Biosci. Biotechnol. Biochem., 69 (11), 2073–2080, 2005

Growth Inhibition by Tungsten in the Sulfur-Oxidizing Bacterium
Acidithiobacillus thiooxidans
Atsunori N EGISHI,1 Tadashi M URAOKA,2 Terunobu M AEDA,3 Fumiaki T AKEUCHI,4
Tadayoshi K ANAO,5 Kazuo K AMIMURA,5 and Tsuyoshi S UGIO2; y1

Technical Research Institute, Hazama Corporation, 515-1 Nishimukai, Karima, Tsukuba 305-0822, Japan
Division of Science and Technology for Energy Conversion, Graduate School of Natural Science and Technology,
Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan
3
Civil Chemical Engineering Corporation, 3411 Sanuki-machi Ryugasaki, Ibaragi 301-0033, Japan
4
Environmental Management and Safety Section, Health and Environment Center, Okayama University,
3-1-1 Tsushima-naka, Okayama 700-8530, Japan
5
Faculty of Agriculture, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan
2

Received May 9, 2005; Accepted July 24, 2005

Growth of ﬁve strains of sulfur-oxidizing bacteria
Acidithiobacillus thiooxidans, including strain NB1-3,
was inhibited completely by 50 M of sodium tungstate(Na2 WO4 ). When the cells of NB1-3 were incubated in
0.1 M -alanine–SO4 2 buﬀer (pH 3.0) with 100 M
Na2 WO4 for 1 h, the amount of tungsten bound to thecells was 33 g/mg protein. Approximately 10 times
more tungsten was bound to the cells at pH 3.0 than at
pH 7.0. The tungsten binding to NB1-3 cells wasinhibited by oxyanions such as sodium molybdenum
and ammonium vanadate. The activities of enzymes
involved in elemental sulfur oxidation of NB1-3 cells
such as sulfur oxidase, sulfur dioxygenase, and sulﬁteoxidase were strongly inhibited by Na2 WO4 . These
results indicate that tungsten binds to NB1-3 cells andinhibits the sulfur oxidation enzyme system of the cells,
and as a result, inhibits cell growth. When portland
cement bars supplemented with 0.075% metal nickel
and with 0.075% metal nickel and 0.075% calcium
tungstate were exposed to the atmosphere of a sewage
treatment plant containing 28 ppm of H2 S for 2 years,
the weight loss of the portland cement bar with metal
nickel and calcium tungstate was much lower than the
cement bar containing 0.075% metal nickel.
Key words:

tungsten; Acidithiobacillus thiooxidans;concrete corrosion; bacteriostatic agent;sulfur oxidation

Corrosion of concrete structures, especially, in sewer
systems and sewage treatment plants, has become a big
social problem. In 1945, Parker ﬁrst presented evidence
that concrete structures are corroded by sulfuric acidproduced by the sulfur-oxidizing bacterium Thiobacillus
concretivourus.1–3) The bacterium isolated by Parkery

was later identiﬁed as Acidithiobacillus thiooxidans. In
the process of concrete corrosion, hydrogen sulﬁdeproduced in sewage pipes by the action of sulfatereducing bacteria is oxidized by sulfur-oxidizing bacteria to give sulfuric acid.1–7) Concrete contains calciumoxide, calcium hydroxide, and calcium carbonate, andthese compounds react with sulfuric acid to give a sandy
calcium sulfate (gypsum).7) After the studies done byParker, many kinds of thiobacilli such as Halothiobacillus neapolitanus8) (formerly Thiobacillus neapolitanus), Thiomonas intermedius8) (formerly Thiobacillus
intermedius), and Thiobacillus novellus, Paracoccus
versutus8) (formerly Thiobacillus versutus), and Acidithiobacillus ferrooxidans8) (formerly Thiobacillus ferrooxidans) were isolated from corroded concrete andmore information was accumulated about the process ofconcrete corrosion. A concrete structure just after
construction contains calcium hydroxide and thus has
a pH of 12–13. The initial step in concrete corrosion is
the neutralization of the concrete structure mainly byatmospheric carbon dioxide or by sulfur-oxidizing
bacteria that can grow under alkaline pH.9) The second
step is a rapid acidiﬁcation of the neutralized concrete
structure by sulfuric acid produced by obligately chemolithotrophic sulfur oxidizers A. thiooxidans and A. ferrooxidans.10)
It is known that oxidation of elemental sulfur by
A. thiooxidans is a complex process involving thecontact of cells with sulfur particles, the oxidation of
sulfur to sulﬁte, and the oxidation of sulﬁte to sulfate.
The ﬁrst step in sulfur oxidation from elemental sulfur to
sulﬁte is catalyzed by sulfur dioxygenase.11–13) Sulﬁte isthought to be the key intermediate in the oxidation of
inorganic sulfur compounds in thiobacilli.14–18) Sulfur isnearly quantitatively oxidized to sulﬁte by A. thioox-

To whom correspondence should be addressed. Tel/Fax: +81-86-251-8306; E-mail: sugio@cc.okayama-u.ac.jp

2074

A. NEGISHI et al.

idans cells when the sulﬁte oxidation is inhibited by 2-nheptyl-4-hydroxyquinoline N-oxide (HOQNO).19) The
sulﬁte produced by sulfur dioxygenase is further
oxidized to sulfate either through sulﬁte:cytochrome coxidoreductase,20) or through adenosine phosphosulfate(APS) reductase.21) A. thiooxidans JCM 7814 has only a
AMP-independent sulfur oxidation pathway, and themembrane-bound sulﬁte dehydrogenase was puriﬁed
from the bacterium.22,23)
Previously we showed that concrete supplemented
with nickel is resistant to corrosion,24) and that nickel
bound to the A. thiooxidans NB1-3 cells inhibits both
sulfur dioxygenase and sulﬁte oxidase, and as a result,inhibits cell growth.9,25,26) We prepared portland cement
bars (40  40  160 mm) supplemented with various
kinds of inorganic and organic compounds, and thesebars were hung from the lid of a sludge tank manhole
and exposed to 5–10 ppm hydrogen sulﬁde atmosphere.24) After 2 years of exposure, it was found thatthe surface of the portland cement bar supplemented
with nickel was especially smooth, while that without
nickel was brittle and rough,24) suggesting the possibility
that nickel can be used as a bacteriostatic agent for theprevention of concrete corrosion. Among the inorganicand organic compounds tested, nickel was the mosteﬀective for the prevention of concrete corrosion. But,
nickel is not the best bacteriostatic agent for preventionof the growth of A. thiooxidans cells, which have an
optimum growth pH at 2–3. We found that the growth of
NB1-3 cells was more strongly inhibited by Ni2þ at
pH 7.0 than at pH 3.0, probably because more nickel
binds to NB1-3 cells at neutral pH than at acidic pH.
Strain NB1-3 cells without nickel treatment contained
1.7 nmol nickel per mg protein, and the cells treated with
10 mM NiSO4 at pH 3.0 and 7.0 contained 35 and
160 nmol nickel per mg of protein respectively.25)
Therefore, to make a concrete structure that does notcorrode easily under attack by A. thiooxidans cells, it isimportant to look for a bacteriostatic agent that can bindto A. thiooxidans cells more tightly under acidic pH thanunder neutral and/or alkaline pH and thus can inhibit
more strongly the sulfur oxidation enzyme system of thebacterium.
In this report we show for the ﬁrst time that a low
concentration of sodium tungstate (50 mM) completely
inhibits the growth and sulfur-oxidation enzyme system
of A. thiooxidans NB1-3 cells, and that in contrast to
Ni2þ , which inhibits completely the growth of A. thiooxidans cells at 5 mM and binds more to NB1-3 cell at
pH 7.0 than at pH 3.0,25) approximately 10 times more
tungsten binds to the NB1-3 cells at pH 3.0 than at
pH 7.0. These results indicate that tungsten can cover
the weak point of Ni2þ as a bacteriostatic agent for theprevention of concrete corrosion caused by A. thiooxidans. The rate of concrete corrosion decreased markedly when the portland cement bars supplemented withboth 0.075% calcium tungstate and 0.075% metal nickel
were incubated in the atmosphere of sewage treatment

plants containing 28 ppm H2 S for 2 years as comparedwith the cement bar supplemented with only 0.075%
metal nickel.

Materials and Methods
Microorganism, medium, and conditions of cultivation. The sulfur-oxidizing bacteria Acidithiobacillus
thiooxidans strain NB1-3, A. thiooxidans JCM 3867,
A. thiooxidans ON106, A. thiooxidans NBRC 13701,
and A. thiooxidans NBRC 137724 were used throughout
this study. Strain NB1-3 was isolated from corroded
concrete in Fukuyama, Japan.25) A 1,000 ml of salt
solution contained 3 g (NH4 )2 SO4 , 0.1 g KCl, 0.5 g
K2 HPO4 , 0.5 g MgSO4 7H2 O, and 0.01 g Ca(NO3 )2 .
The pH of the salt solution was adjusted at pH 2.5 withsulfuric acid. Elemental sulfur was autoclaved byheating for 30 min at 110  C and further incubated for
24 h at 30  C. This sterilization process of elemental
sulfur was repeated three times. Sulfur-salt medium used
for the cultivation of NB1-3 cells composed of 20 ml of
the autoclaved-salt solution (pH 2.5) and the sterilized
elemental sulfur (0.2 g). Growth experiments were doneby shaking 20 ml of sulfur-salt medium inoculated with
NB1-3 culture in a 50-ml ﬂask at 30  C. A large mass of
NB1-3 cells used for the isolation of sulfur dioxygenase
was obtained by culturing the strain in 72-liter sulfursalt medium (pH 2.5) under forced aeration. After
cultivation of the strain for one week at 30  C, theculture ﬂuid obtained was ﬁltered with Toyo no. 2 ﬁlter
paper to remove sulfur particles and centrifuged at
10;000  g to obtain cell pellets. The harvested cells
were washed three times with 50 mM Tris–HCl buﬀer(pH 7.5).

.

Growth rate. Cells were separated from elemental
sulfur particles by ﬁltering cultures with Toyo paper
ﬁlter no. 5B. Almost all of the cells in the culture ﬂuid
passed through the paper ﬁlter. The numbers of cells
after ﬁltration were counted with a hemacytometer(Kayagaki Irika Kogyo, Tokyo, Japan) after dilution
with 0.1 N sulfuric acid, when necessary.
Sulfur oxidase activity. Sulfur oxidase activity wasmeasured at pH 3.0 with resting cells of NB1-3 by theoxygen uptake caused by the oxidation of elemental
sulfur in a Warburg manometer. The reaction vessel
contained 3.0 ml of reaction mixture plus 0.2 ml of 20%
potassium hydroxide in the center well. The gas phase
was air and the temperature was kept at 30  C. The
reaction mixture (pH 3.0) was composed of -alanine(100 mmol), resting cells (5 mg protein), and elemental
sulfur (100 mg).
Sulfur dioxygenase activity. Sulfur dioxygenase activity was measured at pH 6.0 with the cytosol of NB1-3
by the oxygen uptake caused by the oxidation ofelemental sulfur in a Warburg manometer.25) Each

vessel contained 3.0 ml of reaction mixture plus 0.2 mlof 20% KOH in the center well. The composition of thereaction mixture was as follows (in 3.0 ml): 2 mg ofprotein from the 105;000  g soluble fraction (thecytosol) of NB1-3, 200 mmol of MES buﬀer (pH 6.0),
0.5 mmol of HOQNO, 300 mg of glycerol, 100 mg ofelemental sulfur, and 50 mmol of GSH. The gas phase
was air, and the temperature was 30  C.
Sulﬁte oxidase activity. Sulﬁte oxidase activity was
manometrically measured at pH 7.5 with the plasma
membrane by the oxygen uptake caused by the oxidationof SO3 2 in a Warburg manometer.25) Each vessel
contained 3.0 ml of reaction mixture plus 0.2 ml of 20%
KOH in the center well. The gas phase was air, and thetemperature was 30  C. The composition of the reaction
mixture was as follows (in 3.0 ml): 2 mg of plasma
membrane, 200 mmol of MOPS buﬀer (pH 7.5), and
20 mmol of NaHSO3 .
Analysis of tungsten bound to A. thiooxidans NB1-3
cells. The amounts of tungsten bound to resting cells of
A. thiooxidans NB1-3 cells were measured by thethiocyanate method.27,28)
Endurance test of portland cement bar containing
nickel and tungsten. The portland cement bar (40 
40  160 mm) containing 0.075% metal nickel and0.075% calcium tungstate were prepared with a mortar
mixer. After 28 d of standard curing, the portland
cement bars prepared were hung from the lid of asludge tank manhole and exposed to the atmosphere,
containing hydrogen sulﬁde (H2 S).24) The concentrations of H2 S in sewage treatment plants A and B were 42
and 28 ppm respectively. After 2 years of exposure in
the sludge tank manhole, a sandy calcium sulfate on the
cement bar was taken away, and the weights of theconcrete bars were measured.24)
Protein content. Protein was measured by the methodof Lowry et al. with crystalline bovine serum albumin as
the reference protein.29)

Results
Growth of sulfur-oxidizing bacteria in sulfur medium
with sodium tungstate
A. thiooxidans NB1-3 was grown in 20 ml of sulfur
medium (pH 2.5) with or without sodium tungstate(Na2 WO4 ). Growth of NB1-3 was strongly inhibited by
20 mM Na2 WO4 , and completely by 50 mM Na2 WO4(Fig. 1). Five strains of A. thiooxidans, including strain
NB1-3, were cultured in sulfur medium with or without
50 mM Na2 WO4 . None of the strains tested grew in sulfur
medium (pH 2.5) with 50 mM Na2 WO4 (data not shown),
indicating that Na2 WO4 is a strong growth inhibitor
common to A. thiooxidans cells.

2075

Cell growth ( × 107 cells/ml)

Growth Inhibition by Tungsten in A. thiooxidans

Time (d)Fig. 1. Eﬀect of Sodium Tungstate on the Growth of A. thiooxidans
NB1-3.
A. thiooxidans NB1-3 was grown in 20 ml of sulfur medium(pH 2.5) with 1 mM ( ), 10 mM ( ), 20 mM ( ), and 50 mM ( )sodium tungstate, or without sodium tungstate ( ). Values shown
are means of duplicate experiments.

Amounts of tungsten bound to A. thiooxidans NB1-3
cells
To clarify the mechanism of tungsten inhibition more
precisely, the amounts of tungsten bound to A. thiooxidans NB1-3 cells were measured. Resting cells of NB13 were incubated for 1 h in 2.0 ml of 0.1 M -alanine–
SO4 2 buﬀer (pH 3.0) containing 100 mM Na2 WO4 .
After incubation of the cells with 100 mM Na2 WO4 , thecells were washed three times with 0.1 M -alanine–
SO4 2 buﬀer (pH 3.0), and then the concentrations oftungsten bound to the cells were measured. The amountsof tungsten bound to the resting cells increased with theamounts of resting cells used (Fig. 2). When the resting
cells were incubated with 2.0 ml of 100 mM Na2 WO4 at
pH 3.0 for 1 h, the maximum amount of tungsten boundto NB1-3 cells was 33 mg/mg protein. The amounts oftungsten bound to the cytosol and plasma membrane
fractions of NB1-3 were 90 and 88 mg/mg protein
respectively.
Eﬀects of pH on the binding of tungsten to A. thiooxidans NB1-3 cells
The eﬀects of pH on the binding of tungsten to
A. thiooxidans cells were studied with resting cells ofstrain NB1-3. The amount of tungsten bound to NB1-3
cells increased concomitant with pH decrease (Fig. 3).
Approximately 10 times more tungsten bound to the
NB1-3 cells at pH 3.0 than at pH 7.0.
Eﬀect of heavy metal ions on the binding of tungsten
to A. thiooxidans NB1-3 cells
To study the speciﬁcity of tungsten binding to resting
cells of A. thiooxidans, the amounts of tungsten boundto the NB1-3 cells was measured in the presence of
1.0 mM Na2 WO4 and 1.0 mM metal ions other than2076

A. NEGISHI et al.

Na2 WO4 . The same concentration of cupric, cadmium,
zinc, and manganese ions as that of sodium tungstate(1.0 mM) did not disturb tungsten binding to the cells(Fig. 4). Ferric ions slightly increased the amounts oftungsten bound to the cells. Vanadate and molybdenum,
which are oxyanions, inhibited tungsten binding 42 and
68% respectively. These results suggest the existence ofa protein to which oxyanion binds speciﬁcally in
A. thiooxidans NB1-3 cells.

Endurance test of portland cement bar supplemented
with nickel and tungsten
Portland cement bars (40  40  160 mm) without
metal, with 0.075% metal nickel, and with 0.075% metal
nickel plus 0.075% calcium tungstate were prepared.
The portland cement bars thus prepared were hung fromthe lids of sludge tank manholes of sewage treatment( µ g/mg protein)

Amounts of tungsten bound to resting cells

Fig. 2. Eﬀects of Concentration of A. thiooxidans NB1-3 Cells on
Tungsten Binding.
Various concentration of A. thiooxidans NB1-3 cells was incubated in 2.0 ml of 0.1 M -alanine–SO4 2 buﬀer (pH 3.0) with
100 mM Na2 WO4 for 1 h at 30  C. After treatment, the resting cells
were washed three times with 0.1 M -alanine–SO4 2 buﬀer(pH 3.0) to remove free Na2 WO4 , and the amounts of tungsten
bound to the cells were measured, as described in ‘‘Materials andMethods’’. Values shown are means of duplicate experiments.

Eﬀects of sodium tungstate on the enzymes involved inelemental sulfur oxidation
To clarify the mechanism of growth inhibition ofstrain NB1-3 by Na2 WO4 , the eﬀect of tungsten on theactivity of sulfur oxidase, which catalyzes oxidation ofelemental sulfur to yield sulfate, was studied at pH 3.0
with resting cells of NB1-3. The activity of sulfur
oxidase of NB1-3 cells was inhibited 33, 68, and 95% by
0.1, 1.0, and 10 mM of Na2 WO4 respectively (Fig. 5A).
It is known that elemental sulfur is ﬁrst oxidized bysulfur dioxygenase to yield sulﬁte, and that the sulﬁteproduced is further oxidized by sulﬁte oxidase to yield
sulfate in A. thiooxidans.11–20) Sulfur dioxygenase activity is located in the 105;000  g soluble fraction of
A. thiooxidans NB1-3 cells. The activity measured at
pH 6.0 with 105;000  g soluble fraction was inhibited
8, 63, and 64% by 1.0, 3.0, and 10 mM Na2 WO4respectively (Fig. 5B). Sulﬁte oxidase was located in theplasma membrane of NB1-3. Activity measured at
pH 7.5 with the plasma membrane was inhibited 8, 36,
and 49% by 1.0, 3.0 and 10 mM Na2 WO4 respectively(Fig. 5C). These results indicate that tungsten bound to
NB1-3 cells and inhibited the sulfur oxidation enzyme
system of the cells, and as a result, inhibited cell growth.

pH
Fig. 3. Eﬀects of pH on Tungsten Binding to Resting Cells of A. thiooxidans NB1-3.
Resting cells (1 mg of protein) of A. thiooxidans NB1-3 were incubated in 2.0 ml of the buﬀers indicated in this ﬁgure ( , 0.1 M -alanine–
SO4 2 buﬀer; , 0.1 M sodium acetate buﬀer; , 0.1 M sodium phosphate buﬀer; , 0.1 M H3 BO4 –KCl–NaOH buﬀer) with 100 mM Na2 WO4 for
1 h at 30  C. After the treatment, the cells were washed three times with 0.1 M -alanine–SO4 2 buﬀer (pH 3.0) to remove free Na2 WO4 and theamounts of tungsten bound to the cells were measured, as described in ‘‘Materials and Methods’’. Values shown are means of duplicate
experiments.

2077

45
40
35
30
25
20
15
10

Na 2MoO4 + Na 2WO4NH4VO3 + Na 2WO4

FeCl3 + Na 2WO4

FeSO4 + Na 2WO4

CoSO4 + Na 2WO4

MnSO4 + Na 2WO4Al2(SO4)3 + Na 2WO4

ZnSO4 + Na 2WO4

CdSO4 + Na 2WO4

NiSO4 + Na 2WO4

0

CuSO4 + Na 2WO4

5
Na 2WO4

Amount of tungsten bound to resting cellsof A. thiooxidans NB1-3 ( µ g/mg protein)

Growth Inhibition by Tungsten in A. thiooxidans

Fig. 4. Eﬀect of Heavy Metal Ions on the Amounts of Tungsten Binding to Resting Cells of A. thiooxidans NB1-3.
Resting cells (1 mg of protein) of NB1-3 were incubated with 1.0 mM Na2 WO4 for 1 h at 30  C, or with 1.0 mM Na2 WO4 plus 1.0 mM metal ion
such as CuSO4 , NiSO4 , CdSO4 , ZnSO4 , Al2 (SO4 )3 , MnSO4 , CoSO4 , FeSO4 , FeCl3 , NH4 VO3 , or Na2 MoO4 for 1 h at 30  C. After treatment, thecells were washed three times with 0.1 M -alanine–SO4 2 buﬀer (pH 3.0) to remove free metal ion and Na2 WO4 . The amount of tungsten boundto the cells were measured as described in ‘‘Materials and Methods’’. Values shown are means of duplicate experiments.

180
160

120

100

A

90

B

C
100

80

140

O2 uptake ( µ l)

O2 uptake ( µ l)

100
80
60

O2 uptake ( µ l)

70

120

60
50
40

80

60

40

30
40

20
20

20

10

0

0

0

30

60

0

0

Time (min)30Time (min)

60

0

30

60

Time(min)Fig. 5. Eﬀect of Sodium Tungstate on the Activities of Sulfur Oxidase, Sulfur Dioxygenase, and Sulﬁte Oxidase of A. thiooxidans NB1-3.
The activity of sulfur oxidase, which catalyzes oxidation of elemental sulfur to yield sulfate (A), was measured at pH 3.0 with resting cells of
NB1-3. Enzyme activity was measured in the reaction mixtures with 0.1 mM ( ), 1.0 mM ( ), and 10 mM ( ) of sodium tungstate, or without
sodium tungstate ( ). The activity of sulfur dioxygenase, which catalyzes oxidation of elemental sulfur to yield sulﬁte (B), was measured at
pH 6.0 with the cytosol (105;000  g soluble fraction) of NB1-3. Enzyme activity was measured in the reaction mixtures with 0.1 mM ( ),1.0 mM ( ), and 10 mM ( ) of sodium tungstate, or without sodium tungstate ( ). The activity of sulﬁte oxidase, which catalyzes oxidation of
sodium sulﬁte to yield sulfate (C), was measured at pH 7.5 with the plasma membrane of NB1-3. Enzyme activity was measured in the reaction
mixtures with 1.0 mM ( ), 3.0 mM ( ), and 10 mM ( ) of sodium tungstate, or without sodium tungstate ( ). Values shown are means ofduplicate experiments.

plants A and B and exposed to the atmosphere,
containing hydrogen sulﬁde. The concentrations ofhydrogen sulﬁde in sewage treatment plants A and B
were 42 and 28 ppm respectively. After 2 years of

exposure in the sludge tank manholes, the weights of theconcrete bars were measured.
When portland cement bars without metals were hungfor 2 years in the atmosphere of sewage treatment plant

2078

A. NEGISHI et al.

12
Weight loss of concrete (% per 2 years)

A

B

10
8
6
4
2
0

Fig. 6. Endurance Test of Portland Cement Bars Containing Nickel and Tungsten.
Portland cement bars (40  40  160 mm) without metal (I), with 0.075% metal nickel (II), and with 0.075% metal nickel plus 0.075%
tungsten oxide (III) were prepared. They were hung from the rids of manholes of sewage treatment plants A and B and exposed to atmospheres
containing hydrogen sulﬁde for 2 years. The concentrations of hydrogen sulﬁde in sewage treatment plants A and B were 42 and 28 ppm
respectively. After 2 years of exposure in the manhole atmospheres of sewage treatment plants A and B, weight losses of the cement bars were
measured. Values shown are means of duplicate experiments.

A, in which the mean concentration of H2 S per year was
42 ppm, the weight loss of the bar was 4% as comparedwith the bar without H2 S treatment (Fig. 6A). The
addition of metal nickel to the cement bar at 0.075%
decreased the weight loss by 0.8% over 2 years.
Interestingly, the addition of both 0.075% metal nickeland 0.075% calcium tungstate decreased weight loss by
0.2% over 2 years. When the portland cement bars
without metals, with 0.075% metal nickel, and with
0.075% metal nickel plus 0.075% calcium tungsten werealso hung for 2 years in the atmosphere of sewage
treatment plant B, in which mean concentration of H2 S
per year was 28 ppm, the weight losses of these cement
bars were 10, 6, and 1%, respectively (Fig. 6B). These
results indicate that further addition of 0.075% calcium
tungstate to the 0.075% metal nickel-portland cement
bar can prevent weight loss in concrete bars as comparedwith the 0.075% metal nickel-portland cement bars.

Discussion
It was found in this study that (i) 50 mM of Na2 WO4can inhibit completely the growth of ﬁve strains of
A. thiooxidans, including strain NB1-3, (ii) Na2 WO4binds to NB1-3 cells more tightly below pH 5 thanabove pH 6.0, and (iii) the enzymes involved inelemental sulfur oxidation such as sulfur oxidase, sulfur
dioxygenase, and sulﬁte oxidase are strongly inhibitedby Na2 WO4 . These results suggest that inhibition of the
enzyme system involved in elemental sulfur oxidation
by tungsten is probably one of the main causes of thegrowth inhibition by tungsten in A. thiooxidans. Sulfur
oxidase activity measured with resting NB1-3 cells
showed a broad optimum pH curve from 2 to 7.5. The

sulfur oxidase activity of NB1-3 cells measured at
pH 3.0 was inhibited 33, 68, and 95% by 0.1, 1.0, and10 mM of Na2 WO4 respectively (Fig. 5A), but activity
measured at pH 7.0 was inhibited 29% by 10 mM of
Na2 WO4 (data not shown). Since tungsten binds more
tightly below pH 5.0 than above pH 6.0 (Fig. 3), theseresults suggest that the level of inhibition by Na2 WO4 ofsulfur oxidase depends on the level of tungsten bound tothe cells. Sulfur dioxygenase and sulﬁte oxidase activities were measured at pH 6.0 and 7.5 with the cytosol
and the plasma membrane as enzyme sources, respectively (Fig. 5B and C). The activities of sulfur dioxygenase and sulﬁte oxidase measured at the optimum pHs
were inhibited 64% and 49% by 10 mM Na2 WO4 ,(Fig. 5B and C). The low level of inhibition by 10 mM
Na2 WO4 of both enzyme activities is probably due tothe low level of tungsten binding to these enzymes at thepHs used for enzyme analysis.
The most interesting ﬁnding obtained in this study,
however, is that approximately 10 times more tungsten
can bind to NB1-3 cells at pH 3.0 than at pH 7.0. Thisﬁnding appears to be important for the growth prevention of A. thiooxidans cells by tungsten in the environment of sewage treatment plants. The pH-dependence oftungsten-binding to NB1-3 cells was quite diﬀerent fromthat of nickel-binding to NB1-3 cells. Previously we
showed that the growth of NB1-3 cells was inhibited
more strongly by Ni2þ at pH 7.0 than at pH 3.0 and that
more nickel can bind to NB1-3 cells at neutral pH thanat acidic pH.9,25,26) Since A. thiooxidans, which is a main
sulfur-oxidizing bacterium accelerating concrete corrosion, has an optimum pH for growth between 2.0 and
3.0, the discovery of a bacteriostatic agent that can bindwell to A. thiooxidans cells under acidic conditions andGrowth Inhibition by Tungsten in A. thiooxidans

strongly inhibit cell growth is expected to make aconcrete structure that has more resistance to the attackof A. thiooxidans cells.
The eﬀectiveness of the addition of tungsten to
portland cement bars for the prevention of concrete
corrosion was ascertained when the portland cement
bars (40  40  160 mm) supplemented with both0.075% metal nickel and 0.075% calcium tungstate
were exposed to the atmosphere of sewage treatment
plant B containing 28 ppm of H2 S for 2 years. But theaddition of tungsten to the portland cement bars
(40  40  160 mm) did not have any favorable eﬀect
on the prevention of concrete corrosion when the cement
bars were exposed to the atmosphere of a sewage
treatment plant containing H2 S at more than 42 ppm for
2 years. Speciﬁcally, when portland cement bars without
metals, with 0.075% metal nickel, and with 0.075%
metal nickel plus 0.075% calcium tungstate were hungfor 2 years in the atmosphere of sewage treatment plant
C, in which the mean concentration of H2 S per year was
81 ppm, the weight losses of the cement bars were 74%,
22%, and 22% respectively as compared with thecement bars without H2 S treatment (data not shown).
These results suggest that the addition of tungsten to
0.075% metal nickel-portland cement bars is eﬀective
for the prevention of concrete corrosion when the
cement bars are hung in the atmosphere of a sewage
treatment plant containing less than 28 ppm of H2 S.
Tungstate, vanadate, and molybdenum are oxyanions.
When 1.0 mM vanadate or 1.0 mM molybdenum coexisted with 1.0 mM Na2 WO4 and NB1-3 cells, the amountof tungsten bound to the cells decreased to 42 and 68%of the control experiment without vanadate or molybdenum (Fig. 4). Metal ions other than vanadate andmolybdenum did not show the same phenomena as
observed with the addition of vanadate or molybdenum,suggesting the existence of a speciﬁc protein in
A. thiooxidans NB1-3 cells that binds to oxyanions.
Previously we showed existence of tungsten-binding
protein in A. ferrooxidans cells. The protein was puriﬁed
from a plasma membrane of A. ferrooxidans AP19-3 inan electrophoretically homogenous state.27,28) The tungsten-binding protein was composed of two subunits withapparent molecular masses of 12 and 20.7 kDa.28) But
incubation of the tungsten-binding protein for 1 h with
Na2 WO4 plus metal ions, such as NaVO3 , Na2 MoO4 ,CuSO4 , NiSO4 , MnSO4 , CoSO4 , or CdCl2 , did notmarkedly aﬀect the amount of tungsten bound to thetungsten-binding protein.28) These results suggest thepossibility that the protein in A. thiooxidans NB1-3 cells
which can bind oxyanions speciﬁcally, including
Na2 WO4 is diﬀerent from the tungsten-binding protein
isolated from A. ferrooxidans. To clarify these points
and the mechanism of growth inhibition by tungsten in
the genus Acidithiobacillus more precisely, puriﬁcationof oxyanion-binding protein from A. thiooxidans NB1-3
cells is currently underway.

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