ECOLOGICAL ADAPTATIONS OF HETEROTROPHIC AND OLIGOTROPHIC

BACTERIA IN ISKAR RESERVOIR

A. Panova-Karadzhova, R. Dimkov
Sofia University “St. Kliment Ohridski”, Faculty of Biology, Department of General and Applied Hydrobiology, 8 “Dragan
Tzankov” Str., 1164 Sofia, Bulgaria
Correspondence to: Antoaneta Panova-Karadzhova
E-mail: antoaneta.panova@gmail.com

ABSTRACT
A simple cultivation method was used to describe bacterial communities according to the concept of r- and K-strategists.
Bacterial colonies appearing on agar plates were daily quantified for a period of six days. Significant differences were found
in the distribution in relation of water depth layers and of organic content. In general microbial community on the surface was
dominated by r-strategists and following depth gradient was replaced by a community dominated by K-strategists (in the
hypolimnion). Later-on in the bottom layer, bacterial community was dominated again by r-strategists. Eco-physiological
index showed that with depth distribution of r- and K-strategists became more even while in the epilimnion both groups have
uneven distribution.

Keywords: K- and r-strategists, eco-physiological index,
bacterial distribution
Introduction
Some habitats are characterized with low population density
and are dominated by organisms, which are adapted to high
growth levels and rapid colonization. Other habitats possess
high population density and are dominated to survive in these
high competitive conditions. Fundamental difference of these
two types of adaptation is described by MacArthur и Wilson,
1967 (6) in connection to different phases of colonization of
ocean islands. They separate the organisms in r-strategists,
which are the first colonizers of the environment and K-
strategists – adapted to more crowded conditions. Adaptation
of r-strategists to crowded habitats makes them suitable for
non-constant environments, where growth is limited to short
time period and high population levels can not be reached. In
such conditions the ability of these organisms to grow fast
and to explore the possibilities when they appear gives them
competitive advantage. According to these r-strategists are
usually small, with short live cycles and high growth rate.
Other characteristic of r-strategists is their sensibility to
toxicants in the environment and their metabolism is not
adjusted to use recalcitrant substances. On the other side K-
strategists are adapted to stabile conditions, where dense
populations could develop and where fast growth is not an
advantage (2). They possess more effective cell metabolism
and are able to use recalcitrant substances as cellulose and
lignin. They are also more resistant to toxicants (1). Between
freshwater microorganisms there are species from both types.
There are different situations in aquatic habitats (for example
colonization and different phases of community maturation)
when r- and K-strategists are more adapted to the
corresponding conditions. In their research of microbial
diversity in soils De Leij et al. 1993 (4) use simple cultivation
method to describe bacterial communities according to
ecological concept of r- and K-strategists.
Materials and methods
Microbial community structure
Filtered water samples (5ml and 10ml) were placed over
nutrient agar (NA, Scharlau, Brit. Phar. for heterotrophs and
diluted 1:10 NA, Scharlau, Brit. Phar. for oligotrophs) and
incubated for 6 days (144 hours) at 22oC. New colonies were
counted every 24 hours. Six different classes were
differentiated – colonies visible after 24, 48, 72, 96, 120 and
144 hours. Colonies were counted when colonies were
between 5 and 200 in Petri dishes. The number of bacteria in
each class was expressed as a proportion (%) from the total
colony counts. Different distributions present the ratio
between r- and K-strategists in each sample, since

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characteristics of r-strategists include fast growth in response
to environment, in contrast with K-strategists, which can be
characterized by slow growth in response to enrichment. Fast
growers were defined as bacteria that produce visible
colonies at 22oC on NA within 24 and 48 hours. The different
distribution curves were analyzed using multinomial
analyses. To compare the influence of concentration of
carbon source were also studied structure of oligotrophs
community. Here fast growers were defined as bacteria
forming visible colonies after 48 hours because
microorganisms isolated from Iskar reservoir form small,
non-visible colonies on the 24th hour. Water samples were
first filtrated through 0,45µm pore size membrane filters. The
different distribution curves were analyzed using multinomial
analyses (Statistika 8).
Eco-Physiological Index
To express the distribution of microorganisms from different
classes (days of incubation) in a single value, the Shannon-
Weaver diversity index (8) was calculated.
H’ = - Σ (pi * log10 pi),
where pi – representing each of the 7 classes as a
proportion of the total population in that sample;
pi=population in each class i/total population. The more even
the distribution of the classes the higher the Shannon-Weaver
index. Since the Shannon-Weaver diversity index is usually
used as a measure of the number of species and the
abundance of each species of population the name diversity
index is strictly speaking not appropriate for the
quantification of classes of organisms with similar
development characteristics on agar. Therefore, the name
eco-physiological index (EP-index) is used. EP-index values
were compared using analyses of variance.
Results and Discussion
Bacterial communities structure of heterotrophic bacteria was
different for diverse depth layers in Iskar reservoir (Р<0.001).
Fast growing bacteria which formed visible colonies in 24
hours of incubation were not more than 47.5% (Fig.1),
highest numbers grew from samples from the surface layer (0
m) – 28,9% and from the bottom layer. These water layers
were most influenced by the weather conditions on the
surface and at the bottom by the substratum. In that layers
developed mostly fast growing bacteria (r-strategists).

80 a) b) c) d) e)
60
40
20
% CFU.day-1

0
0m 5m 10 m 15 m 20 m
0 1 2 3 4 5 6 70 1 2 3 4 5 6 70 1 2 3 4 5 6 7 0 1 2 3 4 5 6 70 1 2 3 4 5 6 7
80 h) i) incubation time, days
f) g)
60 heterotrophs
40 oligotrophs

20
0
25 m 30 m 40 m 50 m
0 1 2 3 4 5 6 70 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
incubation time, days
Fig.1. Comparison in the microbial population structure of heterotrophs and oligotrophs in relation to carbon source concentration. Data are from CFU on
nutrient agar and on diluted 1:10 nutrient agar for 144 hours (n = 3÷5).

Microorganisms which formed visible colonies on the 48th 80%. Considerable percentage of fast growing bacteria in the
hour of incubation were most dominant in the samples from surface layers again confirmed the predominance of fast
10th, 15th, 10th and 15th meter depth layers – they reached growing microorganisms or r-strategists in these most divers

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conditions, where their ability to grow fast and to exploit the
environment gave them an advantage. In the hypolimnion most
of the bacteria grew after 72th hour, in the 20th and 30th meter
depth just 30% of bacteria grew in the third day of incubation.
Structure of oligotrophs community was analyzed for
comparison of the influence of carbon source concentration.
Bacterial community from different water layers differed
from one to other (Р<0.001). Fast growers which formed
visible colonies within 24 hours of incubation were
significantly fewer than these incubated with higher
concentration of carbon sources and were under 20%. At 48th
hour on incubation (second day) appeared much more
colonies especially from the water samples from 5th, 20th and
25th meter (45%, 35% and respectively 60%). These water
EP-index
EP-index,
Depth, m
n=3
0 0.449
5 0.470
10 0.598
15 0.659
20 0.710
25 0.526
30 0.680
40 0.595
50 0.619
Some genetic differences between the organisms exist
according to the concept of K- and r-strategies in relation to
their ability to exploit and survive in diverse habitats (5; 7).
That means that the environmental conditions select fast
growing organisms or r-strategists in uncrowded, nutrient
rich environments and select other organisms adapted to
exploit resources in crowded conditions – K-strategists. In
general r-strategists are adapted to stable conditions and K-
strategists to unstable (7). From this point of view pelagial
could be considered as an environment where different
conditions could be found, depending on depth, and where
different water layers could be characterized with diverse
conditions, influenced by various factors. Surface water layer
was most influenced by weather conditions and atmosphere
activities and epilimnion during stratification could be
characterized as being most unsteady in relation to different
factors as sun light, temperature, wind, etc. Bottom layer was
also comparatively dynamic environment, influenced by
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layers could be characterized as non-constant environment –
the water from surface to the 5th meter was still influenced by
weather conditions and 20th and 25th were the water
extraction layers in that area of the reservoir where constantly
huge amounts of water are taken for drinking water purposes.
On the third day of incubation most colonies appeared in the
plates from surface layer and from bottom layer, around 40%.
Presence of fewer nutrients in low concentration allowed
development of K-strategists that formed visible colonies
later during the incubation. Growth rate was slower – in
water samples from 10th, 15th and especially 30th and 40th
meter form just 10% visible colonies within fists two days of
incubation.
TABLE. 1
Standard Standard error,
deviation, n=3 n=3
0.00887 0.00512
0.0200 0.0116
0.00963 0.00556
0.000762 0.000440
0.00917 0.00529
0.0201 0.0116
0.0233 0.0134
0.00729 0.00421
0.0205 0.0119
bottom substrate, rising of organic and inorganic substances
from sediments. In the upper part of the hypolimnion
conditions were relatively constant and were weakly
influenced by atmosphere activities. K-strategists possess
active cell metabolism that give them competitive advantage
to compete for less nutrients and to use recalcitrant carbon
sources. In that parts of the water column most of the
available carbon sources were used and most of the
microorganisms were in inactive state. Identification of K-
and r-strategists in situ is impossible so one has to rely on
cultivation methods on agar media to identify them (4). A
Petri dish containing nutrient agar represents a nutrient-rich,
uncrowded environment low in inhabiting substances. Such
an environment should favor development of r-strategists. К-
strategists will also grow but in slower growth rates and the
abundance of r- and K-strategists could be established on a
daily basis quantifying new colonies until no more colonies
appear. Rate of growth is the only characteristic to
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differentiate between r- and K- strategists (4). Ideally
bacterial colonies should be tested against more
characteristics such as ability to grow on recalcitrant
substances, ability to grow under crowded conditions,
sensitivity to a number of inhabiting and toxic substances.
Disadvantage of using more than one parameter against
which colony development is measured is that it increases the
time for analysis and reduces number of analyzed samples. If
it would be accepted that growth rate on its own is a valid
parameter to differentiate between r- and K-strategists, than a
succession would take place in the water column following
the layers from surface to the bottom of the reservoir.
Microbial community dominated by r-strategists (on the
surface) was replaced by a community that become more and
more dominated by K-strategists (in the hypolimnion)
excluding bottom layer where again bacterial community was
dominated by r-strategists.
On the surface of the water body and in the epilimnion
where the conditions were unstable and strongly influenced
by the weather conditions the decrease in nutrient
concentration led to decrease in numbers of fast growing
colonies. But within the second day of incubation just up to
40 % of the colonies grew, while in the richer media quantity
of r-strategists was much more greater (Fig.1.а), б), с), d)).
The same tendency was found in samples from bottom layer
where the sediments influenced significantly environmental
conditions (Fig.1.i). In the middle layers where conditions
were comparatively stable, bacterial growth rate was slower
and not so dependent on nutrient concentration. (Fig. 1. e),
g), h). In the 25th meter depth layer – water extraction layer –
great amount of water was constantly extracted and with it
also nutrients and organic matter. In the samples from that
layer could be found great percentage of fast growers – on the
second day of incubation 60% of microorganisms form
visible colonies, growing on the diluted media.
Eco-physiological index (Table.1) increased with depth
and was greatest in the 20th meter depth. High values of the
EP-index are typical of uniformly distribution of different
classes, its value tells something about the evenness of class
distribution in a community, but no ecological or functional
information about the groups that are affected can be
obtained. Community dominated by r-strategists will have the
same EP-index as a community dominated by K-strategists
although large changes have taken place. The reason for this
loss of information is that EP-index represents community
with one value. Regardless of this could be made the
conclusion that with depth distribution of r- and K-strategists
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became more even while in the epilimnion both groups had
uneven distribution. In general functional diversity in all
depth layers was comparatively high. Such high bacterial
diversity in different environments was also found out in the
research of Verschuere, 1997 (9).
Conclusions
Characterization of microbial communities in relation to
depth using that method is simple, reproducible and highly
sensitive. In that way many samples and great amount of
microorganisms could be analyzed, and also various habitats
in time and space could be compared. Other advantage of the
method is that data are expressed as a percentage so samples
from different habitats – water, soil, sediments, could be
compared. Diversity in microbial communities in general
decreases in response to a stress factor from the environment
or to disturbances of ecological equilibrium of population
relations in a community (3). Microbial community on the
surface was dominated by r-strategists and following depth
gradient was replaced by a community dominated by K-
strategists (in the hypolimnion) and again in the bottom layer
bacterial community was dominated by r-strategists.
Acknowledgements
We would like to express our gratitude to the members of
Department of General and Applied Hydrobiology, Faculty
of Biology for their support and assistance. The authors
would like to thank the team of Drinking Water Treatment
Facility “Bistritsa” especially to the microbiological,
hydrobiological and chemical laboratory staff for their
cooperation and collaboration. The study was supported by
Sofia University “St. Kl. Ohridski” - Project N 098/2007.
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3. Atlas RM et al (1991) Microb Ecol 22: 249-256.
4. De Leij FAAM et al (1993) Microb Ecol 27: 81-97.
5. Luckinbill LS (1978) Science 202: 1201-1203.
6. MacArthur R and Wilson E (1967) The Theory of
Island Biogeography. Princeton University Press,
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 Princeton, USA. 9. Verschuere L et al (1997) J Appl Microb. 83, 603–612.
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