Environmental Pollution 156 (2008) 1069–1074

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Bacteria, hypertolerant to arsenic in the rocks of an ancient gold mine,

and their potential role in dissemination of arsenic pollution
Lukasz Drewniak a, Aleksandra Styczek a, Malgorzata Majder-Lopatka b, Aleksandra Sklodowska a, *
a
Laboratory of Environmental Pollution Analysis, Faculty of Biology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland
b
Laboratory of Measurement of Environment Parameters, Faculty of Fire Safety Engineering, The Main School of the Fire Service (SGSP),
Slowackiego 52/54, 01-629 Warsaw, Poland

The activity of the described heterotrophic bacteria leads to mobilization of arsenic and in this way contributes to
the dissemination of arsenic pollution.

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present study was to find out if bacteria present in ancient gold mine could transform
Received 7 November 2007 immobilized arsenic into its mobile form and increase its dissemination in the environment. Twenty-two
Received in revised form 24 April 2008 arsenic-hypertolerant cultivable bacterial strains were isolated. No chemolithoautotrophs, which could
Accepted 27 April 2008
use arsenite as an electron donor as well as arsenate as an electron acceptor, were identified. Five isolates
exhibited hypertolerance to arsenic: up to 500 mM of arsenate. A correlation between the presence of
Keywords: siderophores and high resistance to arsenic was found. The results of this study show that detoxification
Arsenic pollution
processes based on arsenate reductase activity might be significant in dissemination of arsenic pollution.
Scorodite
Hypertolerant bacteria
It was concluded that the activity of the described heterotrophic bacteria contributes to the mobilization
of arsenic in the more toxic As(III) form and a new mechanism of arsenic mobilization from a scorodite
was proposed.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Arsenic is a toxic element that is widely distributed in the en-
vironment as a result of natural geochemical phenomena and an-
thropogenic activity. Contamination of drinking water supplies by
inorganic hydrolysis species of As(III) (arsenite) and As(V) (arse-
nate) is frequently reported (ATSDR, 2005 toxicological profile for
arsenic). As(III) is more mobile than As(V), which adsorbs to the
surface of minerals such as ferrihydrite and alumina.
Microorganisms have evolved a variety of mechanisms of arse-
nic resistance. Amongst these is minimization of arsenic uptake
through increasing the specificity of phosphate uptake (Cervantes
et al., 1994). Some microorganisms can also oxidize arsenite with
arsenite oxidase, either as a detoxification process alone or using
arsenite as an electron donor (Santini et al., 2000; Batalgia-Brunet
et al., 2002; Muller et al., 2003). Other microorganisms use arsenate
as a terminal acceptor in dissimilatory arsenate respiration (Lav-
erman et al., 1995; Krafft and Macy, 1998; Switzer Blum et al., 1998).
However, the most well known mechanism of arsenic resistance in
microorganisms requires the ars operon and is based on energy-
* Corresponding author. Tel./fax: þ48 22 554 1006.
E-mail addresses: ldrewiak@biol.uw.edu.pl (L. Drewniak), mmajder@sgsp.edu.pl
(M. Majder-Lopatka), asklodowska@biol.uw.edu.pl (A. Sklodowska).
dependent efflux of both arsenate and arsenite from the cell (Cer-
vantes et al., 1994; Oremland and Stoltz, 2003). In this operon the
gene arsC is particularly interesting because its product, a cyto-
plasmic arsenate reductase, catalyzes reduction of less toxic arse-
nate to more toxic arsenite, which may be transported out of the
cell by ArsAB arsenic chemiosmotic efflux system and by ATPase
membrane system (Silver and Phung Le, 2005). In this way, ex-
pression of arsC might increase the toxicity of arsenic species in the
environment.
Elevated levels of arsenic have been observed in effluent from
the ancient Zloty Stok gold mine in Southwest Poland. The Zloty
Stok mine is located in the southern part of a town of the same
name in Lower Silesia. The first true mine at this site was a gold
mine opened in the 13th century. Exploitation of arsenic started in
the 18th century. This element occurs as loellingite (FeAs2) and
arsenopyrite (FeAsS) in serpentinite and marbles. Other minerals
extracted from the same mine contain such metals as iron (mag-
netitedFe3O4, pyrrhotinedFeS, pyritedFeS2), lead (galenadPbS),
zinc (sphaleritedZnS) and copper (chalcopyritedCuFeS2). The
mine was closed in 1962, leaving some 300 km of underground
passages on 21 levels. Most of the underground galleries and shafts
are now filled with water and are partly or totally inaccessible.
Spruce wood beams and other timbering were left.
The aim of this study was to find out if bacteria isolated from this
arsenic-containing site could transform immobilized arsenic

0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2008.04.019
1070 L. Drewniak et al. / Environmental Pollution 156 (2008) 1069–1074
species to its mobile form that can disseminate in the environment.
Such microorganisms either use arsenite as an electron donor or
arsenate as an electron acceptor under aerobic and anaerobic
growth conditions, respectively, or they resist the high concentra-
tions of arsenic species through various detoxification pathways.
2. Materials and methods
2.1. Site description
Gertruda Adit, a gallery within the Zloty Stok mine, was the area examined in the
present study. The first 500 m section of this gallery is fully lit and safe. The deeper
section is unlit, separated off by a low sluice gate, and partially filled with water. The
end and middle sections of Gertruda Adit are characterized by a stable air temper-
ature of 10.4–11.1  C and stable water temperature of 10–12  C throughout the year
(Chlebicki et al., 2005). Some weathering processes can be observed on the walls of
Gertruda Adit and on the surface of waste deposits situated around the mine. Sec-
ondary minerals such as scorodite (FeAsO4 $ 2H2O) (Paszek, 2001) and goethite
occur in these places.
2.2. Sample collection
Samples of rock and biofilm from rocks were collected from the walls of the end
segment of Gertruda Adit that is completely closed to visitors. Mine water samples
were also taken. Air samples were collected from three different locations: at the
end of Gertuda Adit, at the bottom and 1.5 m above the bottom, and 50 m from the
end of the Adit. The rock and biofilm samples were placed in sterile 50 ml tubes, the
mine water into 15 l sterile barrels, and air was collected in 12 l gas sample bags
(TedlarBag, SKC Inc., USA).
2.3. Chemical analysis
Samples of rock were thoroughly dried at 60  C and then 8 ml 65% HNO3, 1 ml
36% H2O2 were added to a dry mass of 0.25–0.30 g and digested in a closed system
with heating in a microwave oven (Milestone Ethos Plus with Lab Terminal 800
Controller, Italy) (Namiesnik et al., 2000). Another 1 ml of 36% H2O2 was then added.
Samples of 0.25–0.30 g (wet weight) of biofilm were processed in the same way.
Water samples (5 ml) were placed in 12 ml glass vials (Agillent, USA) and mixed
with 4 ml 65% HNO3 immediately after sampling. For mineralization, 1 ml 36% H2O2,
was added, then digested before another 1 ml of 36% H2O2 was added.
Total arsenic was analyzed by flame atomic absorption spectrometry (FAAS) and
graphite furnace atomic absorption spectrometry (GFAAS) (AA Solaar M6 Spec-
trometer, TJA Solutions, UK) using an arsenic standard solution (Merck, Darmstadt,
Germany) prepared in 0.5 M HNO3. The detection limit was 1 mg/l for FAAS and
10 mg/l for GFAAS. Arsenic concentration measurements were validated using in-
ductively coupled plasma–mass spectrometry (ICP-MS, ELAN DRC II, Perkin Elmer,
USA).
Quantitative analysis of Al, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, V and Zn were
performed by ICP-MS (ELAN DRC II, Perkin Elmer, USA) with a detection limit of:
0.05 mg/l for Ca, Mg, Cd, Co, Fe and Cu, 0.5 mg/l for Al and Mn, 1.0 mg/l for V and 2.0 mg/
l for As and Cr. Soil S1 (AGH University of Science and Technology, Poland) was used
as a certified reference material for analysis validation.
In the gold mine, levels of oxygen, hydrogen sulfide and combustible gases were
simultaneously monitored using electrochemical and catalytic diffusion sensors
(TMX 412 Multi-Gas Monitor, Industrial Scientific Corporation, USA). The air con-
centration of arsenic hydride was measured by ion mobility spectrometry (IMS,
RAID-M 100, Bruker Daltonics GmbH, Germany). The detailed composition of the air
was analyzed using a Fourier transform infrared spectrometer (FTIR, GASMET Dx-
4010, Temet Instruments Oy, Finland).
2.4. Media and growth conditions
Luria–Bertani (LB) medium at pH 7.0 (Sambrook and Russell, 2001) and a mini-
mal salt medium (MSM) (Santini et al., 2000) were used for bacterial isolation and
culture enrichment. For anaerobic growth in MSM, KNO3 was replaced by NH4Cl
(0.15 g/l). LB or MSM plates contained 20 g agar per liter. Isolates were cultured in
100 ml of medium in 300 ml Erlenmeyer flasks or on agar-containing plates, at
temperatures of 16  C or 22  C. Anaerobic growth was achieved in special anaerobic
jars containing Anaerocult reagent (Merck, Darmstadt, Germany).
2.5. Isolation of arsenic-tolerant bacteria
Samples of rock and rock biofilm were placed in 50 ml of 0.9% NaCl and vigor-
ously mixed for 24 h before each was used to inoculate selective media. For the
isolation of aerobic chemolithoautotrophs, which can gain energy from arsenite
oxidation, MSM supplemented with 10 mM sodium arsenite was used. Modified
MSM containing 5 mM sodium arsenate and 5 mM sodium lactate was used for the
isolation of anaerobic arsenate respiring bacteria. Cultures were incubated for 7 days
at 16  C and then subcultured twice, allowing 7 days for growth after each
inoculation into a fresh medium. The cultures were then diluted and plated on MSM
agar containing the selective substrates. Arsenic-resistant heterotrophs were iso-
lated on LB agar medium supplemented with 5 mM sodium arsenate or 5 mM so-
dium arsenite.
2.6. 16S rRNA gene amplification and sequencing
A colony PCR method (Gathogo et al., 2003) was used for the amplification of 16S
rRNA genes. A single colony of each bacterial isolate was resuspended in 20 ml of PCR
lysis solution (50 mM NaOH; 0.25% SDS) and then boiled for 15 min at 95  C. After
placing on ice, 180 ml of cold, sterile water was added to each sample, which were
then used as DNA templates for PCR. Amplification reactions of 50 ml were composed
of 1 ml of the lysed cell samples, 0,1 nmol of primers 27f and 1492r (Lane, 1991),
0,2 mM of dNTPs and 1 U of Taq polymerase with its supplied 1 buffer (Qiagen,
Germany). The thermal cycle was performed in a Mastercycler (Eppendorf, Ger-
many) and consisted of an initial denaturation step at 94  C for 5 min, then 20 cycles
of 94  C for 3 s, 53  C (in every cycle  1) for 3 s, 72  C for 1.5 min, followed by 15
cycles of 94  C for 3 s, 46  C for 3 s, 72  C for 1.5 min, with a final extension of 72  C
for 10 min. PCR products were examined by agarose gel electrophoresis and then
purified using a PCR Purification Kit (Qiagen) according to the manufacturer’s in-
structions. The amplified 16S rDNA fragments were used as templates for DNA se-
quencing with an ABI Prism 377 automatic sequencer (Applied Biosystems, Foster
City, U.S.A.). Six primers were used in the sequencing: 27f, 515f, 906f, 907r, 519r and
1492r (Lane, 1991).
2.7. Detection of arsC genes
For amplification of arsC genes two primer pairs described by Sun et al. (2004)
were used: amlt-42-f (50 -TCG CGT AAT ACG CTG GAG AT-30 ); amlt-376-r (50 -ACT TTC
TCG CCG TCT TCC TT-30 ) and smrc-42-f (50 -TCA CGC AAT ACC CTT GAA ATG ATC-30 );
smrc-376-r (50 -ACC TTT TCA CCG TCC TCT TTC GT-30 ).
These primers amplified a fragment of w350 base pairs of the arsC sequence.
The PCR conditions were as follows: initial denaturation step at 94  C for 5 min, then
30 cycles of 94  C for 3 s, 54  C (for primer amlt-42-f and amlt-376-r) or 59  C (for
primer smrc-42-f and smrc-376-r) for 3 s, 72  C for 3 s with a final extension of 72  C
for 10 min. Negative controls included a deionized water reagent control. The arsC
gene of Sinorhizobium sp. M14 (Drewniak and Sklodowska, 2007) was used as
a control in the amplification reaction. The presence of arsC gene in a PCR product of
a positive control was confirmed by sequencing.
2.8. Morphological, physiological and biochemical tests
Cell morphology and Gram staining of the isolates were examined after 24 h or
48 h (depending on the tested strain) of incubation on LB agar plates.
Isolates were tested for the ability to utilize acetate or lactate. Filter-sterilized
stocks of sodium acetate or sodium lactate were added to MSM to give a final
concentration of 10 mM. Overnight cultures grown in LB medium were centrifuged
(11,500  g, 5 min, 4  C) and the cells washed twice with MSM. The bacterial sus-
pensions were then added to 5 ml of MSM supplemented with organic substrate.
The cultures were grown at 22  C for 24 h, and the OD600 was measured at the start
and end of incubation.
2.9. Determination of the minimum inhibitory concentrations of metals
The method of Courvalin et al. (1985) was used for determining the minimal
inhibitory concentrations of metal elements. LB medium supplemented with the
respective metal compounds was inoculated with cells from fresh overnight cultures
to a final density of approximately 106 cells/ml and then incubated for 24 h. The
minimum inhibitory concentration (MIC) is defined as the lowest concentration of
Menþ that completely inhibits bacterial growth. The metals and their compounds
used for MIC determination are as follows: As(III) 0.0–15.0 mM; As(V) 0.0–600 mM;
Cr(III) 0.0–12 mM; Zn, Cd, Ni, Co, Cu 0.0–6.0 mM; Se, Mn, V 0.0–20 mM.
2.10. Arsenic respiratory process screening assay
The ability of bacterial isolates to oxidize As(III) or reduce As(V) and use these
arsenic compounds in respiratory processes was tested. MSM agar containing
10 mM sodium arsenite as the electron donor was used for the determination of
As(III) oxidation. MSM agar containing 5 mM lactate as a carbon source, and 5 mM
sodium arsenate as the electron acceptor was used for the determination of As(V)
reduction. Cultures for the arsenate reduction test were grown under anaerobic
conditions. After 5 days of cultivation at 22  C, the agar plates were flooded with
0.1 M AgNO solution. The reaction between AgNO3 and As(III) or As(V) results in the
formation of a colored precipitate (Simeonova et al., 2004). A brownish precipitate
reveals the presence of Ag3AsO4 (silver arsenate) in the medium (colonies
expressing arsenite oxidase), while a yellow precipitate shows the presence of
Ag3AsO3 (silver arsenite) (colonies expressing arsenate reductase).
 L. Drewniak et al. / Environmental Pollution 156 (2008) 1069–1074 1071

2.11. Assay for arsenate reductase activity Table 1

Identification of arsenic-tolerant bacterial isolates in biofilms from the Zloty Stok
For the preparation of cell-free extracts, the isolated strains were grown to gold mine
stationary phase in 300 ml of LB medium containing 10 mM arsenate. Cells were
harvested by centrifugation (11,500  g, 20 min, 4  C) and then washed twice in Isolate number Phylogenetic Phylogenetic position
50 ml of ice-cold reaction buffer (10 mM Tris pH 7.5, 1 mM EDTA, 1 mM MgCl2). The (accession group
Best match in GenBank Identity (%)
cells were disrupted by sonication (600 W, 5–10  3 s, depending on the isolate) and number) based on 16S
database
unbroken cells were removed by centrifugation as above. The supernatants (crude rDNA analysis
(accession number)
cell-free extracts) were used in the arsenate reductase activity assay.
OS1 Actinobacteria Rhodococcus erythropolis 100
Arsenate reductase activity was measured by the method of Anderson and Cook
(EF491951) (EF154264)
(2004). Reactions were performed at 22  C and started by mixing 50 ml cell-free
OS2 Actinobacteria Micrococcus chenggongense 99
extract with 180 ml of reaction buffer (10 mM Tris [pH 7.5]; 1 mM Na2EDTA; 1 mM
strain AS1
MgCl2), then adding sodium arsenate (to a final concentration of 100 mM) and DTT
(EF491952) (DQ660308)
(to a final concentration of 300 mM). The reactions were stopped by the addition of
OS3 g-Proteobacteria Pseudomonas sp. A-13 98
a molybdate reagent, forming a blue arseno-molybdate complex with the remaining
(EF491953) (AY556391)
arsenate (Johnson and Pilson, 1972). The concentration of the formed complex was
OS4 Actinobacteria Arthrobacter sp. J64 99
measured by absorbance at 750 nm using a Beckman DU-65 Spectrophotometer
(EF491954) (AJ864856)
(Beckman Coulter, Fullerton, CA, USA).
OS5 Actinobacteria Micrococcus sp. 98TH11318 99
(EF491955) (AY159885)
2.12. Test for the presence of siderophores OS6 Actinobacteria Micrococcus luteus isolate OS-159 99
(EF491956) (AM237392)
The ability of isolates to produce siderophores was checked by growing the
OS7 g-Proteobacteria Pseudomonas mandelii 99
bacterial strains on modified chrome azurol S (CAS) agar plates as described by
(EF491957) (AY179326)
Schwyn and Neilands (1987). Instead of glucose, yeast extract (to a final concen- OS8 g-Proteobacteria Pseudomonas sp. ‘‘ARDRA PS3’’ 99
tration of 0.04%) was added. The plates were incubated for 4 days in the dark at 22  C (EF491958) (AY364087)
and the formation of halos around colonies was noted. OS9 g-Proteobacteria Serratia grimesii strain ZFX-1 99
(EF491959) (AY789460)
2.13. Protein determination OS10 Actinobacteria Streptomyces sporora(V)eus strain: 99
NBRC 15456
Bradford reagent (Bradford, 1976) was used to determine protein concentra- (EF491960) (AB184682)
tions. Bovine serum albumin (Bio-Rad, CA, USA) was the standard. OS11 Flavobacteria Chryseobacterium sp. JIP 16/96 99
(EF491961) (AY468460)
2.14. Nucleotide sequence accession numbers OS12 Bacilli Bacillus akibai 98
(EF491962) (AB043858)
The 16S rDNA sequences determined in this study have been deposited in OS13 Bacilli Bacillus cohnii 98
GenBank (Table 1). (EF491963) (AF140014)
OS14 Bacilli Desemzia incerta 99
(EF491964) (Y17300)
3. Results and discussion
OS15 Actinobacteria Microbacterium sp. CQ0110Y 98
(EF491965) (DQ852355)
3.1. Site characterization OS16 a-Proteobacteria Brevundimonas sp. KACC 10191 99
(EF491966) (DQ855080)
The deepest section of Gertruda Adit that is closed to tourists is OS17 g-Proteobacteria Stenotrophomonas sp. LQX-11 98
(EF491967) (DQ256392)
dark, separated off by a low sluice gate, and partially filled with OS18 g-Proteobacteria Stenotrophomonas sp. BMC 99
water. It is characterized by a stable air temperature of 10.4–11.1  C. (EF491968) (DQ991144)
A reduced concentration of oxygen, 17.2% compared to the normal OS19 g-Proteobacteria Pseudomonas sp. TM13_11 99
21%, was found. In different locations in the mine the concentration (EF491969) (DQ279323)
OS20 g-Proteobacteria Pseudomonas jessenii strain 99
of arsenic hydride reached 1.52–3.23 mg/m3, greatly exceeding the
HAMBI2391
occupational exposure limit in Poland (0.2 mg/m3). These values, (EF491970) (AF501361)
however, are smaller than the lowest toxic concentration (TCL0) for OS21 a-Proteobacteria Sphingomonas anadarae strain: 99
AsH3, which is 10 mg/m3. Apart from CO2, the other identified KMM 3933
components occurred in trace amounts and none of these com- (EF491972) (AB261013)
OS22 a-Proteobacteria Paracoccus marcusii 99
pounds is normally present in atmospheric air. The presence of (EF491971) (Y12703)
simple organic compounds (aliphatic hydrocarbons 660 mg/m3;
aromatic hydrocarbons 69 mg/m3; volatile alcohols, aldehydes and
acids 8.6 mg/m3), probably originating from wooden detritus, was
variable, with one biofilm sample containing considerably more
shown in the Gertruda Adit air and they represent the source of
(372 mg/kg) than any other sample (12–88 mg/kg).
carbon and energy for the growth of heterotrophic
microorganisms.
Previously, we described the destruction of wood in the Zloty 3.2. Isolation and identification of arsenic-tolerant bacteria
Stok gold mine by specific ‘‘wood fungi’’ (Chlebicki et al., 2005). The
presence of fungi such as Anthrodia serialis, Arcyria sp. (slime Bacteria were isolated from arsenic-containing biofilms on the
mould), Paxillus panuoides and Physisporinus vitreus as well as walls of the Gertruda Adit in the Zloty Stok gold mine. No micro-
bacteria from the genus Pseudomonas was demonstrated and their organisms were isolated that use arsenic compounds in basic
activity and arsenic detoxification processes were thought to be the metabolic processes and directly contribute to the biogeochemistry
probable source of AsH3 in soil air (Cheng and Focht, 1979; Bentley of arsenic in the environment. However, some bacteria were iso-
and Chasteen, 2002) but was not described as far as mine air is lated which could carry out non-energy-generating microbial
concerned. transformations of arsenic. These isolates transformed arsenic in
The rock samples contained more Fe (21,293–31,226 mg/kg) detoxification processes only, and participate in transformations of
than biofilm samples (3614–17,875 mg/kg), while two of the three iron with biogeochemical cycles that intersect these of arsenic.
biofilm samples contained more Ca, Zn and Mn than the rock A total of 22 bacterial strains from rocks and rock biofilms grew
samples. Levels of As were similar in two biofilm samples (22– with different colony morphologies on LB agar medium containing
26 mg/kg) and in one was higher (59 mg/kg), while Cu values were inorganic arsenic compounds. Only four were isolated on LB
1072 L. Drewniak et al. / Environmental Pollution 156 (2008) 1069–1074

medium containing 5 mM sodium arsenite [As(III)]. The others 350 mM). The remaining strains exhibited lower levels of arsenate
were isolated on LB agar medium supplemented with 5 mM so- tolerancedfrom 25 to 100 mM. The highest MICs for As(III) were
dium arsenate [As(V)]. Half of the arsenic-tolerant bacterial isolates found for three strains (up to 15 mM), and up to 12.5 mM for six
were Gram-negative and all were short or long rods. The Gram- strains. Other strains showed lower levels of resistance to
positive isolates varied in morphology from rods, spherical cells As(III)dbetween 5 and 10 mM.
(cocci) to filamentous types. Microbial resistance to arsenic species is widespread in nature
Partial 16S rDNA sequences (w1.4 kbp) were amplified from but resistance to concentration of As(V) higher than 100 mM is
each isolate, then sequenced, and the bacteria classified according considered as very high (Jackson et al., 2005). The tolerance
to their similarity to sequences in the GenBank database (Table 1). reaching 300–500 mM of As(V) should be considered as hyper-
The largest group of strains belonged to the g-Proteobacteria and tolerance. The bacterial strains described here generally show
Actinobacteria. Three isolates belonged to a division of Firmicutes A much higher resistance to As(V) than those isolated from gold
single strain was a member of the Flavobacteria. mines in similar studies (Macy et al., 1996; Battaglia-Brunet et al.,
These results show that the isolated arsenic-tolerant bacteria 2002; Simeonova et al., 2004) and other arsenic-tolerant microor-
form phylogenetically distant clusters, representing only five clas- ganisms (Carlin et al., 1995; Saltikov and Olson, 2002; Lopez-Maury
ses of bacteria, within the ‘‘big four’’ bacterial phyla (Proteobacteria, et al., 2003). Such high resistance to As(V) has been described in the
Firmicutes, Actinobacteria and Bacterioides). It should be appreci- biotechnologically-important bacterium Corynebacterium gluta-
ated that microorganisms that can be isolated using standard cul- micum (Ordonez et al., 2005), which is one of the most resistant
tivation methods are estimated to constitute less than 1% of all microorganisms described to date. High resistance to arsenate
microbial species and may be thought of as the ‘‘weeds’’ of the (>100 mM) was also found in bacteria isolated from the Lake
microbial world (Hugenholtz, 2002). This means that the bio- Pontchartrain estuary in Louisiana, USA (Jackson et al., 2005), but in
diversity of microorganisms living in this extreme environment contrast to our isolates their arsenic-tolerant phenotype was
might be considerably higher and besides heterotrophs, hyper- mainly demonstrated on solid media, which, due to colonial
tolerant to arsenic, other microorganisms including ‘‘arsenic che- growth, may not give an accurate estimate of resistance level. All of
molithoauthotrophs’’ are likely to be present. To gauge the real the Zloty Stok gold mine isolates showed considerable arsenic re-
diversity of arsenic-metabolizing microbes in this gold mine, sistance, but with great variation between strains. The presence of
a functional ‘‘metagenomics’’ study is required to obtain the arsC gene was confirmed in all strains except two with low activity
knowledge of the whole biogeochemical cycle in the described area of arsenate reductase (Table 2). Differences in the levels of tolerance
and it should involve the other parts of mine environment such as to arsenic might be attributed to the position of isolates in the
water and water sediments. biofilm structure. At the top of the biofilm (furthest from the rock
surface), bacteria may receive less exposure to arsenic and so do not
require such high arsenic resistance activity. On the other hand, the
3.3. Phenotypic characterization levels of arsenic tolerance found were generally much greater than
the average arsenic concentration in the examined environment
All of the tested bacteria were resistant to both inorganic arsenic (i.e. rocks and rock biofilms). This might indicate that the genetic
species [As(III) and As(V)], and some of them showed extreme and cellular mechanisms of arsenic resistance evolved many years
tolerance to arsenate (Table 2). The most resistant isolate was strain ago when higher levels of arsenic were present in the natural
OS1 that tolerated arsenate at concentrations up to 500 mM environment.
[37,500 mg/l As, approaching the solubility limit of As(V) in LB To further examine the high level of resistance to As(V), arsenate
medium]. Ten isolates were also hypertolerant to arsenate (150– reductase activity was measured. Arsenate reductase activity was
detected in all of the tested isolates, and four strains showed
Table 2
massive activity (Table 2). Five strains showed lower level arsenate
Tolerance of bacterial isolates for arsenite and arsenate, arsenate reductase activity, reductase activity, while the lowest activity was found in strains
and the presence of siderophores OS14 and OS22. The isolated bacteria were also screened for side-
Isolate MIC for As(III) MIC for As(V) Activity of arsenate Siderophores arsC
rophore production. Sixteen isolates showed the presence of side-
number reductase rophores (Table 2) and this was correlated with the highest activity
(mM) (mM) (mmol As(V)/min/ of arsenate reductase.
mg protein) It was significant that the isolates most highly resistant to As(V)
OS1 10 500 3.38 þ þ showed the presence of siderophores (Table 2). A deficit of iron ions
OS2 12.5 300 0.952 þ þ (ferric and ferrous) in the mine water was noted (data not shown)
OS3 10 200 0.132 þ þ
OS4 10 150 0.125 þ þ
and siderophores play an important role in iron uptake. During this
OS5 5 50 0.036  þ process, As(V) can be mobilized from the solid to the aqueous
OS6 5 75 0.068  þ phase, and in some microsites arsenic concentrations can reach
OS7 15 100 0.500 þ þ much higher levels than the average value in the examined envi-
OS8 12.5 350 0.394 þ þ
ronment. Thus, the presence of siderophores may also provide an
OS9 12.5 350 0.966 þ þ
OS10 5 150 0.055   explanation for the extreme arsenic resistance of isolates and their
OS11 5 25 0.070 þ þ high levels of arsenate reductase activity. A simplified scheme for
OS12 5 75 0.027 þ þ As3þ release from, for example, scorodite, is proposed and this is
OS13 5 100 0.587 þ þ presented in Fig. 1. Microorganisms produce iron-binding com-
OS14 5 50 0.02  þ
pounds (siderophores) that help them to uptake an iron from in-
OS15 5 100 1.299 þ þ
OS16 10 200 0.107 þ þ soluble minerals such as scorodite. Siderophores bind Fe3þ in
OS17 15 100 0.131 þ þ complexes that can be taken up by active transport mechanisms
OS18 15 100 0.136 þ þ into the bacterial cell. During this process, As(V) is mobilized from
OS19 12.5 200 0.315 þ þ
the solid to the aqueous phase and the toxic effects of arsenic for
OS20 12.5 350 1.275 þ þ
OS21 12.5 150 0.275  þ bacterial cells are neutralized by the activity of the arsenate re-
OS22 5 50 0.025   ductase. As a result of this process As(III) is released into the
environment.
 L. Drewniak et al. / Environmental Pollution 156 (2008) 1069–1074
Fig. 1. Proposed mechanism of arsenic mobilization from scorodite. Microorganisms produce iron-binding compounds (siderophores) that help them to uptake an iron from in-
soluble minerals such as scorodite. Siderophores bind Fe3þ in complexes that can be taken up by active transport mechanisms into the bacterial cell. During this process, As(V) is
mobilized from the solid to the aqueous phase and the toxic effects of arsenic for bacterial cells are neutralized by the activity of the arsenate reductase. As3þ is transported out of
the cell by ArsAB arsenic chemiosmotic efflux system and by ATPase membrane systemW.
In addition to arsenic, isolates showed tolerance to other metals
and metalloids. Multiresistance to Cu, Cd, Co, Cr, Zn, V, Mn and Se
was seen in a few. The most resistant for copper (up to 6 mM) were
strains OS9 and OS16; for cadmium (up to 2 mM) OS1, OS4 and OS9;
for nickel (up to 6 mM) OS17 and OS18; for zinc OS1 and OS9; for
chromium (up to 12 mM) OS9, OS15, OS17, OS18, OS19 and OS20;
and for manganese (up to 6 mM) OS1, OS9, OS15, OS17 and OS18.
All of the isolates (except for OS21 and OS22) showed tolerance to
the tested concentrations of vanadium and selenium. The most
highly multiresistant isolate was OS9 and the most sensitive strains
were OS12, OS13 and OS22.
Resistance to elements that did not occur in the examined en-
vironment (i.e. Cd, Co, Se) was not surprising. It is known that many
genetic determinants for heavy metal resistance are evolutionarily
old and widespread in the microbial world (Silver, 1996), and de-
spite the lack of selective pressure they are often present in bac-
terial genomes.
Half of the isolates were able to use lactate and/or acetate as
a carbon and energy source. Strain OS18 could not use organic acids
other than lactate and acetate. Strain OS 14 could use lactate only.
These results showed that simple organic compounds in the air
of Gertruda Adit that most probably originated from detritus might
represent the source of carbon and energy for the growth of het-
erotrophic microorganisms. Enzymatic and assimilation tests con-
firmed that the arsenic-tolerant bacterial isolates were able to use
these compounds or their derivatives.
4. Conclusions
The role of heterotrophs hypertolerant to arsenic in the dis-
semination of arsenic pollution has been largely ignored to date.
However, the results of the present study clearly show that de-
toxification processes based on arsenate reductase activity might
be significant in arsenic-tolerant microorganisms. Described bac-
terial strains were isolated from rocks and rock biofilms that form
part of the mine environment only, but this part contains mainly
1073
environmentally immobilized arsenic and iron. As shown in pre-
vious studies, under optimal conditions some bacterial strains can
deliver around 254 mg As3þ/mg protein per minute into the envi-
ronment. The presence of siderophores is significant because it
leads to the mobilization of ferric ions from insoluble compounds,
in parallel leading to mobilization of arsenic. This phenomenon is
important from an environmental point of view because several
methods of arsenic removal are based on the precipitation of ar-
senate with Fe3þ ions. The activity of the heterotrophic bacteria
described here leads to mobilization of arsenic in the more toxic
As3þ form and so contributes to the contamination of important
drinking water sources situated around the mine and mine waste
deposits.
Acknowledgments
This research was supported by ordered research project No.
PBZ-KBN-111/T09/2004 from the Ministry of Science and Higher
Education, Poland over the period 2005–2008. We gratefully ac-
knowledge the help of Dr. John Gittins for his critical reading of the
manuscript and English correction.
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