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SHORT COMMUNI CATI ON

Isolation and characterization of arsenite-oxidizing bacteria


from arsenic-contaminated soils in Thailand
Saowapar Kinegam Thanvapon Yingprasertchai
Somboon Tanasupawat Natchanun Leepipatpiboon
Ancharida Akaracharanya Kyoung-Woong Kim
Received: 11 March 2008 / Accepted: 14 July 2008 / Published online: 8 August 2008
Springer Science+Business Media B.V. 2008
Abstract Two hundred and eighty-eight arsenic-resistant
bacteria were isolated by an enrichment culture method
from a total of 69 arsenic-contaminated soil-samples col-
lected from Dantchaeng district in Suphanburi province
(47 samples), and from Ron Phiboon district in Nakhon Sri
Thammarat province (22 samples), in Central and Southern
Thailand, respectively. Twenty-four of the 288 isolated
arsenic-resistant bacteria were found to be arsenite-oxidiz-
ing bacteria. On the basis of their morphological, cultural,
physiological, biochemical and chemotaxonomic charac-
teristics, and supported by phylogenetic analysis based upon
their 16S rRNA gene sequences, they were divided into ve
groups, within the genera Acinetobacter, Flavobacterium,
Pseudomonas, Sinorhizobium and Sphingomonas, respec-
tively. Within genera, phylogenetic analysis using the 16S
rRNA gene sequences suggested that they were comprised
of at least ten species, ve isolates being closely related
to known bacteria (Acinetobacter calcoaceticus NCCB
22016
T
, Pseudomonas plecoglossicida FPC951
T
, Ps.
knackmussii B13
T
, Sinorhizobium morelense Lc04
T
, and
Sphingomonas subterranea IFO16086
T
). The other ve
proposed species are likely to be new species closely related
to Flavobacterium johnsoniae, Sinorhizobium morelense,
Acinetobacter calcoaceticus and Pseudomonas plecogloss-
icida, but this awaits further characterization for
conrmation of the taxonomic status. No overlap in isolated
species or strains was observed between the two sites. The
strain distribution and characterization are described.
Keywords Acinetobacter Arsenite-oxidizing bacteria
Flavobacterium Pseudomonas Sinorhizobium
Sphingomonas
Introduction
Arsenic (As), a toxic heavy metal element, is widely
distributed in nature and commonly occurs in a variety of
natural metal-bearing sulphides (Nriagu 2002). The nat-
ural occurrence of arsenic in many subservice drinking
water aquifers around the globe and its subsequent con-
tamination of drinking water or food is a major concern to
human health around the world. Elevated arsenic con-
centrations typically occur from the weathering of
arsenic-bearing minerals and geothermic sources and this
largely depends upon microbial induced reduction, oxi-
dation and methylation, which inuence the availability
and toxicity of this element (Islam et al. 2004). In addi-
tion anthropogenic point sources of local importance that
result in the local contamination of nearby air, soil,
S. Kinegam S. Tanasupawat
Department of Microbiology, Faculty of Pharmaceutical
Sciences, Chulalongkorn University, Bangkok 10330, Thailand
T. Yingprasertchai
Department of Food Science and Technology, Faculty of Science
and Technology, Kanchanaburi Rajabhat University,
Kanchanaburi 71000, Thailand
N. Leepipatpiboon
Department of Chemistry, Faculty of Science, Chulalongkorn
University, Bangkok 10330, Thailand
A. Akaracharanya (&)
Department of Microbiology, Faculty of Science, Chulalongkorn
University, Bangkok 10330, Thailand
e-mail: sanchari@chula.ac.th
K.-W. Kim
Department of Environmental Science and Engineering,
Gwangju Institute of Science and Technology,
Gwangju 500-712, South Korea
1 3
World J Microbiol Biotechnol (2008) 24:30913096
DOI 10.1007/s11274-008-9821-4
sediment and aquatic systems with arsenic include long-
term mining and the associated smelting of the sulphide
ores, coal combustion, runoff from mine tailings, pigment
production for paints, hide tanning waste, pesticide
application and poultry feedlot supplements (Lee et al.
2005). Arsenic exists naturally in both organic and inor-
ganic forms. The two major forms of inorganic arsenic
are the reduced form, arsenite (As III), and the oxidized
form, arsenate (As V). Arsenate acts as a phosphate
analogue and interferes with phosphate uptake and
transport (Tamaki and Frankenberger 1992), and inhibits
oxidative phosphorylation but, although found primarily
in aerobic conditions, it typically remains bound to min-
erals in the solid phase and thus is less available to enter
the higher animal food chains. In contrast, arsenite dis-
rupts the sulfhydryl group of proteins and interferes with
enzyme function (NRC 1999) but, despite being primarily
found in anoxic environments, it is most common in the
aqueous phase, where it is more mobile and can gain
entry into human food chains more easily under most
environmental conditions. Note that even on entry to
aerobic conditions the non-catalysed oxidation rate of
arsenite is slow with an estimated half life of a year.
Arsenite thus shows a greater biological toxicity.
Microbial activity strongly inuences arsenic oxidation
states. More than thirty strains of bacteria from at least nine
genera across the a-, b- and c-, d- and e-Proteobacteria as
well as the Archaea, are known for their ability to trans-
form inorganic arsenic forms by oxidation or reduction
(Gihring and Baneld 2001). This physiologically diverse
grouping includes both heterotrophic and chemolithoauto-
trophic arsenite oxidizers and is consistent with the
hypothesis that arsenic species may have played a crucial
role in the early stages in the development of life prior to
the cyanobacterial evolution and formation of signicant
free oxygen levels (Lebrun et al. 2003). Bacterial oxidation
of arsenite to arsenate represents a potential partial
detoxication mechanism and has applications in bio-
remediation (Simeonova et al. 2005) because it generates
the less toxic and less mobile form of arsenic. Indeed,
potentially suitable arsenite (As III)-detoxication bacteria
have been suggested (Oremland and Stolz 2003). The cli-
matic condition in Thailand is hot and humid which is
highly conducive for microbial growth. In addition, the
relatively diverse soil types and natural high biodiversity of
this region, coupled with both naturally exposed and
recently mined sources of arsenic to provide selection for
existing or derived tolerance mechanisms, makes it a likely
worthwhile region for searching for new isolates of arse-
nite-oxidizing bacteria. The purpose of this study was to
isolate novel arsenite-oxidizing bacteria for subsequent
investigation of their potential application in
bioremediation.
Materials and methods
Isolation of arsenic-resistant bacteria
Sixty-nine arsenic-contaminated soil samples (upper 10 cm
depth, pH 67) were collected from the old tin mines in
Dantchaeng district (eight sites), Suphanburi province and
Ron Phiboon district (six sites), Nakhon Sri Thammarat
province in Central and Southern Thailand, respectively.
Arsenic-resistant bacteria were isolated from each of these
soil samples by enrichment culture. Soil samples were
inoculated into chemically dened medium (CDM; sodium
citrate 0.5 g/l, casamino acids 0.05 g/l, NaNH
4
HPO
4
1.5 g/
l, KH
2
PO
4
1 g/l, MgSO
4
7 H
2
O 0.4 g/l, Vit I solution
(pyridoxal HCl 0.25%, thiamine 1%, calcium pantothenate
1%, riboavin 1%, niacin 1%, p-aminobenzoate 0.5%,
pyridoxine HCl 2%, vitamin B
12
0.002%) 10 ml, Vit II
solution (folic acid 0.0125%, biotin 0.00025%) 2 ml, sup-
plemented with 1.33 mM As [III] and incubated at
30 + 3C (in situ environmental temperature) on a rotary
shaker (200 rev/min) in the dark for 48 h. The cultures were
sequentially puried by streaking on As [III]-supplemented
CDM agar then restreaking on R2A (yeast extract 0.5 g/l,
proteose peptone 0.5 g/l, casamino acids 0.5 g/l, dextrose
0.5 g/l, soluble starch 0.5 g/l, sodium pyruvate 0.3 g/l,
KH
2
PO
4
0.3 g/l, MgSO
4
7 H
2
O 0.05 g/l) agar medium,
and incubated at 30 + 3C for 48 h. Pure cultures were
preserved on R2A agar medium and kept at 4C.
Screening of arsenite-oxidizing bacteria
The presence of arsenite-oxidizing bacteria was screened
for from the isolated arsenic-resistant bacteria by a modi-
ed microplate method (Simeonova et al. 2004). A single
colony of arsenic-resistant bacteria was grown in 12 ml of
CDM medium (without arsenic) at 30 + 3C in the dark
for 72 h on a rotary shaker (200 rev/min). The cultures
were centrifuged at 4,600g for 15 min, washed twice with
sterile deionized water and suspended in 1.2 ml deionized
water. Twenty microlitres of the cell suspension was added
to each of 3 wells of a round-bottomed 96-well plate lled
with 80 ll of 0.2 M TrisHCl buffer (pH 7.4) containing
arsenite or arsenate at a nal concentration of 2.66 and
1.33 mM, respectively, which was a concentration that
precipitate colour clearly differentiated, and then incubated
at 30 + 3C for 72 h. After 72 h, bacterial viability was
investigated by transferring cells to CDM agar plates using
a sterile toothpick and culturing. Colour reaction test was
initiated by adding 0.2 M AgNO
3
(100 ll) into the 72 h
microtiter plate culture. Since arsenite and arsenate form a
light yellow and light brownish precipitates, respectively,
isolates that showed a light brownish precipitate in the
presence of both arsenite and arsenate were selected as
3092 World J Microbiol Biotechnol (2008) 24:30913096
1 3
arsenite-oxidizing bacteria. Each assay was performed as
three independent repeats for each trial.
Bacterial identication methods
Arsenite-oxidizing bacteria isolated as above were grown
on R2A agar medium at 37C for 24 h and the cell mor-
phology, colony appearance, spore formation, and
pigmentation were recorded. Catalase, oxidase, hydrolysis
of aesculin, L-arginine, casein, gelatin, starch, tyrosine and
deoxyribonuclease (DNase) activity; MR-VP, indole test,
nitrate reduction, Simmon citrate test, Triple Sugar Iron
agar (TSI), dihydroxyacetone from glycerol, urease activity
and acid from carbohydrates were determined, as described
by Barrow and Feltham (1993). Growth at different pH (5,
6, 8 and 9), in 3% and 5% (w/v) NaCl, and at different
temperatures (10, 15, 20, 37, 40, 50, 55 and 60C) were
investigated. All tests were carried out using R2A medium
as the basal medium and incubated at 37C, except for the
investigation of the effect of temperatures. Quinones were
extracted from freeze-dried cells with chloroform:methanol
(2:1) and puried with a silica gel TLC (Merck no.
1.05744). The puried quinones were analysed by HPLC
(Komagata and Suzuki 1987).
DNA was isolated from cells grown on R2A agar for
1848 h, and puried by the method of Saito and Miura
(1963). DNA base composition (% GC composition) was
determined by the method of Tamaoka and Komagata
(1984). The 16S rRNA gene was amplied, puried, and
analysed as described previously (Tanasupawat et al.
2004). The derived sequences (9371,501 bp) were
aligned with the selected sequences obtained from the
GenBank/EMBL/DDBJ database employing CLUSTAL_X
version 1.83 (Thompson et al. 1997). The alignment was
manually edited to remove gaps and ambiguous nucleo-
tides prior to construction of phylogenetic trees. The
phylogenetic tree was constructed by using the neighbour-
joining method (Saitou and Nei 1987) in the MEGA pro-
gramme version 2.1 (Kumar et al. 2001). The condence
values of branches of the phylogenetic tree were deter-
mined using bootstrap analyses (Felsenstein 1985) based
on 1,000 resamplings.
Results and discussion
Isolation of arsenite-oxidizing bacteria
From 69 arsenic-contaminated soil samples collected,
288 arsenic-resistant isolates were cloned. From these,
24 arsenite-oxidizing bacterial strains without any
arsenate-reducing activity were isolated and further
characterized.
Identication of the isolates
The twenty-four arsenite-oxidizing bacterial isolates were
categorised into ve different groups based on their mor-
phological, cultural, physiological and biochemical
characteristics. They were all Gram-negative, rod-shaped
or coccobacilli. Across the ve groups, colony morpholo-
gies on R2A agar medium were quite diverse and were thus
described within each group. Most of them showed a
positive reaction for growth on 3% (w/v) NaCl, at pH 59,
and in anaerobic conditions, catalase, citrate utilization;
hydrolysis of L-arginine and starch; and acid from glucose,
but were negative for both growth at 60C and for the
VogesProskauer reaction. The more variable characteris-
tics in physiological and biochemical properties of each
group are summarized in Table 1.
Group 1 contained 13 isolates; AS8-1, AS11-2, AS11-3,
AS18-1, AS18-2, AS18-3, AS19-1, AS19-2, CSP3-1,
S1-8A, S2-2F, S2-4E, and S2-8E with circular, convex,
smooth, slightly opaque and yellowish colour colonies of
0.050.45 cm diameter. The representative isolates (ran-
domly selected), AS11-3 and AS19-1, showed the highest
16S rRNA gene sequence similarity to Acinetobacter
calcoaceticus NCCB 22016
T
(Fig. 1), whilst isolate AS19-1
contained Q-10 as a major ubiquinone and had a genomic
DNA G + C content of 43.6 mol %. Therefore, the repre-
sentative isolate in Group 1, AS19-1 was identied as
Acinetobacter calcoaceticus and, AS11-3 as a newspecies in
the genus Acinetobacter (Carr et al. 2003).
Group 2 contained one isolate, S2-3H with circular,
convex, translucent, entire margin and yellow-orange col-
our colonies of 0.050.2 cm diameter. S2-3H showed
97.4% 16S rRNA gene sequence similarity to Flavobac-
terium johnsoniae DSM 2064
T
(Fig. 1). This isolate
contained MK-6 as a major menaquinone and had a DNA
G + C content of 37 mol %. Therefore, the isolate was
identied as a new species in the genus Flavobacterium
(McCammon and Bowman 2000).
Group 3 contained ve isolates, AS4-1, AS5-1, AS14-3,
AS20-1 and AS20-2, with circular, smooth, regular margin,
white-yellowish colour colonies of 0.10.3 cm diameter
which produced a pigment with light yellow-green uores-
cence on irradiation with U.V. light. The representative
isolates (randomly selected), AS5-1 and AS20-2, showed the
highest 16S rRNA gene sequence similarity to Pseudomonas
plecoglossicida FPC951
T
(99.4%) and P. knackmussii B13
T
(99.3%), respectively (Fig. 1). The isolates AS5-1 and AS20-
2 contained Q-9 as a major ubiquinone and AS5-1 had a DNA
G + Ccontent of 61.7 mol %. The 16S rRNAgene sequence
similarity of AS14-3 was 98.7% to P. plecoglossicida
FPC951
T
. Therefore, the representative isolate AS14-3 was
identied as a new species in the genus Pseudomonas (Ach-
ouak et al. 2000).
World J Microbiol Biotechnol (2008) 24:30913096 3093
1 3
Group 4 contained four isolates, S1-1E, S2-1B, S2-3G
and S3-1C, with circular, smooth, shiny, convex and
creamy-white colonies of 0.10.5 cm diameter. The rep-
resentative isolates (randomly selected), S3-1C, S2-3G and
S1-1E showed the highest 16S rRNA gene sequence sim-
ilarity (99.4%, 98.1% and 97.1%) to Sinorhizobium
morelense Lc04
T
(Fig. 1), respectively. The isolate S3-1C
contained Q-10 as major ubiquinone and a DNA G + C
content of 61.6 mol %. The 16S rRNA gene sequence
similarity of S3-1C was 99.4% to Sinorhizobium morelense
Lc04
T
. Therefore, the representative isolate S2-3G and
S1-1E were identied as a new species in the genus
Sinorhizobium (Wang et al. 2002; Willems et al. 2003).
Group 5 contained one isolate, S2-3F, with circular, low
convex, smooth, entire, opaque and yellow colonies of
0.10.3 cm diameter. With a high 16S rRNA gene
sequence similarity (99.1%) to Sphingomonas subterranea
IFO16086
T
(Fig. 1), containing Q-10 as a major ubiqui-
none and a DNA G + C content of 64.9 mol %, the isolate
in Group 5 was identied as Sphingomonas subterranea
(Balkwill et al. 1997). As outlined above, phylogenetic
analysis revealed that the Gram-negative arsenite-oxidizing
bacteria isolated and grouped biochemically into ve
groups were likely to be members of the ve genera
Acinetobacter, Flavobacterium, Pseudomonas, Sinorhizobium,
and Sphingomonas. However, overlap in the observed
distribution of isolates between the two sites was restricted
to the abundant Acinetobacter which comprised of 40%
and 64% of arsenite-oxidizing bacteria isolates found at
Dantchaeng district, Suphanburi province and Ron Phiboon
district, Nakhon Sri Thammarat province, respectively
(Table 2). Of course, the sampling size means we cannot
exclude the existence of the other species from each district
occurring in the other district, but it seems likely that their
abundance at least is signicantly different. The potential
for bioremediation application of the arsenite-oxidizing
bacteria isolated here and the mechanisms they attain this
by, will now be further studied.
Conclusion
Arsenite-oxidizing bacteria were isolated from two sites in
Thailand with some apparent species-site specicity (at least
at the level of isolatable species abundance). Acinetobacter,
Flavobacterium, Sinorhizobium and Sphingomonas isolates
were distributed in Dantchaeng district, Suphanburi prov-
ince (Central Thailand), whereas Acinetobacter and
Table 1 Phenotypic and chemotaxonomic characteristics of the isolates
Characteristics Group 1
(13 isolates)
Group 2
(1 isolate)
Group 3
(5 isolates)
Group 4
(4 isolates)
Group 5
(1 isolate)
Cell shape Coccobacilli Rods Rods Rods Rods
Growth in 5% (w/v) NaCl +(-2) - +(-1) -(+1) -
Growth at 40C + - + + +
Growth at 50C +(-4) - +(-1) + +
Catalase + + + +(-2) +
Oxidase -(+1) - + + -
Methyl red -(+1) - - - -
DNase -(+3) - +(-1) - +
Urease +(-1) - + + -
Indole production -(+1) - - - -
Citrate utilization + - + + -
H
2
S formation +(-6) - +(-1) +(-1) -
Nitrate reduction -(+3) - -(+1) - -
Hydrolysis
Aesculin -(+4) + -(+1) + +
L-Arginine + + + + -
Casein -(+2) + -(+1) -(+1) -
Gelatin -(+3) - -(+1) - -
Acid from glucose +(-5) + +(-1) +(-2) +
Ubiquinone (Q)* Q-9 MK-6 Q-9 Q-10 Q-10
DNA G + C (mol %)* 43.6 37 61.7 61.6 64.9
*Determined for selected representative strains
3094 World J Microbiol Biotechnol (2008) 24:30913096
1 3
Pseudomonas isolates were found in Ron Phiboon district,
Nakhon Sri Thammarat province (Southern Thailand). The
novel species, Flavobacteriumsp. S2-3H, Sinorhizobiumsp.
S2-3G and S1-1E distributed in Dantchaeng district, Su-
phanburi province, and Acinetobacter sp. AS11-3 and
Pseudomonas sp. AS14-3, distributed in Ron Phiboon
Fig. 1 Phylogenetic tree based
on 16S rRNA gene sequences,
showing the relationship
between strain AS5-1, AS14-3,
AS20-2, AS19-1, AS11-3, S2-
3F, S1-1E, S2-3G, S3-1C and
S2-3H and related bacterial
species. The branching pattern
was generated by the neighbour-
joining method. Based on 1,000
replications, bootstrap
percentages above 50% are
shown. Bar, 0.02, substitutions
per nucleotide position
Table 2 Distribution of the
arsenite-oxidizing bacteria
isolated
a
%SS: Percentage sequence
similarity of the isolates 16S
rRNA gene sequence to the
indicated nearest matching
species (closest species) in
GenBank using BLASTn
searching
Groups are those designated
from phenotypic and
chemotaxonomic characteristics
as outlined in Table 1
Location Isolate no. Closest species %SS
a
Group Identication
Dantchaeng S2-3H Fl. johnsoniae DSM 2064
T
97.4 2 Flavobacterium sp.
S1-1E Si. morelense Lc04
T
97.1 4 Sinorhizobium sp.
S2-3G Si. morelense Lc04
T
98.1 4 Sinorhizobium sp.
S3-1C Si. morelense Lc04
T
99.4 4 Si. morelense
S2-3F Sp. subterranea IFO 16086
T
99.1 5 Sp. subterranea
Ron Phiboon AS11-3 A. calcoaceticus NCCB 22016
T
98.9 1 Acinetobacter sp.
AS19-1 A. calcoaceticus NCCB 22016
T
99.6 1 A. calcoaceticus
AS5-1 P. plecoglassicida FPC
.
951
T
99.4 3 P. plecoglossicida
AS14-3 P. plecoglossicida FPC951
T
98.7 3 Pseudomonas sp.
AS20-2 P. knackmusii B13
T
99.3 3 P. knackmussii
World J Microbiol Biotechnol (2008) 24:30913096 3095
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district, Nakhon Sri Thammarat province, will be further
classied by evaluation of their DNADNA hybridization
and some chemotaxonomic characteristics.
Acknowledgements The authors thank Mr. Paichayon Char-
oenchaisri, Bureau of Environmental Management, Department of
Primary Industries and Mines, Ministry of Industry for providing
assistance in soil sampling and Dr. Robert Butcher for critical reading
of the manuscript. This work was supported by the International
Environmental Research Center (IERC), Gwangju Institute of Science
and Technology, Gwangju, Korea.
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