APPLIED MICROBIOLOGY, Apr. 1975, p. 562-564 Copyright i 1975 American Society for Microbiology
Vol. 29, No. 4 Printed in U.SA.
Effects of Cadmium on the Growth and Uptake of Cadmium by
Microorganisms' J. J. DOYLE,2 R. T. MARSHALL, Arm W. H. PFANDER* Departments of Food Science and Animal Husbandry,* University of Missouri, Columbia, Missouri 65201
Received for publication 15 October 1974
Six species of microorganisms, Escherichia coli, Bacillus cereus, Lactobacillus acidophilus, Staphylococcus aureus, Streptococcus faecalis and Actinomyces niger, were grown under suitable conditions in appropriate media. Cadmium chloride was added to provide 0, 5, 10, 20, 40, and 80 ,g of Cd per ml. At 40 and 80 gg of Cd per ml, E. coli and B. cereus grew well and the other species were repressed. Cd uptake patterns differed significantly among the species tested. The significance of these data with respect to Cd in food chains is discussed. In recent years there have been a number of reports on the adaptation of various microorganisms to toxic quantities of heavy metals. A variety of organisms are known to be tolerant to mercury and copper in particular as well as to chromium, iron, cobalt, nickel, silver, cadmium, arsenic, and selenium (2-4, 6, 10, 14). Tornabene and Edwards (12) found that Micrococcus luteus and Azotobacter sp. immobilized large quantities of lead when these organisms were grown in a media containing lead salts. Novick and Roth (11) and Chopra (5) reported that certain strains of Staphylococcus aureus were resistant to inorganic ions including mercury and cadmium. F. I. McLean et al. (0. J. Lucis, Z. A. Shaikh, and E. R. Jansz, Fed. Proc. 31:699, 1972) found that when Escherichia coli, Anacystis midulans (blue green alga) and Chlorella sp. (green alga) were grown in appropriate media with 109cadmium chloride (10-8 M), cells took up 9, 98, and 80% of the cadmium, respectively, and this uptake paralleled growth of the cultures. As the cadmium content of rivers, lakes, oceans, and sludge increases with time (8) the ability of microorganisms to accumulate large amounts of cadmium seems possible with a subsequent recycling of this metal through the food chains of man (7). This problem may be particularly acute in the Great Lakes region into which thousands of tons of industrial waste are pumped each day and where the cadmium concentration (Lake Erie) ranges up to 1 gg/ liter (7). 'Animal Science Research Center, Agricultural Experiment Station, journal series no. 6893. Approved by the
Director.
'Present address: USDA, ARS, University Station, Grand Forks, N. D. 58201.
The objectives of our experiments were to determine the ability of various microorganisms to accumulate cadmium and to determine what possible effects, in terms of growth, dietary cadmium may have on the microflora commonly found in the intestinal tract of man. Six organisms were tested, viz., E. coli, Bacillus cereus, Lactobacillus acidophilus, S. aureus, Streptococcus faecalis, and Aspergillus niger. Each organism was inoculated (at the rate of 0.1 ml of 24-h brain heart infusion broth culture for the bacteria and 0.2 ml of 120-h brain heart infusion broth for the fungus) into 100 ml of sterile brain heart infusion broth containing one of the following treatments: basal; basal plus 5 ,gg of cadmium per ml; basal plus 10 ug of cadmium per ml; basal plus 20 ug of cadmium per ml; basal plus 40 Mg of cadmium per ml; basal plus 80 MAg of cadmium per ml. The cadmium was added as cadmium chloride. There were eight samples per treatment. Incubation time and temperature were 40 h and 37 C, respectively, for the bacteria and 120 h and 23 C for the fungus. After incubation, growth of the bacteria was quantitated from culture turbidity using the Bausch and Lomb Spectronic 20 and a wavelength of 640 nm. The growth of A. niger was quantitated from the dry weight of organisms recovered from the medium. The bacterial cells were harvested by centrifugation (4,100 x g for 10 min) from the broth and washed twice in physiological saline. The fungal cells were harvested by sieving with cheese cloth and then washing twice in physiological saline. All samples within treatments were bulked, freeze dried, digested in a 5:1 nitric acid/perchloric acid mixture, and analyzed for cadmium in a Perkin Elmer model 403 atomic absorption spectrophotometer.
562
VOL. 29, 1975
563
NOTES
The effects of cadmium on the growth of microorganisms are reported in Table 1. In general, cadmium had a significant repressive effect on growth in broths containing 40 and 80 ,ug of Cd per ml. The effect was greater for S. aureus, S. faecalis, L. acidophilus, and A. niger than for the other two organisms. The cadmium content of the cells increased with increases in cadmium content of the media (Table 2). Mean concentrations of microbial cadmium (milligrams per gram of dry cells) were 53 for B. cereus, 38 for A. niger, and four for E. coli. These levels are low in relation to uptake of lead, 490 and 310 mg/g of whole cells of M. luteus and Azotobacter sp., respectively (12). The data in Table 2 were plotted on a dose response scale. Several patterns of uptake seemed apparent, so regression equations describing the best fit of the data were calculated and are recorded in Table 3. A significant linear and quadratic uptake is shown in B. cereus throughout the range of concentration studied. Cell growth was only slightly depressed. E. coli showed a similar response but uptake was very low and was associated with a gradual decline in growth. S. aureus showed a linear increase in uptake through 20 Mg/ml whereas growth was being
depressed. Two species, L. acidophilus and S. faecalis, showed nonsignificant responses. The growth of both of these species was stimulated by low levels of added cadmium. The best fit for L. acidophilus was curvilinear. A. niger took up Cd slowly from dilute solutions and at an accelerated rate from solutions containing 40 and 80 Mg of Cd per ml. The ability of the microorganisms, especially E. coli and B. cereus, to immobilize large quantities of cadmium from the broth and to grow at relatively high concentrations may indicate possession of specific inherited resistance to inorganic ions by these organisms. Novick and Roth (11) and Chopra (5) reported that penicillinase plasmids in S. aureus carried resistance factors to some inorganic ions including arsenate, lead, mercuric, and cadmium. Novick and Roth (11) stated that the phenomenon of extrachromosomal inorganic resistance is not merely a peculiarity of the staphylococci but was also found in E. coli and others. McLean et al. (Fed. Proc. 31:699, 1972) also reported great resistance by E. coli when grown in media containing cadmium. Chopra (5) reported that the biochemical basis of resistance to cadmium ions is unknown. However, Vaczi et al. (13) reported that resistance to Hg2+ ions is
TABLE 1. Effects of cadmium on growth of microorganisms as determined by absorbance or dry weight0 Treatment
Organism
No. of samples_ L. acidophilus A. niger0 samples S. faecalis B. cereus S. aureus E. coli
110.9 0.49 X 0.011 0.20 ± 0.011 105.7 0.78 ± 0.02' 0.19 ± 0.01' 82.6 0.88 i 0.01' 0.20 ± 0.011 66.4 0.39 ± 0.05' 0.34 0.022 0.06 ± 0.004' 0.03 X 0.002' 45.5 0.3 0.08 ± 0.003' 0.02 + 0.002' ± a Data with different superscripts are significantly different (P < 0.05). Mean standard error of the mean. 'A. niger determinations were by dry weight in milligrams (all other organisms were by absorbance). All eight samples bulked for each treatment.
Control Cd (5ug/ml) Cd (10 tig/ml) Cd (20;Mg/ml) Cd (40Mug/ml) Cd (80Mg/ml)
8 8 8 8 8 8
1.09 i 0.04" 2 1.14 ± 0.051,2 1.23 ± 0.02' 1.04 ± 0.072 1.07 ± 0.06" 2 0.82 ± 0.052
0.90 X 0.011 0.81 ± 0.018 0.85 ± 0.01' 0.78 ± 0.012 0.05 ± 0.01' 0.02 ± 0.003'
0.62 X 0.03' 0.60 ± 0.01"' 0.56 ± 0.012. 0.41 ± 0.01' 0.30 ± 0.01' 0.31 + 0.01'
TABLE 2. Uptake of cadmium by microorganismsa Cadmium concn in cells (mg/g) (dry matter basis) Treatment
Control Cd (5ug/ml) Cd (10 Ag/ml) Cd (20 jg/ml) Cd (40 ,g/ml) Cd (80 ;g/ml)
E. coli
S. aureus
B. cereus
S. faecalis
L. acidophilus
A. niger
Vol. 29, No. 4 Printed in U.SA.
Effects of Cadmium on the Growth and Uptake of Cadmium by
Microorganisms' J. J. DOYLE,2 R. T. MARSHALL, Arm W. H. PFANDER* Departments of Food Science and Animal Husbandry,* University of Missouri, Columbia, Missouri 65201
Received for publication 15 October 1974
Six species of microorganisms, Escherichia coli, Bacillus cereus, Lactobacillus acidophilus, Staphylococcus aureus, Streptococcus faecalis and Actinomyces niger, were grown under suitable conditions in appropriate media. Cadmium chloride was added to provide 0, 5, 10, 20, 40, and 80 ,g of Cd per ml. At 40 and 80 gg of Cd per ml, E. coli and B. cereus grew well and the other species were repressed. Cd uptake patterns differed significantly among the species tested. The significance of these data with respect to Cd in food chains is discussed. In recent years there have been a number of reports on the adaptation of various microorganisms to toxic quantities of heavy metals. A variety of organisms are known to be tolerant to mercury and copper in particular as well as to chromium, iron, cobalt, nickel, silver, cadmium, arsenic, and selenium (2-4, 6, 10, 14). Tornabene and Edwards (12) found that Micrococcus luteus and Azotobacter sp. immobilized large quantities of lead when these organisms were grown in a media containing lead salts. Novick and Roth (11) and Chopra (5) reported that certain strains of Staphylococcus aureus were resistant to inorganic ions including mercury and cadmium. F. I. McLean et al. (0. J. Lucis, Z. A. Shaikh, and E. R. Jansz, Fed. Proc. 31:699, 1972) found that when Escherichia coli, Anacystis midulans (blue green alga) and Chlorella sp. (green alga) were grown in appropriate media with 109cadmium chloride (10-8 M), cells took up 9, 98, and 80% of the cadmium, respectively, and this uptake paralleled growth of the cultures. As the cadmium content of rivers, lakes, oceans, and sludge increases with time (8) the ability of microorganisms to accumulate large amounts of cadmium seems possible with a subsequent recycling of this metal through the food chains of man (7). This problem may be particularly acute in the Great Lakes region into which thousands of tons of industrial waste are pumped each day and where the cadmium concentration (Lake Erie) ranges up to 1 gg/ liter (7). 'Animal Science Research Center, Agricultural Experiment Station, journal series no. 6893. Approved by the
Director.
'Present address: USDA, ARS, University Station, Grand Forks, N. D. 58201.
The objectives of our experiments were to determine the ability of various microorganisms to accumulate cadmium and to determine what possible effects, in terms of growth, dietary cadmium may have on the microflora commonly found in the intestinal tract of man. Six organisms were tested, viz., E. coli, Bacillus cereus, Lactobacillus acidophilus, S. aureus, Streptococcus faecalis, and Aspergillus niger. Each organism was inoculated (at the rate of 0.1 ml of 24-h brain heart infusion broth culture for the bacteria and 0.2 ml of 120-h brain heart infusion broth for the fungus) into 100 ml of sterile brain heart infusion broth containing one of the following treatments: basal; basal plus 5 ,gg of cadmium per ml; basal plus 10 ug of cadmium per ml; basal plus 20 ug of cadmium per ml; basal plus 40 Mg of cadmium per ml; basal plus 80 MAg of cadmium per ml. The cadmium was added as cadmium chloride. There were eight samples per treatment. Incubation time and temperature were 40 h and 37 C, respectively, for the bacteria and 120 h and 23 C for the fungus. After incubation, growth of the bacteria was quantitated from culture turbidity using the Bausch and Lomb Spectronic 20 and a wavelength of 640 nm. The growth of A. niger was quantitated from the dry weight of organisms recovered from the medium. The bacterial cells were harvested by centrifugation (4,100 x g for 10 min) from the broth and washed twice in physiological saline. The fungal cells were harvested by sieving with cheese cloth and then washing twice in physiological saline. All samples within treatments were bulked, freeze dried, digested in a 5:1 nitric acid/perchloric acid mixture, and analyzed for cadmium in a Perkin Elmer model 403 atomic absorption spectrophotometer.
562
VOL. 29, 1975
563
NOTES
The effects of cadmium on the growth of microorganisms are reported in Table 1. In general, cadmium had a significant repressive effect on growth in broths containing 40 and 80 ,ug of Cd per ml. The effect was greater for S. aureus, S. faecalis, L. acidophilus, and A. niger than for the other two organisms. The cadmium content of the cells increased with increases in cadmium content of the media (Table 2). Mean concentrations of microbial cadmium (milligrams per gram of dry cells) were 53 for B. cereus, 38 for A. niger, and four for E. coli. These levels are low in relation to uptake of lead, 490 and 310 mg/g of whole cells of M. luteus and Azotobacter sp., respectively (12). The data in Table 2 were plotted on a dose response scale. Several patterns of uptake seemed apparent, so regression equations describing the best fit of the data were calculated and are recorded in Table 3. A significant linear and quadratic uptake is shown in B. cereus throughout the range of concentration studied. Cell growth was only slightly depressed. E. coli showed a similar response but uptake was very low and was associated with a gradual decline in growth. S. aureus showed a linear increase in uptake through 20 Mg/ml whereas growth was being
depressed. Two species, L. acidophilus and S. faecalis, showed nonsignificant responses. The growth of both of these species was stimulated by low levels of added cadmium. The best fit for L. acidophilus was curvilinear. A. niger took up Cd slowly from dilute solutions and at an accelerated rate from solutions containing 40 and 80 Mg of Cd per ml. The ability of the microorganisms, especially E. coli and B. cereus, to immobilize large quantities of cadmium from the broth and to grow at relatively high concentrations may indicate possession of specific inherited resistance to inorganic ions by these organisms. Novick and Roth (11) and Chopra (5) reported that penicillinase plasmids in S. aureus carried resistance factors to some inorganic ions including arsenate, lead, mercuric, and cadmium. Novick and Roth (11) stated that the phenomenon of extrachromosomal inorganic resistance is not merely a peculiarity of the staphylococci but was also found in E. coli and others. McLean et al. (Fed. Proc. 31:699, 1972) also reported great resistance by E. coli when grown in media containing cadmium. Chopra (5) reported that the biochemical basis of resistance to cadmium ions is unknown. However, Vaczi et al. (13) reported that resistance to Hg2+ ions is
TABLE 1. Effects of cadmium on growth of microorganisms as determined by absorbance or dry weight0 Treatment
Organism
No. of samples_ L. acidophilus A. niger0 samples S. faecalis B. cereus S. aureus E. coli
110.9 0.49 X 0.011 0.20 ± 0.011 105.7 0.78 ± 0.02' 0.19 ± 0.01' 82.6 0.88 i 0.01' 0.20 ± 0.011 66.4 0.39 ± 0.05' 0.34 0.022 0.06 ± 0.004' 0.03 X 0.002' 45.5 0.3 0.08 ± 0.003' 0.02 + 0.002' ± a Data with different superscripts are significantly different (P < 0.05). Mean standard error of the mean. 'A. niger determinations were by dry weight in milligrams (all other organisms were by absorbance). All eight samples bulked for each treatment.
Control Cd (5ug/ml) Cd (10 tig/ml) Cd (20;Mg/ml) Cd (40Mug/ml) Cd (80Mg/ml)
8 8 8 8 8 8
1.09 i 0.04" 2 1.14 ± 0.051,2 1.23 ± 0.02' 1.04 ± 0.072 1.07 ± 0.06" 2 0.82 ± 0.052
0.90 X 0.011 0.81 ± 0.018 0.85 ± 0.01' 0.78 ± 0.012 0.05 ± 0.01' 0.02 ± 0.003'
0.62 X 0.03' 0.60 ± 0.01"' 0.56 ± 0.012. 0.41 ± 0.01' 0.30 ± 0.01' 0.31 + 0.01'
TABLE 2. Uptake of cadmium by microorganismsa Cadmium concn in cells (mg/g) (dry matter basis) Treatment
Control Cd (5ug/ml) Cd (10 Ag/ml) Cd (20 jg/ml) Cd (40 ,g/ml) Cd (80 ;g/ml)
E. coli
S. aureus
B. cereus
S. faecalis
L. acidophilus
A. niger
