Biosci. Biotechnol. Biochem., 62 (6), 1087-1092, 1998

Isolation of a Sulfur-oxidizing Bacterium That can Grow under Alkaline pH,
from Corroded Concrete
Terunobu MAEDA, Atsunori NEGISHI, Yuko OSHIMA, Yasuo NOGAMI,
Kazuo KAMIMURA, and Tsuyoshi Sumo

JlllA

Biosci. Biotechnol. Biochem., 62 (6), 1087 ー 1092, 1998

Isolation of a Sulfur-oxidizing Bacterium That can Grow under Alkaline pH,
from Corroded Concrete
Terunobu MAEDA, Atsunori NEGISHI, Yuko OSHIMA,* Yasuo NooAMI,*
Kazuo KAMIMURA,* and Tsuyoshi Sumo*,tHazama Corporation, Technical Research Institute, 515-1 Nishimukai, Karima, Tsukuba 305-0822, Japan*Department of Biological Function and Genetic Resources Science, Faculty of Agriculture, Okayama University,
1-1-1 Tsushima Naka, Okayama 700-8530, Japan
Received December l, 1997
To study the early stages of concrete corrosion by b.acter­ia, sulfur-oxidizing bacterium strain R0-1, which grows in
an alkaline thiosulfate medium (pH 10.0) was isolated
from corroded concreate and characterized. Strain R0-1
was a Gram negative, rod-shaped bacterium (0.5-0.6 x 0.91.5µm). The mean G + C content of the DNA of strain
R0-1 was 65.0 mol%. Optimum pH and temperature for
growth were 8.0. and 30-37 ° C, respectively. When grownin thiosulfate medium with pH 10.0, growth rate of thestrain was 48% of that observed at the optimum pH for
growth. Strain R0-1 used sulfide, thiosulfate, and glucose,but not elemental sulfur or tetrathionate, as a sole energy
source. Strain R0-1 grew under anaerobic conditions in
pepton-N03 medium containing sodium nitrate as an elec­tron acceptor, and had enzyme activities that oxidized
sulfide, elemental sulfur, thiosulfate, sulfite, and glucose,but not tetrathionate. The bacterium had an activity to as­
similate 14C02 into the cells when thiosulfate was used as anenergy source. These results suggest that strain R0-1 isThiobacillus versutus. Strain R0-1 exuded Ca 2+ from con­crete blocks added to thiosulfate medium with pH 9.0 andthe pH of the medium decreased from 9.0 to 5.5 after 22days of cultivation. In contrast, Thiobacillus thiooxidans
strain NBl-3 could not exude Ca 2+ in the same thiosulfate
medium, suggesting that strain R0-1, but not T. thioox­
idans NBl-3, is involved in the early stage of concrete corro­sion because concrete structures just after construction con­tain calcium hydroxide and have a pH of 12-13.
Key words:

sulfur-oxidizing bacterium; Thiobacillus ver­sutus; corroded concrete

In 1945, Parker presented evidence that concrete struc­tures are corroded by the sulfuric acid produced by thesulfur-oxidizing bacterium Thiobacillus concretivou­
rus.1-3> Concrete in its main structure is composed of calci­
um oxides, hydroxides, and carbonates and these com­pounds can react with sulfuric acid to give calcium
sulfate (gypsum).4> This calcium loss from concrete great­ly diminishes the strength of concrete structures. The
bacterium isolated by Parker was later identified as Thio­
bacillus thiooxidans and thibacilli other than T. thioox­
idans, namely Thiobacil/us neapolitanus, T inter­medius, and Thiobacillus novellus,5-1> were also isolated
from sewer systems and corroded concretes. The resultst Corresponding author.

accumulated up to now strongly suggest that a final rap­
idly corrosive stage of concrete structure is due to the ac­tivity of the obligately acidophilic chemolithoautotroph
T. thiooxidans. However, the explanation for the preli­minary stage of slow corrosion of concrete structures is
not clear.3l Since concrete structures just after construc­tion contain calcium hydroxide and thus have a pH of
12-13, it seems that sulfur-oxidizing bacteria that cangrow under alkaline pH conditions are involved in the in­itial stage of concrete corrosion.
We recently showed that concrete supplemented withnickel is resistant to corrosion8l and nickel bound to theplasma membrane of T. thiooxidans NB1-39l inhibits sul­
fur dioxygenase and sulfite oxidase of the membrane,2
and as a result, inhibits cell growth.10-1 J To study the ear­ly stages of concrete corrosion and identify the biologi­
cal aspects of Ni protection in concrete corrosion more
precisely, we tried to isolate sulfur-oxidizing bacteriathat can grow under alkaline pH conditions. Here, weshow that Thiobacillus versutus is also present in the cor­roded concrete and compared with T. thiooxidans NBI2
3 strongly exudes Ca + from carbonated concrete blocks
at pH 9.5.
Materials and Methods
Microorganisms, media, and conditions of cultiva­tion. The bacteria used in this study was Thiobacil/us
thiooxidans strain NBI-3, 9) Thiobacillus versutus
IF014568, Thiobacil/us novel/us IF014993, and sulfur­
oxidizing bacterium strain RO-I isolated from corroded
concrete of a sewage treatment plant in ltoman City,
Okinawa Prefecture, Japan. The methods for the isola­tion of sulfur-oxidizing bacterium were as follows. The
corroded concrete (1.0 g) was incubated under aerobic
conditions at 30 °C in 20 ml of thiosulfate medium (pHcontaining
sodiumthiosulfate(0.5%),
9.5)
Na2HP04• l2凡0(0.15%),
KH2P04(I.I%),
MgS04·7H20 (0.01%), NH4Cl�0..03%), and yeast ex­
tract (0.03%). When sulfur-oxidizing bacteria grew inthe thiosulfate medium, samples of the culture medium
were plated on gellan gum plates containing sodium
thiosulfate
Na2HP04·12凡0(0.5%),(0.15%),
KH2P04 (1.1%), MgS04·7H20 (0.01%), NH4Cl (0.03%),and yeast extract (0.03%). Colonies appearing on theplate were picked up. This process was repeated more

1088

T. MAEDA et al.

than three times and the final isolates were preserved inthiosulfate medium (pH 7.5). Glucose medium (pH 7.5)
contained glucose (0.2%), Na2 HP04 ·12H20 (0.15%),
KH2P04 (1.1%), MgS04 ·7H2 0 (0.01%), NHぶl(0.03%), and yeast extract (0.03%). Peptone-N03 medi­um (pH 7.5) used for the growth of strain R0-1 underanaerobic conditions was composed of peptone (10%),
NaCl (0.5%), and KN03 (0.1%).
Growth rate. Cells in the medium were counted with ahemacytometer (Kayagaki Irika Kogyo Co. Ltd.,Tokyo) after dilution with water when necessary.
Growth rate was also measured by the increase of absor­
bance of the culture at 660 nm.
Inorganic sulfur-oxidizing activites. The oxidation ac­
tivity for inorganic sulfur compounds was estimatedfrom the oxygen uptake rate in a Warburg manometer.
A Warburg flask contained a reaction mixture (3.0 ml)
in the reaction chamber and 20% potassium hydroxide(0.2 ml) in the center well. The gas phase was air and thetemperature was kept at 30 ° C. The reaction mixture in
the Warburg flask was composed of 0.1 M diethanola­
mine-HCl buffer (pH 9.0, 2.0 ml), washed intact cells(1.0 mg of protein), and sodium sulfide (10µmol) forthe measurement of hydrogen sulfide oxidation activity.
The reaction mixtures were composed of 0.1 M MOPS
buffer (pH 8.0, 2.0 ml), washed intact cells (1.0-5.0 mg
of protein), and elemental sulfur (200 mg) and potassi­um tetrathionate (20µmol), sodium thiosulfate (20µmol), or sodium sulfite (20µmol) for the measure­ments of elemental sulfur, tetrathionate, thiosulfate,and sulfite oxidation activities. The reaction was doneby shaking the reaction mixture at 30 ° C. The amount ofchemical oxidations of each inorganic sulfur compound
was also checked with a reaction mixture containing 10min boiled cells instead of the native cells.
Activity of 14C02 uptake into washed intact cells ofstrain R0-1. The activity of carbon dioxide (CO2 ) fixa­
tion was estimated from the amount of Na214C03 assimi­
lated by the cells. The composition of the reaction mix­
ture was as follows: 4.0 ml of 0.1 M P-alanine-Soi­buffer, pH 3.0; washed intact cells of strain R0-1 grownin thiosulfate- or glucose-medium, 1 mg of protein; car­rier Na2 C03 , 1µmol; Na214C03 , 1µCi; and thiosulfate,20µmol. The total volume of the reaction mixture was
5.0 ml. The reaction mixture except sodium carbonate
and thiosulfate was incubated at 30 ° C for 10 min. The
reaction was started by adding Na宍co3 , carrier
Na2 C03 , and thiosulfate. After 60 min of incubation at
30 ° C, the reaction was stopped by adding 0.5 ml of 20
mM mercuric chloride. The reaction mixture (0.55 ml)
was withdrawn and then passed through a 0.45-µm mem­brane filter. The filter with cells was washed three times
with 10 ml of water and then put into 4.0 ml of a count­ing sol (Scintisol EX-H). After the filter had been com­pletely solubilized in the counting mixture, the radioac­
tivity was measured with an Aloka LSC-635 liquid
scintillation system.

Analysis of Ca2+ exuded from concrete blocks.
Sulfur-oxidizing bacterium strain R0-1 were inoculated
in pH 9.5- and pH 7.0-thiosulfate media with two piecesof carbonated concrete blocks (0.6 x 0.6 x 0.6 mm), andcultured for 24 days at 30 ° C. A sample of the culture
medium was withdrawn, centrifuged at 12,000 x g for
15 min to discard cells, and the amount of Ca 2 + in the
supernatant was measured by atomic absorption spec­
troscopy with a Shimadzu AA-625-01 spectrophotome­
ter using a, air-acetylene flame. The spectral line chosen
was 4227 A. The standard solution of Ca 2 + was pre­pared by dilution of the 1000µg/ ml standard calcium so­lution for atomic absorption spectroscopy (Ishizu Phar­maceutical Co.) with 1 N HCI.
Analysis of DNA base composition. The DNA basecompositon was measured by reversed-phase high-pres­sure liquid chromatography after the DNA was hydro­
lyzed into nucleosides with enzymes.13 J
Results and Discussion
Isolation of sulfur-oxidizing bacteria that can growunder alkaline pH conditions from the corroded con­crete.
Fifty-nine strains of sulfur-oxidizing bacteria were iso­lated from the corroded concrete of sewage treatment
plants in Itoman City, Okinawa Prefecture, Japan. It
has been known that Thiobacillus novel/us and Thia­
bacillus versutus are facultative sulfur-oxidizing bacter­ia that can grow in thiosulfate and glucose media withthe pH from neutral to slightly alkaline.14-1 6J Seven
representative sulfur-oxidizing bacterial strains among
fifty nine isolates, Thiobacillus novel/us IF014993 andThiobacillus versutus IF014568, were grown in thiosul­
fate medium (pH 9.5) for 5 days. The levels of cell
growth in the medium were estimated by the pH
decrease due to the sulfuric acid production by these sul­
fur-oxidizing bacteria and by the increase of absorbanceat 660 nm of culture medium. Strain R0-1, comparedwith the other eight strains, grew in the medium with amarked pH decrease (Table 1). The growth rate of strain
R0-1 in the medium was faster than those of T. novel/us
Table 1. Growth of Sulfur-oxidizing Bacteria Isolated from Corrod­
ed Concrete in Thiosulfate Medium (pH 9.5)
Strainwithout inoculation
R0-1
R0-2
R0-3
R0-4
R0-5
R0-6
R0-7
Thiobacillus versutus IFO 14568
Thiobaci//us novel/us IFO 14993

G’ rowth yield"(A660nm / 5 d)

b
pH

0.000
0.296
0.099
0.112
0.065
0.126
0.092
0.125
0.142
0.087

9.50
6.37
9.10
7.35
9.48
9.45
9.38
9.35
9.31
8.92

'Growth rate was measured by the increase of absorbance at 660 nm of theculture.
b The pH of the culture was measured after 5 days of cutivation.

1089

Isolation of Sulfur-oxidizing Bacterium from Corroded Concrete
and T. versutus. We selected strain R0-1 as the represen­tative of sulfur-oxidizing bacterium isolated from cor­roded concrete that grows well in alkaline thiosulfate
medium.
Growth of sulfur-oxidizing bacterium strain RO-I in
inorganic sulfur media
Figure 1 shows a micrograph of strain R0-1. The
strain was a Gram negative, non-spore-forming, androd-shaped bacterium (0.5-0.6 x 0.9-1.5µm). The mean
G + C content of the DNA of this strain was 65.0 moI%.
Taylor and Hoare reported that the mean G + C con­tents of the DNA of T. novel/us and T. versutus (Thio­
bacillus A2) grown in thiosulfate medium was 68 and 65
mol %, respectively. 15> The growth rate of strain R0-1
was similar to T. novel/us IF014993 and T. versutus
IF014568 when cultivated in the medium containing
thiosulfate or glucose as an energy source (Table 2), in­dicating that strain R0-1 is a facultative chemolithoauto­
troph. Strain R0-1, T. novel/us IF014993, and T. versu­
tus IF014568 consumed thiosulfate in the medium 14.8,
8.0, and 8.0 mM per 7 days, respectively. Only T. novel­/us IFO14993 among the three sulfur-oxidizing bacteria
could use tetrathionate as an energy source for growth.
The amount of tetrathionate in the medium consumed
by strain R0-1, T. novel/us IF014993, and T. versutus
IF014568 were 0.0, 0.0, and 2.2 mM per 7 days, respec­tively. Taylor and Hoare reported that T. versutus does
not use tetrathionate as an energy source.'5l Among
three sulfur-oxidizing bacteria, only T. novel/us
IF014993 used elemental sulfur as an energy source.
Enzyme activities that oxidize reduced inorganic sul­
fur compounds and glucose were measured with washedintact cells of strain R0-1, T. novel/us IF014993, andT. versutus IF014568 grown in thiosulfate and glucose
media (Table 3). When grown in thiosulfate medium,
these three sulfur-oxidizing bacteria had enzyme activi­
ties that oxidize sodium sulfide, sodium thiosulfate, sodi­
um sulfite, and glucose. Strain R0-1 and T. versutus
IF014568, unlike T. novel/us IF014993, could not oxi­dize tetrathionate. These results support the idea that T.

Fig. 1. Electron Micrograph of Strain R0-1.
Bar represents 1.0µm.

Table 2. Growth of Stram RO-I, T. versutus, and T. novel/us on theMedium Supplemented with Inorganic Sulfur or Glucose as an Energy
SourceEnergy- source
StrainStrain RO-I
A660nm/7 d"T. versutus IF014568
A660nm/7 d
T. novellus IF014568
A660nm/7 d

Elemental thiosulfate tetrath1onate Glucose
sulfur
0.000

0.105

0.029

0.497

0.004

0.077

0.030

0.896

0.027

0.055

0.144

1.060

'Growth rate was measured by the increase of absorbance of the medium at
660nm.
Table 3. Enzyme Activities that Oxidize Reduced Inorganic Sulfur
Compounds and Glucose
Substrate
Intact cells grownon thiosulfate medium
Sodium sulfide
Elemental sulfur
Sodium thiosulfate
Potassium tetrathionate
Sodium sulfite
GlucoseIntact cells grownon glucose medium
Sodium thiosulfate
Glucose

Oxidation activity (µI/mg/min)
Strain R0-1

T. versutus
IF014568

T. novellus
IF014993

2.52
0.60
2.33
0.00
0.56
0.84

2.29
0.35
2.25
0.00
0.07
0.12

1.53
0.00
4.02
2.81
3.73
0.28

0.17
0.84

0.18
0.76

0.10
0.38

novel/us IF014993, but not Strain R0-1 and T. versutus
IF014568, grew in tetrathionate medium. When grownin glucose medium, thiosulfate-oxidizing activities ofthese three sulfur-oxidizing bacteria were markedly
repressed.
Activity of carbon dioxide fixation
l ℃ 02 fixation activity of strain R0-1 was studied with
washed intact cells grown in thiosulfate and glucose me­dia (pH 8.0). Thiosulfate grown-cells assimilated 14C02
when thiosulfate was used as an energy source (Fig. 2).
The specific activity of 14C02 fixation was 291 nmol/mg/60 min. However, when glucose was used as anenergy source, the activity markedly decreaased (11nmol/mg/60 min). The glucose grown strain R0-1 did
not have 14C02 fixation activity, indicating that strain
R0-1 grews autotrophically in thio.sulfate medium with
CO2 as a carbon source and heterotrophically in glucose
medium with glucose as a carbon source.
Optimum pH and temperature for growth
Strain R0-1, T. versutus IF014568, and T. novel/us
IF014993 had optimum pHs for growth at 7.0, 7.0, and8.0, respectively (Fig. 3A). It is interesting that strain
R0-1 grew better in the medium with alkaline pH thanT. versutus IF014568 and T. novel/us IF014993. The

T. MAEDA et al.

1090

300
2

0
0

1

0
0

(6E/1
0Eu la�eldn zou゜゜

20
40Time (m)60

Fig. 2. 14C02 Uptake Activity of Sulfur-oxidizing Bacterium Strain
RO-I Isolated from Corroded Concrete.
14C0 uptake activities were measured in the reaction mixture2
with (O) or without (●） 20 mM HgCl2.
0.3

0.3

5
1

0

2
5
. 1
0

2

(PS/09911') 4茎 0ぶ）―
au

5
2
0

5
2

5
0
0

5
0

4

2

1

゜
゜

゜゜

゜

(PS/ 099\f )4l
MO9
」 ―
au

0

0.1

6

8

10

12

pH

゜

10

20 30 40 50
Temperature (℃）

60Fig. 3. Effects of pH (A) and Temperature (B) on the Growth of Sul­
fur-oxidizing Bacterium Strain RO-I, T. versutus, and T. novel/us
in Thiosulfate Medium.
Sulfur-oxidizing bacterium strain RO-I (■ ）， T. versutus
IF014568 (●）， and T. novel/us IF014993 (▲） were statically grownin 20 ml of thiosulfate medium for 5 days.

J

Table 4. Characteristics of Sulfur-oxidizing Bacterium Strain RO­
I Isolated from Corroded ConcreteCharacteristics

negativerod-shaped
motile65
730-35

negativerod-shaped
non-motile
68
7
25-30

sulfidethiosulfate

sulfidethiosulfate

glucoseglucosesulfidethiosulfate
tetrathionate
glucose+＋+-++++-+＋＋十 一

Oxidizing activity for
Sodium sulfide
Elemental sulfur
Sodium thiosulfate
Potassium tetrathionate
Sodium sulfite
GlucoseAbility to growby nitrate respiration

negativerod-shaped
motile65
830-37+ ＋ + —+ + +Gram stain
Morphology
Motility
G+C content (mo!%)Optimum pH for growth
Optimum temperaturefor growth ( 0 C)
Energy source

T. versutus T. novellus
Strain R0-1 IF014568
IF014993

growth rates of strain R0-1 in thiosulfate medium with
pH 9.0 and 10.0 were 75 and 48% of the optimum
growth of this bacterium. The three sulfur-oxidizing bac­teria did not grow in thiosulfate medium with pHs
above 11.0. Strain R0-1, like T. versutus IF014568,
had an optimum temperature for growth at 30-37 ° C(Fig. 3B).
The properties of strain R0-1 are summarlized in Ta­
ble 4. The most remarkable points found in strain R0-1
and T. versultus IFO 14568 were the cells'ability to growunder anaerobic conditions in the medium containing so­dium nitrate as an electron acceptor and its inability tooxidize tetrathionate as an energy source. Strain R0-1
can grow in media with neutral and slightly alkaline pHs
not only autotrophically with CO 2 as a carbon source
and thiosulfate as an energy source but also heter­otrophically with glucose as carbon and energy sources.
It has been known that the facultative sulfur-oxidizing
bacterium Thiobacil/us intermedium grows in media
with pHs from 3.0 to 7.0, but this bacterium cannot
grow under anaerobic conditions. 1 7 > Another facultative
sulfur-oxidizing bacterium, Thiobacil/us acidophilus,
grows in elemental-sulfur media with pHs from 2.0 to
4.0, but the bacterium also cannot grow under anaero­
bic conditions. 18> Thiobacil/us denitrijicans 19> and Thio­
bacillus neapolitanus grow in media with pHs from 5.0
to 8.0, but they are obligate chemolithoautotrophs andcannot use glucose as energy and carbon sources. 14> The
value of the mean G + C content of the DNA of strain
R0-1 corresponded with that of T. versutus. The results
obtained in this report suggest that strain R0-1 is T. ver­sutus.
Effects of nickel sulfate on the growth of strain R0-1
Nickel added to concrete protects the concrete fromcorrosion. 8> The causes of protection by nickel from cor­rosion were studied with the acidophilic, chemolithoau­
totrophic, sulfur-oxidizing bacterium Thiobacil/us
thiooxidans NBl-3 isolated from corroded concrete.9 · 10>
We showed that nickel binds to the plasma membraneof this strain and inhibits sulfur dioxygenase and sulfite
oxidase of the membrane, and as a result, inhibits cell
growth. 10> Therefore, it is very interesting to study
whether strain R0-1, which grows in the medium with aslightly alkaline pH, is inhibited by NiS04 or not.
Growth of strain R0-1 in thiosulfate medium wasstrongly inhibited by 0.3 mM of NiS04 (Fig. 4A).
Growth inhibition was also observed in the cases of T.
versutus IF014568 and T. novel/us IF014993 (data notshown), suggesting that nickel ion has similar inhibitory
effects not only on the growth of acidophilic T. thioox­
idans but also on those of neutorophilic T. versutus andT. novel/us. The thiosulfate oxidizing activity of washedintact cells of strain R0-1 was completely inhibited by
10 mM of NiS04 (Fig. 4B).
Exudation of Ca 2+ from concrete blocks by sulfur-ox­
idizing bacteria
Serious corrosion was noted in some concrete struc­tures used for sewage treatment. 1 -6> In the process of con­crete corrosion, sulfate-reducing bacteria produce

1091

Isolation of Sulfur-oxidizing Bacterium from Corroded Concrete
5

3

z+

l

2

1

0.1 0.2 0.3 0.4 0.5
Cone. of NiSO 4 (mM)

2

3

dn 'O
7)a丑e�
iLI
E\6Eヽ―

4

E\s11
au
:)Il
a:i80 X)£ MOJ
ニPS/I

(B)

(A)

゜゜

2

4

6

8

Cone. of NiSO 4 (mM)

10

Fig. 4. Effects of NiS04 on the Growth in Thiosulfate Medium (A)and on Thiosulfate Oxidation Activity (B) of Sulfur-oxidizing Bae­terium Strain R0-1.
(A) Sulfur oxidizing bacterium strain R0-1 was grown in thiosul­
fate medium (pH 8.0) with or without NiS04 for 5 days and the lev­els of growth were measured by counting cells with a hemacytome­
ter. (B) Thiosulfate oxidizing activity was measured from theoxygen uptake caused by the oxidation of thiosulfate in a Warburg
manometer.

(B)

5

゜

25

0
1

10 15 20
Time (d)51(Edd)paz―
11
qn1
0s +zBu゜

5

5

10 15 20
Time(d)25

Fig. 5. Solubilization of Ca z+ from Concrete Blocks by Sulfur-ox­
idizing Bacterium Strain R0-1 and Thiobacil/us thiooxidans Strain
NBl-3.
Sulfur-oxidizing bacterium strain R0-1 and T. thiooxidans strain
NBl-3 were grown on thiosulfate media (pH 9.0 and pH 7.0) con­taining three pieces of carbonated concrete blocks (6 x 6 x 6 mm).
(A) pH change. (B) Solubilization of calcium ion from concrete
blocks. Ca z+ solubilized from the concrete blocks were measured byatomic absorption spectrophotometer. Symbols: △，without inocu­
lation (pH 9.0); o, without inoculation (pH 7.0); ▲， Strain R0-1
(pH 9.0); ●， Strain RO-I (pH 7.0); ◆， T. thiooxidans strain NBl-3(pH 7.0); ■， Strain R0-1 (pH 7.0) in the presence of 1 mM of
NiS04.

References
1) C. D. Parker, Aust. J. Exp. Biol. Med. Sci., 23, 81-90 (1945).
2) C. D. Parker, Aust. J. Exp. Biol. Med. Sci., 23, 91-98 (1945).
3) C. D. Parker, Nature, 159, 439-44!) (1947).
4) W. Sand, Appl. Environ. Microbiol., 53, 1645-1648 (1987).
5) K. Milde, W. Sand, W. Wolff, and E. Bock, J. Gen. Microbiol.,
129, 1327-1333 (1983).
W. Sand and E. Bock, Environ. Tech. Lett., 5, 517-528 (1984).
N. Yoshida, T. Morinaga, and Y. Murooka, J. Ferment.
Bioeng., 76, 400-402 (1993).
T. Maeda and A. Negishi, in "Fracture and Damage of Concreteand Rock-FDCR-2," ed. by H.P. Rossmanith, ©E & FN Spon,
1993.
T. Maeda, A. Negishi, Y. Nogami, and T. Sugio, Biosci.
Biotech. Biochem., 60, 626-629 (1996).
10) Y. Nogami, T. Maeda, A. Negishi, and T. Sugio, Biosci.
Biotech. Biochem., 61, 1373-1375 (1997).
11) T. Sugio and T. Maeda, Bio Industry (in Japanese), 13, 13-20(1996).
12) T. Maeda, Building Maintenance & Management (in Japanese),
n’
〇,

釘，

9,

hydrogen sulfide from organic compounds and sulfate
ion in the sewage and the hydrogen sulfide thus formedis oxidized by sulfur-oxidizing bacteria on the surface ofsewer pipes to produce sulfuric acid. The concrete struc­ture just after construction contains calcium hydroxide
and thus has a pH 12-13. The calcium hydroxide in theconcrete structure, then, is carbonated with carbon di­oxide in the atmosphere to produce calcium carbonate,decreasing the pH of the concrete structure from 12-13
to 8.5-10. This pH decrease is known as neutralizationof the concrete structure and concrete corrosion is in­creased after this neutralization.20l Sulfuric acid pro­
duced by sulfur-oxidizing bacteria not only prompts theneutralization of concrete but also exudes Ca
from
the concrete structure as calcium salfate, causing a rapid
decomposition of the concrete.1-3> Thus, it seems reason­
able that the level of concrete corrosion caused bysulfur-oxidizing bacteria can be estimated by the concen­
tration of Ca z+ exuded from concrete blocks. Strain
R0-1 had the ability to exude Ca z + from carbonatedconcrete blocks in the thiosulfate medium and the pHsof the media decreased from 9.0 to 5.5 after 22 days ofcultivation (Fig. 5). Exudation of Ca 2+ by strain R0-1
in the pH 9.0 medium was faster than the pH 7.0 medi­um, suggesting that strain R0-1 exudes Ca z+ from con­crete blocks more rapidly undrer alkaline pH than atneutral pH. Thiobacillus thiooxidans strain NBl-3,9>acidophilic sulfur-oxidizing bacterium isolated from cor­roded concrete, could not exude Ca z+ from concrete
blocks in the thiosulfate medium with pH 7.0 and pH
9.0, suggesting that strain R0-1, but not T. thiooxidans
NBl-3, is involved in the early stage of concrete corro­sion. The activity of strain R0-1 to exude Ca z+ fromconcrete blocks was markedly inhibited by 0.1 mM of
NiS04.
A facultative sulfur-oxidizing bacterium, T. versutus
strain R0-1, was first isolated from corroded concreteof a sewage treatment plant. The results that strain RO1, but not T. thiooxidans NBl-3, exudes Ca z+ from car­bonated concrete blocks strongly suggests that theformer is more important in early stages of concrete cor­rosion than the latter. Optimum pH for growth of strain
R0-1 was one pH unit higher than that of T. versutus
IF014568. However, strain R0-1 could not grow inthiosulfate medium with the pH above 11. Thus, strain
R0-1 probably attack concrete structures after they arecarbonated and neutralized by carbon dioxide in the at­mosphere to pH 8.5-10. It is interesting that not only T.
thiooxidans NBl-3 but also strain R0-1 was strongly in­hibited by Ni z+ . In T. thiooxidans NBl-3, the Ni z+
strongly binds to the plasma membrane and inhibits sul­
fur dioxygenase and sulfite oxidase of the membrane,
and as a result, inhibits cell growth.9-1 2> To study the pro­tection of concrete from corrosion by nickel more pre­cisely, the mechanism of growth inhibition of strain
R0-1 by Ni z+ 1s now under exammat10n.

1092
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