JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 85, No. 6, 630-633. 1998

Heavy Metal Particle Resistance in Thiobacillus intermedius 13- 1 Isolated from Corroded Concrete

NAOTO YOSHIDA,‘* YOSHIKATSU MUROOKA,2 AND KIHACHIRO OGAWA’

Department of Biological Resource Sciences, Faculty of Agriculture, Miyazaki University, 1-I Gakuen Kibanadai-Nishi 889-2S and Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1

Yamadaoka, Suita-shi, Osaka 565-0871,2 Japan

Received 6 November 1997/Accepted 18 March 1998

The effect of exposure to heavy metal particles on the growth and survival of bacterial cells was investigated. Thiobacillus intermedius 13-1, Escherichia coli JM109, and Agrobacterium radiobacter IF012665bl were cultured on LB solid medium or in 5 ml of liquid medium containing 0.03 or 0.1 g respectively of the heavy metals, aluminum, cadmium, iron, lead, molybdenum, nickel, and zinc. Cadmium, nickel and zinc strongly inhibited cell growth in the three strains. In contrast, the bacterial cells were not inhibited by aluminum, iron, or lead in the solid or liquid medium. When these bacteria were exposed to heavy metals by vigorous shaking for 10 min, lead and molybdenum, but not nickel and zinc, were markedly toxic to the bacterial cells. Different reaction were thus observed under low-level, long-term and high-level, short-term exposure conditions. T. intermedius showed more resistance to zinc and nickel, and was more sensitive to molybdenum exposure, than E. coli and A. radiobacter.

[Key words: Thiobacillus, heavy metal resistance, particle, exposure]

The largest sources of heavy metal dust and fumes released into the general environment are the burning of fossil fuels (coal, oil etc.) and the incineration of municipal waste materials (1). Heavy metal particles may also escape into the air from zinc, lead, or iron smelters. A number of metals have been found to induce can- cerous growths in various animal species in vivo (2), and to cause mutagenic or chromosomal transformations in cultured cells in vitro (3).

Studies on the relationship between bacteria and heavy metals have revealed the existence of are highly specific bacterial resistance systems, and the basic biochemical mechanisms of these resistances to heavy metals as well as the molecular biology and genetics that determine and govern them have been explained by Silver (4). Bacteria are common inhabitants of metal-contaminated sites, where they accumulate and immobilize heavy metals. The cell walls of gram-positive bacteria have strong metal-binding properties (5). Metals such as manganese, nickel, and iron are absorbed through specific uptake receptors (6, 7). Under ion stress, bacteria produce low- molecular-weight ligands, called siderophores, that bind heavy metal ions and transport them to heavy metal side- rophore receptors (8).

Thiobacillus intermedius strain 13-1, isolated from the corroded concrete of a sewer, was found to exhibit resistance to several heavy metals (9). The environmental stratum around corroded concrete consists of metal parti- cles from construction materials. A number of reports have described the resistance mechanism of bacteria to either the ion or soluble form of heavy metals (4-9), but the behavior of bacteria against insoluble forms of heavy metals has not been reported. The development of heavy metal-resistant bacteria in a stratum has been recognized as an important indication of pollution (10, 11). Recent- ly, we have found that T. intermedius, but not Agrobac- terium radiobacter strain IF012665b1, was adsorbed

* Corresponding author.

aggregately onto the surface of cadmium particles (12). Escherichia coli was used as a common control strain in a wide range of experiments with cadmium- and zinc-sensi- tive strains. In the present study therefore, the effect of exposure to heavy metal particles (aluminum, cadmium, lead, molybdenum, nickel, iron, and zinc) on the growth and survival of T. intermedius was investigated, and compared to the effects on E. coli JM109 and A. radio- batter IF012665b 1.

A. radiobacter was obtained from the Institute for Fermentation, Osaka. Lead (8-60 pm particles), cadmium (10-50 pm particles), molybdenum (4-30 pm particles), nickel (2-4 pm particles), and zinc (2-8 pm particles) were purchased from Nakalai Tesque, Kyoto, aluminum (80-160 pm particles) was obtained from Katayama Chemical Co., Osaka, and iron (lo-135 pm particles) from Yoneyama Chemical Industries, Osaka. All the heavy metal particles were pure elements, not the salt form.

The toxicity of heavy metal particles was detected on LB agar plates. Each metal (0.03 g) was placed onto a plate directly after it had been streaked with the test strains, which had been grown in LB liquid medium for 24 h. After 48-h incubation at 3O”C, clear inhibition zones were observed around heavy metal particles. The distance from the edge of each metal particle to zone boundary was measured (Fig. 1). Clear inhibition zones of 5.3, 4.0, and 3.0mm around cadmium were observed in the growth of T. intermedius, E. coli, and A. radio- batter, respectively. Zinc was also toxic, resulting in respective inhibition zones of 6.0, 3.9, and 8.0mm, which the zones of inhibition produced by nickel were 1.9, 3.0, and 2.6 mm. Interestingly, molybdenum gave a 4.0-mm inhibition zone in the case of T. intermedius, but it did not inhibit E. coli and inhibited A. radiobac- ter only a little. T. intermedius was thus more sensitive to molybdenum than E. coli and A. radiobacter. Alumi- num, iron, and lead produced no inhibition zones under the conditions used with any of the three kinds of bac-

630

VOL. 85, 1998 NOTES 631

P E 7 ;6

3s

54 E g 3

E 2 ‘s

8 ’ 50 c Cb FL Pb zn Al Cd Fe MO Nl Pb Zn dl C-d F’e MO N’i P’b Zk

FIG. I. Inhibition zone radii of T. intermedius 13-1 (A), E. coli JM109 (B), and A. rudiobacter IF012665bl (C) on LB plates caused by heavy metal particles.

teria. Although lead is not known to have any beneficial effect, it did not appear to be toxic to these bacteria. When 10 ,~l of the upper phase obtained from LB (50 ~1) which had been exposed to 0.03 g of each heavy metal for 48 h at 30°C was spotted onto an LB plate, no inhibi- tion zone appeared around the spot, showing that inhibi- tion zones were formed due to the toxicity of the heavy metal particles, but not by the ionic form derived from the unstable region of the metal surface.

Figure 2 shows the effects of heavy metal particles on cell growth in LB liquid medium containing T. inter- medius, E. co/i, or A. radiobacter. In the liquid medium, the frequency of exposure of bacterial cells to heavy metal particles was quite low because the weight of the particles caused them to sediment the tube bottom dur- ing shaking. This experiment was designed to model low-level, long-term exposure. The full growth of 50/11 of pre-cultured cells of each bacterium was added to 5 ml LB liquid medium containing 0.1 g of several heavy metal particles-aluminum, cadmium, lead, molybdenum, nickel, or zinc. Each bacterial strain was cultured in the medium with the heavy metal particles at 30°C on a rota- tory shaker (150rpm/min). The growth of each strain was evaluated by measuring the optical density of the upper phase of the culture medium at 550 nm at intervals during the cultivation. All the experiments were per- formed over 42 h under exactly the same conditions.

There was no significant difference in cell growth from the controls in the presence of aluminum or lead, suggest- ing that particles of these heavy metals had no effect on the test bacterial strains under the experimental condi- tions used in the case of either a liquid or solid medium (Figs. 1 and 2). Although T. intermedius grew as well as the control during the early phase, its growth was retarded to 66 and 74% of that of the control after 42 h in the

presence of nickel and zinc, respectively. On the other hand, nickel and zinc strongly inhibited cell growth in early phase in E. coli and A. radiobacter. Cadmium was the most toxic heavy metal, completely inhibiting the growth of the three strains. Interestingly, the E. coli and A. radiobacter strains grew as well as the controls in the presence of molybdenum, but the growth of T. inter- medius was strongly inhibited by this heavy metal. These results show that the three bacteria were resistant to aluminum and lead, but not to cadmium, zinc and nickel. Although T. intermedius had resistance to zinc and nickel, it was more sensitive to molybdenum than E. coli and A. radiobacter. The turbidity was not measured in the medium containing iron because colored complexes were formed.

The survival of bacterial cells highly exposed to heavy metal particles was compared to controls not exposed to such particles (Fig. 3). In this experiment, bacterial cells were strongly exposed to heavy metal particles for 10min as a model of high-level, short-term exposure. Bacterial cell suspensions (3 x lo9 cells/ml), which had been washed and suspended in 300 ~1 of sterilized water, were added to tubes containing 0.1 g of heavy metal par- ticles which had been washed twice with 100 times the volume of distilled water. The mixture was vigorously shaken with a vortex mixer for 10min at room tempera- ture or not shaken as a control, and then 1OOmM of citric acid was added to release bacteria adsorbed onto the heavy metal particles (12). Aqueous phase samples were diluted lo5 to 10’ times with sterile distilled water. Spread-plating on a series of LB agar plates was done using lOO/*l of diluted samples. After 24-h incubation at 3O”C, the number of colonies formed was counted. Growth was expressed relative to the growth on a con- trol plate without any heavy metal particles. The control

B

10 20 30 40 500 10 20 30 40 0 10 20 30 40 I

Time (h) Time (h) Time(h)

FIG. 2. Growth of T. intermedius 13-1 (A), E. coli JM109 (B), and A. rudiobucter IF012665bl (C) in LB liquid medium in the presence of 0.1 g of the following heavy metal particles: aluminum (0), cadmium (A), lead (A), molybdenum ( n ), nickel ( q ), and zinc (v), or in the absence of any heavy metal particle as a control (0).

632 YOSHlDA ET AL. J. FERMENT. BIOENG.,

100

z 75

1 *L 50 L

8 25

0 NoneAl Cd Fe MO NI Pb Zn NoneAi Cd Fe MO NI Pb Zn NoneAl Cd Fe MO Ni Pb Zn

FIG. 3. Effect of exposure to heavy metal particles on viability of T. intermedius 13-1 (A), E. co/i JM109 (B), and A. radiobacter lF012665bl (C). Values are shown relative to the survival of the control grown in the absence of heavy metal particle exposure, which was taken as 100%. Each bacterial strain was exposed to aluminum, iron, lead, molybdenum, nickel, or zinc for 10 min at room temperature, and the viability was assessed by the ability of the samples to form colonies on LB plates.

invariably contained loo+- 10 colonies per duplicate plate. Surviving cells were detected on the plate as colo- nies.

Despite the fact that, except for T. intermedius, the test bacteria could grow in LB liquid medium containing lead and molybdenum to the same extent as the control (Fig. 2), significant toxicity was observed when the cells of these strains were strongly exposed to lead or molyb- denum. Different reactions were thus observed in response to low-level, long-term and high-level, short- term exposure conditions. When exposed to lead, the survival of T. intermedius, E. coli, and A. radiobacter strain was severely inhibited to only 1 .O, 2.1, and 0.1% of the control, respectively, showing that the bacteria were extremely sensitive to lead under high-level, short- term exposure. When exposed to molybdenum, the survival of these bacteria was also arrested to between 5 and 20% of the controls. On the other hand, whereas all the bacteria were repressed in liquid medium containing nickel or zinc, these heavy metal particles did not com- pletely inhibit cell survival under high-level, short-term conditions, and in the case of T. intermedius these heavy metals did not influence cell survival. The survival of T. intermedius, E. coli, and A. radiobacter cells exposed to cadmium was inhibited to less than 0.5% of the con- trols, suggesting that the bacteria are extremely sensitive to cadmium in both long- and short-term exposure. These results also show that T. intermedius was resistant to nickel and zinc particles more than E. coli and A. radiobacter. Except for a small effect of iron on A. radiobacter, no serious aluminum or iron toxicity were observed, suggesting that these metals had little or no effect on cell growth. If vortexing is required for toxicity to occur, vigorous mixing will have more affect on the survival of bacteria. Also, the wide and diverse range of metal particles sizes indicates an even greater diversity in exposure between metal particles. Smaller particles imply a greater surface area and hence more exposure. However, we did not find any physical effect of exposure to friction, regardless of the vortex time (lo-30 min).

The effects of heavy metal particles on T. intermedius, E. coli, and A. radiobacter growth were determined in terms of long-term exposure to low doses (Fig. 2), and short-term exposure to high doses (Fig. 3). Figure 2 shows that apart from the case of T. intermedius ex- posed to molybdenum, aluminum, lead, and molybde- num did not inhibit bacterial growth in LB liquid medium. When the bacteria were exposed to heavy metal particles by vigorous shaking for 10 min, lead, and molybdenum, but not nickel and zinc, displayed marked toxicity to

cell growth (Fig. 3). Nickel and zinc affected growth only on low-level, long-term exposure, while molybde- num and lead affected it only on high-level, acute ex- posure. Individual bacterial strain differ in their sensitiv- ity to zinc and nickel under the high-level, short-term exposure conditions. Consequently, the results suggest that lead and molybdenum strongly influence bacterial viabil- ity under high-level, short-term exposure but not under low-level, long-term exposure conditions. T. intermedius was more resistant to zinc and nickel, and more sensitive to molybdenum-exposure than E. coli and A. radiobac- ter. It is likely that at least some of the metals do not remain elemental upon exposure to water and air. For example, the iron on the plates in the first experiment had a prominent orange color indicative of Fe3+ (data not shown). Undefined portions of some of the metals proba- bly become soluble, and a contaminating portion of the added metals is already in the ionic form and thus affects the cells. Bacterial cells may uptake fine metal particles such as by endocytosis, resulting in inhibition of cell growth by the action of metal solubilization in the cells. Some surface metal particles may also dissolve in the medium and form complexes with medium compo- nents. Such complexes are thought to be a toxic to bac- terial cells.

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