A rapid analysis of tungsten in a tungsten-mixed
portland cement by use of sulfur-oxidizing bacteria（硫黄酸化細菌を利用したタングステン含有コンクリート中のタングステンの迅速分析）○Tsuyoshi Sugio1, Rie Sugio1, Atsunori Negishi2, Hideki Igawa3
1Sugio Inst. of Chemolithoautotrophy,. 2Tech Res. Inst. Hazama Ando Corp,
3Tech. Res. Lab. Nippon Hume Corp.

Tcmry-sugio@nifty.com
Abstract Corrosion of concrete structures, especially, in sewer system and sewage treatment plants, has become a big
social problem. In the process of concrete corrosion, hydrogen sulfide produced in sewage pipes by the action of sulfatereducing bacteria is oxidized by sulfur-oxidizing bacterium Acidithiobacillus thiooxidans to produce sulfuric acid, Portland
cement contains calcium oxide, calcium hydroxide, and calcium carbonate, and these compounds react with sulfuric acidto give a sandy calcium sulfate (gypsum). Gypsum production being dependent on A. thiooxidans markedly weakens
strength of concrete structures. Author has clarified that tungsten (W) strongly inhibits growth of A. thiooxidans underacidic conditions and nickel (Ni) inhibits bacterial growth at neutral and slightly alkaline pHs. A portland cement mixed
with tungsten and nickel as a bacteriostatic agent was produced to protect concrete corrosion. An effectiveness of theconcrete supplemented with W and Ni on concrete corrosion has been reported. In this paper we tried to develop a rapid
method to analyze tungsten in a tungsten-mixed portland cement by an activity of sulfur-oxidizing bacteria to produce
sulfuric acid.
Method & Results Analysis of tungsten was carried out in a transparent plastic tube with screw cap. A portland
cements that contains tungsten (0, 4, 8, and 20 kg/m3) were prepared and powdered. The reaction mixture (25.5 ml)
contains powdered concrete (100 mg), 9K salt medium pH 3.5 (20 ml), 0.1 M β-alanine buffer pH 2.0 (3.0 ml), EDTA-2Na(2 μmol), elemental sulfur (250 mg), tetrathionate-K (6.6 μmol); thiosulfate-Na (0.4 mmol), 0.001% bromocresol green(1.0 ml), and cell suspensions of sulfur-oxidizing bacteria (A. thiooxidans, Thiomonas intermedia, Paracoccus versutus,Starkeya novella). The reaction was started by addition of concrete. Both pH and color changes were traced every 30 minfor 2 h. After 2 h, the reaction mixture containing a cement without tungsten showed greenish yellow (pH 3.92). In
contrast, reaction mixtures containing 4, 8, and 20 kg tungsten/m3 showed greenish blue (pH 4.22), blue (pH 4.57), andblue (pH 4.68), respectively, indicating that this analysis is useful to distinguish rapidly whether a concrete sample
contains tungsten or not. Addition of sodium cyanide or sodium tungstate (inhibitor for sulfur oxidase of A. thiooxidans)
to reaction mixtures increased the speed of blue color development.

Concrete No.1
Concrete No.2
Concrete No.3
Concrete No.4
Biccrete No.1
Biccrete No.2
Biccrete No.3
Biccrete No.4

Tungsten oxide(mg/kg)

14.8
16.0-

Cement(kg/m3)

2.4
4.2
-

102
206
458
936

163
202
4841010A. thiooxidans
4.5

387
387
387
387

A. thiooidans plus Starkeya
novella3.5

A. thiooxidans, Starkeya
novella, plus 2 mM NaCN

2.5
03060

9012003060

pH Increase of a Reaction Mixture Containing Concrete Sample
and A. thiooxidans
Concrete sample addedA, Concrete No.1

5.3

C, Concrete No.3

4.9

D, Concrete No.4

H. Biccrete No.4

4.7
4.5
4.3
4.1

4.1

3.5

4.9

pHF, Biccrete No.2

4.3

Concrete No.1
Biccrete No.2
Biccrete No.3
Biccrete No.4

5.1

E, Biccrete No.1

4.7

A, Concrete No.1
B, Concrete No.2
C, Concrete No.3
D, Concrete No.4
E. Biccrete No.1
F, Biccrete No.2
G, Biccrete No.3
H, Biccrete No.4

5.3

B, Concrete No.2

5.1

3.9120

pH Increase of a Reaction Mixture Containing Concrete Sample,A. thiooxidans and Paracoccus versutus
Concrete sample added

5.5

5.5

Concrete samples
added to Sulfur medium

90Time (min)

3.9
3.7

3.7

3.503060

900120306090120120 min120 min

Time (min)Time (min)

0.2
0.15

pH Increase of a Reaction Mixture Containing Concrete Sample,
A. thiooxidans , andThiomonas intermedia

0.1
0.05
00510

4.5

pH

4.3
4.1

4.8

A, Concrete No.1
B, Concrete No.2

4.9

C, Concrete No.3
D, Concrete No.4

4.7

E, Biccrete No.1

4.5

F, Biccrete No.2

4.3

G. Biccrete No.3
H. Biccrete No.4

4.1

3.9

4.6

Concrete sample added

5.1

A, Concrete No.1
B, Concrete No.2
C, Concrete No.3
D, Concrete No. 4E, Biccrete No.1
F, Biccrete No.2
G, Biccrete No.3
H, Biccrete No.4

4.7

pH Increase Caused by Concrete Addition was Repressedby the Exsistence of Acidithiobacillus thiooxidans

pH Increase of a Reaction Mixture Containing Concrete Sample,
A. thiooxidans and Starkeya novella
5.3

Concrete sample added

4.9

Time (days)

3.9

3.7

4.4

3.7

3.5

4.2

pH

1.5 mM

2

Strain NB1- 3 was grown on sulfur - salt medium (pH 2.5) statically at 30 ℃ .

0.25

1.0 mM

3Time (min)G. Biccrete No.3

0.3

0.5 mM

3.5

2.5

2

4.5

0.35

0.2 mM

4pHpH

4Medium Supplemented with 25 mg of Concrete Sample

0.4

Con. of Na2WO4

4.5

A. thiooxidans plus 2 mM NaCN

30.45

387
44044043555pH

Nickel(mg/kg)Cell growth (A660 nm)

Concrete

0.5

5.5

5.5

Growth of Acidithiobacillus thiooxidans strain NB1-3 in Sulfur

Tungsten Oxide, Nickel, and Cement Contentsin Concrete Samples Used in this Study

Effect of Na2WO4 on pH Increase of a Reaction Mixture Supplemented withBiccrete No.1, A. thiooxidans, and Starkeya novella

Effect of NaCN on pH Increase of a Reaction Mixture Containing
Biccrete No.1 and Sulfur- Oxidizing Bacteria

03060903.5

1200430120 min

Time (min)6090120

Time (min)120 min

3.8
3.6
3.4

pH Increase of a Reaction Mixture Containing Concrete Sample,
A. thiooxidans , Thiomonas intermedia and Starkeya novella
Concrete sample added

3.2
3030

6090120

Time (min)A, Concrete No.1, without A. thiooxidans

B, Concrete No.1 with A. thiooxidans

C, Biccrete No1 without A. thiooxidans

D, Biccrete No.1 with A. thiooxidans

5A, Concrete No.1
B, Concrete No.2
C, Concrete No.3
D, Concrete No.4
E, Biccrete No.1
F, Biccrete No.2
G, Biccrete No.3
H, Biccrete No.4

4.5

Composition of a Reaction Mixture used for Analysis of Tungsten in Concrete
The analysis of tungsten in concrete sample was done in a transparent plastic tube with screw cap.
The composition of a reaction mixture was shown below. The total volume was 25.5 ml.

4

Acidithiobacillus thiooxidans NB1-3

・・・・・・・・・・・・・・・・ 0.1 ml

Thiomonas intermedia NBRC 14564

・・・・・・・・・・・・・・・・ 0.2 ml

pH

１．Sulfur-Oxidizing Bacteria

3.5

Paracoccus versutus

NBRC 14568

・・・・・・・・・・・・・・・・ 0.2 ml

Starkeya novella

NBRC 14993

・・・・・・・・・・・・・・・・ 0.2 ml

32.5

２．Energy Source for sulfur-oxidizing bacteria
Elemental sulfur

・・・・・・・・・・・・・・・・ 250 mg

Patassium tetrathionate

・・・・・・・・・・・・・・・・ 6.6 μmolSodium thiosulfate・・・・・・・・・・・・・・・・ 0.4 mmol

2

３． 0.1 M β-alanine-SO42- buffer (pH 2.0)

・・・・・・・・・・・・・・・・ 3.4 ml

４． 9K basal salt medium (pH 2.5)

・・・・・・・・・・・・・・・・ 20 ml

５． EDTA-2Na

・・・・・・・・・・・・・・ 0.002 mmol

６． 0.001% Bromocresol green solution

・・・・・・・・・・・・・・・・・ 1.0 ml

７． Concrete sample powdered・・・・・・・・・・・・・・・・・100 mg

Color Change of a Reaction Mixture after 120 min of Incubation

90120120 minConclusion
Four plain concretes (Concrete No.1, 2, 3 and 4) and four concretes supplemented with tungsten and nickel(Biccretes No. 1, 2, 3 and 4) were prepared. These eight concretes were powdered.
Each of the powdered concretes (100 mg) was added to the acid solution (25.5 ml) containing an acid-baseindicator (BCG), sulfur-oxidizing bacteria, energy sources for sulfur-oxidizing bacteria, and EDTA-2Na.
After a vigorous shaking in a transparent plastic tube with a screw cap, both color and pH changes of these acidsolutions were traced every 30 min for 2 h.

2.

We found the best condition to distinguish a plain concrete from Biccrete by use of a color change ofacid-base indicator (BCG). The results obtained from color change were confirmed by use of a pH meter.
After incubation of eight concrete samples for 2 h, four plain concretes gave greenish yellow (pH 3.8～ 4.1).
In contrast, four Biccretes containing tungsten gave blue (pH 4.4～5.1).

3.

The following results indicate that a rapid analysis of tungsten in tungsten-mixed portland cements isbased on a microbiological activity of sulfur-oxidizing bacteria to produce sulfuric acid from inorganic
sulfur compounds and this bacterial activity is inhibited by tungsten dissolved from a tungsten
mixed concrete, but not from a plain concrete without tungsten.
(a) pH increase or blue color development in an acid solution was accelerated by 2 mM of sodium cyanide
and 1.0 mM of tungstate.
These chemicals are known as a potent inhibitor for the enzymes that is involved in sulfuric acidproduction in Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans.
(b) It was found that A. thiooxidans, Thiomonas intermedia, Starkeya novella, but not Paracoccus versutus,
are most preferable sulfur-oxidizing bacteria for the analysis of tungsten in concrete.

4.54120min

3.5pH0 min60

1.

5E, Biccrete No.1
F, Biccrete No.2
G, Biccrete No.3
H, Biccrete No.430Time (min)pH Increase of a Reaction Mixture Containing Sulfur- Oxidizing
Bacteria and Concrete

Concrete sample used
A, Concrete No.1
B, Concrete No.2
C, Concrete No.3
D, Concrete No.4

03
2.5
2030

60

90120Time (min)A, Concrete No.1

B, Concrete No.2

C, Concrete No.3

D, Concrete No.4

E, Biccrete No.1

F, Biccrete No.2

G, Biccrete No.3

H, Biccrete No.4

Sulfur-oxidizing bacteria used
Acidithiobacillus thiooxidans
Thiomonas intermedia
Paracoccus versutus
Starkeya novella