Previous Article | Next Article 
Applied and Environmental Microbiology, June 1999, p. 2577-2584, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Temperature Dependence of Inorganic Nitrogen Uptake: Reduced
Affinity for Nitrate at Suboptimal Temperatures in Both Algae
and Bacteria
David S.
Reay,1,*
David B.
Nedwell,1
Julian
Priddle,2 and
J. Cynan
Ellis-Evans2
Department of Biological Sciences, University
of Essex, Wivenhoe Park, Colchester CO4 3SQ,1
and British Antarctic Survey, Natural Environment Research
Council, High Cross, Cambridge CB3 0ET,2 United
Kingdom
Received 16 November 1998/Accepted 11 March 1999
 |
ABSTRACT |
Nitrate utilization and ammonium utilization were studied by using
three algal isolates, six bacterial isolates, and a range of
temperatures in chemostat and batch cultures. We quantified affinities
for both substrates by determining specific affinities (specific
affinity = maximum growth rate/half-saturation constant) based on
estimates of kinetic parameters obtained from chemostat experiments. At
suboptimal temperatures, the residual concentrations of nitrate in
batch cultures and the steady-state concentrations of nitrate in
chemostat cultures both increased. The specific affinity for nitrate
was strongly dependent on temperature (Q10 
3, where
Q10 is the proportional change with a 10°C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect
of low temperature on affinity for nitrate has been identified for
psychrophilic, mesophilic, and thermophilic bacteria and algae. The
different responses of nitrate uptake and ammonium uptake to
temperature imply that there is increasing dependence on ammonium as an
inorganic nitrogen source at low temperatures.
| |
INTRODUCTION |
The forms of inorganic nitrogen used
most commonly by bacteria and algae are nitrate and ammonium
(82). In many estuarine and marine systems the nitrogen
concentration is limiting (14, 23), and nitrate
concentrations are generally much higher than ammonium concentrations,
although ammonium is invariably the preferred nitrogen source when it
is available (46, 82). Despite the relatively low
concentrations of ammonium, the great importance of ammonium to the
global nitrogen cycle has been increasingly recognized (60,
61). Information concerning the mechanisms of uptake of the two
forms of nitrogen is limited (19), and most data have been
derived from measurements of uptake systems in higher plants (12,
42, 43) rather than measurements of uptake systems in algae and bacteria.
Previous work has shown that the affinity of bacteria for organic
substrates can be highly temperature sensitive (51) and that
a low temperature exacerbates any effect of nutrient limitation by
making it increasingly difficult to sequester substrates
(83). It has been pointed out that decreased affinity for
inorganic substrates, such as nitrate, at low temperatures could have
profound effects on the productivity of low-temperature environments,
such as the Southern Ocean (51). Relatively little is known
about the temperature sensitivity of inorganic nitrogen uptake by
microorganisms, but it has been suggested that there is a difference
between the temperature dependence of the uptake system for nitrate and
the temperature dependence of the uptake system for ammonium
(13).
A low affinity for inorganic nutrients, as indicated by a high
half-saturation constant (Ks) for uptake of
silicate and nitrate, has been reported previously for Southern Ocean
phytoplankton at low temperatures (36, 69). However, no
consistent trend of changing affinity for inorganic nitrogen with
temperature has been identified previously by using
Ks values alone. The use of Ks to measure affinity can be misleading as this
parameter does not necessarily reveal changes in substrate affinity at
low concentrations (9). On the other hand, specific affinity
(aAo) is a more robust measure of substrate
affinity. This parameter is the initial slope of the rectangular
hyperbola (Michaelis-Menten or Monod) function relating growth rate
(µ) to substrate concentration and is given by
aAo = µmax/Ks, where µmax
is the maximum growth rate and Ks is the concentration at which µ = 0.5µmax.
aAo is the slope of the hyperbola at zero
concentration and thus provides an unambiguous measure of the ability
of cells to accumulate substrate and grow at very low concentrations,
and this parameter is independent of the uptake mechanism (7, 8,
30). Because aAo for growth is a rate
divided by a concentration, it has the dimensions time
1 · (mass · liter3)1 and in our calculations has the
units liters per micromole per hour. Such a measurement of affinity is
related to growth through the cell yield.
In this study we investigated the influence of temperature on affinity
for nitrate and ammonium in a range of algae and bacteria by
using aAo to describe changes in the
affinity of an organism for inorganic nitrogen. Note that ammonium is
used below to indicate both ammonia (NH3) and ammonium
(NH4+), except where a distinction between the
two is required, when the appropriate chemical formulae are used.
| |
MATERIALS AND METHODS |
Bacterial and algal isolates were chosen so that a wide range of
physiological and taxonomic types was represented (Table 1). All cultures were monospecific and
axenic and were checked regularly for contamination (6).
The µmax values for both bacterial and algal isolates
were determined by using a temperature gradient block incubator
(51, 77). The temperature range was adjusted so that it was
suitable for each organism (Table 1). For algal isolates a temperature gradient block with illuminated wells was used. Illumination was provided by a bank of Triton fluorescent tubes (38 W; 400 to 710 nm,
with peaks at 450, 550, and 620 nm; Interpet Ltd., Dorking, England.)
placed immediately below the temperature gradient block (well
illumination, 200 µmol of quanta · m2 · s1). Optically standardized test tubes containing 10 ml
of sterile FC2 medium for bacterial isolates (51, 52) or
modified f/2 medium for algae (27) were prepared with either
80 µM NH4Cl or 80 µM NaNO3 as the nitrogen
source. Two diatoms, Chaetoceros curvisetum and
Chaetoceros sp., were not grown on NH4Cl in
batch cultures. Preliminary experiments showed that at a concentration of 80 µM, nitrogen was the first nutrient to be depleted, which induced the stationary phase. Subsequent aseptic addition of either a
sterile nitrate solution or a sterile ammonium solution resulted in a
further increase in the optical density, which confirmed that N
limitation occurred. Tubes were placed in the wells of the temperature
gradient block, and each tube was aerated continuously; the airstream
was humidified to prevent evaporation. After all of the tubes had
become equilibrated to the temperature in the block, each tube was
inoculated with 0.2 ml of an exponential-phase culture grown on the
same medium at the optimum growth temperature of the organism.
After inoculation, growth at each temperature was monitored by periodic
measurement of turbidity with a nephelometer (model EEL Unigalvo DS29;
Diffusion Systems, London, United Kingdom). The µmax at
each temperature was obtained from a first-order linear regression
analysis which determined the slope of the linear part of the
semilogarithmic plot of optical density versus time.
Batch cultures were regarded as being in the stationary phase when the
variation (standard error) in the optical densities at four successive
times over a 24-h period was <2% of the mean optical density for the
same period. The residual concentration of the limiting nutrient
(nitrate or ammonium) during the stationary phase in batch cultures
reflected the affinity of the uptake system for the substrate
(55). Changes in the residual substrate concentration with
temperature provide an indication of changes in affinity (51), although Ks values cannot be
calculated directly. When cultures reached the stationary phase, they
were removed from the temperature gradient block and centrifuged at
6,000 × g for 15 min. Supernatant (triplicate 1-ml
samples) was then removed and analyzed to determine either the residual
nitrate content or the residual ammonium content. Nitrate contents were
determined colorimetrically (72). The method used was linear
for concentrations ranging from 1 to 20 µM, and the limit of
detection was 0.25 µM. Ammonium contents were analyzed by the
indophenol blue method (29), which was modified by
substituting sodium dichloroisocyanuric acid for the original unstable
chlorine donor, hypochlorite (37). The ammonium values were
linear for concentrations between 1 and 40 µM, and the limit of
detection was 0.5 µM. Samples were diluted when necessary in order to
obtain concentrations within the detection ranges of the colorimetric
methods used.
Chemostat cultures were used to measure µmax independent
of batch cultures for all isolates. The use of chemostats also allowed us to measure the Ks and
aAo values over a range of temperatures. The
chemostat incubation temperatures were set to give a range up to and
including the optimum temperature for each isolate. Bacterial (FC2) and
algal (f/2) media containing either 80 µM NH4Cl or 80 µM NaNO3 were used for all chemostat experiments so that
nitrogen was the growth rate-limiting nutrient. Dilution rates were set
at 0.018 h1 for all chemostats, which were continuously
mixed and aerated. All algal cultures were also continuously
illuminated (200 µmol of quanta · m2 · s1) with twin banks of Triton fluorescent tubes.
Each chemostat was inoculated with 1 ml of an exponential-phase culture
grown on the same medium at the optimum growth temperature for the
isolate. After inoculation, the growth of each isolate to the steady
state was monitored by periodically aseptically removing a 1-ml
subsample, whose optical density at 550 nm (OD550) was then
determined with a spectrophotometer. A chemostat was considered to be
in a steady state when the variation in the standard error for at least
six optical densities determined over a period of at least 60 h
was <2% of the mean OD550 (51). Nitrogen
limitation was confirmed by aseptically adding to a chemostat either 5 ml of a sterile 10 mM nitrate solution or 5 ml of a sterile 10 mM ammonium solution. A subsequent increase in the optical density confirmed that N limitation occurred under steady-state conditions.
Under steady-state conditions Ks values were
determined by removing 5-ml subsamples from the chemostats. Each
subsample was filtered through a Whatman cellulose acetate filter
(nominal particle retention size, 0.2 µm), and residual nitrate or
ammonium content in the filtrate was measured.
Ks values were calculated by using the following
equation: Ks = s(µmax 
D)/D, where s is the residual substrate
concentration and D is the dilution rate (per hour) (55, 69). Note that the Ks values
derived in this way were Ks values for growth
and thus are not always equivalent to Ks values
determined for uptake (25).
µmax values in the chemostats were determined by
increasing the dilution rate to values that were higher than the
critical dilution rate in order to induce washout. µmax
was calculated from the slope of a plot of ln OD550 versus
time during washout. aAo values were then
calculated by using µmax values determined for the same
chemostat cultures rather than µmax values determined for
batch cultures.
Data were analyzed by performing box plots, F tests, one-way analyses
of variance (ANOVAs), and first-order linear regression analyses (LRAs)
(21). Statistical analysis and data plotting were performed
by using the data analysis packages supplied in Systat version 5.04 (Systat Inc.), Excel version 7.0 (Microsoft), and SigmaPlot version
3.0. (Jandel Scientific).
| |
RESULTS |
Batch cultures.
The bacterial and algal isolates used grew at
a wide range of temperatures and, consequently, there was a wide range
of optimum incubation temperatures (Table
2). The residual concentrations of
nitrate and ammonium at the stationary phase in batch cultures exhibited a similar trend with all of the bacterial and algal species
investigated (Fig. 1). At or near the
optimum temperatures for growth the residual concentrations of either
nitrate or ammonium were below the sensitivity of the analyses. As the
temperature deviated from the optimum temperature, the residual
concentration of either nitrate or ammonium generally increased,
although the residual nitrate concentrations tended to increase more
than the residual ammonium concentrations increased. As the incubation temperature approached the minimum growth temperature of an organism, the residual concentrations of both N sources increased rapidly. The
maximum concentrations (80 µM, equivalent to the starting N
concentration in the medium) were observed at temperatures at which
growth ceased.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 1.
Residual concentrations of nitrate ( ) and ammonium
( ) in batch cultures of three bacterial isolates grown at a range of
temperatures. The arrows indicate the optimum growth temperature for
each of the isolates.
|
|
µ
max values exhibited considerable interspecies
variation. Psychrotolerant bacteria (
Hydrogenophaga
pseudoflava,
Brevibacterium sp.,
Vibrio
logei) had lower µ
max values over their growth
temperature
ranges than meso- and thermophilic bacteria
(
Klebsiella oxytoca,
Escherichia coli,
Bacillus stearothermophilus) had. The flagellate
alga
Dunaliella tertiolecta had µ
max values that
were significantly
higher than the µ
max values obtained
for the diatoms
C. curvisetum and
Chaetoceros sp.
The µ
max values for all of the bacterial and
algal
isolates were significantly dependent on temperature (
P < 0.05, as determined by LRA). We observed no significant difference
(
P > 0.2, as determined by ANOVA) between the
µ
max values for
nitrate-limited and ammonium-limited
batch cultures of the isolates
grown on both
substrates.
Chemostat cultures.
There were no significant differences
between the µmax values measured in batch cultures and
the µmax values measured in chemostat cultures for any of
the bacterial or algal cultures, as determined by ANOVA (P > 0.5). The steady-state nitrate concentrations exhibited a
significant negative correlation with temperature for all of the
isolates (Tables 3 and
4), which reflected the substantial increases in the residual nitrate concentrations that occurred with
decreasing temperature in batch cultures. The half-saturation constant
for nitrate (Knit) increased as the incubation
temperature decreased for most of the bacterial and algal isolates,
although no consistent trend was identified for E. coli or
B. stearothermophilus. The half-saturation constant for
ammonium (Kamm) did not consistently increase as
the temperature decreased for any isolate.
The specific affinity for nitrate (
anito)
values calculated for the temperatures by using the relevant
µ
max and
Ks values consistently
decreased as the temperature decreased. An LRA showed that there
was a
significant linear relationship between
anito and temperature for all isolates
(
P < 0.05, as determined by
LRA), although there were
large interspecies variations among
anito
values. The response of
anito to temperature
change was greater in the psychrotolerant species
(whose
Q
10 values ranged from 3 to 5, where Q
10 is the
proportional
change with a 10°C temperature increase) than in the
thermotolerant
species (whose Q
10 values ranged from 1.8 to
2.5). The specific
affinity for ammonium
(
aammo) values also varied greatly among
species but generally showed
little temperature dependence (the
Q
10 values for individual species
ranged from 0.75 to 2.1).
Only one isolate (a
Brevibacterium sp.
isolate) showed any
significant dependence on temperature (
P <
0.05, as
determined by LRA; Q
10,
2). It must be noted that in
some cases steady-state concentrations of ammonium approached
the limit
of detection, and the specific affinity values in such
cases must be
regarded as lower
limits.
To illustrate the consistent response of
aAo
to temperature change,
aAo data for nitrate
and ammonium were normalized to express the
aAo value at a given temperature as a
percentage of the value at
15°C (Fig.
2). (The psychrophilic diatom
Chaetoceros sp. and the
thermophilic bacterium
B. stearothermophilus did not grow at 15°C,
so these organisms were
omitted from this comparison.) The normalized
plot showed that there
was significant dependence of
anito on
temperature (
P < 0.001, as determined by LRA) but no
significant
temperature dependence of
aammo
(
P = 0.73, as determined by LRA). The Q
10
values for normalized
aAo were 2.9 ± 0.43 (mean ± standard error;
n = 21) and
0.98 ± 0.6
(
n = 25) for nitrate and ammonium,
respectively. These normalized
data again indicated that the
temperature response of affinity
for nitrate was consistently greater
than the temperature response
of affinity for ammonium in all of the
bacterial and algal isolates
examined.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 2.
Plot of normalized (to 15°C)
aammo and anito
values versus incubation temperature for a range bacteria and
microalgae. The dashed line is the first-order linear regression line
through normalized ammonium data (n = 25;
r2 = 0.01; P = 0.73). The solid line is
the first-order linear regression line through normalized nitrate data
(n = 21; r2 = 0.71; P <0.001). The
dotted lines are the 95% confidence limits for the regression lines.
|
|
| |
DISCUSSION |
Relationship between affinity and temperature.
The mechanisms
that limit growth at high temperatures (protein denaturation, etc.) are
well-documented (16, 64), but the mechanisms responsible for
limiting µ at low temperatures are still a source of contention. Our
data provide consistent evidence that growth of both bacteria and
marine algae under N-limited conditions at low temperatures is
restricted by the reduced ability of the organisms to sequester
inorganic nitrogen. (A similar decreased affinity for uptake of organic
substrates at low temperatures has been reported previously
[51].) Although inhibition of membrane transport at
low temperatures that leads to growth limitation has been suggested
before (35, 49), consistent evidence which supports the
hypothesis has not been presented previously.
In the N-limited batch cultures that were grown at or near the optimum
growth temperatures of species, the residual concentrations
of both
nitrate and ammonium were below the analytical limits,
indicating that
utilization of both N sources is most effective
at these temperatures.
However, at suboptimal temperatures the
increases in the residual
concentrations of nitrate and ammonium
during the stationary phase
illustrated that bacteria and algae
became less able to take up
inorganic nitrogen as the temperature
decreased, whether the isolate
was psychrophilic, mesophilic,
or thermophilic. This implied that the
efficiency of the uptake
system for both nitrate and ammonium decreased
with temperature
and so reduced the ability of the organisms to
sequester inorganic
nitrogen from the surrounding medium
(
55). However, the greater
effect of suboptimal temperatures
on residual nitrate concentrations
than on residual ammonium
concentrations indicated that low temperatures
had different effects on
nitrate utilization and ammonium
utilization.
The µ
max values varied with temperature, as reported
previously for N-limited cultures (
62,
73). The
generally lower µ
max values for the psychrotrophic
and psychrophilic bacteria (
H. pseudoflava,
Brevibacterium sp.) than for mesotrophic isolates (e.g.,
K. oxytoca and
E. coli) and the greater
µ
max values for the microflagellate
alga
D. tertiolecta than for the two diatoms are both previously
identified trends (
7).
Nitrate uptake and temperature.
An examination of the measured
Ks values indicated that affinity for nitrate
was reduced to a greater extent by low temperature than affinity for
ammonium was, but when Ks was used alone as a
measure of affinity, this trend was by no means consistent. Such
inconsistency is common when Ks alone is used as
a measure of substrate affinity (15, 48). Such use of
Ks has been criticized as it does not adequately
reflect changes in the ability of cells to sequester substrate at low
concentrations (8), which depends on both
Ks and µmax. Alternatively,
aAo, approximated by
µmax/Ks, has been employed
(9, 25, 30, 40, 51). aAo provides
an unambiguous measure of the ability of an organism to sequester
substrate at low concentrations and is independent of uptake mechanism
(26). Measurements of aAo,
therefore, allow examination of the effect of temperature on affinity
for a substrate and identify temperature effects on uptake alone
(8). Using aAo, we demonstrated
for the first time that a temperature below the optimum growth
temperature consistently resulted in decreased affinity for nitrate in
a number of bacteria and marine algae. This trend was consistent for
psychrophilic, mesophilic, and thermophilic species despite the
different growth temperature ranges of the organisms. These
findings suggest that there is a common mechanism that is responsible
for the decrease in affinity for nitrate that occurs as the temperature decreases.
Our
Ks values for the algae were high compared
to the few other values reported previously for algal chemostat
cultures (
7).
Many of the
Ks values
reported previously for algal growth were
determined with fed-batch
cultures (
17,
44,
81) and cannot
be compared with our data
because they are
Ks values for uptake
rather
than
Ks values for growth and do not represent
steady-state
conditions (
25). It must also be asked whether
Ks values by
themselves provide any useful
information about affinity for a
substrate, which might be why coherent
trends of
aAo with temperature are not seen
with
Ks alone. The
anito values that we determined for algae
are similar to the few other
values that are available in the
literature with comparable units
(we have not been able to find any
such values for bacteria).
For example, our values for
anito near the optimal growth temperatures
for
D. tertiolecta (0.004
µmol liter
1
h
1 at 25°C),
C. curvisetum (0.0165 µmol
liter
1 h
1 at 22°C), and
Chaetoceros sp. (0.013 µmol liter
1
h
1 at 5°C) are similar to the values reported for
Scenedesmus sp.
(0.02 µmol liter
1
h
1 at 20°C [
62]) and
Chaetoceros
neglectum (0.015 µmol
1 liter
1
h
1 at 0°C [
69]).
Despite the interspecies variations in actual
anito values, the Q
10 values
were similar (mean, 3.1; standard error, ±0.48;
n = 8), and this finding corroborated the idea that the temperature
responses for nitrate uptake by algae and bacteria are similar.
Although no previously published Q
10 values for
anito were found, our Q
10 value
for
anito normalized to 15°C agrees well
with the relatively high Q
10 values
reported for nitrate
uptake by microorganisms (
39,
59,
74,
75), a feature which
is characteristic of active, carrier-mediated
transport systems
(
39). An important consequence of the decrease
in affinity
for nitrate that occurs as the temperature decreases
is the fact that
at a low temperature there is an increase in
the growth rate-limiting
concentration of nitrate (
55) and therefore
an increased
degree of nitrate limitation imposed by the low temperature.
In other
words, at a low temperature there is exacerbation of
nitrate limitation
for both algae and bacteria. A corollary of
this is the hypothesis that
addition of more nitrate should reverse
the nitrate limitation imposed
by low affinity at a low temperature,
as demonstrated for organic
substrates by Wiebe et al. (
83).
This implies that nutrient
limitation bioassays must be carried
out at the in situ temperature or
the effective availability of
external substrate pools may
change.
The temperature dependence of nitrate use agrees with what is known
about the nitrate uptake system. Nitrate is apparently
taken up by an
active transport system in bacteria, algae, and
higher plants, and this
transport system appears to be ATP driven
rather than directly
dependent on an electrochemical gradient
(
12,
18,
71,
79).
There is additional evidence that active
nitrate uptake is strictly
Na
+ dependent (
39), is highly sensitive to
metabolic inhibitors
(
78), and may be strongly inhibited by
the presence of ammonium
(
13,
39). Such active uptake
systems can be very responsive
to temperature changes (
66).
Although the temperature dependence
of nitrate uptake has been
demonstrated most clearly in higher
plants (
22,
42), it
seems increasingly apparent from our studies
and other studies
(
39,
41,
74) that nitrate uptake by bacteria
and microalgae
is also highly temperature dependent. Furthermore,
uptake of other
inorganic algal nutrients which are primarily
sequestered by active
transport is also likely to be adversely
affected by low temperatures
because of decreased
affinity.
The data in the literature for µ
max and
Ks values in chemostats at different
temperatures is extremely restricted, but the
data which is available
tends to support our paradigm. Uptake
of nitrate at different
temperatures in nitrate-limited cultures
of
Scenedesmus sp.
(
62), uptake of phosphate in phosphate-limited
cultures of
Scenedesmus sp. (
1), and silicate uptake in
silicate-limited
cultures of the ice alga
Pseudonitzschia
seriata (
70) all indicate
that decreases in affinity
occur at temperatures below the optimum
temperature when affinity is
measured by determining
aAo. It has been
pointed out (
5) that phosphate, a nutrient primarily
acquired through active uptake (
4), is not utilized
efficiently
in cold high-latitude
waters.
Ammonium uptake, N preference, and temperature.
The more
constant concentrations of ammonium than of nitrate in steady-state
chemostats at a range of incubation temperatures indicated that
ammonium uptake is less temperature dependent than nitrate uptake is.
The fact that low temperature has a greater inhibitory effect on
nitrate uptake than on ammonium uptake has been documented previously
in higher plant roots by several workers (42, 43), but until
now this difference has not been established for phytoplankton and
bacteria (53). The fact that the response of affinity for
ammonium to decreased temperature is consistently less than the
response of affinity for nitrate in bacteria, algae, and higher plants
suggests that there are fundamental differences in the ammonium and
nitrate uptake mechanisms across a broad phylogenetic range. This is
not surprising since the differing biochemical requirements for
assimilation of nitrate compared with that of ammonium apply to all
organisms and are therefore likely to be evolutionarily highly conserved.
The low Q
10 value (
1) of normalized
aammo agrees with data from previous studies
of ammonium uptake in higher plants (
42,
43). However,
higher Q
10 values have been reported for ammonium
uptake by
phytoplankton in the field (
56,
68). The low Q
10 values found for
aammo are characteristic of
channel-mediated ion fluxes (
66), but
the exact mechanisms
involved in ammonium uptake and control of
uptake are poorly
understood. In the last 20 years several uptake
pathways, including
pathways for both active and passive uptake
of ammonium, have been
suggested (
2,
12,
34,
38,
79,
80). Ammonia (NH
3)
can diffuse freely through cell membranes
(
32), but at
neutral pH >99% of NH
3 is protonated as
NH
4+ (
38). In slightly
alkaline environments, such as marine systems,
the proportion of
NH
3 may increase to >10% of the total ammonium
(NH
3 and NH
4+ combined)
(
67). The concentration gradient of NH
3 across a
cell membrane may be maintained by NH
3 protonation within
the
cell and by equilibration between ammonium and
NH
3 outside the
cell (
31). Passive uptake of
NH
3 may therefore make a significant
contribution to
the N requirements of bacteria and algae (particularly
organisms
with large surface area/volume ratios). Several studies
have
confirmed that there is a preference for, or selection by,
small
phytoplankton for ammonium (
28,
41,
57). In a
comparison
of ammonium preference in diatoms, dinoflagellates,
cyanobacteria,
chlorophytes, and other organisms (
13), it
was found that the
greatest contrast was between the diatoms and the
other organism
category, which consisted mainly of small flagellates.
The microflagellate
preference for ammonium was much
greater than the preference in
diatoms, while the larger diatoms showed
greater preference for
nitrate (
45,
54).
Low temperature probably affects nutrient uptake by causing alterations
in physical characteristics of the cell membrane.
Such changes
associated with low temperature may control active
nutrient uptake
across cell membranes in several ways (
10,
58).
Low
temperature may hinder conformational changes in membrane
transport
proteins and thus prevent solute molecules from combining
with their
carrier proteins, or it may result in reduction in
the substrate supply
to a transport protein and inactivation of
carrier proteins. Low
temperature may also result in a reduction
in membrane fluidity; it is
known that reductions in membrane
fluidity decrease the activity of
transporter and respiratory
proteins (
3,
20,
76) embedded in
the membrane phospholipids,
and indeed the embedded proteins may
reciprocally influence the
membrane fluidity (
47). Thus,
while different species may adapt
their membranes to be functional over
different ranges of temperature
by changing the ratios of saturated,
unsaturated, or branched-chain
membrane lipids (see references
63 and
65 for reviews), we
hypothesize that within the range of temperature for each species
there
is decreased affinity for substrates taken up by active
transport as
the temperature decreases below the optimum temperature
for growth
(
50). As passive uptake is less affected by temperature
than
active uptake is (
38), any significant passive component
should make overall ammonium sequestration less dependent on
temperature
than active nitrate uptake
is.
Nitrogen nutrition at low temperature and its ecological
implications.
We demonstrated that a reduction in temperature
results in reduced affinity for nitrate and decreased utilization in
several algal and bacterial isolates representing a wide range of
physiological types. Ammonium uptake and affinity in the same isolates
did not appear to be affected by a reduction in temperature to the same extent. This suggests that in low-temperature aquatic plankton ecosystems, microbial nitrogen utilization tends to be biased away from
nitrate and that ammonium is a more important substrate.
In the Southern Ocean, surface water temperatures reach as low as
1.8°C, and the maximum summer temperature is around 4.0°C
(
33). In such a low-temperature environment, we would expect
that nitrate uptake by phytoplankton would be reduced and that
ammonium
would be increasingly important as a nitrogen source.
This hypothesis
is consistent with the generally low
f ratios,
0.2 to 0.6 (
13,
53), reported for the Southern Ocean (the
f
ratio is the uptake of nitrate expressed as a proportion of
the total
inorganic nitrogen uptake). The low
f ratios occur despite
nitrate concentrations which are often more than 40 times the
ammonium
concentrations (
11). The importance of ammonium may
be
further increased by the competitive effects of ammonium and
nitrate;
even relatively low concentrations of ammonium may inhibit
nitrate
uptake by microorganisms (
13,
24,
82).
The temperature dependence of nitrogen preference in microbial
plankton, especially in marine phytoplankton, could have far-reaching
implications for biogeochemical nutrient cycling on a global scale.
Several large regions of the World Ocean have been characterized
as
high-nutrient-low-chlorophyll regions, where annual primary
production
is too low to exhaust the supply of inorganic nutrients.
Any changes in
the environmental controls which result in suboptimal
use of nitrate in
these regions could result in major alterations
in the pattern of
primary production and thus accumulation of
organic
material.
| |
ACKNOWLEDGMENTS |
We thank Peter J. le B. Williams of the University of Bangor for
his constructive comments on early drafts of the manuscript.
This work was carried out during research studentship GT4/94/339/L from
the Natural Environment Research Council, United Kingdom, to David S. Reay. This studentship was CASE funded in conjunction with the British
Antarctic Survey, Cambridge, United Kingdom.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 35Q, United Kingdom. Phone: 01206 872818. Fax: 01206 873416. E-mail: dsreay{at}essex.ac.uk.
| |
REFERENCES |
| 1.
|
Ahlgren, G.
1987.
Temperature functions in biology and their application to algal growth constants.
Oikos
49:177-190.
|
| 2.
|
Balch, W. M.
1986.
Exploring the mechanism of ammonium uptake in phytoplankton with an ammonium analogue, methylamine.
Mar. Biol.
92:163-171.
|
| 3.
|
Baldassare, J.,
G. Brenckle,
M. Hoffman, and D. Silbert.
1977.
Modification of membrane lipid: functional properties of membrane in relation to fatty acid structure.
J. Biol. Chem.
252:8797-8803[Free Full Text].
|
| 4.
|
Bieleski, R. L., and I. B. Ferguson.
1983.
Physiology and metabolism of phosphate and its compounds, p. 422-445.
In
A. Läuacli, and R. L. Bieleski (ed.), Inorganic plant nutrition, new series. Springer-Verlag, Berlin, Germany.
|
| 5.
|
Broecker, W., and T.-H. Peng.
1993.
What caused the glacial to intergalcial CO2 change?, p. 95-115.
In
M. Heimann (ed.), The global carbon cycle. Springer-Verlag, Berlin, Germany.
|
| 6.
|
Butcher, R. W.
1959.
Plymouth algal collection no. 83.
Fish. Invest. London Ser.
4:22-23.
|
| 7.
|
Button, D. K.
1985.
Kinetics of nutrient-limited transport and microbial growth.
Microb. Rev.
49:270-297[Free Full Text].
|
| 8.
|
Button, D. K.
1993.
Nutrient-limited microbial growth kinetics: overview and recent advances.
Antonie Leeuwenhoek
63:225-235.
|
| 9.
|
Button, D. K. A.
1986.
Affinity of organisms for substrate.
Limnol. Oceanogr.
31:453-456.
|
| 10.
|
Clarkson, D. T.,
M. J. Earnshaw,
P. J. White, and H. D. Cooper.
1988.
Temperature dependent factors influencing nutrient uptake: an analysis of responses at different levels of organisation.
Symp. Soc. Exp. Biol.
42:281-309[Medline].
|
| 11.
|
Cota, G. F.,
W. O. Smith,
D. M. Nelson,
R. D. Muench, and L. I. Gordon.
1992.
Nutrient and biogenic particulate distributions, primary productivity and nitrogen uptake in the Weddell-Scotia Sea marginal ice zone during winter.
J. Mar. Res.
50:155-181.
|
| 12.
|
Cruz, C.,
S. H. Lips, and M. A. Martins-Loução.
1993.
Uptake of ammonium and nitrate by carob (Ceratonia siliqua) as affected by root temperature and inhibitors.
Physiol. Plant.
89:532-543.
|
| 13.
|
Dortch, Q.
1990.
The interaction between ammonium and nitrate uptake in phytoplankton.
Mar. Ecol. Prog. Ser.
61:183-201.
|
| 14.
|
Dugdale, R. C., and J. J. Goering.
1967.
Uptake of new and regenerated forms of nitrogen in primary productivity.
Limnol. Oceanogr.
12:196-206.
|
| 15.
|
Ellis-Evans, J., and D. Wynn-Williams.
1985.
The interaction of soil and lake microflora at Signy Island, p. 662-668.
In
W. Siegfried, P. Condy, and R. Laws (ed.), Antarctic nutrient cycles. Springer-Verlag, Berlin, Germany.
|
| 16.
|
Eppley, R. W.
1972.
Temperature and phytoplankton growth in the sea.
Fish. Bull. (Dublin)
70:1063-1085.
|
| 17.
|
Eppley, R. W.,
J. N. Rogers, and J. J. McCarthy.
1969.
Half-saturation constants for uptake of nitrate and ammonium by marine phytoplankton.
Limnol. Oceanogr.
14:912-920.
|
| 18.
|
Falkowski, P. G.
1975.
Nitrate uptake in marine phytoplankton (nitrate, chloride) activated adenosine triphosphate from Skeletonema costatum (Bacillariophyceae).
J. Phycol.
11:323-326.
|
| 19.
|
Flynn, K. J.,
M. J. R. Fasham, and C. R. Hipkin.
1997.
Modelling the interactions between ammonium and nitrate uptake in marine phytoplankton.
Phil. Trans. R. Soc. London B Biol. Sci.
352:1-22.
|
| 20.
|
Foot, M.,
R. Jeffcoat,
M. Barratt, and N. Russell.
1983.
The effect of growth temperature on the membrane lipid environment of the psychrophilic bacterium Micrococcus cryophilus.
Arch. Biochem. Biophys.
224:718-727[Medline].
|
| 21.
|
Fry, J. C.
1993.
Biological data analysis: a practical approach.
IRL Press, Oxford, United Kingdom.
|
| 22.
|
Glass, A. D. M.,
M. Y. Siddiqim,
T. J. Ruth, and T. W. J. Rufty.
1990.
Studies in the uptake of nitrate in barley. II. Energetics.
Plant Physiol.
93:1585-1589[Abstract/Free Full Text].
|
| 23.
|
Glibert, P. M.
1988.
Primary productivity and pelagic nitrogen cycling, p. 3-31.
In
T. H. Blackburn, and J. Sørensen (ed.), Nitrogen cycling in coastal marine environments. John Wiley and Sons Ltd., New York, N.Y.
|
| 24.
|
Glibert, P. M.,
D. C. Biggs, and J. J. McCarthy.
1982.
Utilization of ammonium and nitrate during austral summer in the Scotia Sea.
Deep Sea Res.
29:837-850.
|
| 25.
|
Goldman, J. C., and P. M. Glibert.
1983.
Kinetics of inorganic nitrogen uptake, p. 233-275.
In
E. J. Carpenter, and D. G. Capone (ed.), Nitrogen in the marine environment. Academic Press, New York, N.Y.
|
| 26.
|
Gottschal, J. C.
1985.
Some reflections on microbial competitiveness among heterotrophic bacteria.
Antonie Leeuwenhoek
51:473-494.
|
| 27.
|
Guillard, R. R. L., and J. H. Ryther.
1962.
Studies of marine planktonic diatoms. I. Cyclotella nana (Hustedt) and Detonula confervacla (Cleve) Gran.
Can. J. Microbiol.
8:229-239[Medline].
|
| 28.
|
Harrison, W. G.
1983.
The time-course of uptake of inorganic and organic nitrogen compounds by phytoplankton from the Eastern Canadian Arctic: a comparison of temperate and tropical populations.
Limnol. Oceanogr.
28:1231-1237.
|
| 29.
|
Harwood, J. E., and A. L. Kuhn.
1970.
A colorimetric method for ammonia in natural waters.
Water Res.
4:805-811.
|
| 30.
|
Healey, F. P.
1980.
Slope of the Monod equation as an indicator of advantage in nutrient competition.
Microb. Ecol.
5:281-286.
|
| 31.
|
Henderson, P. J. F.
1971.
Ion transport by energy-conserving biological membranes.
Annu. Rev. Microbiol.
25:393-428[Medline].
|
| 32.
|
Henderson, P. J. F.
1991.
Studies of translocation analysis.
Biosci. Rep.
11:477-525[Medline].
|
| 33.
|
Herbert, R. A., and M. Bhakoo.
1979.
Microbial growth at low temperatures, p. 1-16.
In
A. D. Russell, and R. Fuller (ed.), Cold tolerant microbes in spoilage and the environment. Academic Press, London, United Kingdom.
|
| 34.
|
Hoch, M. P.,
M. L. Fogel, and D. L. Kirchman.
1992.
Isotope fractionation associated with ammonium uptake by a marine bacterium.
Limnol. Oceanogr.
37:1447-1459.
|
| 35.
|
Ingraham, J. L., and G. F. Bailey.
1959.
Comparative effect of temperature on metabolism of mesophilic and psychrophilic bacteria.
J. Bacteriol.
77:609-613[Free Full Text].
|
| 36.
|
Jacques, G.
1983.
Some ecophysiological aspects of Antarctic phytoplankton.
Polar Biol.
2:27-33.
|
| 37.
|
Kirkwood, D. S.
1996.
Nutrients: practical notes on their determination in seawater.
ICES, Copenhagen, Denmark.
|
| 38.
|
Kleiner, D.
1981.
The transport of NH3 and NH4+ across biological membranes.
Biochim. Biophys. Acta
639:41-52[Medline].
|
| 39.
|
Lara, C.,
R. Rodriguez, and M. G. Guerrero.
1993.
Nitrate transport in the cyanobacterium Anacystis nidulans.
Physiol. Plant.
89:582-587.
|
| 40.
|
Law, A. T., and D. K. Button.
1977.
Multiple-carbon-source-limited growth kinetics of a marine coryneform bacterium.
J. Bacteriol.
129:115-123[Abstract/Free Full Text].
|
| 41.
|
Le Bouteiller, A.
1986.
Environmental control of nitrate and ammonium uptake by phytoplankton in the Equatorial Atlantic Ocean.
Mar. Ecol. Prog. Ser.
30:167-179.
|
| 42.
|
Macduff, J. H.,
M. J. Hopper, and A. Wild.
1987.
The effect of root temperature on growth and uptake of ammonium and nitrate by Brassica napus L. cv. Bien venu in flowing solution culture.
J. Exp. Bot.
38:53-66[Abstract/Free Full Text].
|
| 43.
|
Macduff, J. H.,
M. J. Hopper,
A. Wild, and F. E. Trim.
1987.
Comparison of the effects of root temperature on nitrate and ammonium nutrition of oilseed rape (Brassica napus L.) in flowing solution culture.
J. Exp. Bot.
38:1104-1120[Abstract/Free Full Text].
|
| 44.
|
MacIsaac, J. J., and R. C. Dugdale.
1972.
Interactions of light and inorganic nitrogen in controlling nitrogen uptake in the sea.
Deep Sea Res.
19:209-232.
|
| 45.
|
Malone, T. C.
1980.
Algal size, p. 433-464.
In
I. Morris (ed.), The physiological ecology of phytoplankton. Blackwell, London, United Kingdom.
|
| 46.
|
McCarthy, J. J., and E. J. Carpenter.
1983.
Nitrogen cycling in near-surface waters of the open ocean, p. 487-512.
In
E. J. Carpenter, and D. G. Capone (ed.), Nitrogen in the marine environment. Academic Press, New York, N.Y.
|
| 47.
|
McGibbon, L.,
A. Cossins,
P. Quinn, and N. Russell.
1985.
A differential scanning calorimetry and fluorescence polarisation study of membrane lipid fluidity in a psychrophilic bacterium.
Biochim. Biophys. Acta
820:115-121.
|
| 48.
|
Mechling, J. A., and S. S. Kilham.
1983.
Temperature effects on silicon limited growth of the Lake Michigan diatom Stephanodiscus minultus (Bacillariophyceae).
J. Phycol.
18:119-205.
|
| 49.
|
Morita, R. Y., and G. E. Buck.
1974.
Low temperature inhibition of substrate uptake, p. 124-129.
In
R. R. Colwell, and R. Y. Morita (ed.), Effects of the ocean environment on microbial activities. University Park Press, Baltimore, Md.
|
| 50.
| Nedwell, D. Effect of low temperature on microbial
growth: lowered affinity for substrates limits growth at low
temperature. Submitted for publication.
|
| 51.
|
Nedwell, D. B., and M. Rutter.
1994.
Influence of temperature on growth rate and competition between two psychrotolerant Antarctic bacteria: low temperature diminishes affinity for substrate uptake.
Appl. Environ. Microbiol.
60:1984-1992[Abstract/Free Full Text].
|
| 52.
|
Ogilvie, B. G.,
M. Rutter, and D. B. Nedwell.
1997.
Selection by temperature of nitrate-reducing bacteria from estuarine sediments: species composition and competition for nitrate.
FEMS Microb. Ecol.
23:11-22.
|
| 53.
|
Olson, R. J.
1980.
Nitrate and ammonium uptake in Antarctic waters.
Limnol. Oceanogr.
25:1064-1074.
|
| 54.
|
Owens, N. J. P.,
J. Priddle, and M. J. Whitehouse.
1991.
Variations in phytoplanktonic nitrogen assimilation around South Georgia and in the Bransfield Strait (Southern Ocean).
Mar. Chem.
35:287-304.
|
| 55.
|
Pirt, S. J.
1975.
Principles of microbe and cell cultivation.
Blackwell Scientific Publications, Oxford, United Kingdom.
|
| 56.
|
Priscu, J. C.,
A. C. Palmisano,
L. R. Priscu, and C. W. Sullivan.
1989.
Temperature dependence of inorganic nitrogen uptake and assimilation in Antarctic sea-ice microalgae.
Polar Biol.
9:443-446.
|
| 57.
|
Probyn, T. A.
1985.
Nitrogen uptake by size-fractionated phytoplankton populations in the southern Benguela upwelling system.
Mar. Ecol. Prog. Ser.
22:249-258.
|
| 58.
|
Quinn, P. J.
1988.
Effects of temperature on cell membranes, p. 237-258.
In
S. P. Long, and F. I. Woodward (ed.), Plants and temperature. Company of Biologists, Cambridge, United Kingdom.
|
| 59.
|
Raimbault, P.
1984.
Influence of temperature on transient-response in nitrate uptake and reduction by 4 marine diatoms.
J. Exp. Mar. Biol. Ecol.
84:37-53.
|
| 60.
|
Raven, J. A.,
B. Wolenweber, and L. L. Handley.
1992.
Ammonia and ammonium fluxes between photolithotrophs and the environment in relation to the global nitrogen cycle.
New Phytol.
121:5-18.
|
| 61.
|
Raven, J. A.,
B. Wollenweber, and L. L. Handley.
1993.
The quantitative role of ammonia/ammonium transport and metabolism by plants in the global nitrogen cycle.
Physiol. Plant.
89:512-518.
|
| 62.
|
Rhee, G.-Y., and I. J. Gotham.
1981.
The effect of environmental factors on phytoplankton growth: temperature and the interactions of temperature with nutrient limitation.
Limnol. Oceanogr.
26:635-648.
|
| 63.
|
Russell, N.
1992.
Psychrophilic microorganisms, p. 203-224.
In
R. Herbert, and R. Sharp (ed.), Molecular biology and biotechnology of extremophiles. Blackie, Glasgow, United Kingdom.
|
| 64.
|
Russell, N. J.
1990.
Cold adaptation of microorganisms.
Phil. Trans. R. Soc. London B Biol. Sci.
326:595-611.
|
| 65.
|
Russell, N. J., and N. Fukunaga.
1990.
A comparison of thermal adaptation of membrane lipids in psychrophilic and thermophilic bacteria.
FEMS Microbiol. Rev.
75:171-182.
|
| 66.
|
Serrano, R.
1991.
Transport across yeast vacuolar and plasma membranes, p. 523-585.
In
J. R. Broach, J. R. Pringle, and E. W. Jones (ed.), The molecular and cellular biology of the yeast Saccaromyces: genome dynamics, protein synthesis, and energetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 67.
|
Sillen, L. C., and A. E. Martell.
1964.
Stability constants of metal ion complexes.
Chemical Society, London, England.
|
| 68.
|
Smith, W. O., and W. G. Harrison.
1991.
New production in polar regions: the role of environmental controls.
Deep Sea Res.
38:1463-1479.
|
| 69.
|
Sommer, U.
1986.
Nitrate and silicate competition among Antarctic phytoplankton.
Mar. Biol.
91:345-351.
|
| 70.
|
Stapleford, L. S., and R. E. H. Smith.
1996.
The interactive effects of temperature and silicon limitation on the psychrophilic ice diatom Pseudonitszchia seriata.
Polar Biol.
16:589-594.
|
| 71.
|
Stewart, W. D. P.
1980.
Transport and utilization of nitrogen by algae, p. 577-606.
In
J. W. Payne (ed.), Microorganisms and nitrogen sources. Wiley and Sons Ltd., New York, N.Y.
|
| 72.
|
Strickland, J. D. H., and T. R. Parsons.
1972.
A practical handbook of seawater analysis.
Fisheries Research Board of Canada, Ottawa, Canada.
|
| 73.
|
Thomas, W. H., and A. N. Dodson.
1974.
Effect of interactions between temperature and nitrate supply on the cell division rates of two marine phytoflagellates.
Mar. Biol.
24:213-217.
|
| 74.
|
Tischner, R.
1984.
Interaction between chloroplast-cytoplasm vacuoles with respect to the regulation of nitrogen metabolism in Chlorella, p. 157-163.
In
W. Weissner, D. Robinson, and R. C. Starr (ed.), Compartments in algal cells and their interaction. Springer-Verlag, Berlin, Germany.
|
| 75.
|
Tischner, R., and H. Lorenzen.
1981.
Nitrate uptake and reduction in Chlorella. Characterisation of nitrate uptake in nitrate grown and nitrogen-starved Chlorella sorokiniana, p. 252-259.
In
H. Bothe, and A. Trebst (ed.), Biology of inorganic nitrogen and sulfur. Springer-Verlag, Berlin, Germany.
|
| 76.
|
Tsukagoshi, N., and C. Fox.
1973.
Transport system assembly and the mobility of membrane lipids in Escherichia coli.
Biochemistry
12:2822-2829[Medline].
|
| 77.
|
Upton, A. C.,
D. B. Nedwell, and D. D. Wynn-Williams.
1990.
The selection of microbial communities by constant or fluctuating temperatures.
FEMS Microbiol. Ecol.
74:243-252.
|
| 78.
|
Watt, D. A.,
A. M. Amory, and C. F. Cresswell.
1993.
Constitutive and inducible aspects of nitrate-nitrogen uptake by Chlamydomonas reinhardtii.
Physiol. Plant.
89:507-511.
|
| 79.
|
Wheeler, P. A.
1983.
Phytoplankton nitrogen metabolism, p. 309-346.
In
E. J. Carpenter, and D. G. Capone (ed.), Nitrogen in the marine environment. Academic Press, New York, N.Y.
|
| 80.
|
Wheeler, P. A.
1979.
Uptake of methylamine (an ammonium analogue) by Macrocystis pyrifera (Phaeophyta).
J. Phycol.
15:12-17.
|
| 81.
|
Wheeler, P. A.,
P. M. Glibert, and J. J. McCarthy.
1982.
Ammonium uptake and incorporation by Chesapeake Bay phytoplankton: short term uptake kinetics.
Limnol. Oceanogr.
27:1113-1128.
|
| 82.
|
Wheeler, P. A., and S. A. Kokkinakis.
1990.
Ammonium recycling limits nitrate use in oceanic subarctic Pacific.
Limnol. Oceanogr.
35:1267-1278.
|
| 83.
|
Wiebe, W. J.,
W. M. J. Sheldon, and L. R. Pomeroy.
1993.
Evidence for an enhanced substrate requirement by marine mesophilic isolates at minimal growth temperatures.
Microb. Ecol.
25:151-159.
|
Applied and Environmental Microbiology, June 1999, p. 2577-2584, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Tadonleke, R. D., Lazzarotto, J., Anneville, O., Druart, J.-C.
(2009). Phytoplankton productivity increased in Lake Geneva despite phosphorus loading reduction. J PLANKTON RES
31: 1179-1194
[Abstract]
[Full Text]
-
Hernandez-Leon, S., Fraga, C., Ikeda, T.
(2008). A global estimation of mesozooplankton ammonium excretion in the open ocean. J PLANKTON RES
30: 577-585
[Abstract]
[Full Text]
-
Koschorreck, M., Tittel, J.
(2007). Natural Alkalinity Generation in Neutral Lakes Affected by Acid Mine Drainage. J. Environ. Qual.
36: 1163-1171
[Abstract]
[Full Text]
-
Morgan-Kiss, R. M., Priscu, J. C., Pocock, T., Gudynaite-Savitch, L., Huner, N. P. A.
(2006). Adaptation and Acclimation of Photosynthetic Microorganisms to Permanently Cold Environments. Microbiol. Mol. Biol. Rev.
70: 222-252
[Abstract]
[Full Text]
-
Weston, K., Fernand, L., Mills, D. K., Delahunty, R., Brown, J.
(2005). Primary production in the deep chlorophyll maximum of the central North Sea. J PLANKTON RES
27: 909-922
[Abstract]
[Full Text]
-
Kaartokallio, H., Laamanen, M., Sivonen, K.
(2005). Responses of Baltic Sea Ice and Open-Water Natural Bacterial Communities to Salinity Change. Appl. Environ. Microbiol.
71: 4364-4371
[Abstract]
[Full Text]
-
Thomas, D. N., Dieckmann, G. S.
(2002). Antarctic Sea Ice--a Habitat for Extremophiles. Science
295: 641-644
[Abstract]
[Full Text]
-
McMinn, A., Bleakley, N., Steinburner, K., Roberts, D., Trenerry, L.
(2000). Effect of permanent sea ice cover and different nutrient regimes on the phytoplankton succession of fjords of the Vestfold Hills Oasis, eastern Antarctica. J PLANKTON RES
22: 287-303
[Abstract]
[Full Text]
Top
Abstract
Introduction
