FEMS Microbiology Ecology 74 (1990) 243-252
Published by Elsevier
243
FEMSEC 00280
The selection of microbial communities by constant
or fluctuating temperatures
A.C. Upton I, D.B. Nedwell * and D.D. Wynn-Williams
*
' Universiv of Essex, Department of Biologv, Colchester. Essex, U.K. and 'British Antarcric Suroey,
Natural Environment Research Council. Cambridge, U.K.
Received 15 March 1990
Revision received 30 April 1990
Accepted 14 June 1990
Key words: Chemostat; Diversity of enriched communities; Cardinal growth temperatures
1. SUMMARY
The diversity of bacterial communities isolated
from Antarctic lake sediment in chemostats under
constant low temperature (8" C) or diurnally
fluctuating temperature (1" C to 16 " C) was examined. The median optimum temperature for
growth of the freshwater bacteria isolated from
the fluctuating chemostat was significantly lower
( P< 1%)than that for those from the constant
temperature chemostat. The diversity of the enriched bacterial community isolated in the chemostat culture subjected to short-term temperature
fluctuations was greater than that enriched under
constant temperature. At least 4 different groups
of bacteria, that occupied separate 'temperature
niches', were isolated from the fluctuating chemostat compared to only one group isolated from
the stable chemostat. Furthermore, a pseudomonad from the fluctuating chemostat was shown
to out-compete another pseudomonad from the
Correspondence to: A.C. Upton, University of Essex, Department of Biology, Wivenhoe Park,Colchester, Essex C04 3SQ,
U.K.
0168-6496/0168-6496/90/$03.50
Q
stable chemostat when both were subjected to the
fluctuating temperature regime. However, the
pseudomonad of constant ( 8 O C) temperature
origin out-competed that isolated under fluctuating conditions when subjected to a stable temperature regime.
2. INTRODUCTION
Increasing variability in an environment increases the diversity of its microbiota which in
turn increases the resilience of its community to
environmental stress [I]. A multiplicity of niches,
in which microbial species can co-exist in close
proximity, will reduce inter-species competition
thus enhancing diversity. Heterogeneity of the
physico-chemical environment occurs spatially,
often over very small distances (km) and its effect
on a microbial community has been studied with
multi-stage culture systems [l]. Variation in a
habitat may also occur in time - temporal heterogeneity. Non-steady-state conditions are probably
prevalent in nature so that physiological adaptation of a microbial species to steady-state con&
tions may be less relevant than the ability to
1990 Federation of European Microbiological Societies
244
respond quickly to changing environmental conditions.
Harris [2] emphasised that periodic perturbations in marine planktonic habitats maintains high
community diversity, which declined if the perturbations were eliminated. An intermittent supply
of nutrients has been shown to maintain co-existence of two Thiobacillus species, whereas one
was eliminated if the substrates were supplied at
one time [3].
Changes in microbial community structure in
response to seasonal temperature variations [4-81
are probably derived from changes in the competitive ability of the constituent species. Thus, while
microbial communities are broadly adapted to the
prevailing temperature range of their environment,
short-term microbial variations may result from
seasonal or diurnal temperature changes. Cairns
[9] showed that temperature perturbation of river
water due to thermal pollution resulted in the
selection of specific algal groups. As temperature
increased from 20 O C to 40 O C there was progressive selection of diatoms, green algae and cyanobacteria, respectively. The selection was reversed
with decreasing temperature. However, there has
been little research into the effect of short-term
temperature fluctuations on microbial competition, survival, and community adaptation.
Many natural environments such as those in
Antarctic fellfields [101 and inter-tidal sediments
[ill may be subjected to considerable diurnal or
semi-diurnal temperature fluctuations. Studies of
short-term environmental temperature changes
have until now been limited to temperature shift
experiments [12,13]. Shaw [12] found that when
the incubation temperature of mesophilic and
p s y c ~ o p h i f yeast
i ~ cultures was shifted within the
range in which the temperature characteristic ( p )
was relatively constant (moderate temperatures),
growth resumed immediately at the exponential
rate for the new temperature equal to the rate of a
culture grown solely at this temperature. In experiments with freshwater bacteria Hofle [13]
showed a similar response by one psychrotroph to
elevated temperature. However, another psychrotroph showed a higher growth rate than the rate
of a culture grown solely at the new temperature,
a third one showed a lower growth rate than
the rate of a culture grown solely at the new
temperature.
The present study concerns the diversity of
bacterial communities enriched from Antarctic
freshwater sediment in chemostats under different
temperature regimes. The response of bacteria isolated from these communities to transient environmental changes, especially temperature fluctuation
is also investigated.
3. MATERIALS AND METHODS
3.1, Source of microorganisms
Freshwater sediment was collected during
February 1985 from Heywood Lake on Signy 1sland in the maritime Antarctic [14,15], by members of the British Antarctic Survey. Sediment was
taken aseptically from the shallow ( < 1 m) shelf
area at the edge of the lake, transferred to a sterile
plastic bag and immediately frozen to be stored
and transported at - 20 C . Prior to use the Sample was thawed overnight at 4°C. No overlyin
water from the lake was present in the sample. g
3.2. Continuous culture apparatus
TWOsingle-stage chemostats [16,17J were emplayed. The medium was fed into each culture b
Y
peristaltic pumps (Multiperpex; LKB & o m a ,
Sweden) through a double line-breaker system to
ensure that no back contamination of the medium
reservoir occurred. The working volume of each
chemostat was approximately 510 d,
and a dilution rate ( D ) of 0.02 h-' was applied. The retention time of each culture was therefore approxi,
mately 50 h. Each culture was stirred with a
magnetic bar (45 mm X 8 mm) on a reversible
stirrer (Baird and Tatlock, U.K.) to optimise the
homogeneity of each culture and increase aeration. Spatial heterogeneity in the chemostat vessel
was therefore minimised and was assumed to be
negligible. The glass of each culture vessel was
coated with a silicone layer (Repelcote; Hopkins
and Williams, U.K.) prior to each experiment to
prevent wall growth of the culture.
Each culture was aerated by filter-sterilised air.
The air flow rate was controlled at approximately
60 ml min-' by needle valves (Dralim, U.K.) and
-
245
a calibrated flow meter (RS1 type with stainlesssteel float; Meterate, U.K.). The dissolved oxygen
concentration in the steady-state cultures was determined using a dissolved oxygen meter (Kent
Industrial Measurements Ltd., U.K.).
Each culture overflowed via a weir and was
collected in 10-litre aspirators. Samples of each
culture were collected from the overflow in sterile
Universal bottles (FSA Laboratory Supplies, U.K.)
kept in ice during sampling.
Each culture system was autoclaved for 1 h at
121OC and cooled before filling with approximately 500 ml of the medium. After setting the air
flow rate and the temperature regime, they were
left for at least 2 d prior to inoculation to ensure
that neither the medium nor vessels were contaminated. This also allowed the culture medium
to equilibrate to the temperature selected and for
the medium to become equilibrated with air.
The temperature of both culture vessels was
controlled by circulating anti-freeze solution in
water jackets surrounding each vessel. The solution was passed through continuous cooling units
(Grant Instruments Ltd., U.K.) working against
heater units (Grant Instruments Ltd., U.K.). The
analogue heater unit of chemostat 1 was interfaced to a microcomputer (BBC B; Acorn Computers Ltd., U.K.) programmed to cycle the culture temperature in a sine wave pattern over a
24-h period. The temperature of the chemostat
medium was monitored continuously using a 30
cm long thermistor thermometer in a glass sleeve
protuding into the centre of the culture. The data
was recorded on a digital thermometer (Kane-May
Ltd., U.K.) connected to a chart recorder. The
digital heater unit of chemostat 2 was set to maintain the culture at constant temperature. The temperature was periodically monitored using the
thermometer from chemostat 1.
3.3. Culture media
Modified FC2 medium from Tanner [18-201
was used for each of the chemostat enrichment
and competition cultures, and the batch cultures
in which the isolated bacteria were maintained.
The growth-limiting carbon source was 0.1% w/v
glycerol. Cycloheximide (50 mg ~ 1 - l )was added
to inhibit fungi. The same medium was also used
as agar plates, or as sloppy agar in Universal
bottles (FSA Laboratory Supplies, U.K.). Small
volumes of the medium were autoclaved while the
large volume (20 1) used for the chemostats was
filter-sterilised and stored in pre-sterilised 20-litre
aspirators. Aliquots of all media, both liquid and
agar, were incubated whilst un-inoculated to test
for contamination and as controls.
3.4. Inoculation of the chemostat cultures
Each culture was inoculated by injection using
a 10-cm, 19-gauge stainless steel needle inserted
through a Suba-seal (FSA Laboratory Supplies,
U.K.) that covered the inoculation port on each of
the chemostats. Each culture was held in batch
growth for 3 d after inoculation to ensure that
growth had been established before the continuous culture commenced. The turbidity of each
culture was routinely measured using a nephelometer zeroed against an uninoculated tube of the
liquid medium. Direct counts of the bacteria present in samples collected from the chemostat cultures were made using a computerized version of
the third nearest neighbour method of Roser et al.
[21,22].
3.5. Enrichment isolation cultures
Two 5-g sub-samples of thawed sediment were
aseptically transferred to sterile Universal bottles
and 12-ml aliquots of sterile FC2 medium minus
the carbon source were added as the diluent. Before inoculation the samples were shaken vigorously on a vertical shaker (Stuart Flask Shaker)
for 10 min. The sediment was allowed to stand for
2 min to permit settling of particulates before 5-ml
aliquots of the dilution were used as the inoculum
for chemostat 1 and 2.
The culture temperature of chemostat 1 was set
to cycle in a sine wave pattern between 0.9O C and
16.1OC ( + / - 0.4O C) over a 24-h period. Chemostat 2 was set to maintain the culture at constant
temperature of 8.2OC ( + /- 0.2OC). The dilution
rate ( D ) of both cultures was 0.02 h-'.
The turbidity measurements of chemostat 1
were all taken at the same point in the temperature cycle (around the median temperature on the
ascending limb of the cycle).
246
Each continuous culture was incubated at its
respective temperature regime for 24 d allowing
11.7 turnovers of the culture volume. The final
samples (approximately 10 ml) were taken aseptically from the overflow tube and stored overnight
at -2OOC. The selective effect of this freezing
was considered to be minor as the freshwater
sample employed as the inoculum had been transported back from Antarctica at - 20 " C.
The final samples were then thawed at 4" C
and 10-fold dilution series were made aseptically
with FC2 diluent. All routine handling of the
samples and isolates was carried out on an ice tray
[23] in a laminar flow cabinet (M.D.H. Ltd., U.K.)
to maintain low temperatures and to minimise
contamination. Sub-samples (0.1 ml) of each dilution were spread on to triplicate pre-cooled agar
plates of FC2 medium using glass spreading rods.
3.6. Isolation and description of the enriched
bacterial communities
The spread plates from the fluctuating and
stable temperature enrichment cultures were incubated at 8" C for 28 d. The resulting colony-forming units (cfu) were then counted. All the colonies from a plate, or colonies chosen at random
were then picked off and sub-cultured in FC2
liquid media at 8" C. These isolates were routinely
sub-cultured and stored at between 0 " C and 4" C.
The optimum and maximum growth temperatures for each isolate in liquid FC2 medium were
determined using aluminium temperature gradient
block incubators [24]. Growth at each temperature
was determined from the increase in turbidity of
the culture at each temperature.
FC2 streak plates were used to test for growth
at 0 ° C and to check for purity. For each isolate
numerical taxonomic profile codes were determined from API NE rapid identification test
galleries (API Laboratory Products Ltd., U.K.).
The test galleries employed 12 assimilation tests
and 9 biochemical tests. The 7-digit numerical
profile code is unique for each combination of
positive and negative results. Modifications to the
standard API method included the use of a saline
suspension [O.l% (w/v) NaCl] and incubation
temperature at 10 O C. The strips were read after
set time intervals until the profile codes from
sequential readings did not change.
Representatives of each group of isolates with
the same API profile codes were identified to
generic level by conventional taxonomic tests [25].
The tests carried out in addition to those of the
API NE strips, were the Gram strain; motility (by
hanging drop); flagella stain; Hugh and Leifson's
test for oxidative or fermentative metabolism of
glucose; and sensitivity to the vibriostatic agent
pteridine 0/129 (2,4-diamino-6,7-diisopropylpteridine phosphate; B.D.H. Ltd., U.K.). All of
these tests were conducted as described by Skinner
and Lovelock [26]. The latter two tests required
incubation for two weeks at 10°C.
3.7. Competition cultures
One isolate (Isolate 1) from the fluctuating
temperature regime enrichment culture and one
(Isolate 2) from the constant 8" C temperature
regime were used to investigate their relative cornpetitive ability under different temperature regimes. The isolates were chosen primarily as they
had cardinal growth temperatures that were substantially different. The optimum growth tempera,
ture of Isolate 1 was 9.7"C and that of Isolate 2
was 20.9 " C. Their maximum growth temperatures
were 26.5 " C and 32.0 O C, respectively. The taxonomic tests showed both isolates belonged to the
genus Pseudomonas.
Two chemostats (chemostats 1 and 2) were
both inoculated with the two bacterial cultures.
The population density of cultures grown at 8 O c
was determined by direct counting. Each Pure
culture was then diluted with sterile FC2 medium
and each chemostat inoculated with 5 ml of each
culture containing approximately 1.2 x IO* cells.
The turbidity and direct counts of these two competition cultures were routinely measured.
The temperature regimes were similar to those
of the enrichment cultures. Chemostat 1 was set to
fluctuate its temperature in a sine wave form
between 1.4"C and 17.2"C ( + / - 0.3"C) over 24
h. Chemostat 2 was set to maintain a constant
8.1"C (+/-0.2"C). The dilution rates ( D ) of
both competition cultures were slightly higher than
those for the enrichment cultures. The dilution
rate of the fluctuating temperature regime culture
247
was 0.0225 h-' and for the 8°C temperature
regime was 0.0216 h-'. Each culture was allowed
to remain in batch growth for 3 d before the
continuous cultures commenced.
Samples were taken aseptically after approximately every turnover, and 10-fold dilution series
made with FC2 diluent. Aliquots (0.1 ml) of each
dilution were spread plated immediately, onto triplicate pre-cooled FC2 agar plates, and also onto
triplicate pre-cooled agar plates of FC2 medium
which had 0.1% (w/v) mannitol replacing the
glycerol as the carbon source. These two media
were used to enumerate the cfu of the two isolates.
These spread plates were incubated at 8OC for
2-3 weeks before counting. API NE tests had
previously shown Isolate 1 to be unable to grow
on mannitol whereas Isolate 2 was able to utilise
it. Both isolates were able to metabolise glycerol.
The total viable count of both isolates and the
relative proportion of each isolate was determined
by comparing the total count on the glycerol
medium and the count of Isolate 2 on the mannito1 medium. The outcome of the competition under each temperature regime was therefore assessed.
4. RESULTS
4.1. The enrichment isc itions
The progress of the freshwater enrichment cultures relative to steady-state conditions are shown
in Fig. 1 and appeared to be in steady-state after 9
volume turnovers. In each culture the oxygen in
the medium was found to be above 80% saturated
with respect to air and therefore not oxygenlimited. The number of cfu isolated from the
.-Jc
3
L
400 350 -
2
300 -
-0
250
-
200
-
2
a
v
0-0
I°C to 16OC culture
0-0
aoc
culture
150 100 -
-w
.-
? ;F0 o 5
1
2
3
4
s
6
7
a
9 1 0 1 1 1 2
Number of Turnovers Completed
Fig. 1. Change in bacterial density (as turbidity) in fluctuating
temperature regime and constant 8 C freshwater enrichment
cultures.
steady-state cultures after 11.7 turnovers, and the
median optimum and maximum growth temperatures of the two series of isolates are summarised
in Table 1. The median optimum growth temperatures of the freshwater isolates from each series of
steady-state enrichment cultures were compared
for significant differences using a non-parametric
two-tailed Mann-Whitney U-test [27,28) (Table
1). The median optimum growth temperature of
the isolates from the fluctuating temperature regime enrichment culture was significantly lower
than that for the isolates from the 8°C regime
culture ( P-c 1%).
None of the isolates were found to be strict
psychrophiles according to the definition of Morita
[29], as all had maxima above 20°C. All the
isolates from the two enrichment cultures possessed the ability to grow at 0 O C and were therefore considered to be psychrotrophic.
Table 1
Total number of colony-forming units (cfu), and the median optimum and maximum growth temperatures, of the bacteria isolated
from steady-state freshwater chemostat enrichment cultures under the fluctuating and constant 8 O C temperature regimes
Temperature
regime
Total cfu
at steady-state
on-9
Number of
isolates
tested
Median optimum
temperature of
isolates ( O C)
Median maximum
temperature of
isolates ( C)
Fluctuating (1-16OC)
1.53 X lo8
16
16.0
27.0
Constant ( 8 O C)
1.57 x 10'
15
21.0
36.0
O
248
numerical profile codes for each series of isolates.
It was assumed that each unique profile code
represented one 'group'. These 'groups' are summarised in Table 2. Approximately 70% of the 16
isolates from the fluctuating temperature regime
culture appeared to have been adapted to or
selected by the lower temperatures of the cycle,
with optima about or below 16°C; hence a lower
median optimum growth temperature than for the
isolates from the constant 8°C culture (Table 1).
The API codes showed there to be at least 4
separate groups present. There may possibly have
been 5, with 2 groups having the same profile code
(14 6 2 3 4 2) but separated by the isolates having
different optimum growth temperatures. One
group had an optimum below 10O C and the other
group an optimum at about 16°C. Three other
groups were present with higher optima, that presumably had been selected by Or were adapted t o
the higher part of the temperature cycle. The
proportion of isolates in these latter 3 groups was
about 30% of the total. The 15 isolates from the
8 O C regime culture had one common API profile
code and hence only one group was Present.
The bacteria representative of the numerical
groups that were isolated from the enrichment
cultures were identified to generic level. Of isolates from the fluctuating temperature regime culture, 80% were found to belong to the s n u s
pseudomonm, while the remainder were members
Table 2
The number of groups of freshwater bacteria and their final
proportion in each of the enrichment cultures selected by the
fluctuating and constant temperature regimes
Temperature
regime
Group API
code
Approx.
optimum
temps. of
group ( O C)
1462342
1462342
1067777
1243277
1043277
<10.0
16.0
23.5
21.0
23.0
No. of
isolates
as % of
total
Fluctuating
(1-16OC)
23.5
47.1
5.8
11.8
11.8
Constant
(8 C)
1457273
19.5-24.7
100
The turbidity readings for each steady-state
were approximately 115 nephelometer units for
the fluctuating temperature regime culture and 80
for the constant 8 ° C r e g h e culture. This indicates the relative steady-state biomass in each
culture. These are in agreement with the mean
total number of cfu in each culture (Table 1)
which was approximately 10-fold higher in the
fluctuating temperature culture than in the constant 8" C regime culture.
The diversity of bacteria present in each of the
enrichment cultures after 11.7 turnovers was assessed by the number of different API NE
Table 3
Population density and relative proportion of freshwater Isolates 1 and 2 when in competition in continuous culture under a
fluctuating and a constant temperature regime
Temperature
regime
Fluctuating (1-17OC)
Constant (8OC)
Percentage of
total count
Number of volume
turnovers in culture
(x107ml-')
(D= 0.022 h-')
1
2
1
2
at inoculation
12.0
4.2
8.6
11.0
11.4
12.0
7.1
4.4
3.1
5.7
50.0
4.96
6.04
7.12
9.21
50.0
62.8
33.8
22.0
33.3
at inoculation
12.0
2.7
3.0
5.7
3.0
12.0
7.2
8.3
11.9
10.4
4.75
5.79
6.83
8.83
Viable count (Cfu)
37.2
66.2
78.0
66.7
50.0
27.3
26.5
32.4
22.4
50.0
72.7
73.5
67.6
77.6
249
of the genus Vibrio. Three out of the 15 isolates
from the 8°C regime culture that were numerically coded were further identified and also found
to belong to the genus Pseudomonas.
4.2. The competition cultures
The proportion of each of the freshwater isolates competing under the selected temperature
regimes showed that in both the fluctuating and
constant temperature regime approximately 70%
of the culture consisted of the isolate that had
been enriched originally under the prevailing temperature regime (Table 3). In the fluctuating regime approximately 70% of the culture after 6
turnovers was Isolate 1, that had been isolated
from a similar fluctuating temperature regime.
However, in the constant 8°C regime culture approximately 70% of the culture after only 4.75
turnovers was Isolate 2, that had been isolated
from a constant 8 ” C temperature regime. The
mean viable count and relative proportion of each
of the two isolates in the two competition cultures
are presented in Table 3.
5. DISCUSSION
5.1. The enrichment isolations
The experimental results clearly showed that
the steady-state microbial community selected under conditions of variable temperature was more
diverse than the community selected under a constant temperature. The turbidity of each culture
was stable after 9.3 turnovers (Fig. 1) and the
bacteria isolated were therefore assumed to be
representative of the microbial communities
selected under each temperature regime. It is normally assumed that a continuous culture has
reached steady-state conditions after approximately 5 turnovers [30]. Steady-state for a culture
under cycling conditions must be defined as a
state where all parameters relating to growth, such
as growth rate and cell numbers, are constant
relative to specific points in the cycle but not
necessarily constant between different points
within one cycle i.e. a stable but not necessarily
constant state. This has been termed a limit cycle
1311.
Spatial heterogeneity with significant environmental variation over perhaps very small distances
permits the co-existence in close proximity of microbial species which would’ otherwise compete
with each other [l].Maintenance of species within
an environment under conditions of reduced competition enhances community diversity. Our data
emphasise that the imposition of temporal heterogeneity on microbial communities also increases
diversity. The temporally changing temperature
regime presumably provided a series of ‘temperature niches’ within which different bacterial species
were favoured. For any one species to be maintained within the selected steady-state community
it was necessary for it to survive the adverse part
of the temperature cycle without being washed-out
before its own ‘temperature niche’ returned. In a
chemostat the balance between the dilution rate
( D ) and the time required for a complete temperature cycle therefore will have a marked effect on
the diversity of the selected steady-state culture.
However, under the conditions used in the present
work fluctuating temperature certainly contributed to increased diversity, showing that species
were not washed-out before their period of active
growth re-occurred.
The 15 isolates from the constant 8°C temperature regime culture possessed one common API
numerical profile code, showing that only one
numerically defined group was present at steadystate. Chemostat theory predicts that only the
single most efficient bacterium will survive in a
steady-state culture in competition for a single
growth rate-limiting substrate [30], and our data
conform to t h s hypothesis. However, where there
is environmental heterogeneity, more bacteria may
survive because of the increased variety of niches
within which different micro-organisms may be
competitively successful. In the chemostat with
the fluctuating temperature regime at least 4 distinct groups of bacteria were able to co-exist, each
apparently having a different optimum temperature for growth (Table 2). These data support the
concept of selection of bacteria physiologically
adapted to different ‘temperature niches’ within
the temperature cycle. The higher turbidity readings (Fig. 1) and increased viable count (Table 1)
at steady-state in the variable temperature chem-
250
ostat, above the constant temperature chemostat,
again emphasised the larger and more diverse
microbial community selected in the former. Similarly, Gottschal et al. [3,32] have shown that temporal heterogeneity (in the form of alternating
supplies of acetate and thiosulphate) maintained
Thiobacillus uersutus (T. A 2 ) in co-culture in a
chemostat with Thiobacillus neopolitanus and the
heterotrophic spirillum G7. In contrast, when
grown under conditions of continuous supply of
either substrate, the T. uersutus was always competitively excluded by one of the other species at
steady-state. Again therefore temporal heterogeneity, in this case of substrate supply, enhanced the
diversity of the steady-state community.
Full conventional identification of the freshwater isolates from the two enrichment cultures
was not carried out. In conventional taxonomy
different weightings may be given to different test
characters. A difference in only one test result
therefore may or may not result in a group of
isolates being regarded as homogenous or being
sub-divided, thereby influencing the apparent diversity [33,34]. The API numerical profile codes
assign equal weightings to every characteristic and
two isolates were regarded as different if only one
test result differed. These numerical profile codes
showed that several distinct groups of isolates
were selected under the fluctuating temperature
regime, each group apparently with a different
' temperature niche'.
For any one species, survival will be enhanced
by it being able to grow and compete successfully
to as low a temperature as possible, thereby
broadening its ' temperature niche' within the temperature cycle. The bacteria in the fluctuating
temperature culture must have been able to survive
at the lowest temperature reached within the cycle.
The median optimum of the isolates from the
fluctuating temperature regime community was
lower than that from the constant 8°C regime,
implying that the majority of those isolates were
selected by the low temperature part of the cycle
rather than by the high temperature part. This was
confirmed by the data presented in Table 2 which
shows that approximately 70% of the isolates had
optima at 16OC or below. Furthermore, the
median maximum growth temperature of the iso-
lates from the fluctuating regime was significantly
lower than that for the isolates from the constant
regime ( P< 1%). This, too, implied that the
majority of the isolates were adapted to or selected
by the low temperature part of the cycle rather
than the high part of the cycle over the range of
temperature used in this work.
5.2. The competition cultures
The enrichment experiments had yielded physiologically distinct isolates under the different temperature regimes used. The two isolates selected
for use in subsequent competition experiments
reflected physiological types that perhaps exhibited different survival strategies in relation to
temperature. Isolate 1 was a Pseudomonas sp.
which derived from the variable temperature
chemostat and was the nearest approximation to
an obligate psychrophile isolated during these experiments. Its optimum temperature for growth
was 9.7 C , although its maximum growth temperature (26.5 O C ) was hgher than that convention,
ally defined for a psychrophile 1291. In contrast,
Isolate 2 from the constant 8 ° C culture, which
was also a Pseudomonas sp. was a PsYchrotroph,
able to grow at 0 ° C but optimum growth at
2 0 . 9 " ~and maximum at 32°C. The majority of
Antarctic heterotrophs appear to be PsYchrotrophic rather than psychrophific W351The competition experiments between these two
cultures (Table 3) showed that the psyChrOtrophic
Isolate 2 was indeed able to out-compete Isolate 1
at a constant 8 ° C . The proportion of Isolate 1 in
the culture fell to O d Y 22% within 9 turnovers. In
contrast, under a temperature regime fluctuating
between 1 and 17" c Isolate 1 seemed to be cornpetitively more successful and was the predominant organism after 9 volume turnovers. This implied that the reason Isolate 1 was more successful
under the fluctuating temperature regime was that
it was favoured during the low temperature part of
the cycle.
An isolate growing under a temperature regime
similar to its initial isolation therefore appeared to
possess a competitive advantage over another isolate enriched under a different temperature regime. In the two experiments presented in this
paper the competitive advantage was not complete
O
251
as the ‘disadvantaged’ isolate was not completely
washed-out even after 9 turnovers of each culture.
A mixed culture therefore resulted in both cases.
This may have been due to the cultures not actually being carbon-limited, and not always competing for limiting substrate. If they were simultaneously temperature-limited during the low temperature part of the cycle competition for substrate would be reduced and co-existence enhanced. The two isolates could then co-exist and
persist at the temperature of the chemostat culture
as long as their growth rates were equal to or
greater than the dilution rate.
Our data reported above illustrate that the
principle of environmental heterogeneity enhancing community diversity applies also to temporal heterogeneity. The competition experiments
support the concept of physiological specialists
adapted to, and successful for, specific niche requirements, compared to physiological generalists
able to be active over a wider range of conditions.
These observations describe the phenomenon, but
the mechanism of competition and selection by
temperature must operate through the effect of
changing temperature on growth rate. The ability
of one micro-organism to out-compete other micro-organisms in an environment is the result of
its higher growth rate. The growth rate will be
determined by efficiency of nutrient uptake and
utilisation (cell yield). Growth rates can be defined by the growth constants - maximum specific
growth rate ( urnax), the half saturation constant
(K,), and cell yield (Y). In fact urnax,K , and Y
are not constants but are functions of temperature, varying exponentially with temperature
change [36]. Harder and Veldkamp [37] showed
that the out-come of competition between a
psychrophle and a psychrotroph, in steady-state
chemostats with a growth-limiting substrate at different constant temperatures, depended on the
interaction between temperature and the growth
rate constants urn, and K,, and cell yield (Y).
Therefore substrate concentration and temperature interact to influence the growth rate of a
microbial species at any particular combination of
these factors, and hence the outcome of its competition with another species. The reason why
changes in environmental temperature influence
the competitive success of different microorganisms is because of differential effects of temperature upon their growth rate constants. The
outcome of competition will therefore depend on
the temperature interacting with nutrient concentrations, and in non-steady-state environments,
where the temperature fluctuates with time and a
complex situation develops. As many environments [10,11] have seasonally and diurnally variable temperature regimes, this interaction is ecologically important.
ACKNOWLEDGEMENTS
This work was supported by a Natural
Environment Research Council Special Topic
C.A.S.E. studentship (GT4/84/BAS/6)
for
A.C.U. to D.B. Nedwell of the University of Essex
and D.D. Wynn-Williams of the British Antarctic
Survey. We gratefully acknowledge the British
Antarctic Survey for providing the samples from
Heywood Lake.
REFERENCES
[ l ] Wimpenny, J.W.T., Lovitt, R.W. and Coombs. J.P. (1983)
Laboratory model systems for the investigation of spatially and temporally organised microbial ecosystems. In
Microbes in Their Natural Environments (Symposium of
the Society for General Microbiology, No. 34) (Slater,
J.H.. Whittenbury, R. and Wimpenny, J.W.T., Eds.), pp.
67-1 17. Cambridge University Press, Cambridge.
[2] Harris, G.P. (1985) Opinion: the answer lies in the nesting
behaviour. Freshwater Biol. 15. 375-380.
[3] Gottschal, J.C., Nanninga, H.J. and Kuenen, J.G. (1981)
Growth of Tbiobacillus A2 under alternating growth conditions in the chemostat. J. Gen. Microbiol. 126, 85-96.
[4] Sieburth, J. McN. (1967) Seasonal selection of estuarine
bacteria by water temperature. J. Exp. Mar. Biol. E d . 1,
98-1 21.
[5] Nedwell, D.B. and Floodgate, G.D. (1971) The seasonal
selection by temperature of heterotrophic bacteria in an
intertidal sediment. Mar. Biol. 11, 306-310.
161 Inniss, W.E. and Mayfield, C.1. (1979) Seasonal variation
of psychrotrophicbacteria in sediment from Lake Ontario.
Water Res. 13, 481-484.
[7] Tison, D.L., Pope, D.H. and Boylen, C.W. (1980) Influence of seasonal temperature on the temperature optima of bacteria in sediments of Lake George, New York.
Appl. Env. Microbiol. 39, 675-677.
252
(81 King, D. and Nedwell, D.B. (1984)Changes in the nitrate
[24]Upton, A.C. and NedwelI, D.B. (1989) Temperature re-
reducing community of an anaerobic saltmarsh sediment
in response to a seasonal selection by temperature. J. Gen.
Microbiol. 130,2935-2941.
[9]Cairns, (1972)Coping with heated waste water discharges
from steam-electricpower plants. Bioscience 22,411-420.
[lo]Walton, D.W.H. (1982)The Signy Island terrestrial reference sites, XV. Micro-climate monitoring, 1972-1974. Br.
Antarct. Sum. Bull. 55, 111-126.
[ll]Nedwell, D.B. and Gray, T.R.G. (1987) Soils and sediments as matrices for microbial growth. In Ecology of
Microbial Communities (Symposium of the Society for
General Microbiology, No. 41)(Fletcher, M., Gray, T.R.G.
and Jones, J.G., Eds.), pp. 21-45. Cambridge University
Press, Cambridge.
[12]Shaw, M.K. (1967) Effect of abrupt temperature shift on
the growth of mesophilic and psychrophilic yeasts. J.
Bacteriol. 93,1332-1336.
[13]Hdfle, M.G. (1979) Effects of sudden temperature shifts
on pure cultures of four strains of freshwater bacteria.
Microb. Ecol. 5, 17-26.
(141 Ellis-Evans, J.C. (1981)Freshwater Microbiology at Signy
Island, South Orkney Islands, Antarctica. Ph.D. thesis,
CNAA (unpublished).
I151 Ellis-Evans, J.C. (1981) Freshwater microbiology in
Antarctica, 11. Microbial numbers and activity in
nutrient-enriched Heywood Lake, Signy Island. Br.
Antarct. Surv. Bull. 54,105-121.
[16]Monod, J. (1950)La technique de culture continue: thtorie
et applications. Ann. Inst. Pasteur 79, 390-410.
[17]Novick, A. and W a r d , L. (1950) Description of the
chemostat. Science 112,714-716.
[IS]Tanner, A.C. (1981)The Microbiology of Antarctic Marine
Sediments. Ph.D. thesis, University of Dundee (unpublished).
[19]Upton, A.C. (1988) Comparative Physiological Adaptation of Selected Antarctic Microbial Communities to Low
Temperature. Ph.D. thesis, University of Essex (unpublished).
[ZO]Upton, A.C. and Nedwell, D.B. (1989)Nutritional flexibility of oligotrophic and copiotrophic Antarctic bacteria
with respect to organic substrates. FEMS Microbiol. Ecol.
sponses of bacteria isolated from different Antarctic environments. In University Research in Antarctica (Proceedings of British Antarctic Survey Antarctic Special
Topic Award Symposium, 9-10 November 1988) (Heywood, R.B., Ed.), pp. 97-101. British Antarctic Survey,
Cambridge.
[25J b e g , N.R. and Holt, J.G. (1984) Bergey’s Manual of
Systematic Bacteriology, Vol. 1. Williams & Wilkins, Baltimore, MD.
126) Skinner, F.A. and Lovelock, D.W. (1979) Identification
Methods for Microbiologists (Soc.Appl. Bact. Tech. Ser.
14). 2nd edn. Academic Press, London.
[27]Elliott, J.M. (1977)Some Methods for the Statistical Analysis of Samples of Benthic Invertebrates (Freshwater B i e
logical Association Scientific Publication No. 25). 2nd
edn., pp. 112-115.Titus Wilson and Son Ltd., Kendal.
[28]Neave, H.R.(1981)Elementary Statistics Tables, 2nd a n .
George Allen and Unwin (Publishers) Ltd., London.
[29]Morita, R.Y. (1975)Psychrophilic bacteria. Bacteriol. Rev.
62, 1-6.
[21]Roser, D., Nedwell, D.B. and Gordon, A. (1984) A note
on ‘plotless’ methods for estimating bacterial cell densities. J. Appl. Bacteriol. 56,343-347.
1221 Roser, D., Nedwell, D.B. and Gordon, A. (1985)A note
on ‘plotless’ methods for estimating bacterial cell densities
- Erratum. J. Appl. Bacteriol. 57,298.
(231 Wiebe, W.J. and Hendricks, C.W. (1971) Simple, reliable
cold tray for the recovery and examination of thermosensitive organisms. Appl. Microbiol. 22, 734-735.
39,144-167.
[M] Pirt, S.J. (1975) Principles of Microbe and Cell Cultivation. pp. 274. Blackwell Scientific Publications, Oxford.
(311 B&, M.J. (1981)Mixed culture kinetics. In Mixed Culture Fermentations (Society for General Microbiology,
Special Publication, No. 5 ) (Bushell, M.E. and Slater, J.H.,
E~s.),pp. 25-51. Academic Press, London.
[32]Gottxhal, J.C., De Vries, S. and Kuenen, J.G. (1979)
Competition between the facultatively chemolithotrophi,
Thiobaci/h A2, an obligately chemolithotrophicThiobacik
/US and a heterotrophic spirillum for inorganic substrates.
Arch. Microbiol. 121,241-249.
(331 Gtiffiths, A.J. and Lovitt, R. (1980) Use of numerical
profiles for studying bacterial diversity. Microb. Ecol. 6,
35-43.
[34]flhs-Evans, J.C. and WYnn-Wilfiams, D.D. (1985) The
interaction of soil and lake microflora at SiWY Island. I,,
Antarctic Nutrient Cycles and Food Webs (Siegfried,
w.R., condy, P.R. and Laws, R.M.) pp. 662-~6~.
Springer-Verlag, Berlin.
Delille, D. and Perret, E. (1989)Influence of temperature
on the growth potential of southern polar marine bacteriaMicrob. Ecol. 18, 117-123.
(361 Topiwala, H. and Sinclair, C.G. (1971)Temperature relationship in continuous culture. Biotech. Bioeng. 13, 795-
813.
[37)Harder, W. and Veldkamp, H. (1971) Competition of
marine psychrophilic bacteria at low temperatures.
Antonie van Leeuwenhoek J. Microbiol. 37, 51-63.