- Email: [email protected]
Cadmium accumulation by marine bacteria
Marine Em'ironmental Research 17 (1985) 159-162
Cadmium Accumulation by Marine Bacteria
G. N. Flatau & M. J. G a u t h i e r INSERM Unit6 40, I Avenue Jean Lorrain. F-06300 Nice, France
Complexation of metals by marine bacteria is a well known phenomenon, t In the marine ent:ironment it may play a determining role in the mobilisation of metals and their transfer into sediments 2 or through food chains. 3 However, only a limited number of studies have analysed metal binding mechanisms in marine bacteria, the cell structure of which depends more closely on the ionic composition of the environment. ~ The aim o f this work was to investigate the mechanisms responsible f o r the accumulation of cadmium by Gram negative marine bacteria. Previous studies on a large number of strains showed that cadmium accumulation was dependant on the respiratory type of the strain. 5 Cadmium uptake by resting cells of a marine pseudomonad was a linear function of time. The accumulation was lower in cells previously killed by ethanol or potassium cyanide, but was faster during the first hours of the experiment. These variations in metal accumulation suggest that physiological mechanisms are necessary for its adsorption on to the cell wall or its cytoplasmic incorporation. 6 Cadmium accumulation increased in more permeable and unstable cells after a previous treatment by 200 #~ EDTA and then by 200/~M MgCI 2 to complex the excess of EDTA (Fig. l(a)). However, cadmium loaded cells lost the major part of the metal when the medium was added with 10mM/litre EDTA (Fig. l(b)): complexation of calcium and magnesium probably induced a permeabilisation of the cell wall and enhanced the exchange of cadmium. It was also possible that EDTA exposed new binding sites, which were 159 Marine Environ. Res. 0141-1136/85/$03-30 ~ Elsevier Applied Science Publishers Ltd,
England, 1985. Printed in Great Britain
G. .V. Flatau, .U. J. Gauthier
160 ng~ ~.mg'tdry cell S
pg Ca. mg~dry ceils Isoo A
1500
/
I I I / I I I I P I I
1000
1000
I / I I
/'j' /
I
500
11
500
kx
,
8
(a)
hours
(b)
Fig. I. (a) Cadmium accumulation by marine pseudomonad previously treated by EDTA and MgCI, ( - - - ) , or non treated (--). (b) Cadmium release by marine pseudomonad in artificial sea-water containing EDTA (--) or without EDTA (- - -). Cells previously accumulated Cd from 3 litre artificial sea-water added with 1 mg Cd/li~re.
progressively saturated by cadmium; because EDTA decreased the respiration of the cells, the additional cadmium might only be adsorbed on these new sites. In a medium containing 5/JM/litre of the uncoupling agent carbonylcyanide, p-trifluoromethoxy phenylhydrazone (FCCP), cellular respiration rate and cadmium uptake decreased. These also immediately and strongly decreased when FCCP was added 8 h after the beginning of the experiment. Inhibition of protein synthesis by chloramphenicol had only slight effects on cadmium uptake whether it was added at the beginning of the experiment or 8h after. These results suggest that cadmium accumulation did not require induced protein synthesis and probably involved an energy dependent transfer related to respiration processes.
Cadmium accumulation by marine bacteria
161
Cadmium accumulation was carried out with untreated cells, mureinoplasts (cells covered with their murein layer) and spheroplasts (cells partly covered with their murein layer) for 4 h. At the first hour, cadmium accumulation was about 2 times greater in spheroplasts than in mureinoplasts and about 3 times greater than in the untreated cells. This ratio decreased with time, probably due to the rapid breakdown of
ng Cd. mg'~dry cells f
3000
/
,4
I
2000
/
/
[
1000
'2
& hours
Fig. 2. Cadmium accumulation by whole cells (--), mureinoplasts (---) and spheroplasts (---).
spheroplasts (spheroplast life time did not exceed 3 h). After 4 h of contact with cadmium, mureinoplasts had accumulated 3 times more metal than whole ceils and about 1.5 times more than spheroplasts (Fig. 2). These results suggest that the cadmium accumulation could either be due to an increase of exchanges through the naked cytoplasmic membrane or to a further fixation of metal on new superficial binding sites. In either respect, it seems that the triple layered cell wall of G r a m negative in marine bacterial cells acts as an active ion filter.
162
G.N. Flatau, M. J. Gauthier REFERENCES
1. Tornabene, T. G. & Edwards, H. H. Science, 1976, 1334-5 (1972). 2. Ramamoorthy, S., Springthorpe, S. & Kuschner, D. J. Bull. Environ. Contain. Toxicol., 17, 505-11 (1977). 3. Flatau, G. N. & Gauthier. M. J. Can. J. Microbiol., 29. 210-17 (1983). 4. McLeod, R. A. In Advances in Microbiology of the Sea, Vol. 1, (M. R. Droop & E. J. F. Wood, eds), Academic Press, London, pp. 95-126, 1968. 5. Gauthier, M. J., Flatau, G. N., Breittmayer, J. P., Mathieu, A. & Clement, R. Ent'iron. Technol. Lett., 5, 441-52 (1984). 6. Flatau. G. N., Clement, R. L. & Gauthier, M. J. Chemosphere, 13 (12), 1397-400 (1984).
Cadmium Accumulation by Marine Bacteria
G. N. Flatau & M. J. G a u t h i e r INSERM Unit6 40, I Avenue Jean Lorrain. F-06300 Nice, France
Complexation of metals by marine bacteria is a well known phenomenon, t In the marine ent:ironment it may play a determining role in the mobilisation of metals and their transfer into sediments 2 or through food chains. 3 However, only a limited number of studies have analysed metal binding mechanisms in marine bacteria, the cell structure of which depends more closely on the ionic composition of the environment. ~ The aim o f this work was to investigate the mechanisms responsible f o r the accumulation of cadmium by Gram negative marine bacteria. Previous studies on a large number of strains showed that cadmium accumulation was dependant on the respiratory type of the strain. 5 Cadmium uptake by resting cells of a marine pseudomonad was a linear function of time. The accumulation was lower in cells previously killed by ethanol or potassium cyanide, but was faster during the first hours of the experiment. These variations in metal accumulation suggest that physiological mechanisms are necessary for its adsorption on to the cell wall or its cytoplasmic incorporation. 6 Cadmium accumulation increased in more permeable and unstable cells after a previous treatment by 200 #~ EDTA and then by 200/~M MgCI 2 to complex the excess of EDTA (Fig. l(a)). However, cadmium loaded cells lost the major part of the metal when the medium was added with 10mM/litre EDTA (Fig. l(b)): complexation of calcium and magnesium probably induced a permeabilisation of the cell wall and enhanced the exchange of cadmium. It was also possible that EDTA exposed new binding sites, which were 159 Marine Environ. Res. 0141-1136/85/$03-30 ~ Elsevier Applied Science Publishers Ltd,
England, 1985. Printed in Great Britain
G. .V. Flatau, .U. J. Gauthier
160 ng~ ~.mg'tdry cell S
pg Ca. mg~dry ceils Isoo A
1500
/
I I I / I I I I P I I
1000
1000
I / I I
/'j' /
I
500
11
500
kx
,
8
(a)
hours
(b)
Fig. I. (a) Cadmium accumulation by marine pseudomonad previously treated by EDTA and MgCI, ( - - - ) , or non treated (--). (b) Cadmium release by marine pseudomonad in artificial sea-water containing EDTA (--) or without EDTA (- - -). Cells previously accumulated Cd from 3 litre artificial sea-water added with 1 mg Cd/li~re.
progressively saturated by cadmium; because EDTA decreased the respiration of the cells, the additional cadmium might only be adsorbed on these new sites. In a medium containing 5/JM/litre of the uncoupling agent carbonylcyanide, p-trifluoromethoxy phenylhydrazone (FCCP), cellular respiration rate and cadmium uptake decreased. These also immediately and strongly decreased when FCCP was added 8 h after the beginning of the experiment. Inhibition of protein synthesis by chloramphenicol had only slight effects on cadmium uptake whether it was added at the beginning of the experiment or 8h after. These results suggest that cadmium accumulation did not require induced protein synthesis and probably involved an energy dependent transfer related to respiration processes.
Cadmium accumulation by marine bacteria
161
Cadmium accumulation was carried out with untreated cells, mureinoplasts (cells covered with their murein layer) and spheroplasts (cells partly covered with their murein layer) for 4 h. At the first hour, cadmium accumulation was about 2 times greater in spheroplasts than in mureinoplasts and about 3 times greater than in the untreated cells. This ratio decreased with time, probably due to the rapid breakdown of
ng Cd. mg'~dry cells f
3000
/
,4
I
2000
/
/
[
1000
'2
& hours
Fig. 2. Cadmium accumulation by whole cells (--), mureinoplasts (---) and spheroplasts (---).
spheroplasts (spheroplast life time did not exceed 3 h). After 4 h of contact with cadmium, mureinoplasts had accumulated 3 times more metal than whole ceils and about 1.5 times more than spheroplasts (Fig. 2). These results suggest that the cadmium accumulation could either be due to an increase of exchanges through the naked cytoplasmic membrane or to a further fixation of metal on new superficial binding sites. In either respect, it seems that the triple layered cell wall of G r a m negative in marine bacterial cells acts as an active ion filter.
162
G.N. Flatau, M. J. Gauthier REFERENCES
1. Tornabene, T. G. & Edwards, H. H. Science, 1976, 1334-5 (1972). 2. Ramamoorthy, S., Springthorpe, S. & Kuschner, D. J. Bull. Environ. Contain. Toxicol., 17, 505-11 (1977). 3. Flatau, G. N. & Gauthier. M. J. Can. J. Microbiol., 29. 210-17 (1983). 4. McLeod, R. A. In Advances in Microbiology of the Sea, Vol. 1, (M. R. Droop & E. J. F. Wood, eds), Academic Press, London, pp. 95-126, 1968. 5. Gauthier, M. J., Flatau, G. N., Breittmayer, J. P., Mathieu, A. & Clement, R. Ent'iron. Technol. Lett., 5, 441-52 (1984). 6. Flatau. G. N., Clement, R. L. & Gauthier, M. J. Chemosphere, 13 (12), 1397-400 (1984).