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The Role of The Bacterial Community in the Radionuclide Transfers in Freshwater Ecosystems.
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THE ROLE OF THE BACTERIAL COMMUNITY IN THE RADIONUCLIDE TRANSFERS IN FRESHWATER ECOSYSTEMS.
F.Hambuckers-Berhin, A.Hambuckers, J.Remacle Microbial Ecology, Department of Botany B22, University ot Liege, B 4000 LIEGE, BELGIUM.
INTRODUCTION
During the last forty years extensive industrial use of radioactive and fissile materials has taken place in Europe and elsewhere. Large scale nuclear facilities, for civil and defence purposes, were developed some of which giving rise to significant radioactive contamination of the biosphere. Therefore several questions are to be asked concerning human health hazards following releases of radioactive substances in the environment either continuously or accidentally. Four nuclear plants are settled in two sites along the river Meuse, Chooz (1) and Tihange (3). Moreover two new plants are being build in the site of Chooz (France). The river Meuse flows through France, Belgium and Netherlands and play a great role for the human welfare of the populations living in these countries. The river Meuse supplies five millions people with drinking water. Eight hundred km2 (agricultural lands, meadows) are flooded with water pumped from the river. It must also be mentioned that seventy tons of fishes were caught in 1982 in the river and its tributaries. Our laboratory is involved in a multidisciplinary research programme dealing with the study of the impact of nuclear power plants on the aquatic ecosystem more especially on rivers [l-2-31. Two neutron activation products contained in the waste effluents of the above nuclear facilities are mainly studied 6oCo and 134Cs. For example, the nuclear plant, Tihange 2, released lo00 MBq of 334Cs in 1982 [3]. It can be supposed that the transfer and the immobilization of these radionuclides could occur along the aquatic food webs since: (i) Co ion is a trace metal required for normal growth of all living organisms, (ii) Cs competes with K and ammonium ions [4] which are important metabolites. Moreover they could effectively radiocontaminate the living components of the food web owing to their periods (60Co: 5.2 years; 134Cs: 2.05 years). The final aim of the programme is to elaborate a general model of radionuclide tranfers through the food webs. Unfortunately the role of bacteria was neglected up to the recent years which impeded the finalization of the model. It is indeed obvious that bacteria could take a significant part in the radionuclide cycle at three levels. Firstly, they are vectors of radionuclide transport because they are able to take up, store or immobilize radionuclides generally in organic molecules or to precipitate them as insoluble salts e.g. carbonates,
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sulphides as observed for Cd and Zn ions 15-61, Besides under some conditions bacteria release the radionuclides. Secondly bacteria can be an important step in further intake of radionuclide by the other living components of the food web. Thirdly bacteria are responsible, through the biodegradation, of the release of radionuclides sequestered in the organic matter.
w I I
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T
Ll I
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BOM
A T E R
CRAZING 2 /'
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CATABOLISM
3
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Figure 1. Conceptual scheme of the radionuclide fluxes by considering the interactions between bacteria and the organic matter. POM: Particulate Organic Matter ROM: Refractory Organic Matter UDOM: Utilizable Dissolved Organic Matter BOM: Bacterial Organic Matter
Radionuclides Organic matter transfers Radionuclide transfers The radionuclide fluxes (Figure 1) can be conceived by considering the interactions between bacteria and the organic matter described by Billen and Fontigny 171. These fluxes occur in the water column and in the interstitial water of the sediments. The particulate organic matter or polymeric organic matter (POM) is hydrolyzed by bacterial
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ectoenzymes and transformed in utilizable dissolved organic matter (UDOM) i. e. small molecules such as amino acids, organic acids, mono- and oligosaccharides which can be easily taken up by hcterotrophic bacteria. A part of POM, not or slowly metabolizable by bacteria, constitutes the refractory organic matter (ROM). A part of this durable organic matter arises from cell envelopes and walls of degraded bacteria. These components can remain in individual form or can be adsorbed on clays (specially smectite or kaolinite). In this case, the radionuclide-binding capacity of ROM would he reduced because the sites normally available to radionuclides-binding would be masked or chemically neutralized as shown for metal-binding capacity of walls- and cell envelopes-clays composites 18-91. During organic matter hydrolysis, radionuclides sequestered in POM are either transfered to the water phase, or complexed with UDOM. On the other hand, bacterial yields were evaluated in the river Meuse [lo] and averaged 30%. It means that 70% of consumed substrate are catabolyzed while the remaining 30% are transformed into bacterial biomass (BOM). It results that a part of sequestered radionuclides is soluhilized through bacterial activities. Finally bacterial biomass can be destroyed by cell lysis or grazed by aquatic predators. It leads to organic matter recycling and transfers of immobilized radionuclides either to the water column or to POM or to higher trophic levels of the food web. The density of the bacterial community is difficult to assess in river ecosystems. However it can be assumed that 30% of the primary production is consumed by planctonic bacteria [ 1 I]. In steady state conditions the bacterial concentration remains relatively unchanged due to grazing which is of the same order of magnitude as bacterial growth rate 0.1 h-l 110-121. Moreover, the bacterial density is controlled by environmental conditions. So, a pulse of UDOM provokes bacterial blooms [13], and the initial bacterial level will be reached again through predation. All the above processes exemplify the significant bacterial contribution to the radionuclide cycling. The first part of this paper deals with the comparison of the aerobic bacterial communities growing in the water column and the sediments in order to evaluate the homogeneity of bacterial colonization of the river and to know whether it is allowed to extrapolate the radionuclide flux kinetics to the two main compartments of the river. The second part is devoted to the study of 6oCo and 134Cs fluxes between bacteria and water in the river Meuse.
COMPARISON OF THE AEROBIC BACTERIAL COMMUNITIES COLONIZING THE WATER COLUMN AND THE SEDIMENTS Material and methods Water and sediments were collected at Hastiere along the river Meuse. The samples were aseptically taken, stored in sterile bottles until analysis. The bacterial strains were collected from the water column following the scheme depicted in Figure 2 .
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100 ml Meuse water (5 aliquots)
100 ml Meuse water (5 aliquots)
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filtration (0.22pm pore size)
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filter on PCA
solution spread on PCA (3 replicates)
bacterial film dispersed in 15 ml
10 fold'dilution
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100 pl of dilution spread on PCA (3 replicates)
Figure 2. Analysis of the bacterial communities of the river Meuse. Isolation of bacterial strains from the water column (PCA = Plate Count Agar).
The bacterial strains were harvested from the sediments following the scheme of figure 3. The absence of contamination was checked by following the same schemes with sterile Meuse water and sterile distilled water. The Petri dishes were incubated at 30°C during maximum 5 days. The strains were then isolated and purified. Fifty strains were selected and characterized by their morphology, gram-stain and biochemical profile ( API strip, Bio-Merieux, Oxy-Ferm tube, Roche). The characters were coded 1 for positive or present, 0 for negative or absent and 1 for missing test. Strain similarity was estimated by the simple matching coefficient of Sokal and Mitchener [14]. Cluster analysis was carried out by using the average linkage method (procedure CLUSTER, UPGMA [15]). The strains showing the highest similarity coefficient are proximally gathered in a hierarchical structure of more and more large groups. The results of the cluster analysis are graphically represented by dendrograms using the TREE procedure [ 151.
+
34 1
sediments (250 g)in suspension in 400 ml of sterile Meuse water (5 aliquots) shaking 48 h, room temperature
/ / 100 PI of siispension
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decantation
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spread on PCA (3 replicates)
filtration of supernatant (20 ml, 0.22 pm pore size)
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( 3 replicates)
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100 pI on PCA
(3 replicates)
bacterial film dispersed in 15 ml of sterile Meuse water
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10 fold dilution 100 pl bn PCA (3 replicates)
Figure 3. Analysis of the bacterial communities of the river Meuse. Isolation of the bacterial strains from the sediments (PCA = Plate Count Agar).
Results The dendrogram (Figure 4) is composed of all the strains (50) which are gathered by hierarchical level. The minimal distance between the strains is 0 which means full similarity between the strains, on the ground on the chosen tests. So, the dendrogram gives an evaluation of the similarity between the strains and each strain is characterized by its level of aggregation I 161. All the 50 strains were recovered in two main clusters. Cluster 1 which joined 70% of all the strains, is composed of 63 % of water column strains and 37 % of sediment strains. Cluster 2 contains the remaining 30 % with 1 strain from the water column and 14 strains from the sediments. The strains clustered in this group were different from the strains of cluster 1 by the ability to use carbohydrates such as saccharose, mannitol, sorbitol and rhamnose (Table 1). The cluster analysis shows that the aerobic bacterial community isolated from the sediment was constituted of two groups : one group presenting the same biochemical features as the
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aerobic bacterial community isolated from the water column, and an other group composed of strains only present in the sediments.
Average d i s t a n c e between c u s t e r s 0.0 w U Y U
S
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c\l L
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1.4
20 21
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14 19 3 13 19
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15 17 26 24 16
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0.8
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0.6
18 6 15 8
5
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0.4
1 10 9 17
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0.2
10
27 18
Figure 4. Dendrogram of the cluster analysis of 50 strains isolated from the water column (w) and the sediments (s). I t appears that a great proportion of the bacteria colonizing the sediment and the bacterial community of the water column share similar biochemical features. It could be due to the fact that a part of the bacteria of the water column settle down and colonize the interstitial water and the sediments.
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'Table 1 Main characteritics of strains of clusters 1 and 2 (see Figure 4) CLUSTER 1 number of strains total from water from sediment..
35 22
13
CLUSTER 2 15 1
14
% of positive responses in each cluster
Gram + Gram-
34 66
20 80
oxidase catalase arginine dihydrolase indole ONPG~@)
63 86 29 0 14
20 80 53 20 100
degradation of gelatin
60
13
acetate malate citrate
0 0
0 0 60
glucose saccharose arabinose mannitol sorbitol melibiose rhamnose
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100 67 93 87 80 80 87
utilisation of
?dl
51
0 6 0 0 6 0
ONPG, o-Nitrophenyl---galactopyranoside
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Consequently the role of bacteria in the radionuclide transfers in aquatic ecosystem will be investigated by analyzing the aerobic bacterial community isolated from the sediments. It will be considered in a first step that the responses could be representative of the aerobic bacterial communities of the river.
KINETICS OF 6oC0 AND 134CS TRANSFERS MEDIATED BY BACTERIA. It was decided to study the radionuclide transfers in a bacterial community instead of in a monospecific bacterial culture in order to obtain more reliable results from an ecosystemic point of view. The inoculum of the bacterial community was isolated from sediments sampled in the river Meuse at Hastikre (Belgium). The sediment! were stored in sterile bottles at 4'C until the experiments. A new preculture was carried out before each experiment in order to obtain a bacterial community as close as possible to the natural community. It is well known indeed that several strains cannot develop in laboratory conditions so that subcultured bacterial communities lose a lot of strains with uncontroled changes of the specific composition.
a. Radianuclide immobilisation by the bacte rial community, Material and methods
Preculture and cultures conditions. Precultures are carried out by inoculating sterile Meuse water with sediments. The Meuse water is supplemented with starch (2 g.1-l) and bacto-peptone (10 g.1-l) in order to improve the bacterial growth. The preculture vessels are shaked and incubated at 20'C until the mid exponential phase of bacterial growth i.e. during 48 h. At this stage, the bacterial community of the sediment is harvested by centrifugation (10 min, 2,OOOxg). The pellet is discarded and the supernatant is again centrifugated 15 min at 15,200xg. The last pellet is used to inoculate the experimental cultures which are carried out in the same Meuse water medium as preculture. The initial O.D. of cultures are f0.06. The cultures are shaked 24h at 20'C. The bulk growth rate of the bacterial community rankes between 0.13 and 0.15 h'l which is of the same order of magnitude as mentioned in earlier observations [lo].
Radionuclide uptake by bacteria. The culture medium is contaminated with 6oCo and 134Cs (up to 2,000 Bq.1-l) before the inoculation. At regular time intervals, an aliquot of the culture is centrifugated
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(3 min, 12,500xg). The dry weight of the bacterial biomass is determined and after ashing in nitric acid, the radioactivity level is detected by gamma spectrometry.
Results The uptake of 6oCo and 334Cs by the bacterial communities collected from the Meuse river was investigated in presence of increasing radiocontamination (up to 2000 Bq ml-l) in a Meuse water medium. When the data were expressed in the form of a double-reciprocal Lineweaver-Burk plot, two distinct phases were observed for 6oCo. One phase characterized the low contamination levels betwen 24 and 90 Bq.ml-' whereas another phase occured in the high contamination levels between 90 and 2,000 Bq.rn1-l (Figure 5). Each phase followed the Michaelis-Menten kinetics: 1/V = (l/Vmax) + (Km/Vmax). (l/[S]) where, V was the rate of uptake (Bq.g-I.hl), [S] was the activity of the medium (Bq.ml-'), Vmax was the maximal rate and K, was the Michaelis constant. Tdbk 2 (Bq.g-I.h-I) values obtained from Lineweaver-Burk plots of the Km (Bq,ml-l) and V uptake of 6oCo and ly%s.
134cs
Radiocontamination range of w ter (Bq.ml-B)
Vmax
24 - 90 90 - 2,000
2,500 18,692
22 - 295
1,286
Km 63.70 1,145.79 240
Vmax and Km values were calculated from the Lineweaver-Burk plot (Table 2). Km value was significantly greater in the high contamination levels. The two different phases of 6oCo uptake were therefore assumed to reflect high and low affinity uptake systems. The level of radiocontamination respectively explained 58% and 98% of the variation of the uptake rates. The 134Cs uptake kinetic only showed one phase (Figure 6). 73% of the variation of the contamination rate were explained by the level of radiocontamination of the water column. The Km values of Table 2 show that the highest affinity transport system is observed for %3in the low radiocontamination levels. During the radiocontamination,
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33% and 24% of the variations of the concentration factor could be explained by the contact time between the biomass and the radionuclides for 6oCo (from 22 to 2,000 Bq 60Co.m1-1) and 134Cs (from 16 to 300 Bq 134Cs.ml-1) respectively. At the end of the uptake experiment (24 h; bacterial biomass: 500 mg dw.l-I), the activity remaining in the water column ranked between 25% and 40% of the initial activity in the case of 6oCo (initial activity: 0 to 2000 Bq.ml-I) and between 45 and 95% in the case of 134Cs (initial activity: 0 to 300 Bq.ml-I). For the same radiocontamination levels e.g.fl50 Bq.ml-l, the activity level of the bacterial community was +2.5 times higher for 6oCo (+50,000 Bq.g-' d.w.) than for 334Cs(+20 OOO Bq.g-l d.w.). Thus, an important part of &Co and 134Cs can be stored by the aerobic bacterial community which constitutes therefore a pool of radionuclides in the river ecosystem.
Figure 5. Lineweaver-Burk plot of 6oCo uptake by the bacterial community. [S] : radiocontamination eve s of the medium (Bq.mi-l) V : rate of uptake (Bq.g' .h- ) : maximal uptake rate (fjq.g-l.h-l) ::?affinity constant (Bq.ml- ) Equations: (1) low radiocon 'nation lev sy = 0.4 loy+ 23.8 lo9 x (r = 0.7595) (2) high radioconta ination leve : y = 53.5 61.3 10-?fx (r = 0.9924) (r : correlation coefficient)
\ I
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l/CSI .lo-3
Figure 6. Lineweaver-Burk plot of 134Cs uptake by the bacterial community. IS] : radiocontamination eve s of the medium-based Meuse water (Bq.ml-l) V : rate of uptake (Bq.g' .h ) Equation: y = 0.8 + 186.7 x (r = 0.8570) (r : correlation coefficient)
I -\
b. Radionuclide release by the bacterial community.
Material and methods The bacterial community is contaminated with 6oCo and 134Cs during the exponential growth phase. Bacteria are harversted by centrifugation (3 min, 12,5OOxg), washed and suspended in a dialysis tube with sterile Meuse water (Spectrapor 5 ; thickness 0.09 mm; molecular sieve 14,000 D). The dialysis tubes are incubated in running sterile Meuse water at different temperatures (13'C, 20'C) and pH (6.5;7.0;7.5;8.0;8.5;9.0). At time intervals, the radioactivity levels detected either in the bacterial biomass or in the Meuse water outside the dialyse tubes.
Results The decontamination of the bacterial community loaded with 6oCo and 134Cs was investigated in relation to two environmental parameters, the temperature and the pH. The
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activity levels of bacterial biomass at time zero ranked between 200 Bq.g-' d.w.and 900 MBq.g-l d.w. for 6oCo and between 1 and 50 P4Bq.g-l d.w. for 134Cs. When the temperature of the water column wdS maintained at 20°C, the kinetic of the decontamination of the bacterial community was described by a double negative exponential model. It was fitted using the procedure NLIN of SAS 1151 following the DUD method of Raltson and Jennrich [17]: y = m e-ax + n e-bx, where y : 6oCo or 134Cs levels in bacteria (Bq.g-I d.w.) x : time (h) m,n : ordinate values for each exponential (Bq.g-l .d.w.) a : slope of the line with equation In y = In m + ax b : slope of the line with equation In y = In n + bx a and b parameters depended on the desorption rate and were considered as an estimation of the biological half-lives (Tbl and Tb2) calculated as follow: Tbl = In 2 I a Tb2 = In 21 b Examples of adjusted curves were given for 6oCo (Figure 7) and 134Cs (Figure 8).
Figure 7. Desorption of 6oCo by the aerobic bacterial community. Observed values: square; Predicted values: line; y = 69 e-350.347x + 165 e-o.046x (r = 0.9121) y : 6oCo levels in bacteria (Bq.gl d.w.) x : time (h)
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A
0,
1500
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500 0
0
200
100
300
400
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Time (h)
Figure 8. Desorption of 134Cs by the aerobic bacterial community. Observed values: square; Predicted values: line; y = 916 e-7.82x 879 e-o.018x r = 0.9504) y : 134Cs levels in bacteria (Bq.g' d.w.) x : time (h).
+
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For both radionuclides, the biological half-lives Tbl were found to be extremely short, of the order of a few seconds or minutes whereas the biological half-lives Tb2 were longer (1Sh to 461h for 6oCo and 39h to 8,976h for 134Cs). Except for a high value of Tb2 observed with 134Cs (374 d) and a very small value observed with 6oCo (0.6 d), both corresponding with the lowest radiocontamination level of the medium during the radiocontamination of the bacteria communities, the values of biological half-lives were of the same magnitude as observed for a cyanobacterium Scenedesmus sp . [ 18- 191. When the temperature of the water column was maintained at 13OC (average temperature of the Meuse river), the results showed that radionuclides fixed by bacteria were not released. At 20°C, the decontamination of the bacterial community was followed as the pH increased from 6.5 to 9. The chosen criteria was the increase of radioactivity in the water column which was a consequence of the decontamination of bacterial biomass. In any cases, the data were fitted by the following mathematical relation: y = m(l-e-ax) + n(1-ebx), where y : 6oCo or the 134Cs levels in the water column (Bq.ml-l) x : time (h) m,n : ordinate values (Bq.g-l .d.w.) a : slope of the line with equation In y = In m + ax
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b : slope ofthe line with equation In y = In n + bx This mathematical adjustment showed that the evolution of radiocontamination level of the water column was adequately described by a biphasique process with a first phase characterized by a rapid desorption of radioactivity from the bacterial biomass into the water column and a second phase during which the radioactivity level of the water column increased slowly. The parameters a and b depended on the contamination rate of the water column and allow to compare the biological half-lives of the bacterial biomass, Tbl and Th2 respectively. They were calculated as follow: Tbl = I n 2 l a Tb2 = In 21 b. In the case of 60Co,the longest Tbl and Tb2 were found at pH 8, the average pH of the Meuse river (Figures 9 and 10). For the other pH values of the water column, Tbl and Tb2 were shorter which means a more rapid desorption of the 6oCo by the bacterial biomass and consequently an increasin radioactivity level in the water column. The same observations were made in the case of f34Cs. Given that the mean temperature in the Meuse river is 13'C and pH averages 8 , bacteria are likely to act as radionuclide sinks for much of the time but, when the temperature increases in summer they could act as sources.
6.5
7.0
7.5
8.0
t
8.5
a v e r a g e Meuse value
9.0 PH
Figure 9. Biological half-lives Tbl (h) of the 6oCo desorption of the bacterial community vs the water column pH.
35 I
Figure 10. Biological half-lives Tb2 (h) of the "Co desorption of the bacterial community vs the water column pH values.
CONCLUSlON AND SUMMARY. The radionuclide fluxes between the bacteria and the water in aquatic ecosystem were studied by examining the bulk transfers mediated by a bacterial community isolated From the river sediments. The comparison of the aerobic bacterial communities colonizing the sediments and the water column shows that the bacterial community of the sediments is composed of two sub-communities. The fist one is similar to the water column community by its biochemical feahires; the other one displays quite different characteristics and appears to be more representative of the sediments.
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An important part of 6oCo and 134Cs can be immobilized by the bacterial biomass which therefore constitutes a pool of radionuclides, their transfers to the water column being controlled by tern erature and pH. The uptake of 68Co and 134Cs by bacteria can be described by the Michaelis Menten model. The uptake kinetics depend on the type of radionuclide and the level of radiocontarnination in the water column. The highest affinity uptake system is observed for 6oCo at low radiocontarnination levels. The decontamination of bacterial biomass develops in two phases. The first phase is characterized by a very short biological half-life, a few seconds or minutes while the second phase is longer, the biological half-lives reach between 15 h to 461 h for 6oCo and between 39 h to 8,976 h for 134Cs. It could be inferred from the study of the influence of temperature and pH that in the river Meuse where temperature averages 13’C and H is around 8, the rates of radionuclide releases are very low so that 6oCo and lY4Cs are effectively trapped by bacterial biomass. When the conditions change i.e. the temperature raises, the radionuclides immobilized by the bacterial biomass are released and radioactivity consequently increases in the water column. It appears thus that the environmental parameters play an important role in the radionuclide transfers mediated by bacteria and are to be more deeply investigated in the further.
REFERENCES
De Clercq-Versele H, Kirchmann R. L’impact des rejets de la centrale nuclkaire de Tihange (Belgique) sur l’kcosysteme Meuse: cinq annCes d’ktude in situ et d’approche expdrimentale (1976-1980). BLG 555, 1982. KirchmaM R. L’irnpact des rejets de la central nuclCaire de Tihange (Belgique) sur I’Ccosystkme Meuse: dtudes in situ et recherches expkrimentales durant la pCriode 1981-1984. BLG 573, 1985. Kirchrnann R, Vandecasteele CM, Foulquier L, Lambinon J., Sombre L. La radiokcologie des grands fleuves: des donnCes de sites et de l’expkrirnentation B la moddlisation (application la Meuse et au RhOne). BLG 635, 1992. Crerners A. Modelling the transport of radionuclides through the freshwater environment. CCE Project Bi7-008, Final Report, 1992. Rernacle J, Vercheval C. Can J Microbiol 1991: 37: 875-877. Remacle J, Muguruza I, Fransolet M. Wat Res 1992: 26: 923-926. Billen G, Servais P. in Bianchi M, Marty D, Bemand JC, Caumette P, Gauthier M eds Micro-organismes dans les Ccosystkmes ocdaniques. Paris: Masson, 1989; 2 19-245. Walker SG, Flemming CA, Ferris FG, Beveridge TJ, Bailey GW. Appl Environ Microb 1989: 55: 2976-2984.
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Flemming CA, Ferris FG, Bevendge TJ, Bailey GW. Appl Environ Microb 1990: 56: 3191-3203, Servais P, Billen G, Hascoet MC. Wat Res 1987: 21 : 445-450. Azarn F, Fenchel T, Field JG, Gray JS, Meyer-Reil LA, Thingstad F. Mar Ecol Progr Ser 1983: 10: 257-263. Servais P. Etude de la degradation de la matikre organique par les bacteries hiterotrophes en rivkre. DCveloppement d’une demarche methodologique et application a la Meuse belge. Thkse de Doctorat. Universitk Libre de Bruxelles, 1986. Daurnas R, Bianchi M. Arch Hydrobiol Beih Ergebn Limnol 1984: 19: 289-294. Sokal RR, Michener CD. Univ Kansas Sci Bull 1958: 38: 1409-1458. SAS. User’s Guide: Statistics. Cary NC: Sas Institute Inc., 1985. Le Minor L, Viron M. Bacteriologie medicale. Paris: Flarnarion MedecineSciences, 1982. Ralston ML, Jennrich RI. Technometrics 1979: 1: 7-14 Nucho R, Baudin JP. Sci Eau 1986: 5: 361-376. Sombre’ L. Contribution h 1’Ctude du transfert du radiocesium (Cs- 134 et Cs- 137) dans une chaine alimentaire d’eau douce simplifiee: eau - algue verte (Scenedesmus obtiquw) - mollusque filtreur (Dreissena polymorpha) - Poisson (Barbus harhus). These de Doctorat. Universitk de Provence (Aix-Marseille I), 1987.
THE ROLE OF THE BACTERIAL COMMUNITY IN THE RADIONUCLIDE TRANSFERS IN FRESHWATER ECOSYSTEMS.
F.Hambuckers-Berhin, A.Hambuckers, J.Remacle Microbial Ecology, Department of Botany B22, University ot Liege, B 4000 LIEGE, BELGIUM.
INTRODUCTION
During the last forty years extensive industrial use of radioactive and fissile materials has taken place in Europe and elsewhere. Large scale nuclear facilities, for civil and defence purposes, were developed some of which giving rise to significant radioactive contamination of the biosphere. Therefore several questions are to be asked concerning human health hazards following releases of radioactive substances in the environment either continuously or accidentally. Four nuclear plants are settled in two sites along the river Meuse, Chooz (1) and Tihange (3). Moreover two new plants are being build in the site of Chooz (France). The river Meuse flows through France, Belgium and Netherlands and play a great role for the human welfare of the populations living in these countries. The river Meuse supplies five millions people with drinking water. Eight hundred km2 (agricultural lands, meadows) are flooded with water pumped from the river. It must also be mentioned that seventy tons of fishes were caught in 1982 in the river and its tributaries. Our laboratory is involved in a multidisciplinary research programme dealing with the study of the impact of nuclear power plants on the aquatic ecosystem more especially on rivers [l-2-31. Two neutron activation products contained in the waste effluents of the above nuclear facilities are mainly studied 6oCo and 134Cs. For example, the nuclear plant, Tihange 2, released lo00 MBq of 334Cs in 1982 [3]. It can be supposed that the transfer and the immobilization of these radionuclides could occur along the aquatic food webs since: (i) Co ion is a trace metal required for normal growth of all living organisms, (ii) Cs competes with K and ammonium ions [4] which are important metabolites. Moreover they could effectively radiocontaminate the living components of the food web owing to their periods (60Co: 5.2 years; 134Cs: 2.05 years). The final aim of the programme is to elaborate a general model of radionuclide tranfers through the food webs. Unfortunately the role of bacteria was neglected up to the recent years which impeded the finalization of the model. It is indeed obvious that bacteria could take a significant part in the radionuclide cycle at three levels. Firstly, they are vectors of radionuclide transport because they are able to take up, store or immobilize radionuclides generally in organic molecules or to precipitate them as insoluble salts e.g. carbonates,
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sulphides as observed for Cd and Zn ions 15-61, Besides under some conditions bacteria release the radionuclides. Secondly bacteria can be an important step in further intake of radionuclide by the other living components of the food web. Thirdly bacteria are responsible, through the biodegradation, of the release of radionuclides sequestered in the organic matter.
w I I
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BOM
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Figure 1. Conceptual scheme of the radionuclide fluxes by considering the interactions between bacteria and the organic matter. POM: Particulate Organic Matter ROM: Refractory Organic Matter UDOM: Utilizable Dissolved Organic Matter BOM: Bacterial Organic Matter
Radionuclides Organic matter transfers Radionuclide transfers The radionuclide fluxes (Figure 1) can be conceived by considering the interactions between bacteria and the organic matter described by Billen and Fontigny 171. These fluxes occur in the water column and in the interstitial water of the sediments. The particulate organic matter or polymeric organic matter (POM) is hydrolyzed by bacterial
339
ectoenzymes and transformed in utilizable dissolved organic matter (UDOM) i. e. small molecules such as amino acids, organic acids, mono- and oligosaccharides which can be easily taken up by hcterotrophic bacteria. A part of POM, not or slowly metabolizable by bacteria, constitutes the refractory organic matter (ROM). A part of this durable organic matter arises from cell envelopes and walls of degraded bacteria. These components can remain in individual form or can be adsorbed on clays (specially smectite or kaolinite). In this case, the radionuclide-binding capacity of ROM would he reduced because the sites normally available to radionuclides-binding would be masked or chemically neutralized as shown for metal-binding capacity of walls- and cell envelopes-clays composites 18-91. During organic matter hydrolysis, radionuclides sequestered in POM are either transfered to the water phase, or complexed with UDOM. On the other hand, bacterial yields were evaluated in the river Meuse [lo] and averaged 30%. It means that 70% of consumed substrate are catabolyzed while the remaining 30% are transformed into bacterial biomass (BOM). It results that a part of sequestered radionuclides is soluhilized through bacterial activities. Finally bacterial biomass can be destroyed by cell lysis or grazed by aquatic predators. It leads to organic matter recycling and transfers of immobilized radionuclides either to the water column or to POM or to higher trophic levels of the food web. The density of the bacterial community is difficult to assess in river ecosystems. However it can be assumed that 30% of the primary production is consumed by planctonic bacteria [ 1 I]. In steady state conditions the bacterial concentration remains relatively unchanged due to grazing which is of the same order of magnitude as bacterial growth rate 0.1 h-l 110-121. Moreover, the bacterial density is controlled by environmental conditions. So, a pulse of UDOM provokes bacterial blooms [13], and the initial bacterial level will be reached again through predation. All the above processes exemplify the significant bacterial contribution to the radionuclide cycling. The first part of this paper deals with the comparison of the aerobic bacterial communities growing in the water column and the sediments in order to evaluate the homogeneity of bacterial colonization of the river and to know whether it is allowed to extrapolate the radionuclide flux kinetics to the two main compartments of the river. The second part is devoted to the study of 6oCo and 134Cs fluxes between bacteria and water in the river Meuse.
COMPARISON OF THE AEROBIC BACTERIAL COMMUNITIES COLONIZING THE WATER COLUMN AND THE SEDIMENTS Material and methods Water and sediments were collected at Hastiere along the river Meuse. The samples were aseptically taken, stored in sterile bottles until analysis. The bacterial strains were collected from the water column following the scheme depicted in Figure 2 .
340
100 ml Meuse water (5 aliquots)
100 ml Meuse water (5 aliquots)
/
\
filtration (0.22pm pore size)
/
\
filter on PCA
solution spread on PCA (3 replicates)
bacterial film dispersed in 15 ml
10 fold'dilution
I
100 pl of dilution spread on PCA (3 replicates)
Figure 2. Analysis of the bacterial communities of the river Meuse. Isolation of bacterial strains from the water column (PCA = Plate Count Agar).
The bacterial strains were harvested from the sediments following the scheme of figure 3. The absence of contamination was checked by following the same schemes with sterile Meuse water and sterile distilled water. The Petri dishes were incubated at 30°C during maximum 5 days. The strains were then isolated and purified. Fifty strains were selected and characterized by their morphology, gram-stain and biochemical profile ( API strip, Bio-Merieux, Oxy-Ferm tube, Roche). The characters were coded 1 for positive or present, 0 for negative or absent and 1 for missing test. Strain similarity was estimated by the simple matching coefficient of Sokal and Mitchener [14]. Cluster analysis was carried out by using the average linkage method (procedure CLUSTER, UPGMA [15]). The strains showing the highest similarity coefficient are proximally gathered in a hierarchical structure of more and more large groups. The results of the cluster analysis are graphically represented by dendrograms using the TREE procedure [ 151.
+
34 1
sediments (250 g)in suspension in 400 ml of sterile Meuse water (5 aliquots) shaking 48 h, room temperature
/ / 100 PI of siispension
\
\
decantation
I
spread on PCA (3 replicates)
filtration of supernatant (20 ml, 0.22 pm pore size)
I
( 3 replicates)
/
100 pI on PCA
(3 replicates)
bacterial film dispersed in 15 ml of sterile Meuse water
I
10 fold dilution 100 pl bn PCA (3 replicates)
Figure 3. Analysis of the bacterial communities of the river Meuse. Isolation of the bacterial strains from the sediments (PCA = Plate Count Agar).
Results The dendrogram (Figure 4) is composed of all the strains (50) which are gathered by hierarchical level. The minimal distance between the strains is 0 which means full similarity between the strains, on the ground on the chosen tests. So, the dendrogram gives an evaluation of the similarity between the strains and each strain is characterized by its level of aggregation I 161. All the 50 strains were recovered in two main clusters. Cluster 1 which joined 70% of all the strains, is composed of 63 % of water column strains and 37 % of sediment strains. Cluster 2 contains the remaining 30 % with 1 strain from the water column and 14 strains from the sediments. The strains clustered in this group were different from the strains of cluster 1 by the ability to use carbohydrates such as saccharose, mannitol, sorbitol and rhamnose (Table 1). The cluster analysis shows that the aerobic bacterial community isolated from the sediment was constituted of two groups : one group presenting the same biochemical features as the
342
aerobic bacterial community isolated from the water column, and an other group composed of strains only present in the sediments.
Average d i s t a n c e between c u s t e r s 0.0 w U Y U
S
w
w
W
7
L
Q) 4-J
UY 3
-
0
S
w
11 5
Y W
w
w
x S
Y S
S S U Y S
w
S 9 S Y S S
c\l L
0
+ m
3 -
0
3 S S
1.4
20 21
2 8
3 4
5 11
la
21 23 6
12 1
14 19 3 13 19
ao
23 22
15 17 26 24 16
7 14
S S
25
S S
22 9
S
13
S
1.2
7
S S
S
1 .o
16 I
w
0.8
2
5
Y
0.6
18 6 15 8
5
W W
0.4
1 10 9 17
S
w
0.2
10
27 18
Figure 4. Dendrogram of the cluster analysis of 50 strains isolated from the water column (w) and the sediments (s). I t appears that a great proportion of the bacteria colonizing the sediment and the bacterial community of the water column share similar biochemical features. It could be due to the fact that a part of the bacteria of the water column settle down and colonize the interstitial water and the sediments.
343
'Table 1 Main characteritics of strains of clusters 1 and 2 (see Figure 4) CLUSTER 1 number of strains total from water from sediment..
35 22
13
CLUSTER 2 15 1
14
% of positive responses in each cluster
Gram + Gram-
34 66
20 80
oxidase catalase arginine dihydrolase indole ONPG~@)
63 86 29 0 14
20 80 53 20 100
degradation of gelatin
60
13
acetate malate citrate
0 0
0 0 60
glucose saccharose arabinose mannitol sorbitol melibiose rhamnose
43
100 67 93 87 80 80 87
utilisation of
?dl
51
0 6 0 0 6 0
ONPG, o-Nitrophenyl---galactopyranoside
344
Consequently the role of bacteria in the radionuclide transfers in aquatic ecosystem will be investigated by analyzing the aerobic bacterial community isolated from the sediments. It will be considered in a first step that the responses could be representative of the aerobic bacterial communities of the river.
KINETICS OF 6oC0 AND 134CS TRANSFERS MEDIATED BY BACTERIA. It was decided to study the radionuclide transfers in a bacterial community instead of in a monospecific bacterial culture in order to obtain more reliable results from an ecosystemic point of view. The inoculum of the bacterial community was isolated from sediments sampled in the river Meuse at Hastikre (Belgium). The sediment! were stored in sterile bottles at 4'C until the experiments. A new preculture was carried out before each experiment in order to obtain a bacterial community as close as possible to the natural community. It is well known indeed that several strains cannot develop in laboratory conditions so that subcultured bacterial communities lose a lot of strains with uncontroled changes of the specific composition.
a. Radianuclide immobilisation by the bacte rial community, Material and methods
Preculture and cultures conditions. Precultures are carried out by inoculating sterile Meuse water with sediments. The Meuse water is supplemented with starch (2 g.1-l) and bacto-peptone (10 g.1-l) in order to improve the bacterial growth. The preculture vessels are shaked and incubated at 20'C until the mid exponential phase of bacterial growth i.e. during 48 h. At this stage, the bacterial community of the sediment is harvested by centrifugation (10 min, 2,OOOxg). The pellet is discarded and the supernatant is again centrifugated 15 min at 15,200xg. The last pellet is used to inoculate the experimental cultures which are carried out in the same Meuse water medium as preculture. The initial O.D. of cultures are f0.06. The cultures are shaked 24h at 20'C. The bulk growth rate of the bacterial community rankes between 0.13 and 0.15 h'l which is of the same order of magnitude as mentioned in earlier observations [lo].
Radionuclide uptake by bacteria. The culture medium is contaminated with 6oCo and 134Cs (up to 2,000 Bq.1-l) before the inoculation. At regular time intervals, an aliquot of the culture is centrifugated
345
(3 min, 12,500xg). The dry weight of the bacterial biomass is determined and after ashing in nitric acid, the radioactivity level is detected by gamma spectrometry.
Results The uptake of 6oCo and 334Cs by the bacterial communities collected from the Meuse river was investigated in presence of increasing radiocontamination (up to 2000 Bq ml-l) in a Meuse water medium. When the data were expressed in the form of a double-reciprocal Lineweaver-Burk plot, two distinct phases were observed for 6oCo. One phase characterized the low contamination levels betwen 24 and 90 Bq.ml-' whereas another phase occured in the high contamination levels between 90 and 2,000 Bq.rn1-l (Figure 5). Each phase followed the Michaelis-Menten kinetics: 1/V = (l/Vmax) + (Km/Vmax). (l/[S]) where, V was the rate of uptake (Bq.g-I.hl), [S] was the activity of the medium (Bq.ml-'), Vmax was the maximal rate and K, was the Michaelis constant. Tdbk 2 (Bq.g-I.h-I) values obtained from Lineweaver-Burk plots of the Km (Bq,ml-l) and V uptake of 6oCo and ly%s.
134cs
Radiocontamination range of w ter (Bq.ml-B)
Vmax
24 - 90 90 - 2,000
2,500 18,692
22 - 295
1,286
Km 63.70 1,145.79 240
Vmax and Km values were calculated from the Lineweaver-Burk plot (Table 2). Km value was significantly greater in the high contamination levels. The two different phases of 6oCo uptake were therefore assumed to reflect high and low affinity uptake systems. The level of radiocontamination respectively explained 58% and 98% of the variation of the uptake rates. The 134Cs uptake kinetic only showed one phase (Figure 6). 73% of the variation of the contamination rate were explained by the level of radiocontamination of the water column. The Km values of Table 2 show that the highest affinity transport system is observed for %3in the low radiocontamination levels. During the radiocontamination,
346
33% and 24% of the variations of the concentration factor could be explained by the contact time between the biomass and the radionuclides for 6oCo (from 22 to 2,000 Bq 60Co.m1-1) and 134Cs (from 16 to 300 Bq 134Cs.ml-1) respectively. At the end of the uptake experiment (24 h; bacterial biomass: 500 mg dw.l-I), the activity remaining in the water column ranked between 25% and 40% of the initial activity in the case of 6oCo (initial activity: 0 to 2000 Bq.ml-I) and between 45 and 95% in the case of 134Cs (initial activity: 0 to 300 Bq.ml-I). For the same radiocontamination levels e.g.fl50 Bq.ml-l, the activity level of the bacterial community was +2.5 times higher for 6oCo (+50,000 Bq.g-' d.w.) than for 334Cs(+20 OOO Bq.g-l d.w.). Thus, an important part of &Co and 134Cs can be stored by the aerobic bacterial community which constitutes therefore a pool of radionuclides in the river ecosystem.
Figure 5. Lineweaver-Burk plot of 6oCo uptake by the bacterial community. [S] : radiocontamination eve s of the medium (Bq.mi-l) V : rate of uptake (Bq.g' .h- ) : maximal uptake rate (fjq.g-l.h-l) ::?affinity constant (Bq.ml- ) Equations: (1) low radiocon 'nation lev sy = 0.4 loy+ 23.8 lo9 x (r = 0.7595) (2) high radioconta ination leve : y = 53.5 61.3 10-?fx (r = 0.9924) (r : correlation coefficient)
\ I
347
l/CSI .lo-3
Figure 6. Lineweaver-Burk plot of 134Cs uptake by the bacterial community. IS] : radiocontamination eve s of the medium-based Meuse water (Bq.ml-l) V : rate of uptake (Bq.g' .h ) Equation: y = 0.8 + 186.7 x (r = 0.8570) (r : correlation coefficient)
I -\
b. Radionuclide release by the bacterial community.
Material and methods The bacterial community is contaminated with 6oCo and 134Cs during the exponential growth phase. Bacteria are harversted by centrifugation (3 min, 12,5OOxg), washed and suspended in a dialysis tube with sterile Meuse water (Spectrapor 5 ; thickness 0.09 mm; molecular sieve 14,000 D). The dialysis tubes are incubated in running sterile Meuse water at different temperatures (13'C, 20'C) and pH (6.5;7.0;7.5;8.0;8.5;9.0). At time intervals, the radioactivity levels detected either in the bacterial biomass or in the Meuse water outside the dialyse tubes.
Results The decontamination of the bacterial community loaded with 6oCo and 134Cs was investigated in relation to two environmental parameters, the temperature and the pH. The
348
activity levels of bacterial biomass at time zero ranked between 200 Bq.g-' d.w.and 900 MBq.g-l d.w. for 6oCo and between 1 and 50 P4Bq.g-l d.w. for 134Cs. When the temperature of the water column wdS maintained at 20°C, the kinetic of the decontamination of the bacterial community was described by a double negative exponential model. It was fitted using the procedure NLIN of SAS 1151 following the DUD method of Raltson and Jennrich [17]: y = m e-ax + n e-bx, where y : 6oCo or 134Cs levels in bacteria (Bq.g-I d.w.) x : time (h) m,n : ordinate values for each exponential (Bq.g-l .d.w.) a : slope of the line with equation In y = In m + ax b : slope of the line with equation In y = In n + bx a and b parameters depended on the desorption rate and were considered as an estimation of the biological half-lives (Tbl and Tb2) calculated as follow: Tbl = In 2 I a Tb2 = In 21 b Examples of adjusted curves were given for 6oCo (Figure 7) and 134Cs (Figure 8).
Figure 7. Desorption of 6oCo by the aerobic bacterial community. Observed values: square; Predicted values: line; y = 69 e-350.347x + 165 e-o.046x (r = 0.9121) y : 6oCo levels in bacteria (Bq.gl d.w.) x : time (h)
349
A
0,
1500
\
D
m
x
+.> .4
u
1000
500 0
0
200
100
300
400
500
Time (h)
Figure 8. Desorption of 134Cs by the aerobic bacterial community. Observed values: square; Predicted values: line; y = 916 e-7.82x 879 e-o.018x r = 0.9504) y : 134Cs levels in bacteria (Bq.g' d.w.) x : time (h).
+
I
For both radionuclides, the biological half-lives Tbl were found to be extremely short, of the order of a few seconds or minutes whereas the biological half-lives Tb2 were longer (1Sh to 461h for 6oCo and 39h to 8,976h for 134Cs). Except for a high value of Tb2 observed with 134Cs (374 d) and a very small value observed with 6oCo (0.6 d), both corresponding with the lowest radiocontamination level of the medium during the radiocontamination of the bacteria communities, the values of biological half-lives were of the same magnitude as observed for a cyanobacterium Scenedesmus sp . [ 18- 191. When the temperature of the water column was maintained at 13OC (average temperature of the Meuse river), the results showed that radionuclides fixed by bacteria were not released. At 20°C, the decontamination of the bacterial community was followed as the pH increased from 6.5 to 9. The chosen criteria was the increase of radioactivity in the water column which was a consequence of the decontamination of bacterial biomass. In any cases, the data were fitted by the following mathematical relation: y = m(l-e-ax) + n(1-ebx), where y : 6oCo or the 134Cs levels in the water column (Bq.ml-l) x : time (h) m,n : ordinate values (Bq.g-l .d.w.) a : slope of the line with equation In y = In m + ax
350
b : slope ofthe line with equation In y = In n + bx This mathematical adjustment showed that the evolution of radiocontamination level of the water column was adequately described by a biphasique process with a first phase characterized by a rapid desorption of radioactivity from the bacterial biomass into the water column and a second phase during which the radioactivity level of the water column increased slowly. The parameters a and b depended on the contamination rate of the water column and allow to compare the biological half-lives of the bacterial biomass, Tbl and Th2 respectively. They were calculated as follow: Tbl = I n 2 l a Tb2 = In 21 b. In the case of 60Co,the longest Tbl and Tb2 were found at pH 8, the average pH of the Meuse river (Figures 9 and 10). For the other pH values of the water column, Tbl and Tb2 were shorter which means a more rapid desorption of the 6oCo by the bacterial biomass and consequently an increasin radioactivity level in the water column. The same observations were made in the case of f34Cs. Given that the mean temperature in the Meuse river is 13'C and pH averages 8 , bacteria are likely to act as radionuclide sinks for much of the time but, when the temperature increases in summer they could act as sources.
6.5
7.0
7.5
8.0
t
8.5
a v e r a g e Meuse value
9.0 PH
Figure 9. Biological half-lives Tbl (h) of the 6oCo desorption of the bacterial community vs the water column pH.
35 I
Figure 10. Biological half-lives Tb2 (h) of the "Co desorption of the bacterial community vs the water column pH values.
CONCLUSlON AND SUMMARY. The radionuclide fluxes between the bacteria and the water in aquatic ecosystem were studied by examining the bulk transfers mediated by a bacterial community isolated From the river sediments. The comparison of the aerobic bacterial communities colonizing the sediments and the water column shows that the bacterial community of the sediments is composed of two sub-communities. The fist one is similar to the water column community by its biochemical feahires; the other one displays quite different characteristics and appears to be more representative of the sediments.
352
An important part of 6oCo and 134Cs can be immobilized by the bacterial biomass which therefore constitutes a pool of radionuclides, their transfers to the water column being controlled by tern erature and pH. The uptake of 68Co and 134Cs by bacteria can be described by the Michaelis Menten model. The uptake kinetics depend on the type of radionuclide and the level of radiocontarnination in the water column. The highest affinity uptake system is observed for 6oCo at low radiocontarnination levels. The decontamination of bacterial biomass develops in two phases. The first phase is characterized by a very short biological half-life, a few seconds or minutes while the second phase is longer, the biological half-lives reach between 15 h to 461 h for 6oCo and between 39 h to 8,976 h for 134Cs. It could be inferred from the study of the influence of temperature and pH that in the river Meuse where temperature averages 13’C and H is around 8, the rates of radionuclide releases are very low so that 6oCo and lY4Cs are effectively trapped by bacterial biomass. When the conditions change i.e. the temperature raises, the radionuclides immobilized by the bacterial biomass are released and radioactivity consequently increases in the water column. It appears thus that the environmental parameters play an important role in the radionuclide transfers mediated by bacteria and are to be more deeply investigated in the further.
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