Bacterioplanktonic biomass and production in the river Meuse (Belgium). Pierre Servais. Groupe de Mcrobiologie des Milieux Aquatiques, Universitk Libre de ...
Hydrobiologia 174: 99-l 10, 1989 0 1989 Kluwer Academic Publishers. Printed in Belgium
99
Bacterioplanktonic biomass and production in the river Meuse (Belgium) Pierre Servais Groupe de Mcrobiologie des Milieux Aquatiques, Universitk Libre de Bruxelles, CP221, Campus de la Plaine, Boulevard du Triomphe, 1050 Bruxelles, Belgium Received 18 June 1987; in revised form 21 October
1987; accepted 20 January 1988
Key words: river Meuse, bacterial biomass, thymidine
incorporation,
bacterial production,
growth yield
Abstract This paper presents results of bacterial biomass determination by epifluorescence microscopy after acridine orange staining and 3H-thymidine incorporation measurements in the river Meuse. Bacterial production is calculated from thymidine incorporation using an experimental conversion factor (0.5 lo’* bacterial cells produced per mole of thymidine incorporated into macromolecules). Seasonal variations of bacterial biomass and production at two stations are presented. Biomass ranges between 0.05 mgC * 1- ’ (in winter) and 0.8 mgC * l- * (in summer). The variations of bacterial production seem to be closely linked to those of primary production; values lower than 1 PgC * l- ’ * h- ’ are found in winter and high values (> 5 PgC. 1- l* h - ‘) in summer. Longitudinal profiles in the Belgian course of the river show important increase of biomass and production from upstream to downstream. Bacterial growth yield (Y) has been determined (Y = 0.3) in order to calculate bacterial carbon uptake from bacterial production.
Introduction The considerable methodological improvements gained in the field of aquatic bacteriology now allow accurate determinations of bacterial biomass and activity in natural aquatic ecosystems. Direct microscopical methods and particularly epilluorescence microscopy after fluorochrome staining (Hobbie et. al., 1977; Porter & Feig, 1980) have replaced the classical plate counting technique which undervalues bacterial biomass by several orders of magnitude. Several methods have been introduced for estimating the activity of planktonic bacteria; most of them are based on the utilization of labeled products as for example 35S-sulfate (Cuhel et. al., 1981, 1982), 3H-adenine (Karl, 1979, 1982) or 3H-thymidine ( Fuhrman and Azam, 1980, 1982).
In marine ecosystems, many studies, using several of the above mentioned methods, have been carried out, showing that bacterial biomass is a significant part of the total biomass (Hobbie et. al., 1977) and that bacterioplankton plays a quantitatively significant r61e in the carbon cycle (Pomeroy, 1974; Azam and Hodson, 1977; Hagstrom et. al., 1979; Williams, 1981). Some of these new bacteriological techniques, as thymidine incorporation, have also recently been applied in lakes (Riemann et. al., 1982; Bell et. al., 1983; Riemann and Sondergaard, 1984; Lovell and Konopka, 1985). However, only few applications in river ecosystems have already been published (Edwards and Meyer, 1986; Edwards, 1987; Palumbo et. al., 1987) in spite of the fact that bacterial heterotrophic activity causes serious problems of oxygen deficit in a lot
100 of important rivers and is therefore an important process to study and control. The purpose of this paper is to present measurements of bacterial biomass (determined by epifluorescence microscopy after acridine orange staining) and bacterial production (measured with the 3H-thymidine incorporation method) in a river ecosystem. Seasonal and longitudinal variations of biomass and activity in the Belgian part of the river Meuse are presented. Bacterial carbon uptake is compared to primary production measured at the same station; results are discussed in term of carbon fluxes.
NETHERLANDS ,-- -. , \ II
.. ‘.
\I II *\
BELGIUM
Biotopes and methods Study sites The river Meuse rises in the East of France and flows through Belgium and the Netherlands, where it meets the river Rhine, forming the Dutch Delta, which opens in the North Sea. The total length of the river is 885 km, and its catchment area is about 36 000 km*, 40 y0 of which being in the Belgian territory (Fig. 1). Two stations have been regularly studied in the Belgian course of the river: the first one is located 15 km downstream the French-Belgian border at Waulsort (W), the second 65 km downstream the first station, at Huy (H). At some occasions, longitudinal profiles have been performed all along the Belgian course of the Meuse with measurements in twelve different stations. The Belgian Meuse can be divided in three reaches : -the first one, between the French-Belgian border and Namur - in which station W is located - is called the High Meuse and characterized by a drainage basin with a low population density and land mainly devoted to forestry and agriculture ; -the second one, between Namur and Huy, is influenced by the polluted waters of a tributary, the river Sambre, which receives important industrial etIluents and sewage; - the third one, is highly polluted by the emuent of the important industrial area around Liege.
FRANCE
Fig. 1. Map of the Meuse drainage basin (the shaded area corresponds to the Belgian part of the basin) and the studied stations.
Results of physico-chemical analysis of river Meuse at Waulsort and Huy are presented by Servais (1986) and Descy et. al. (1987). Bacterial biomass was determined by epifluorescence microscopy after acridine orange staining (AODC) following the procedure of Hobbie et. al.
101 (1977). Enumeration of bacteria was performed with an epifluorescence Leitz microscopy equipped with a 100 W Hg lamp. The numbers of bacteria per ml. were estimated from enumeration on 10 fields; at least 100 cells were counted on each field. With this procedure the standard deviation of bacterial enumeration did not exceed ten percent. Bacterial sizes were estimated visually by comparison to a calibrated grid, and biovolumes were calculated by treating rods and cocci respectively as cylinders and spheres (Watson 1977). A conversion factor of et. al., 1.2 lo- l3 gC * prnp3 (Watson et. al., 1977) was used for calculating biomass from biovolume. This value is the median of those cited in the literature which are based on theoretical assumptions (range 0.87 lo- 13-1.65 lo- I3 gC * prnp3) (Ferguson and Rublee, 1976; Hagstrom et. al., 1979; Jordan and Likens, 1980; Pedros-Alio and Brock, 1982) but is rather low by comparison to some recent experimental values (range 1.06-5.8 lo- l3 gC*pmp3) (Bratbak and Dundas, 1984; Bratbak, 1985; Nagata, 1986; Lee and Fuhrman, 1987). was estimated from Bacterial production measurement of (methyl-3H) thymidine incorporation into cold TCA insoluble material, performed according to the procedure proposed by Fuhrman and Azam (1982) (Amersham-specific radioactivity 42 to 50 Curies * mmole-’ ). Incubations were performed at 20 nM 3H-thymidine, a concentration shown to saturate the bacterial incorporation process in the river Meuse. After about 1 hour incubation at in situ temperature, 5 % trichloroacetic acid (TCA) insoluble fraction was collected on a 0.2 pm membrane filter (Sartorius) and its radioactivity was measured by liquid scintillation. Usually, triplicates were performed and standard deviation did not exceed 5 percent. On basis of both theoretical and empirical arguments, Fuhrman and Azam (1982) suggested to calculate the bacterial cells production using a conversion factor of 1.7 1018 to 2.4 1018 bacteria formed per mole of thymidine incorporated in the TCA insoluble material in coastal sea water. There has been much controversy in the literature
regarding the validity of this factor (Bell et. al., 1983; Moriarty, 1984; Pollard and Moriarty, 1984). Conversion of tritiated thymidine incorporated measurement into bacterial cells production poses in fact different problems: isotopic dilution by intracellular thymidine (Rosenbaum-Oliver and Zamenhof, 1972; Moriarty and Pollard, 1981, 1982; Fuhrman and Azam, 1982), proportion of tritiated thymidine incorporated into other TCA insoluble macromolecules beside DNA (Karl, 1982; Witzel and Graf, 1984; Servais et. al., 1987), variation in natural bacteria DNA content and in the proportion of thymidine acid residues (Mandelstam and Mac Quillen, 1973). To avoid the difficult theoretical calculation, some authors proposed to determine an experimental conversion factor. In order to determine this conversion factor for the bacteria of the river Meuse, the following experiment was carried out in some occasions. River water sample was sterilized by filtration on a 0.2 pm pore size membrane and inoculated with a small amount of 2 pm pore size membrane filtered water and incubated at 20 “C. A rapid bacterial growth was observed; bacterial numbers and thymidine incorporated were followed every 3 hours during 24 h. Increase in cells number was plotted against integrated thymidine incorporated (Fig. 2). From experimental results, a conversion factor of 0.5 1018 bacteria produced per mole of thymidine incorporated in the TCA insoluble material is deduced. This value is at the lower side of the range of experimental conversion factor using different concentration of 3H-thymidine found for various ecosystems mentioned in the literature (0.5-290 1018) (Kirchman et. al., 1982; Bell et. al., 1983; Ducklow and Hill, 1985; Scavia et. al., 1986; Billen and Fontigny, 1987; Riemann et. al., 1987). This low conversion factor found in the river Meuse can be explained by the weak percentage of 3H-thymidine incorporated into DNA, 30 to 38% of the incorporation in the cold TCA insoluble material (Servais et. al., 1987) and by low isotopic dilution due to the high 3H-thymidine concentration (20 nM) used in this work. To convert bacterial cells production into biomass production, we used the value of mean
Bacteria
formed
lO?l-’
Fig. 2. Relationship between the number of bacteria produced during incubations of reinoculated 0.2 pm filtered Meuse river water and cumulated thymidine incorporation into cold TCA insoluble material. The straight line shows the correlation which corresponds to a conversion factor of 0.5 1Or8 bacteria produced per mole of thymidine incorporated in the cold TCA insoluble material.
biovolume determined by microscopy and the carbon/biovolume factor above-mentioned. Growth yield defined as the ratio between bacterial biomass produced and organic matter utilized was determined by comparing direct measurement of biomass formed and D.O.C. (dissolved organic carbon) utilized during shortterm (lo-20 h) incubation of 0.2 pm filtered and reinoculated river Meuse water samples. D.O.C.
was measured with a Dohrman 80 Total Carbon Analyzer using U.V. promoted persulfate oxidation of organic carbon and determination of the produced CO, by i.r. spectrometry. Phytoplanktonic primary production was meastechnique ured by the 14C0, incorporation according to the procedure of Lancelot (1982); daily primary production was calculated using Vollenweider’s (1965) model.
103
Ol JFMAMJJA
Fig. 3. Seasonal variations - bacterial abondance, - mean biovolume, - bacterial biomass.
in 1984 at station W (0) and H (0) of
SON
D
4
104 Results Bacterial biomass
During the year 1984, enumerations of bacteria were carried out monthly at the stations W and H. Figure 3a shows the seasonal variations of the bacterial abundance at the two stations. The variations present minimum values (around lo9 bacteria per liter) during winter, and maxima in the range 3 to 5 lo9 bact * l- ’ during the period June-September. No significant difference in abundance is observed between the two stations. Fig. 3b presents the seasonal variations of the mean bacterial biovolumes. At both stations, during winter, bacteria are primarily small and spherical (diameter in the range 0.5 to 1 pm); during spring, besides cocci, rods appear (diameter: 0.5 pm-length: 1.5 pm); in summer, rods are predominant in number and their size reaches 1.5 pm in diameter and 2.5 ,um length. During September, little cocci become again predominant. From results of bacterial enumeration and mean bacterial biovolume, seasonal variations of biomass have been calculated (Fig. 3~). Biomass presents minimum values in winter (around
0.05 mgC * l- ‘) and maximum values (about 0.8 mgC . l- ‘) in the period of July-August. The high summer values are due both to increase in cells number and in size. Biomass determinations have also been carried out on samples from longitudinal profiles along the Belgian course of the river. Figure 4 shows a typical example of biovolume and biomass profile measured in May 1985. Upstream Namur, the mean biovolume is close to 0.3 pm3 and the biomass to 0.07 mgC * 1- ‘. Between Namur and Liege, bacteria are slightly bigger (biovolume around 0.5 ,um3) and their biomass is higher (around 0.15 mgC +l- ‘). Downstream Liege, bacterial biovolumes quickly increase to reach maximal values (1.12 pm3) some kilometers upstream the Belgian-Dutch border. As the bacterial abundance is relatively constant in this stream, biomass profile follows the biovolume profile.
Bacterial production
Bacterial production has been measured by the (methyl-3H) thymidine incorporation method during 1983 to 1984 at station W. (Fig. 5). The seasonal variations show low values during winter
loo Fig.
4. Longitudinal
Km
variations of the mean bacterial volume (0) and bacterial biomass (0) along the Belgian course of the Meuse river (May 30, 1985) (kilometer zero at the French Belgian border).
J 1983 Fig. 5. Seasonal variations
A’S’0
of bacterial production
N
D
;
F M A’M
(0-0) and phytoplanktonic
(bacterial production < 1 PgC * l- ’ * h- ‘) and high values during summer (bacterial production > 5 PgC * 1- ’ * h - ’ ). The seasonal variations of bacterial production seem to be closely linked to those of primary production. For the two years, the beginning of the high bacterial period follows the increase of primary production. Figure 6 shows the seasonal variation of bacterial production at station H between November 1983 and October 1984; it presents the same kind of seasonal profile but the spring and summer values are higher than at station W. From figures 5 and 6, the annual bacterial production was calculated by integration ; values of 20 mgC * 1- ’ * y - ’ at station W. and 34 mgC * l- ’ *y- ’ at station H were obtained. Longitudinal profiles of bacterial production measurement have also been carried out in the Belgian course of the river Meuse. Figure 7 shows a typical profile (30-5-85) with values in the range 5 to 10 PgC * l- ’ *h- ’ upstream Liege; down-
J J 1984
A
primary production
S
0 (---)
N C at station W.
stream, the production quickly increases to reach a maximum value (26pgC*l-‘*h-l) some kilometers upstream the Belgian-Dutch border. This profile looks like the biomass profile (Fig. 4) measured at the same date.
Discussion Bacterial abundance measured in the Meuse river are in the range 1 to 5 lo9 bacteria per liter. Most of these bacteria are free-living, bacteria attached to particules are in all cases below five percent of the total. To our knowledge, there are no published results of bacterial enumeration by epifluorescence microscopy performed in rivers water to compare with the river Meuse values. Nevertheless, we performed enumerations in some Belgian and French rivers; the range of values is quite the same 0.6 lo9 to 5 lo9 bacteria per liter (Table 1) in the river Oise and Sambre but
106
1983
1984
Fig. 6. Seasonal variations ofbacterial phytoplanktonic primary production
production (e-0) and (---) at station H.
are significantly higher in the highly polluted rivers Schelde and Rupel. Bacterial biovolume estimated by scanning electron microscopy or the A.O.D.C. method performed in sea, lake or estuary (Gocke, 1975,1977; Ferguson and Rublee, 1976; Hagstrom et. al., 1979; Holhbaugh et. al., 1980; Krambeck et. al., 1981; Pedros-Alio and Brock, 1982; Bell et. al., 1983; Palumbo et. al., 1984; Simon, 1985) generally ranges between 0.01 and 0.3 pm3 per bacteria. The bacterial biovolumes observed in the Meuse river are slightly higher, in the range 0.3 to
1.8 pm3/ bacteria. Some authors have mentioned bacterial biovolumes in the same range; for example, Lewis et. al. (1986) found a mean cell biovolume of 0.66pm3 in an eutrophic lake in Venezuela. In a general way, it is thought that larger cells are simply in better nutritional conditions and so found in rich medium (Pedros-Alio and Brock, 1983). In the Meuse for instance, bacteria are larger during summer when the availability of substrates is higher; and biovolumes increase along the Belgian course of the river with the increase of organic matter sewage. In this ecosystem, the variations of biovolumes play a greater role in the temporal and spatial fluctuations of bacterial biomass than the variations of bacterial numbers themselves. The recent suggestion by some authors (Lee and Fuhrman, 1987) that the biomass/biovolume conversion factor is higher than previously thought, and variable in function of the cell volume, throw some uncertainty on our biomass estimations. From the values of bacterial production and bacterial biomass, growth rate @) can be calculated by dividing the former by the latter; the values of p for the Meuse river range between 0.007 and 0.1 h- i, corresponding to generation times between 7 and 100 h. These values are not affected by the uncertainty on the value of the carbon/biovolume conversion factor as this factor affects equally both biomass and production estimates. In order to compare bacterial uptake of organic matter to primary production, it is necessary to know the bacterial growth yield (Y) to calculate bacterial carbon uptake from bacterial produc-
Table 1. Bacterial enumeration by epifluorescence microafter a&dine orange staining in some important Belgian and French rivers. scopy
Rivers
Bacteria per liter
Meuse Sambre Schelde Ruper Oise
0.6- 5 lo9 3 -5 109 2 -19 lo9 5 -12 lo9 0.6- 1.3109
107
30.
%- 20. ‘s 7 * 3 2 10.
0* 0 Fig. 7. Longitudinal
50
profile of bacterial production
Km
along the Belgian course of the river Meuse (May 30 1985).
tion. From the comparison of bacterial biomass formed and carbon utilization in batch experiments, a value of Y = 0.3 was deduced (Fig. 8). This value, which is of course dependent on the carbon/biovolume conversion factor used, is consistent with the other values determined by comparison of bacterial cells production (from micro-
DOC
100
scopic determination) and carbon consumption in other environments (Lucas et. al., 198 1; Newell et. al., 1981; Linley et. al., 1983; Lancelot and Billen, 1984, 1985; Bjornsen, 1986), which are in the range 0.1 to 0.4, the lowest values being generally observed in nitrogen limited environments.
utilized
mg C.I-’
Fig. 8. Relationship between the amount of bacterial biomass formed and dissolved organic carbon (DOC) utilization during bacterial growth in short-time incubation of sterilized and reinoculated Meuse river water. Determination of the growth yield.
108 Table 2. Annual values of primary production carbon uptake in the river Meuse. Primary production gc.m-Z.y-’
STATION W. 1983 492 1984 547 STATION H. Nov. 1983416 Oct. 1984
Bacterial
and bacterial
carbon
uptake
gC.m-2.y~’
238 231 393
With this value of the growth yield, bacterial carbon uptake in the Meuse river can be calculated as the ratio between bacterial production and growth yield and compared to primary production (Table 2). It is very important to mention that the data ofbacterial carbon uptake calculated by this way are not affected by the uncertainty on the carbon/biovolume conversion factor used, because this factor has the same influence on bacterial production and growth yield. At station W, the annual bacterial carbon uptake represents 48% in 1983 and 43 % in 1984 of the annual primary production. At this station, where sewage and other external organic matter inputs are negligible, about half of the carbon fixed by primary production is thus used by planktonic bacteria. At station H, the carbon consumption which includes consumption of domestic and industrial sewage inputs represents 94 yOof the primary production. The results presented in this paper show the importance of planktonic bacteria in the carbon cycle in a river ecosystem; further investigations in the dynamic of this population are required to understand the working of the first levels of the food web in lotic environments.
Acknowledgment This work was partly supported by the Ministhe de la Rbgion Wallonne pour 1’Eau. I’Environnement et la Vie Rurale. The author is very grateful to Dr. G. Billen for aid and discussion.
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