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Seasonal changes of microbial populations in the sediments of the basins of Lugano and Agno
PEDUZZI, Raffael, DEMARTA, Antonella, TONOLLA, Mauro Amedeo
Abstract
This research was carried out with the aim to explore the heterotrophic microbial population of two sediments in different oxic conditions of the Lake of Lugano (Lago di Lugano). The values of the viable bacterial counts found in our sediment samples were typical for an eutrophic lake. The increase in the proportion of anaerobic to aerobic bacteria at 30 °C observed in the sediment samples of Agno may reflect the depletion of oxygen concentrations in the water column. The genera Aeromonas, Pseudomonas, Bacillus and Clostridium, as well as strains belonging to the Coryneform-group, represented the major taxonomic groups of heterotrophic bacterial communities in the water-sediment interface.
PEDUZZI, Raffael, DEMARTA, Antonella, TONOLLA, Mauro Amedeo. Seasonal changes of microbial populations in the sediments of the basins of Lugano and Agno. Aquatic Sciences , 1992, vol. 54, no. 3-4, p. 331-337
DOI : 10.1007/BF00878145
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Aquatic Sciences 54, 3/4, 1992 1015-1621/92/040331-07 $1.50 + 0.20/0 O 1992 Birkh/iuser Verlag, Basel
Seasonal changes of microbial populations in the sediments of the basins of Lugano and Agno
Raffaele Peduzzi, Antonella Demarta and Mauro Tonolla
Istituto Cantonale Batteriosierologico and Laboratoire d'Ecologie Microbienne de l'Universit~ de Gen~ve, via Ospedale 6, 6904 Lugano, Switzerland
Key words: Heterotrophic bacteria, sediments, taxonomy, seasonal variations, aerobic bacterial population, anaerobic bacterial population, Lake Lugano (Lago di Lugano).
ABSTRACT
This research was carried out with the aim to explore the heterotrophic microbial population of two sediments in different oxic conditions of the Lake of Lugano (Lago di Lugano). The values of the viable bacterial counts found in our sediment samples were typical for an eutrophic lake.
The increase in the proportion of anaerobic to aerobic bacteria at 30 °C observed in the sediment samples of Agno may reflect the depletion of oxygen concentrations in the water column.
The genera Aeromonas, Pseudornonas, Bacillus and Clostridium, as well as strains belonging to the Coryneform-group, represented the major taxonomic groups of heterotrophic bacterial communities in the water-sediment interface.
Introduction
In freshwater lakes, sediments are known to be the zone with the highest degree of biodegradations. These activities allow for the recycling of nutrients, and are performed by specific bacterial or fungal species (Brock, 1979).
In general, studies on the microbiology of sediments focused mainly on the products of microbial activities. However, this kind of research provides only little information about the taxonomic bacterial groups which might be involved in these processes of biodegradation.
Our research was carried out between May 1989 and March 1990 with the aim to explore the heterotrophic microbial population in the sediments of the Lake of Lugano.
Two areas were sampled seasonally: the bay of Lugano, where sediments are permanently anoxygenic, and the bay of Agno, where they are periodically oxygenated. We analysed our samples both quantitatively, counting bacteria and fungi able to develop in culture media, and qualitatively, by taxonomic determina- tions of bacterial and fungal isolates.
332 Peduzzi et al.
Materials and methods
From May 1989 to March 1990, two sediment cores were taken seasonally at depths ranging from 182 to 224 and from 72 to 79 meters in the bays of Lugano and Agno respectively (Fig. 1, in Barbieri and Polli, this issue), with the aid of the submarine
"F.A.-Forel (PX-28)" of Dr. J. Piccard, Cully, Switzerland.
Every sediment core was processed (two for each basin).
Qualitative and quantitative microbial analyses were performed on both water and mud. The water sub-samples consisted in 30 ml of water removed by a sterile produce at about 15 cm over the sediment-water interface, whereas the mud sub- samples were obtained by taking the first 3 cm of the sediment surface.
The following series of dilutions were prepared for each sample: 1/10, 1/100, 1/1000 for water sub-samples, and 1/10, 1/100, 1/1000, 1/10000, 1/100000 for mud sub-samples. The heterotrophic bacteria were cultured using a pour plate method, pipetting 1 ml of each dilution into two replicate Petri dishes (APHA., 1985). The PCA (Plate Count Agar, Difco) medium was added at 44 °C and mixed carefully.
Bacterial and fungal species were determined after culture on Blood Agar medium (Columbia Agar Base, Oxoid, 5% sheep's erythrocytes) and Sabouraud Agar (Biolife), respectively. These media were inoculated with 100 gl of each dilution using a sterile spreader. Bacterial cultures were incubated either aerobically at 30 °C, 20 °C and 4 °C for 2, 3 and 28 days, respectively, or anaerobically for 2 days at 30 °C.
After obtaining pure cultures, the Gram staining and the systematic determi- nations were carried out following classical schemes of bacteriology. Biochemical reactions needed for the taxonomic classification of bacteria were obtained using the API systems (BioM6rieux-Suisse; API 20A, API 20E, API 20NE, API 50, API STAPH, API 20 STREP, API ID 32 E, API ID 32 GN), as well as specially prepared media according to the Bergey's Manual of Systematic Bacteriology (1984).
Fungi were determined after 1 week growth at room temperature to the genus level both by morphological characterisation (Von Arx, 1981) and by biochemical reactions, using the Mycotube (Roche) and API ID32C (BioM6rieux-Suisse) systems.
Results
Viable counts of aerobic and anaerobic heterotrophic bacteria are summarized in Tab. 1. Quantitative values, expressed as colony forming units per millilitre of water or per gram of wet mud (CFU/ml and CFU/g, respectively) changed with incubation conditions. Counts obtained at 20 °C and 4 °C were higher than those at 30 °C in 66 % and 46 % of our samples, respectively.
Incubation temperatures influenced quantitative analyses but didn't changed the results of taxonomic analyses.
The ratio between the anaerobic and aerobic bacterial populations increased during the year in the bay of Agno. This trend was especially marked in the mud (Fig. 1).
Seasonal changes of microbial populations in sediments 333 Table 1. Viable counts of aerobic and anaerobic heterotrophic bacteria at different incubation temperatures, isolated from the water overlaying the sediments and the mud during 4 seasonal campaigns. The values represent colony forming units (CFU) per millilitre of water or per gram of wet mud
Lugano Agno
Water Mud Water Mud
[CFU/ml] [CFU/g] [CFU/ml] [CFU/g]
May-June Aerobic cultures
30 °C 20 °C 4°C
Anaerobic cultures
30 °C 250 _+ 38
September Aerobic cultures
30 °C 20 °C 4°C
Anaerobic cultures
30 °C 83 _+ 12
December Aerobic cultures
30°C 19-+4
20 °C 30 -+ 5
4°C 21-+3
Anaerobic cultures
30 °C 4-+ 1
February-March Aerobic cultures
30 °C 330 +_ 72
20 °C 520-+. 83
4 °C 652 -+ 91
Anaerobic cultures
30 °C 67-+ 10
7860_+1730 1.7 x I011-+0.27 x 1011 7847+_1726 8.5 x 107-+ 1.40 x 107 900___144 4.9 x 10~°___0.54 x 101° 5130-+820 1.1 x 108___0.12 x 10 a 1845___258 2.3 x 101°-+0.30 x 101° 2470+_346 3.0 x 107-+0.40 x 107
3.6 x 109-+0.32 x 109 530_+80
650___ 140 8.1 x 105+_1.30 x 105 1438_+316 872_+ 140 1.3 x 106_+0.14 x 106 2220_+355 976_+137 1.0 x 106_0.13 x 106 848___119
6.9 x 104-+0.62 x 104 25_+4
5.8 x 105-+0.92 x 105 700-+154 1.7 x 106-+0.19 x 106 144___23 1.6 x 106-+0.21 xl06 117_+16 1.4x I05_+0.13 x 105 28-1-4
1.1 x 106-}-0.18 x 106 17_+4 5.3 × 106-+0.53 x 106 43_+7 1.6x 106_+0.21 x 106 54+8 2.8 x I05_0.25 x 105 7-1-I
2.7 x 106-+0.24 x 106
3.7 x 106 _+0.59 x 106 3.3 x 106_+0.36 x 106 7.4x 106-+0.96 x 106 8.7 x 105 _+0.78 x 105
8.5 x 104_+1.40 x 104 3.1 x 10s_+0.34 x l0 s 2.0 x 105 _+0.26 x 105 3.2 x 104_+0.29 x 104
2.9 x 106___0.46 x 106 2.7 x ][06_+0.30 x 106 2.9 x 106_+0.38 x 106 2.1 x 106_+0.19 x 106
The n u m b e r of different bacterial genera determined in the basin of Lugano was 27 in sediments (6 anaerobic genera) and 25 in water (4 anaerobic genera). In the basin of Agno we found 28 genera in sediments (6 anaerobic) and 22 in water (3 anaerobic). The 9 predominant bacterial genera isolated from mud and water samples during the 4 seasonal campaigns are summarized in Tab. 2.
Strains belonging to the genera Aeromonas, Pseudomonas and Bacillus were
present in all samples of water and m u d analysed. Acinetobacter, Flavobacterium and
Clostridiurn were found in all samples of mud, as well as most of the water samples.
The presence of Corynebacterium, Micrococcus and Pasteurella in water and m u d was
334 Peduzzi et al.
I
[] WATER LUGANO [] MUD LUGANO ANAEROBIC/AEROBIC [] WATER AGNO COLONY COUNTS • MUD AGNO1.0
0.8 0.6
0.4 0.2
0.0 May-June September December February-March
Figure 1. Seasonal variations of the proportion of anaerobic/aerobic colony forming units in the water overlaying the sediments and in mud in the basins of Lugano and Agno
Table 2. Presence (P) and absence (A) of the principal bacterial genera isolated from the water overlaying the sediments and from the mud in the basins of Lugano and Agno during the 4 sampling campaigns
Basin Bacterial May-June September Decemper February-March genera
Water Mud Water Mud Water Mud Water Mud Lugano
Agno
Aeromonas P P P P P P P P
Acinetobacter P P P P P P P P
Corynebacterium P P P A A P P P
Flavobacterium P P P P A P P P
Pasteurella P P P P P P P P
Pseudomonas P P P P P P P P
Clostridium P P P P P P P P
Bacillus P P P P P P P P
Micrococcus A P A P A A P P
Aeromonas P P P P P P P P
Acinetobacter P P P P A P P P
Corynebacterium P P A A A P A P
Flavobacterium P P P P P P P P
Pasteurella A P P P P P P A
Pseudomonas P P P P P P P P
Clostridium A P A P P P A P
Bacillus P P P P P P P P
Micrococcus A A A A A A A P
Seasonal changes of microbial populations in sediments 335
[] Pseudomonas [] Aeromonas [] Acinetobacter [] Flavobacterium [] Gram + [] Other genera
[%] 80 WATER-LUGANO
6O
4O
20
0 May-June September December February-March
[%] 80 MUD-LUGANO
60
4O
2O
0 May-June September December February-March
[ ] 80 WATER-AGNO
60
4O
20
0
[Yo] 8o i~ h
60
40
20
0 May-June September December February-March Figure 2. Seasonal variations in percent of the predominant aerobic microflora in mud and in water overlaying the sediments in the basins of Lugano and Agno
336 Peduzzi et al.
less frequent. The quantitative relations among some of these taxonomic groups are depicted in Fig. 2.
Pseudomonas was the predominant genus isolated in both basins during the 4 seasonal campaigns, particularly in the water samples, where it represented more than 50 % of the microbial population determined in the Agno bay.
The muds taken in Lugano were characterised by a marked presence of Gram positive isolates.
Bacteria of the genus Aeromonas represented about lO 20 % of the heterotrophic population considered. The relative number of these microorganisms followed a seasonal trend, reaching the maximum level during the summer months.
Fungi were always isolated in samples taken in the bay of Agno, and were absent only in the spring samples of Lugano. Their isolation from water was connected to their presence in the sediments.
Taxonomic analyses led to the determination of the following groups: Absidia, Acremonium, Candida, Fusarium, Geotrichum, Gongronella, Mucor, Paecilomyces, Penicillium, Rhodotorula, Saccharomyces, Torulopsis, Trichoderma, Trichosporon,
Verticillum, Zygorhynchus.
Discussion
Heterotrophic bacteria play an important role in the remineralization of organic materials in aquatic ecosystems (Horsley, 1979; Nedwell, 1982; Choi, 1987).
The analysis of mud samples resulted in viable counts ranging from 8.5 x 104 _+ 1.40 x 104 to 1.7 x 1011 _+ 0.27 x 1011 CFU/g of aerobic, and from 3.2 x 104 _+ 0.29 x l04 to 3.6 x 109_+ 0.32 x 109 CFU/g of anaerobic bacteria.
These high values are typical for eutrophic lakes.
The activity of bacteria in situ remains to be demonstrated because bacterial cells may be physiologically active or in a dormant state (Collins, 1977). The temperature doesn't seem the major factor affecting the physiological activity of the found cells, because the majority of our strains were able to grow at both incubation temperatures (30 °C and 4 °C), although the environmental temperature is of about 4 °C.
The important viable counts we could detect in the spring samples may be explained, according to Jonas (1990), by an increased bacterial growth due to the sedimentation of planktonic biomass which reached the highest level during the period from March to May (LSA, 1990).
The proportion of the anaerobic to the aerobic bacteria at 30 °C rised during the year in the samples of Agno, especially in muds, where the increase of the ratio observed during the 4 campains was statistically significant (ltl = 6.75; p ____ 0.005 between the first and the last campain). The seasonal variations observed could be due to a progressive reduction of oxygen concentration in the water column, particularly marked in this basin (LSA, 1990).
The substantial presence of Gram positive strains, especially in the mud samples, is in agreement with similar findings by other authors (Yoshimizu, 1983). This Gram positive sub-population was mainly composed of the spore forming bacteria Bacillus and Clostridium, as well as isolates belonging to the Coryneform-group.
Seasonal changes of microbial populations in sediments 337 ACKNOWLEDGEMENTS
The authors would like to thank the °'Banca Unione di Credito" of Lugano, and especially Mr.
Keller, as well as Dr. Piccard and his team.
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Received 14 February 1992;
Revised manuscript accepted 16 July 1992.
