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Cadmium uptake by freshwater bacterial communities
14"mer Research VoL 15. pp. 67 to 71 © Pergamon Press Lid lqSl. Printed in Great Britain
0043-1354/81/0101-0067102.00~
CADMIUM UPTAKE BY FRESHWATER BACTERIAL COMMUNITIES J. Rmo, ct~ Departement de Botanique, Universit~ de Liege, Sart-Tilman, B4000 Liege, Belgium (Received March 1980) AINtmet--Microcosm experiments in chemosiat incubated at 20°C showed that cadmium contamination does not greatly affect bacterial communities in cultures contaminated with up to 1 mg Cd 1- 5 Bacterial productivity remains unchanged and cadmium-resistant strains arise quickly and in great number. The cadmium accumulation by bacteria depends on the bacterial productivity. The free bacteria can accumulate up to 1200 ppm cadmium whereas the adhering bacteria concentrate up to 6100 ppm. At a steady state. 11-29% cadmium is removed from the water phase of cultures.
INTRODUCTION
tone 900 rag; Winogradski saline solution 20 ml; distilled water 1000ml; pH 6.5--7. The medium was phosphate-free in order to avoid cadmium precipitation. When cultures had reached a steady state checked by turbidity, they were contaminated by a continuous flux of cadmium ion (1 ppm) which was similar to the cadmium concentration of an industrial effluent (Patterson. 1979). The behaviour of contaminated bacterial cultures was followed by noting the bacterial productivity, the recovery of cadmium resistant strains, the cadmium concentration in bacteria and in the warm phase. Total microflora enumeration was accomplished by the plate-dilution frequency technique (Harris & Sommers, 1968). The cadmium-resistant strains were enunm'ated by the same technique except for the medium supplemented with 20 ppm cadmium. Bacteria were sampled periodically. They were harvested by centrifuption. After discarding the sulx'matam, the bacterial pellet was oven-dried at 80°C and the dry bacterial biomass weighted. The bacterial productivity is evaluated in rag bact. 1-1 h-1. To each bacterial pellet, 5 ml 6M HNO3 were added and the mixture was left for 3 h. Then it was placed in a hot sand bath at 120°C to release cell.associated metal ions. The mixture was then diluted in 25 ml of distilled water. The cadmium concentrations were determined in the digested bacteria and in the supernatant (i.e. the water phase) by atomic absorption spectrophotometry. Quantities of cadmium are expressed in nag Cd (ks dry bacteria)- 1 and mg Cd I- 1 The oxygen consumptions of the strains were evaluated in an electrolytic respirometer (Sapromat).
Researches are now being made in our laboratory with a view to assess the impact of cadmium upon freshwater bacterial communities and therefore on the fate of this heavy metal in aquatic systems. Indeed. two rivers in the countryside near Li6ge (Belgium) are polluted to a great extent by cadmium and it is important to know the behaviour of cadmium in this environment. As a first step, freshwater bacterial communities were analysed on the ground of their resistance a~ainst cadmium and other toxicants: heavy metals and drugs. So, cadmium-sensitive and cadmium-resistant strains were detected and their physiological characteristics determined (Houba & Remacle, 1980), The cadmium-resistant strains mainly belonged to a G r a m negative genus, Pseudomonas, as observed by Barkay et al. (1979). Besides, the resistance factor proved to be linked to plasmids (Mergeay et al., 1978) which also agrees with the observations of Barkay et al. (1979). In the second step. Cd-sensitive and Cd-resistant strains were analysed in regard to their competitive ability (Remacle & Houba, 1980). In fact, one of the main features when examining the growth of bacteria in presence of cadmium was their ability to accumulate cadmium in their cells. Therefore, from the point of view of aquatic systems it appears important to know more accurately the fate of cadmium mediated by microorganisms. It is the reason why the fate of cadmium is now followed in an open system, the chemostat, in order to mimic the river conditions as closely as possible.
RESULTS Let us examine the bacterial productivity. As shown in Table 1, the bacterial productivity was not Table 1. Comparison of bacterial productivities in continuous cultures before and during cadmium contamination Bacterial productivity (Control at steady state) Dilution rate (h- 1) Time of contamination (days)
MATERIALS AND METHODS . The continuous cultures 'performed in chemostat were seeded by bacterial communities collected in the river Ourthe (Liege, Belgium). All cultures were incubated at 20:C. A gradient of different bacterial productivities was obt~i-ned by modifying the dilution rates, D: 0.16-0.01 h- 1; therefore they ranked from 2 to 50 mg d.w. h-~ 1-1. The composition of the nutritive flux was: glucose 180 rag; pep67
0.1 0,05 0.03
-ld 1 l 1
0.01
1
+ld 0.43 1.13 1
+4d 1.07 0.87 0.80
1
1.2
+30d 1.07 1.2 1.06 1.2
68
J.
REMACLE
affected to a great extent by cadmium contamination even in cultures at low dilution rate (D -- 0.01 h - t) where a low viability might have been expected (Depauw-Gillet & Remacle, 1975). It will also be noted that the recovery of Cd-resistant strains was rising quickly and, in crowded populations, in all cultures (Fig. 1). So, in some cases, nearly all strains became resistant against 20 mg Cd I-t. Moreover, the growth characteristics of Cd-resisrant strains were compared with Cd-sensitive ones in a Cd-free medium. Two typical Cd-sensitive strains, E2s and Et~ and two typical Cd-resistant strains, E, and E79 were selected for this purpose. They were isolated in a sedimentation pond of a zinc-copper factory (Houba & Remade, 1980). The relationship between maximum specific growth rate vs substrate concentration (as C) is shown in Fig. 2, The graph was drawn by the Monod model, the growth parameters /~ and K, being determined by classical methods. It can be noted that the growth of the Cdresistant strains is close to the Cd-sensitive ones at low substrate concentrations. In all cultures, cadmium was accumulated up to high levols in bacteria. In steady state cultures, the maximum conematration reaches 1260 mg k g - t in free bacteria, which rr~ans a concentration factor of 1260 (Table 2). lk'sides, the cadmium concentration in bacteria are not correlated with the dilution rat~; the lowest cadmium concentration in bacteria being recorded at the lowest and the highest dilution rates (Table 2). Nevertheless, by gathering all data at 20°C (9 cultures) where the bacterial productivities ranged from 2 to 50 mg d.w. 1-t h - t , the plot of these productivities vs the rate of cadmium accumulation (#g 1-t h - t ) showed a good correlation (Fig. 4). The relation is VA = 0.93 P~ -'0.74 R = 0.98
VA = rate of cadmium accumulation in cells. #g l-t h-t P, = bacterial productivity, mg d.w. 1-t h-1 In fact, the rate of accumulation implies the rate of cadmium removal from the culture. It could also be noted that the highest cadmium concentrations are not recorded in free bacteria. Indeed, when bacteria films were allowed to develop on the walls of culture vessels, these adhering bacteria could concentrate up to 6100 mg kg- t cadmium. The removal of cadmium from the water phase was very effective just after the beginning of cadmium contamination (Table 3, Fig. 5), it reached then 75-990/0 one day after contamination. But, the removal percentage dropped quickly and after one month contamination` when cultures had reached a steady state again, it equalled 11-290/o. A typical cadmium budget can be established in continuous cultures (Table 4). It can be seen that free bacteria counted in the 14.3%. The difference of 10.6°/0 between input and output might be due to cadmium immobilized by bacteria growing on the walls.
DISCUSSION
The quick recovery of Cd-resistant strains was also observed by Charley & Bull (1979) in silver intoxication. Ik'cause of their resitance, Cd-resistant strains are not greatly affected by intoxication (Fig. 3), and therefore exhibited coionisation capacities similar to sensitive strains. This characteristic and the high proportion of Cd-resistant strains recorded in culture contaminated with cadmium could explain the good homeostasis of bacterial communities exposed to cadmium contamination. This fact was already noted
0 - o 0 3 h"~
O'o.oI h-t
R
, ~
Log -~
II
,oE
IO
9
9
8
8
7
7
6
6
5
5
4
4
2 I Id.
inafion
J Id
/ 4d
,
tin" t
30d Conlamination
Fig. 1. Recovery of cadmium-resistant strains (resistant against 20ppm Cd! in two cultures (D = 0.03 h - ' . 0.01 h -t) contaminated with I POre cadmium, at 20¢C (B: Cd-resistant strain concentration after contamination - b : Cd.resistant strain concentration before contamination).
Cadmium uptake by freshwater bacterial communities Table 2. Cadmium concentrations in bacteria (ppm) vs dilution rates of culture (D = h-1)
E79
h-'
69
0.06
Dilution rate
Cadmium concentration
0.16 0.15 0.10 0.06 0.05 0.04 0.03 0.01
717 947 834 945 1083 1260 673
0.07 E+v
0.06 0.05
760
/
(Singleton & Guthrie, 1977; Sherard et at., 1979). This is true also when shock doses of cadmium (50 mg l- 1) were administered (Lester et al., 1979). Therefore, we may assume that the biodegradation processes are not substantially affected by heavy metals in polluted rivers. Another interesting fact appears to be the cadmium
IO20 40 60 OO I00
uptake by free and adhering bacteria, which accumulate up to 1260 and 6100rag Cd kg -I respectively.It corroborates earlier observation (Chopra, 1970; Remacle & Houba, 1980). High concentration of other heavy metals have also been recorded in bacteria (Guthrie et al., 1977; Patrick and Loutit, 1977; Charley & Bull, 1979). However, when compared with cadmium uptake by bacteria in batch cultures, these cadmium accumulations are rather low. Indeed, in batch cultures,bacteria are able to concentrate up to
004 003
,/ 0.01
mamC t
..
200
Fig. 2. Growth rate vs substrate concentrations (ppm C) of cadmium-tmnmtive strains (EI~, E',s) and cadmium resist. ant strains (EL, E~9) calculated by the Monod model. /z (h- x) k, (ppm C)
EI-7
0.09
E~8 E, E~9
0.09 0.085 0.17
82.6
13,000 mg Cd kg -1 (Remade & Houba, 1980). Besides, it must be pointed out that the higher the dilution rate, the lower the cadmium concentration in bacteria, Therefore, it can be assumed that the period
88 125 207
i?
x.c~s~
BO i
7o!
5°if
6o I i
40
30
20 I0 Cd conc.pprn ,5
I0
15
20
25
30
35
40
(pare) Fig. 3. Oxygen consumption of a Cd-sensitive (E~) and a Cd-resistant strain (E+) in cultures contaminated with cadmium at 20°C (control 100°o). E~ ~---Q: E,--A.
70
J. REMACLE
YA
'~Cd
I-~61
Table 3. Cadmium removal (~,,) in three cultures by comparison with the cadmium influent concentration Cadmium removal Time of contamination (days)
Dilution rates (h - t)
48
42
+ ld 98 98
~1 0.05 0.03
36
+4d 97 97 96
+ 30d 29 11 12
30
Table 4. Cadmium budget in continuous cultures (average of 4 cultures)
24
093x
R -0.97
18
Input
12
100
, iO
I 20
i 30
I 40
/
50
Culture and output Water phase Free bacteria
75.1 14.3
The cadmium accumulation in bacteria leads to the disappearance of the heavy metal from the water phase. In fact, the high cadmium removal percentage observed at the beginning of contamination slows down very quickly up to 11-29%. Stoveland et aL (1979) noted higher cadmium removal, 33-35%, but their cultures were intoxicated by lower cadmium concentrations. At the beginning of exposure to cadmium, no bacterial cells were saturated by cadmium and therefore, all cells could accumulate it. But, when cultures had reached a new steady state i.e. when all cells had stored cadmium, the removal of cadmium from the water phase was achieved by new cells. So cadmium removal is dependent on bacterial productivity. Despite the high cadmium concentration recorded in bacteria, their impact, is of little significance in cadmium removal. This appears logical because of the low weight of bacterial production in comparison with the volume of cultures. Now, we can assume what happens in a river. Consick:ring that at a steady state, bacterial sedimentation equals bacterial production, it can be expected that cadmium is immobilized in the river sediment. It is proved by the high concentration of cadmium (up to
-~
rnqf'fi~
Fig. 4. Accumulation rate of cadmium. Y~,: #g 1- t h- t vs bacterial productivity, P,: mg 1-~ h - ' at 20°C in continuous mixed cultures. of contamination of bacterial cells controls the level of cadmium accumulation. This is exemplified by the fact that the heaviest concentrations are recorded in batch cultures where the residence time of cells is longest. However, at the very low dilution rates (D = 0.01-0.03 h -t) it must be mentioned that the cadmium concentration in bacteria reaches low level. In this case, it is assumed that desorption processes hinder the net accumulation of cadmium by bacteria. This aspect will be investigated in the future. In continuous culture, the highest cadmium coneentrations are observed in adhering bacteria. This might partially be due to the synthesis of glycocalyx (Costerton et al., 1978). This mucilagenous product yielded by adhering bacteria, is composed ,of polysaccharides and peptides which could trap cadmium. Moreover, the duration of contact between cadmium and the adhering bacteria must be very long and thus play a part in cadmium accumulation. Cd removal, %
1(3o
50 251 J
J
'
"
'
'
J
'
I
2
3
4
5
6
7
8
~/7
ID°ysafter 3Ocorit~11~fi~;~,~
Fig. 5. C a d m i u m removal (o~ influent) from the water phase in continuous mixed culture contaminated with I p p m cadmium, at 20°C (D ffi0.04 h-t).
Cadmium uptake by freshwater bacterial communities 400 mg Cd kg-~) observed in sediments of polluted river (NihouI & Bollen, 1976) and by the correlation between cadmium concentration and organic matter concentration in river sediments (Suzuki et a L 1979). These results could be explained to a certain extent b y bacterial sedimentation and also by taking into account the adhering bacteria (e.g. bacterial films on rocks), which are able to accumulate higher levels of cadmium than free bacteria. In this respect, the next step will consist in determining the rate of cadmium desorption from bacteria. Acknowledoement--The author gratefully acknowledges financial support of C.E.C. for this work. REFERENCES
Barkay T., Olson B. H. & Colwell R. R. (1979) Heavy metats transformations mediated by estuarine bacteria. Manaoement and Control of Heat,y Metals in the Environ. ment (Edited by Perry R.L pp. 356-363. Imperial College London. Charley R. C. & Bull A. T.(1979) Bioaccumulation of silver by a multispecies community of bacteria. Arch. Microbiol. 123, 239-244. Chopra I. (1970) Decreased uptake of cadmium by a resistant strain of Staphylococcus aureus. J. oen. Microbiol. 63, 265-267. Costerton J. W., Geesey G. C. & Chenoy K. J. (1978) How bacteria stick'? Scient. Am. 238~ 86-95. Depauw-Gillet M. C. & Remacle J. (1978) The kinetics of microbial population. Microbial Ecology (Edited by Loutit M. W. & Miles J. A. R.L pp. 51-56. SpringerVerlag, Berlin. Guthrie R. K., Singleton F. L. & Cherry D. S. (1977) Aquatic bacteria populations and heavy metals--II. Influence of chemical content of aquatic environments on bacterial uptake of chemical elements. Water. Res. 11, 643--646. Harris R. F. & Sommers L. E. (1968) Plate-dilution frequency technique for assay of microbial ecology. Appl. Microbiol. 16, 330-334.
71
Houba C. & Remacle J. (1980) The composition of saprophytic bacterial communities in freshwater systems contaminated by heavy metals. Microbial Ecol. 6, 55--69. Lester J:N., Perry R. & Dadd A. H. (1979)The influence of heavy metals on a mixed bacterial population of sewage origin in the chemostat. Water. Res. 13, 1055-1063. Mergeay M., Houba C. & Gents J. (1978) Extrachromosoreal inheritance controlling resistance to cadmium, cobalt and zinc ions evidence from curing in a Pseudomoans. Arch. int. Physiol. Biochim. 86, 440-441. Nihoul J. C. J. & Bollen C. (1976) Niveau de pollution du reseau hydrographique et de la zone cotiere beiges. Vol. I1 tome A. Prog. Nat. Rech. Develop. Bruxeiles, Belgique, 280 pp. Patrick F. M.& Loutit M. W. (1977) The uptake of heavy metals by epiphytic bacteria in Alisma plantago aquatica. Water Rex. I!, 699-703. Patterson J. W. (1979) Industry sources and control of metals. Manaoement and Control of Heavy Metals in the Environment (Edited by Perry R.). pp. 617-625. Imperial College, London. Remacle J. & Houba C. (1980) The influence of cadmium upon freshwater saprophytic bacteria. Eat, it. Technol. Lett. 1, 193-200. Sherrad J. H.. Weber A. S., Bagly M. M. & Sujarittanouta S. (1979) The influence of cadmiun~ nickel and cadmium nickel solution on activated sludge process performance. Mana#ement and Control of Heavy Metals in the Environment (Edited by Perry R.), pp. 633-636. Imperial College, London. Singleton F. L. & Guthrie R. K. (1977) Aquatic bacterial populations and heavy metals--l. Composition of aquatic bacteria in the presence of copper and mercury salts. Wmer Res. 11,639--642. Stoveland S., Lester J. N. & Perry R. (1979) Biodcgradation of nitrilotriacetic acid in the activated sludge process and its influence on heavy metal transfer. Mann#emeat and Control of Heal>), Melats in the Environment (Edited by Perry R.), pp. 626-632. Imperial College, Low don. Suzuki M., Yamada T., Miyazaki T. & Kawazee K. (1979) Sorption and accumulation of cadmium in the sediment of the Tama River. Water Res. 13, 57-63.
0043-1354/81/0101-0067102.00~
CADMIUM UPTAKE BY FRESHWATER BACTERIAL COMMUNITIES J. Rmo, ct~ Departement de Botanique, Universit~ de Liege, Sart-Tilman, B4000 Liege, Belgium (Received March 1980) AINtmet--Microcosm experiments in chemosiat incubated at 20°C showed that cadmium contamination does not greatly affect bacterial communities in cultures contaminated with up to 1 mg Cd 1- 5 Bacterial productivity remains unchanged and cadmium-resistant strains arise quickly and in great number. The cadmium accumulation by bacteria depends on the bacterial productivity. The free bacteria can accumulate up to 1200 ppm cadmium whereas the adhering bacteria concentrate up to 6100 ppm. At a steady state. 11-29% cadmium is removed from the water phase of cultures.
INTRODUCTION
tone 900 rag; Winogradski saline solution 20 ml; distilled water 1000ml; pH 6.5--7. The medium was phosphate-free in order to avoid cadmium precipitation. When cultures had reached a steady state checked by turbidity, they were contaminated by a continuous flux of cadmium ion (1 ppm) which was similar to the cadmium concentration of an industrial effluent (Patterson. 1979). The behaviour of contaminated bacterial cultures was followed by noting the bacterial productivity, the recovery of cadmium resistant strains, the cadmium concentration in bacteria and in the warm phase. Total microflora enumeration was accomplished by the plate-dilution frequency technique (Harris & Sommers, 1968). The cadmium-resistant strains were enunm'ated by the same technique except for the medium supplemented with 20 ppm cadmium. Bacteria were sampled periodically. They were harvested by centrifuption. After discarding the sulx'matam, the bacterial pellet was oven-dried at 80°C and the dry bacterial biomass weighted. The bacterial productivity is evaluated in rag bact. 1-1 h-1. To each bacterial pellet, 5 ml 6M HNO3 were added and the mixture was left for 3 h. Then it was placed in a hot sand bath at 120°C to release cell.associated metal ions. The mixture was then diluted in 25 ml of distilled water. The cadmium concentrations were determined in the digested bacteria and in the supernatant (i.e. the water phase) by atomic absorption spectrophotometry. Quantities of cadmium are expressed in nag Cd (ks dry bacteria)- 1 and mg Cd I- 1 The oxygen consumptions of the strains were evaluated in an electrolytic respirometer (Sapromat).
Researches are now being made in our laboratory with a view to assess the impact of cadmium upon freshwater bacterial communities and therefore on the fate of this heavy metal in aquatic systems. Indeed. two rivers in the countryside near Li6ge (Belgium) are polluted to a great extent by cadmium and it is important to know the behaviour of cadmium in this environment. As a first step, freshwater bacterial communities were analysed on the ground of their resistance a~ainst cadmium and other toxicants: heavy metals and drugs. So, cadmium-sensitive and cadmium-resistant strains were detected and their physiological characteristics determined (Houba & Remacle, 1980), The cadmium-resistant strains mainly belonged to a G r a m negative genus, Pseudomonas, as observed by Barkay et al. (1979). Besides, the resistance factor proved to be linked to plasmids (Mergeay et al., 1978) which also agrees with the observations of Barkay et al. (1979). In the second step. Cd-sensitive and Cd-resistant strains were analysed in regard to their competitive ability (Remacle & Houba, 1980). In fact, one of the main features when examining the growth of bacteria in presence of cadmium was their ability to accumulate cadmium in their cells. Therefore, from the point of view of aquatic systems it appears important to know more accurately the fate of cadmium mediated by microorganisms. It is the reason why the fate of cadmium is now followed in an open system, the chemostat, in order to mimic the river conditions as closely as possible.
RESULTS Let us examine the bacterial productivity. As shown in Table 1, the bacterial productivity was not Table 1. Comparison of bacterial productivities in continuous cultures before and during cadmium contamination Bacterial productivity (Control at steady state) Dilution rate (h- 1) Time of contamination (days)
MATERIALS AND METHODS . The continuous cultures 'performed in chemostat were seeded by bacterial communities collected in the river Ourthe (Liege, Belgium). All cultures were incubated at 20:C. A gradient of different bacterial productivities was obt~i-ned by modifying the dilution rates, D: 0.16-0.01 h- 1; therefore they ranked from 2 to 50 mg d.w. h-~ 1-1. The composition of the nutritive flux was: glucose 180 rag; pep67
0.1 0,05 0.03
-ld 1 l 1
0.01
1
+ld 0.43 1.13 1
+4d 1.07 0.87 0.80
1
1.2
+30d 1.07 1.2 1.06 1.2
68
J.
REMACLE
affected to a great extent by cadmium contamination even in cultures at low dilution rate (D -- 0.01 h - t) where a low viability might have been expected (Depauw-Gillet & Remacle, 1975). It will also be noted that the recovery of Cd-resistant strains was rising quickly and, in crowded populations, in all cultures (Fig. 1). So, in some cases, nearly all strains became resistant against 20 mg Cd I-t. Moreover, the growth characteristics of Cd-resisrant strains were compared with Cd-sensitive ones in a Cd-free medium. Two typical Cd-sensitive strains, E2s and Et~ and two typical Cd-resistant strains, E, and E79 were selected for this purpose. They were isolated in a sedimentation pond of a zinc-copper factory (Houba & Remade, 1980). The relationship between maximum specific growth rate vs substrate concentration (as C) is shown in Fig. 2, The graph was drawn by the Monod model, the growth parameters /~ and K, being determined by classical methods. It can be noted that the growth of the Cdresistant strains is close to the Cd-sensitive ones at low substrate concentrations. In all cultures, cadmium was accumulated up to high levols in bacteria. In steady state cultures, the maximum conematration reaches 1260 mg k g - t in free bacteria, which rr~ans a concentration factor of 1260 (Table 2). lk'sides, the cadmium concentration in bacteria are not correlated with the dilution rat~; the lowest cadmium concentration in bacteria being recorded at the lowest and the highest dilution rates (Table 2). Nevertheless, by gathering all data at 20°C (9 cultures) where the bacterial productivities ranged from 2 to 50 mg d.w. 1-t h - t , the plot of these productivities vs the rate of cadmium accumulation (#g 1-t h - t ) showed a good correlation (Fig. 4). The relation is VA = 0.93 P~ -'0.74 R = 0.98
VA = rate of cadmium accumulation in cells. #g l-t h-t P, = bacterial productivity, mg d.w. 1-t h-1 In fact, the rate of accumulation implies the rate of cadmium removal from the culture. It could also be noted that the highest cadmium concentrations are not recorded in free bacteria. Indeed, when bacteria films were allowed to develop on the walls of culture vessels, these adhering bacteria could concentrate up to 6100 mg kg- t cadmium. The removal of cadmium from the water phase was very effective just after the beginning of cadmium contamination (Table 3, Fig. 5), it reached then 75-990/0 one day after contamination. But, the removal percentage dropped quickly and after one month contamination` when cultures had reached a steady state again, it equalled 11-290/o. A typical cadmium budget can be established in continuous cultures (Table 4). It can be seen that free bacteria counted in the 14.3%. The difference of 10.6°/0 between input and output might be due to cadmium immobilized by bacteria growing on the walls.
DISCUSSION
The quick recovery of Cd-resistant strains was also observed by Charley & Bull (1979) in silver intoxication. Ik'cause of their resitance, Cd-resistant strains are not greatly affected by intoxication (Fig. 3), and therefore exhibited coionisation capacities similar to sensitive strains. This characteristic and the high proportion of Cd-resistant strains recorded in culture contaminated with cadmium could explain the good homeostasis of bacterial communities exposed to cadmium contamination. This fact was already noted
0 - o 0 3 h"~
O'o.oI h-t
R
, ~
Log -~
II
,oE
IO
9
9
8
8
7
7
6
6
5
5
4
4
2 I Id.
inafion
J Id
/ 4d
,
tin" t
30d Conlamination
Fig. 1. Recovery of cadmium-resistant strains (resistant against 20ppm Cd! in two cultures (D = 0.03 h - ' . 0.01 h -t) contaminated with I POre cadmium, at 20¢C (B: Cd-resistant strain concentration after contamination - b : Cd.resistant strain concentration before contamination).
Cadmium uptake by freshwater bacterial communities Table 2. Cadmium concentrations in bacteria (ppm) vs dilution rates of culture (D = h-1)
E79
h-'
69
0.06
Dilution rate
Cadmium concentration
0.16 0.15 0.10 0.06 0.05 0.04 0.03 0.01
717 947 834 945 1083 1260 673
0.07 E+v
0.06 0.05
760
/
(Singleton & Guthrie, 1977; Sherard et at., 1979). This is true also when shock doses of cadmium (50 mg l- 1) were administered (Lester et al., 1979). Therefore, we may assume that the biodegradation processes are not substantially affected by heavy metals in polluted rivers. Another interesting fact appears to be the cadmium
IO20 40 60 OO I00
uptake by free and adhering bacteria, which accumulate up to 1260 and 6100rag Cd kg -I respectively.It corroborates earlier observation (Chopra, 1970; Remacle & Houba, 1980). High concentration of other heavy metals have also been recorded in bacteria (Guthrie et al., 1977; Patrick and Loutit, 1977; Charley & Bull, 1979). However, when compared with cadmium uptake by bacteria in batch cultures, these cadmium accumulations are rather low. Indeed, in batch cultures,bacteria are able to concentrate up to
004 003
,/ 0.01
mamC t
..
200
Fig. 2. Growth rate vs substrate concentrations (ppm C) of cadmium-tmnmtive strains (EI~, E',s) and cadmium resist. ant strains (EL, E~9) calculated by the Monod model. /z (h- x) k, (ppm C)
EI-7
0.09
E~8 E, E~9
0.09 0.085 0.17
82.6
13,000 mg Cd kg -1 (Remade & Houba, 1980). Besides, it must be pointed out that the higher the dilution rate, the lower the cadmium concentration in bacteria, Therefore, it can be assumed that the period
88 125 207
i?
x.c~s~
BO i
7o!
5°if
6o I i
40
30
20 I0 Cd conc.pprn ,5
I0
15
20
25
30
35
40
(pare) Fig. 3. Oxygen consumption of a Cd-sensitive (E~) and a Cd-resistant strain (E+) in cultures contaminated with cadmium at 20°C (control 100°o). E~ ~---Q: E,--A.
70
J. REMACLE
YA
'~Cd
I-~61
Table 3. Cadmium removal (~,,) in three cultures by comparison with the cadmium influent concentration Cadmium removal Time of contamination (days)
Dilution rates (h - t)
48
42
+ ld 98 98
~1 0.05 0.03
36
+4d 97 97 96
+ 30d 29 11 12
30
Table 4. Cadmium budget in continuous cultures (average of 4 cultures)
24
093x
R -0.97
18
Input
12
100
, iO
I 20
i 30
I 40
/
50
Culture and output Water phase Free bacteria
75.1 14.3
The cadmium accumulation in bacteria leads to the disappearance of the heavy metal from the water phase. In fact, the high cadmium removal percentage observed at the beginning of contamination slows down very quickly up to 11-29%. Stoveland et aL (1979) noted higher cadmium removal, 33-35%, but their cultures were intoxicated by lower cadmium concentrations. At the beginning of exposure to cadmium, no bacterial cells were saturated by cadmium and therefore, all cells could accumulate it. But, when cultures had reached a new steady state i.e. when all cells had stored cadmium, the removal of cadmium from the water phase was achieved by new cells. So cadmium removal is dependent on bacterial productivity. Despite the high cadmium concentration recorded in bacteria, their impact, is of little significance in cadmium removal. This appears logical because of the low weight of bacterial production in comparison with the volume of cultures. Now, we can assume what happens in a river. Consick:ring that at a steady state, bacterial sedimentation equals bacterial production, it can be expected that cadmium is immobilized in the river sediment. It is proved by the high concentration of cadmium (up to
-~
rnqf'fi~
Fig. 4. Accumulation rate of cadmium. Y~,: #g 1- t h- t vs bacterial productivity, P,: mg 1-~ h - ' at 20°C in continuous mixed cultures. of contamination of bacterial cells controls the level of cadmium accumulation. This is exemplified by the fact that the heaviest concentrations are recorded in batch cultures where the residence time of cells is longest. However, at the very low dilution rates (D = 0.01-0.03 h -t) it must be mentioned that the cadmium concentration in bacteria reaches low level. In this case, it is assumed that desorption processes hinder the net accumulation of cadmium by bacteria. This aspect will be investigated in the future. In continuous culture, the highest cadmium coneentrations are observed in adhering bacteria. This might partially be due to the synthesis of glycocalyx (Costerton et al., 1978). This mucilagenous product yielded by adhering bacteria, is composed ,of polysaccharides and peptides which could trap cadmium. Moreover, the duration of contact between cadmium and the adhering bacteria must be very long and thus play a part in cadmium accumulation. Cd removal, %
1(3o
50 251 J
J
'
"
'
'
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2
3
4
5
6
7
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Fig. 5. C a d m i u m removal (o~ influent) from the water phase in continuous mixed culture contaminated with I p p m cadmium, at 20°C (D ffi0.04 h-t).
Cadmium uptake by freshwater bacterial communities 400 mg Cd kg-~) observed in sediments of polluted river (NihouI & Bollen, 1976) and by the correlation between cadmium concentration and organic matter concentration in river sediments (Suzuki et a L 1979). These results could be explained to a certain extent b y bacterial sedimentation and also by taking into account the adhering bacteria (e.g. bacterial films on rocks), which are able to accumulate higher levels of cadmium than free bacteria. In this respect, the next step will consist in determining the rate of cadmium desorption from bacteria. Acknowledoement--The author gratefully acknowledges financial support of C.E.C. for this work. REFERENCES
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