JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 76, No. 5,400-402. 1993

Characterization and Identification of Bacterial Strains Isolated from Corroded Concrete in the Accumulation Stratum

and Their Resistance Levels to Heavy Metals NAOTO YOSHIDA, TSUTOMU MORINAGA,* AND YOSHIKATSU MUROOKA

Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama 1-4-1, Higashi-Hiroshima 724, Japan

Received 3 February 1993/Accepted 2 August 1993

Twenty thiosulfate-utilizing bacteria that may participate in the corrosion of concrete fabrications, were isolated from the accumulation stratum in the delta area of Hiroshima. Five of the isolated strains were selected for characterization, and four out of the five were classified into the genus Thiobacillus. Three of the five strains were further identified as Thiobacillus intermedius. The growth of these five strains in LB medium containing heavy metals was investigated. They showed strong resistance to cadmium and zinc ions.

Underground concrete sewers in several cities are being decomposed by corrosion (1), which is caused mainly by the action of sulfur bacteria (2). We have isolated several bacterial strains from corroded concrete in the accumula- tion stratum of Hiroshima city, and have found that some metabolites of aerobic non-sulfur utilizing bacteria are solubilized calcium ions from mortar (3).

The accumulation stratum consists of sand and fossil shellfish, but the stratum in a delta area such as the site of Hiroshima city contains abundan t sulfur compounds that come from shells (4). The development of heavy-metal resistant bacteria in the accumulation stratum has been recognized as an important indication of pollution (5, 6). Some strains of Thiobacillus ferrooxidans have the ability to oxidize Fe 2+ to Fe 3+, and show resistance to heavy metals (7). In this paper, we report on the isolation of a number of thiosulfate-utilizing bacteria from the same

TABLE 1. Morphological and physiological

location in Hiroshima city as used previously, and examine their resistance to heavy metals, especially cadmium and zinc ions.

A hole was bored in the accumulation stratum - 1 2 to - 2 0 in a depth near corroded concrete in Hiroshima city and samples of the subterranean soil were collected. From those samples, thiosulfate-utilizing bacteria were isolated using a synthetic medium (8). Twenty bacilliform bacteria strains were isolated that were capable of autotro- phic and chemoorganotrophic growth. They were obligate aerobes and exhibited a pH optima of 6 to 8 for growth. To test their resistances to heavy metals, a complete me- dium (LB medium) consisting of 1% Bacto trypton, 0.5%0 yeast extract (Difco, Michigan, USA), and 0 . 5 ~ NaC1 was used. Cadmium chloride, cobalt chloride, copper sul- fate, mercury chloride, nickel chloride and zinc chloride were used as sources of heavy metals. Various amounts

characteristics of five heavy-metal-resistant strains

5 3 12 13 16 T. intermedius Gram-stain Cell shape Rod Size length 1.5-1.8

width (/~m) 0.6 Spore formation Flagella Single polar Growth at pH 3.0

pH 5.0 + pH 8.0 + 30°C + 50oc .~

Optimum pH 6-8 Optimum temp. (°C) 25-30 Oxygen relation Aerobe NO3 reduced to NO2 Autotrophic growth with thiosulfate + Autotrophic growth with sulfur + Obligate chemolithoautotroph Facultative autotroph + Mol% GC of DNA 68.8 Contains ubiquinone Q-8?

Rod Rod Rod Rod Rod 1.2-1.8 1.0-1.8 1.3-2.0 1.5-2.2 1-4

0.8 0.7-0.9 0.8 0.6 0.5

Single polar Single polar Single polar Single polar

+ + + + + + + + + + + + + + +

6-8 6-8 6-7 6-8 5-7 25-30 25-30 25-30 25-37 30-35

Aerobe Aerobe Aerobe Aerobe Aerobe

+ + + + + + + + + +

+ + + + + 61.9 64.4 68.5 60.7 64.8-67 Q-6 Q-8 Q-8 Q-8 Q-8

* Corresponding author.

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FIG. 1. Growth curves in LB broth of cadmium-resistant strains no. 3 (A), 12 (B), and 13 (C), copper-resistant strain no. 5 (D), mercury- resistant strain no. 16 (E) and T. intermedius IFO 14564 (F). Culture turbidity were measured at 25°C in the presence of cadmium 110 ppm ( o ) , zinc 65 ppm (A), zinc 200 ppm (A), copper 320 ppm ( [] ), copper 640 ppm ( • ), mercury 80 ppm (O), and mercury 100 ppm (~ ) . Growth curves for control cells ( o ) growing in the absence of heavy metals are also shown.

of these chemicals were added to liquid LB medium. The medium without heavy metals was used as a control. Five strains from among the twenty isolates and three reference strains, Thiobacillus thiooxidans IFO 13701, T. ferrooxidans IFO 14245 and Thiobacillus intermedius IFO 14564, were used in the experiment. Each strain was cultured in 5 ml LB medium without heavy metals until its Klett became about 300. Then, 100 pl of the culture was inoculated to 5 ml LB medium with various concen- trations of heavy metals. All the strains were incubated at 25°C. The growth was monitored by measuring the turbidity with a Klett-Summerson's photometer. Time courses of the culture of each strain are shown in Fig. I. As T. thiooxidans IFO 13701 and T. ferrooxidans IFO 14245 grew very slowly, they could not be compared directly with our isolated strains. The time courses for T. intermedius IFO 14564 are shown in Fig. IF. Isolated strains numbered 3, 12 and 13 showed resistance to cad- mium (Fig. 1A, B and C). In particular, strain no. 13 was not affected by the addition of cadmium (110 ppm) in the stationary phase. These three strains also grew in LB broth containing zinc (65 ppm). Strain no. 5 showed re- sistance to copper and grew in the presence of 320 ppm of copper sulfate (Fig. 1D). Number 16 was a mercury- resistant strain and grew in the presence of 80 ppm of mer-

cury chloride (Fig. 1E). As shown in Fig. 1F, T. interme- dius IFO 14564 had lower resistance levels against all the heavy metals used than our isolated strains.

The five isolated strains were identified according to their morphological and biological characteristics, which were investigated by the methods described in Bergey's Manual of Systematic Bacteriology (9). Bacterial cells were grown in LB medium and harvested at the stationary phase. After collecting the cells, chromosomal DNAs were prepared by the procedure of Marmur (10). The G + C contents of the DNAs were determined by the thermal renaturation method (11, 12). Calf thymus DNA was used as the reference. Quinone was extracted according to the method of Yamada et al. (13). Bacterial cells were negatively stained with uranyl acetate and an electron micrograph was taken by a H-600A transmission electron microscope (Hitachi Co. Ltd.). From the characteristics listed in Table 1, all five isolates except no. 3 could be classified into the genus Thiobacillus. In particular, nos. 5, 12 and 13 were T. intermedius.

There are some reports of the isolation of bacteria that can grow in a medium containing a high concentration of cadmium and zinc. Belliveau et aL (5) isolated Bacillus strains from coastal marine sediments and tested their minimum inhibitory concentrations (MICs) to heavy

402 YOSHIDA ET AL. J. FERMENT. BIOENC.,

metals . Bacillus cereus strains were resis tant to c a d m i u m , cobal t , copper and zinc, wi th B. cereus U G F 1374 having the highest M I C to c a d m i u m (10 m M ) . B. subti l is strains were also resis tant to cobal t , copper and zinc, but the M I C s were lower than those o f the B. cereus strains. B. p u m i l u s strains showed resis tance to copper (10 raM) and zinc (20 m M ) . Our isolates had M I C s to c a d m i u m , copper and zinc o f 7 m M , 1 0 m M and 1 0 m M , respec- t ively (da ta no t shown) .

R E F E R E N C E S

I. Meyer, A. H. and Ledhetter, W. B.: Sulfuric acid attack on con- crete sewer pipe. Proc. ASCE SA, 5, 1167-1182 (1970).

2. Thornton, H. T.: Acid attack of concrete caused by sulfur bac- teria action, ACI J., 75, 577-584 (1978).

3. Morinaga, T., Teranishi, S., Kawai, K., Douzono, A., and Tazawa, E.: Concrete deterioration caused by microorganisms. J. Antibact. Antifung. Agents, 20, 485--488 (1992).

4. Aboshi, H.: The history of reclamation in Hiroshima. Tsuchi to Kiso, 396, 46-50 (1991). (in Japanese)

5. Belliveau, B.H., Starodub, M.E., and Trevors, J.T.: Occur- rence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment. Can. J. Microbiol., 37, 513-520 (1991).

6. Baya, A.M., Brayton, R.P., Brown, V.L., Grimes, D.J.,

Russek-Cohen, E., and Colwell, R.R.: Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples. Appl. Environ. Microbiol., 51, 1285-1292 (1986).

7. Olson, G. J., Porter, F. D., Rubinstein, J. , and Silver, S.: Mer- curic reductase enzyme from a mercury-volatilizing strain of Thio- bacillus ferrooxidans. J. Bacteriol., 151, 1230-1236 (1982).

8. Kanagawa, T., Dazai, M., and Fukuoka, S.: Degradation of o,o- dimethyl phosphorodithionate by Thiobacillus thioparus TK-1 and Pseudomonas AK-2. Agric. Biol. Chem., 46, 2571-2578 (1982).

9. Kelly, D. P. and Harrison, A. P.: Genus Thiobacillus, p. 1842- 1858. In Staley, J .T. , Bryant, M. P., Pfenning, N., and Holt, J. G. (ed.), Bergey's manual of systematic bacteriology, vol. 3. Williams & Wilkins, Co., Baltimore (1989).

10. Marmur, J.: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol., 3, 208-218 (1961).

11. Marmur, J. and Doty, P.: Thermal renaturation of deoxy- ribonucleic acids (DNA). J. Mol. Biol., 3, 585-594 (1961).

12. De Ley, J.: Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxy- ribonucleic acid. J. Bacteriol., 101, 738-754 (1970).

13. Yamada, Y., Aida, K., and Uemura, T.: Ubiquinone of acetic acid bacteria and its relation to classification of genera Gluconobacter and Acetobacter, especially of the so-called inter- mediate strains. J. Gen. Appl. Microbiol., 15, 181-196 (1969).