute of Plant Sciences,
325
Swiss Federal Institute of Technology (ETH), Zurich,
Switzerland).
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