Biosci. Biotech Biochem., 60 (4). 626-629, 1996

Nickel Inhibition of the Growth of a Sulfur-oxidizing Bacterium Isolated from Corroded
Concrete
Terunobu MAEDA, Atsunori NEGISHI, Yasuo NoGAMI,* and Tsuyoshi Sumo*-t
Hazama Corporation, Technical Research Institute, 515-1 Nishimukai, Karima, Tsukuba 305, Japan* Department of Biological Function and Genetic Resources Science, Faculty of Agriculture, Okayama University, 1-1-1
Tsushima Naka, Okayama 700, Japan
Received October 11, 1995■

A sulfur-oxidizing bacterium strain NBl-3 isolated from corroded concrete was a Gram negative,
non-spore-forming, and rod-shaped bacterium (0ふ1.0xlふ2.0µm) with a polar flagellum. Strain NBl-3
had its optimum temperature and pH for growth at 30° C and 3.0---4.0, respectively. Strain NBl-3 had
enzyme activities that oxidized elemental sulfur, thiosulfate, tetrathionate, and sulfide and the activity toincorporate 14C0 2 into the cells. The mean G+C content of the DNA was 52.9mol%. These results
indicate that strain NB1-3 is Thiobacillus thiooxidans. Since nickel has been known to protect concrete
from corrosion, the effect of Ni on the growth of strain NBl-3 was studied. The cell growth on tiosulfate-,elemental sulfur-, or tetrathionate-medium was completely inhibited by 0.1 % metal nickel or 5 mM NiSO がBoth cellular activities of elemental sulfur oxidation and CO 2 incorporation were strongly inhibited by
5 mM NiSO 4. The amounts of Ni in cells with or without nickel treatment were l. 7 and 160.0 nmol/mg
protein, respectively. These results indicate that nickel binds d:o strain NBl-3 cells and inhibits enzymes
involved i11 sulfur oxidation of this bacterium, and as a result, inhibits cell growth.
Key word§:

sulfur-oxidizing bacterium; Thiobacillus thiooxidans; corroded concrete; inhibition by Ni

Serious corrosion was noted in some concrete structures
constructed for sewage treatment. 1 - 7l Two kinds ofbacteria, namely sulfate-reducing and sulfur-oxidizing
bacteria, have been known to be mainly involved in thecorrosion of concrete. 2 - SJ In the process of concrete
corrosion, sulfate-reducing bacteria produce hydrogensulfide from organic compounds and sulfate ion in thesewage and the hydrogen sulfide thus formed is oxidized
by sulfur-oxidizing bacteria on the surface of sewer pipes
to give sulfuric acid. In addition to the production ofhydrogen sulfide by sulfate-reducing bacteria, heterotrophic
ノacteria can also produce hydrogen sulfide from sulfur­
containing organic compounds under anaerobic conditions.
The importance of hydrogen sulfide in concrete corrosion
has been stressed. 4, B J The concrete structure just after
construction contains calcium hydroxide and thus has a
pH of 12-13. Carbon dioxide in the atmosphere neutralizes
the concrete structure and prompts concrete corrosion.
However, sulfuric acid produced by sulfur-oxidizing
bacteria aiso prompts the neutralization of the concrete anddamages the concrete structures.
Parker first isolated a sulfur-oxidizing bacterium fromthe corroded concrete and named the bacterium Thiobacillus
concretivourus, which was later identified as Thiobacillus
thiooxidans. i . z J Thiobacilli other than T. thiooxidans werealso isolated from sewer systems and corroded concretes,
and identified as Thiobacillus neapolitanus, T. intermedius,
and T. novellus. 5, 9 J However, there have been few reports
on practical methods to protect concrete from this cor­rosion. Duecker et al. reported that sulfur cement with/3-naphthol and selenium in it resisted a sulfur-oxidizing
bacterium 1 OJ and Emmel et al. described biocides that inhibit
the corrosion by biogenic sulfuric acid in sewage pipelines. 11 l■

t Corresponding author

Recently, we have prepared portland cement bars (4 x 4 x16 cm) containing various kinds of inorganic and organic
compounds, and these bars were hung from a lid of a sludge
tank manhole and exposed to 5-10 ppm hydrogen sulfide
atmosphere.1 2 1 After 2 years of exposure, it was found thatthe surface of the portland cement containing nickel was
especially smooth, while that without nickel was brittle andrough, 121 However, the precise mechanism of this protection
from concrete corrosion by nickel addition were not known.
To study the biological bases of this protection of portland
cement corrosion by nickel, we isolated a sulfur-oxidizing
bacterium from corroded concrete, identified it, and studied
the effects of nickel on the cell growth and the activities ofboth sulfur oxidation and 14C0 2 恥ation in the cells. The
amounts of nickel in the cells after nickel treatment werealso measured.
M磁eirfa!§ 釘llll«Ji f\1位et加螂
Microorganism, medium, and conditions of cultivation. The bactenum
used in this study was isolated from corroded concrete in Fukuyama City,
Japan. The methods for the isolation of sulfur-oxidizing bacterium wereas follows. The corroded concrete (l .O g) was incubated under aerobic
conditions at 30℃ in 20 ml of thiosulfate-salt medium (pH 7.0) containing
sodium thiosulfate (0.2%), yeast extract (0.03%), (NH山so. (0.3%},MgS0 4 ·7H 2 0 (0.05%), 氏HP0 4 (0.05%), KC! (0.01 %), and Ca(NO山(0.001 %). Thiosulfate is unstable at low pH, but stable at neutral pH.
Thus 會 the pH of the medium was adjusted to 7.0. When sulfur-oxidizing
bacteria grew on the thiosulfate-salt medium, the pH of the medium
decreased to 2.0 because of the production of sulfuric acid by the bacteria.
After culturing 5 times in the thiosulfate-salt medium, the culture medium
were spread over the thiosulfate-salt medium with gellan gum (pH 7.0)
A cream-colored colony appearing on the plate was isolated. This isolation
process was repeated three times and the final isolate (strain NB 1-3) weremaintained on the thiosulfate-salt medium and used throughout this study
Growth ra/e. Cells were separated from a solid elemental sulfur byNickel Inhibition o「T. thiooxidans Isolated from Con odeJ Concrete

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Cell Growth of Strain NBl-3 on 20ml of (A) Elemental Sulfur-. (B) Th10sulfate-, and (C) Tetrathionate-salt Media

Symbols A, pH ol the medium; ● cell growthfiltering cultures with a Toyo paper filter No. SB. The numbers of cells m
the filtrates were counted with a hernacylometer (Kayagaki Irika KogyoCo., Ltd.) after dilution with 0.1 N sulfuric acid, when necessary
Oxidative activities on sulfur. thiosu/fale. sulfite, tetrathionate, and su/fide.
The oxidative activities on sulfur, thiosulfate, sulfite, tetrathionate, andsulfide were measured by the oxygen uptake caused by the oxidation ofthese inorganic sulfur compounds in a Warhnre m�nnmeter. Each vessel
contained 3.0 ml of reaction mixture plus 0.2 ml of 20% potassiumhydrooxide in the center well. The gas phase was air and the temperaturew� ぺk<"pt at 30℃ The reaction mixture (pH 3.0) wu3 composed of―{]-alanine-SO i (IOOµmol}, washed intact cells (5 mg of protein), andsubstrate. As substrates. sulfur (] 00 mg), thiosulfate (20µmol), sodium
sulfite (20µmol), potassium tetrathionate (20µmol). and sodium sulfide（勺,mnl) were added to the reaction mixture. The totnl volume of thereaction mixture was 3.0 ml.
Artivi「)·nl 14(0 1 uptake into washed in/act cells. The activity of carbon
dioxide (CO 2 ) fixation was measured by the amount of Na,'4C0 3incorported into the cells. The composition of the reaction mixture was―as follows: 4.0 ml of 0.1 M /J-alanine-SOi buffer, pH 3.0; washed intact
cells of strain NBl-3 grown on sulfur-salt medium, I mg of protein; carrier
Na L CO ., , 1µmol; Na戸C0 3 , lµCi; clcmcntal sulfur (S"), 200 mg. The
total volume of the reaction mixture was 5.0 ml. The reaction mixtmf',
except sodium carbonate and s 0 , was incubated at 30 ° C for IO min. The
reaction was started by adding Na戸C0 3 , carrier Na 2 C0 3 , and S 0 . AfterI h of incubation at 30 ° C, the reaction was stopped by adding 0.5 ml of20 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 IO ml of water and then put into 4 ml of acounting sol (Scintisol EX-H). After the filter had been completely
solubilized in the counting mixture, the radioactivity was measured withan Aloka LSC-635 liquid scintillation system
Nickel measurement. Strain NB 1-3 cells (20 mg protein) were incubatedm 5 ml of 0.1 M 3-(N-morpholino)propanesulfonic acid buffer (MOPS―buffer), pH 7.0 or 0.1 M /J-alanine-SOi buffer, pH 3.0 、 with IOmM NiS0 4
for 10 h at 30℃ The cells treated with NiS0 4 were washed three times
with 20 ml of MOPS buffer, pH 7.0, and ashed in a crucible at about 800℃
for 2 h. The ashes were dissolved in I N HCI. The amount of nickel wasmeasured by atomic absorption spectroscopy with a Shimadzu AA-625-01
spectrophotometer using an air-acetylene flame. The spectral line chosen
was 2320 A. The standard solution of nickel ion was prepared by dilutionof a IOOOµg/ml standard nickel solution for atomic absorption
spectroscopy (ishizu Pharmaceutical Co., Ltd.) with IN HCIfAnalysis u DNA base composition. The DNA base compos1t1on wasmeasured by reversed-phase high-pressure liquid chromatography after
the DNA was hydrolyzed into nucleosides with enzymes t 3 I
Results a!!lld Dis�璽sion
Morphology and physiological characteristics of suりur­oxidizing bacterium isolated什om the corroded concrete
Sulfur-oxidizing bacterium (strain NB 1-3) was isolated

Table I.
NBl-3

Specific Activity of Sulfur Compound Oxidation by Stra1r

Substrate

Activity of sulfur compound oxidatton
0 2 uptake (µ!/mg protein/min)

Sodium sulfide
Elemental sulfur
Sodium thiosulfate
Potassium tetrathionat�
Sodium sulfide
Ferrous sulfate
Glucose

I.JU
0.43
2.0
2.0
2.8
0.00.0from the corroded concrete of a sewage lrealmenl planl inFukuyama City, Japan. Strain NB 1-3 was a Gram-negative,
non-spore-forming, rod-shaped bacterium (0.5-1.0 x 1.5―2.0µm) with a polar flagellum. The mean G+C eontent of
the DNA of strain NBl-3 was 52.9mol%. The b叫eriumh;,rl the optimum temperature and pH for the gro\.vth onelemental sulfur-salt medmm at 30℃ and 3-4, respeC:tively.
Though elemental sulfur, thiosulfate, and tetrathionak
eould be used as a sole source of energy for growth (Fig.
1), the baeterium could not use Fe2+or glueose. Washedintaet eells of strain NB 1-3 had enzyme activities to oxidize
elementalsulfur,thiosulfate,
2tetrathionate,and sulfide(Table I). However, Fe + and glueose were not oxidized
by the strain. The baeterium had a high sulfur-oxidizing
activity under the pH range between 2-8 (Fig. 2). Ap­
proximately 46% of sulfur-oxidizing aetivity still remain­
ed at pH 9.0.
Strain NBl-3 had the activity to incorporate 14C02 into
the cells. Optimum pH for CO2 fixation with elemental
sulfur as an electron donor was 5.0 (data not shown). And
approximately 46% of CO2 fixation activity still remained
1at pH 8.0. 4C0 uptake activity was not observed when2

washed intact cells were incubated in the reaetion mixture
without elemental sulfur (Fig. 3) and strongly inhibited by
0.1 mM 2,4-dinitrophenol and IOµM carbonylcyanide m­chlorophenylhydrazone (CCCP), suggesting that the in­eorporation of 14C02 into the cells is energy dependent.
The results described above strongly indicate that sulfur­
oxidizing bacterium strain NB 1-3 is a chemolithotrophic,
sulfur-oxidizing bacterium Thiobacillus thiooxidans (TableII).

■

■

T

Nickel Inhibition of T. thiooxidans Isolated from Corroded Concretemuch more nickel binds to the cells at higher pH than alower pH.
Binding of nickel to strain NBJ-3 cells
To investigate whether nickel binds to strain NBl-3 cells
or not, the amount of nickel in the cells after nickel treat­ment was measured by the methods shown in Materials andMethods. Strain NB 1-3 cells without nickel treatment
contained 1.7 nmol nickel per mg protein. In contrast, thecells treated with lOmM NiS0 4 al pH 3.0 and 7.0 contained
35 and 160 nmol nickel per mg of protein, respectively,indicating that nickel can bind to strain NBl-3 cells andmuch more nickel binds to the eells at neutral pH than
acidic pH. This findings correspond with the results thatthe bacterium was much more strongly inhibited by Ni 2+
at pH 7.0 than pH 3.0.
We previously showed that the portland cement con­tai n i ng nickel was especially effective for protecting concrete
from corrosion. From the results obtained in this report,
the protective effect of Ni 2+ on concrete corrosion canbe explained as follows: Ni 2 + binds to sulfur-oxidizing
bacterium Thiobacillus thiooxidans cells and inhibits theenzyme system involved in sulfur oxidation of this bac­terium, and as a result, inhibits cell growth and sulfuric
acid production. To purify the protein(s) that is involved

629

in nickel bindi11g from T. thiooxidans strain NHl-3 and toclarify the mechanism of inhibition by Ni of sulfur oxidationof this bacterium are quite important and now underinvestigation.
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