RESEARCH ARTICLE
Adaptive phosphate uptake behaviour of phytoplankton to
environmental phosphate £uctuations
Luis Aubriot1, Sylvia Bonilla1 & Gernot Falkner2
1
Phytoplankton Ecology and Physiology Group, Limnology Division, Faculty of Sciences, Universidad de la República, Montevideo, Uruguay; and 2Cell
Biology Department, Plant Physiology Division, University of Salzburg, Salzburg, Austria
Correspondence: Luis Aubriot, Iguá 4225,
Sección Limnologı́a, Facultad de Ciencias,
Universidad de la República,
Montevideo11400, Uruguay. Tel.: 1598 2
525 86 19, ext. 7149; fax: 1598 2 525 86 17;
e-mail: [email protected]
Received 10 June 2010; revised 13 February
2011; accepted 21 February 2011.
Final version published online 17 March 2011.
DOI:10.1111/j.1574-6941.2011.01078.x
MICROBIOLOGY ECOLOGY
Editor: Patricia Sobecky
Keywords
phosphate uptake; physiological adaptation;
phytoplankton; cyanobacteria; information
processing; coexistence.
Abstract
When phytoplankton growth in lakes is limited by the available phosphate, the
external phosphate concentration fluctuates around a threshold value at which
available energy is insufficient to drive phosphate incorporation into a polyphosphate pool. As a result, occasional increases in the external concentration are
experienced by phytoplankton as a series of phosphate pulses. Based on [32P]
phosphate uptake experiments with lake phytoplankton, we show that a community is able to process information about the experienced pattern of phosphate
pulses via a complex regulation of the kinetic and energetic properties of cellular
phosphate uptake systems. As a result, physiological adaptation to alterations of
ambient phosphate concentration depends on the pattern of phosphate fluctuations to which the community had been exposed during its previous growth. In
this process, the entire community exhibits coherent uptake behaviour with
respect to a common threshold value. Thereby, different threshold values result
from different antecedent pulse patterns, apparently unrestrained by the amount
of previously stored phosphate. The coherent behaviour observed contradicts the
basic assumptions of the competitive exclusion principle and provides an
alternative perspective for explaining the paradoxical coexistence of many phytoplankton species.
Introduction
In many lakes, the formation of phytoplankton biomass is
frequently limited by the amount of discharged phosphate
(Schindler, 1977; Hudson et al., 2000). When this is the case,
the concentration of this nutrient usually decreases to such
low levels that the uptake system does not have sufficient
energy to drive the transport against the transmembrane
electrochemical gradient. Under this condition, algae and
cyanobacteria are able to grow continuously by means of a
special mechanism that allows efficient exploitation of
transient increases in the ambient phosphate concentration,
for example after the excretion of faeces by aquatic animals
(Rigler, 1956). When the external concentration exceeds a
characteristic threshold value, above which the available
energy suffices to drive the transport into the cell (Falkner
et al., 1989, 1994), phosphate is rapidly accumulated by an
activated uptake system and stored in polyphosphate granules (Kulaev & Vagabov, 1983). Because of the uptake
FEMS Microbiol Ecol 77 (2011) 1–16
activity of the entire community, the external concentration
decreases after a transient phosphate supply more or less
rapidly to the threshold value and further uptake is only
possible when the external concentration increases again. As
a result of this energetic constraint, fluctuations of the
external phosphate concentration are experienced by cells
as a pattern of phosphate pulses, in which short-term
increases in the external concentration are interrupted by
periods without phosphate uptake. The growth rate is then
somehow related to the size of the polyphosphate granules;
in continuous cultures, the amount of polyphosphates
increases with an increase in the growth rate (Droop, 1974).
By this mechanism, phosphate uptake and growth are not
directly coupled: phosphate uptake can occur in nongrowing cells and growth is possible without phosphate uptake, at
the expense of stored phosphate. The biological need to
conform the growth rate to the amount of stored polyphosphate gives rise to a regulatory problem. On the one hand,
during transient increases in the external phosphate
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
2
concentration, sufficient phosphate must be incorporated to
sustain growth in periods without phosphate supply. On the
other hand, excessive storage of phosphate must be avoided,
because the polyphosphate granules otherwise become so
large that cellular structures can be disrupted. A direct effect
of granule size on the activity of the uptake system is not
conceivable, because the granules are segregated in the
cytoplasm as osmotically inert structures. Because the
chemical activity of polyphosphates is not affected by
differences in the chain length, the amount of stored
polyphosphates is not determined by any intracellular constraint (this also explains why after excessive phosphate
storing polyphosphates can be found in the periplasmic
space; see e.g. Kulaev & Vagabov, 1983). In this scenario, an
unfavourable waste of incorporated phosphate and energy
can be avoided by a mechanism that regulates the activity of
the phosphate uptake system by some sort of ‘memory’
based on previous phosphate accumulations. The impact
of this memory should be revealed in a concatenation
of adaptive events: thereby, adaptations to former phosphate
pulses had to regulate subsequent ones, such that the
amount of accumulated phosphate meets the demand
at a growth rate, which, in turn, depends on the amount
of accumulated phosphate. It is obvious that such a
memory provides a means to sense the intracellular phosphorus status.
An insight into such a connectivity of adapted events has
been gained from studies with cyanobacteria that were
exposed to different sequences of phosphate pulses (Wagner
et al., 1995; Falkner & Falkner, 2003; Falkner et al., 2006).
These studies revealed that the adapted states attained
during the antecedent pulses were maintained in the subsequent period without phosphate uptake and influenced
the uptake behaviour in the final pulse in a distinct way.
Interestingly, this was also the case when that pulse came
after the subsequent cell division. The capacity to perceive
differences in phosphate pulses and to transcribe them into
distinct adaptive responses can be interpreted as a kind of
cellular information processing in the sense of Bateson
(2000); accordingly, an elementary unit of information is
defined as a difference (in the environment) that makes a
difference (in the self-constitution of the system).
Exposure to different patterns of phosphate pulses frequently resulted in different threshold values, even when the
same amount of phosphate had been incorporated into
these patterns. For this reason, the apparent threshold value
reflects former adaptive processes and can also not be
explained by an exhausted capacity to store phosphate,
which, for theoretical reasons, also does not exist. Because
the threshold value depends on cellular energy conversion,
information processing about the pattern of pulses has an
energetic foundation. An analysis of the regulation of the
phosphate uptake system has showed that for every external
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
L. Aubriot et al.
phosphate concentration and growth rate only one defined
state of optimal efficiency exists, characterized by a distinct
threshold value and an extended range of validity,
over which there is a linear relationship between the
phosphate flow into the cells and the driving force of this
process. The wide linear range, revealed in a linear dependence of uptake rates on the logarithm of the external
concentration, reflects the environmental phosphate concentrations, to which the uptake system has conformed
(Falkner et al., 1989, 1993, 1994).
In a previous study, we found that the phosphate uptake
of a natural community in Lago Rodó obeyed the linear
flow–force relationship over a wide concentration range
after exposure to an elevated phosphate concentration. The
entire community composed by cyanobacteria, diatoms,
green algae, flagellates and bacterioplankton thereby developed coherent adaptive behaviour with respect to a common threshold value (Aubriot et al., 2000). In this study, we
investigated the conditions under which such an energetically favourable state emerges in a natural community.
During the course of these investigations, we also found that
a natural community is potentially able to perceive differences in the pattern of phosphate fluctuations and to
transcribe information about these patterns into distinct
adaptive responses. This finding is of ecological relevance:
coherent attainment of adapted states of a whole community contradicts the competitive exclusion principle and
provides another perspective for explaining the paradoxical
coexistence of many phytoplankton species under nutrientlimiting growth conditions (Hutchinson, 1961).
Theoretical considerations
From what has been described above, it is evident that a
theory of information processing about external phosphate
fluctuations must account for the interdependence of adaptive events, occurring during responses of the phosphate
uptake system to an intermittent phosphate supply. An
adaptive event is a self-organization process, in which the
uptake system passes, via an adaptive operation mode, from
one adapted state to the next. Adaptive operation modes are
initiated, when adapted states, attained during antecedent
pulses, are disturbed in subsequent pulses by further additions of phosphate. In cyanobacteria, adapted states refer to
stationary states, in which at the prevailing ambient concentration, the phosphate transport system in the cell
membrane and the ATP-synthase are conformed to each
other in an energetically favourable manner. Because of this
energetic constraint, any persistent change in the external
phosphate concentration induces an adaptive operation
mode, in which the two energy-converting subsystems are
reconstructed, such that a new and energetically efficient
adapted state emerges.
FEMS Microbiol Ecol 77 (2011) 1–16
3
Adaptive phosphate uptake behaviour of phytoplankton
Nonequilibrium thermodynamics provides the theoretical
means to distinguish between adapted states and adaptive
operation modes. Accordingly, stationary-adapted states are
characterized by a linear dependence of the phosphate uptake
rate JP on the driving force of this process, at least close to
thermodynamic equilibrium. In contrast, adaptive operation
modes, initiated when the uptake system is forced to operate
in regions far from equilibrium, reflect some sort of selforganization, in which the phosphate flow into the cell can be
expected to be a nonlinear function of the driving force.
Because the onset of such a self-organization depends on the
intensity of the phosphate flow, resulting from a previously
attained adapted state, the nonlinear features of an adaptive
operation mode carry information about the physiological
response to antecedent environmental changes. This is the
leitmotif for an investigation of information processing via a
concatenation of adaptive events.
The phytoplankton community of Lago Rodó consists
predominantly of cyanobacteria. Adaptive self-organization
processes, occurring during alterations of the external
phosphate concentration, can be investigated by relating
the phosphate uptake rate to the driving force for the
incorporation of external phosphate into the polyphosphate
pool, comprising three biochemical reactions: (1) Transport
of external phosphate Pe into the cell. (2) Conversion of the
incorporated internal phosphate Pi to ATP via photophosphorylation or respiration. (3) Formation of polyphosphates (Pn, Pn11) from ATP. For an estimation of the
driving force, it is not necessary to consider the individual
steps of polyphosphate formation, as long as we assume that
proportionality exists between the external phosphate concentration and the activities of the reactants, involved in the
consecutive reactions described above. When this condition
is fulfilled, the overall process of polyphosphate formation
from external phosphate can be summarized as follows:
Translocation through the cell membrane: Pe 2Pi
ð1Þ
þ
ATP formation: Pi þ ADP þ nHþ
T 2ATP þ nHC
ð2Þ
Transphosphorylation to polyphosphates:
ATP þ Pn 2Pnþ1 þ ADP
Sum reaction for the overall process:
þ
Pe þ Pn þ nHþ
T 2Pnþ1 þ nHC
ð3Þ
ð4Þ
The sum reaction indicates that the incorporation of
phosphate is coupled either indirectly or directly with the
flow of n protons across the thylakoid membrane. H1
T and
refer
to
the
protons
in
the
thylakoid
and
cytoplasmic
H1
C
space, respectively.
At low external concentrations, an energy source is
needed for the translocation against the existing electrocheFEMS Microbiol Ecol 77 (2011) 1–16
mical gradient at the cell membrane. In the cyanobacterium
Anacystis nidulans, the necessary amount of energy is
provided by an ATPase (Wagner & Falkner, 1992). Because
Eqn.(2) describes the conversion of internal phosphate into
ATP, involved in both the subsequent elongation of the
polyphosphate chain [Eqn.(4)] and the active transport
process [Eqn.(1)], n stands for the number of protons,
required for both processes.
Using the equation for the driving force of chemical
reactions, X = DG = RT lnK[S]/[P], where [S] and [P] refer
to the corresponding substrate and product concentrations,
the sum reaction allows calculating the driving force for
polyphosphate formation from external phosphate:
X ¼ 2:3 RT logðK0 ½Pe ð½Hþ T =½Hþ C Þn Þ
¼ 2:3 RTðlogð½Pe K 0 Þ þ nDpHT Þ
ð5Þ
Pn and Pn11 have the same activity and can be cancelled out.
K 0 is the equilibrium constant of the conversion of external
phosphate to polyphosphate under the prevailing external
conditions. DpHT is the pH gradient across the thylakoid
membrane, depending on the photosynthetic or respiratory
electron transport chain. In this calculation, the comparatively small electric term in the proteomic potential has been
neglected and the uptake process was assumed not to be
affected by the membrane potential due to electroneutral
cotransport of phosphate with protons (Rosenberg, 1987).
In case of an electrogenic transport, additional terms would
have been introduced, but because they can assumed to be
constant under steady-state conditions, they can be included
in the equilibrium constant K 0 .
A linear dependence of the rate of polyphosphate formation from external phosphate JP on the overall process can be
expressed by the equation:
JP ¼ LP ðlogð½Pe K 0 Þ þ nDpHT Þ
The term 2.3RT is included in the proportionality factor
LP, representing a conductivity coefficient that describes the
dependence of the phosphate flow on the overall driving
force of this process. LP is for the simple pore model, when
the internal phosphate concentration is properly chosen and
the driving force is varied only by a change of the external
phosphate concentration, proportional to the maximum
velocity of the uptake process (Rottenberg, 1979).
In case of incomplete coupling between the phosphate
and the proton flows, the concentration dependence of the
uptake rate on the driving force of polyphosphate formation
from external phosphate attains the following form (Falkner
et al., 1994):
JP ¼ LP ðlogð½Pe K 0 Þ þ n0 q2 DpHT Þ
ð6Þ
q is the degree of coupling between the driven phosphate
flow JP and the driving proton flow JH (q is 1 for complete
coupling and becomes 0, when the two processes are totally
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
4
L. Aubriot et al.
uncoupled) and n 0 is a stoichiometric factor that reflects the
flux ratio JH/JP at very high external phosphate concentrations. At the threshold, the value of JP becomes 0; this allows
calculating the logarithmic threshold value from Eqn.(6):
log½Pe A ¼ log K 0 n0 q2 DpHT
ð7Þ
Eqn.(7) shows that the threshold value is increased with a
decrease in the degree of coupling. Following the terminology of Kedem & Caplan (1965), we can define the state at the
threshold value as a ‘static head’ for phosphate uptake.
Replacing the term (log K 0 1n 0 q2DpHT) in Eqn.(6) by
log[Pe]A leads to the simple flow–force relationship that
now contains a threshold value that depends on the degree
of coupling between the phosphate flow and the driving
force of this process:
JP ¼ d½Pe =dt ¼ LP ðlog½Pe log½Pe A Þ
ð8Þ
Within the validity range of this function, a plot of JP vs.
log[Pe] yields a straight line that intercepts the log[Pe] axis at
the logarithmic threshold value, as has already been pointed
out by Thellier (1970) (for a more recent treatment of this
plot, also for other uptake processes, see Thellier et al.,
1993). The slope of the resulting straight line reveals the
proportionality factor LP.
In adaptive operation modes, the extended validity range
of the linear flow–force relation of a distinct adapted state is
lost. The dependence of the uptake rate on the driving force
during this self-organization process can be modelled, when
nonlinear terms are added to Eqn.(8), leading to a function
that was also proposed by Thellier (1970):
JP ¼ d½Pe =dt
¼ LP ðlog½Pe log½Pe A Þ þ Lðlog½Pe log½Pe A Þm
ð9Þ
where m 4 1.
The exponential number m characterizes (in addition to
the phenomenological coefficients L and LP) the dependence
of the phosphate influx rate on the driving force during an
adaptive self-organization process. An adequate value for m
must be found in the fitting procedure. Because even
numbers are meaningless from a physical point of view,
because they would imply an onset of phosphate uptake at
concentrations below the threshold value, m must be an odd
number. For the data from Lago Rodó, a satisfactory fit
(R2Z0.9) could be achieved with either m = 3 or m = 5; the
parameters given in the legends indicate which of the two
values resulted in a better R2. Note that the nonlinear term
in Eqn.(9) becomes negligible when the external phosphate
concentration approaches the threshold value.
A semi-logarithmic plot of the concentration dependence
of uptake rates yielded an important result: in adapted
states, this linear function is valid over a wide concentration
range, extending from the threshold value to the external
concentration, to which the uptake system has been ad2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
justed, even if that concentration exceeded the threshold
value by several orders of magnitude (Falkner et al., 1993,
1995; Wagner et al., 1995). The extension of a linear
flow–force relationship into regions far from equilibrium
contradicts the basic propositions of nonequilibrium thermodynamics, but can be explained by the simultaneous
operation of several low- and high-affinity uptake systems
with different kinetic and energetic parameters (Falkner
et al., 1995; Wagner et al., 1995; Pitt et al., 2010).
An energetic analysis of the adaptive behaviour using the
energy converter model showed that in states of optimal
efficiency, the degree of coupling between the driven phosphate flow and the driving proton flow is tuned like a sensor
to the external phosphate concentration that has initiated
this adaptive process (Falkner et al., 1989, 1994). As a
consequence, both the degree of coupling and the conductivity coefficient LP are diminished when the external concentration is increased, which affects the threshold value [see
Eqn.(7)]. Naturally, the adjusted optimal efficiency is always
below the maximal possible efficiency, corresponding to the
state of thermodynamic equilibrium. Apart from that stationary state of minimum entropy production, the phosphate uptake system attains a second stationary state of least
energy dissipation at the corresponding threshold value,
where the driven flows disappear and hence no work is
invested in the incorporation process (Katchalsky & Curran,
1975); both states have a certain stability that is lost after
persistent exposure to an altered phosphate concentration,
remote from the level at which the system operates with least
energy dissipation.
Owing to these energetic constraints, an uptake system
displays an extremely complex behaviour, when a population is exposed to a series of phosphate pulses, in which the
external concentration is first abruptly increased and then
diminished by the uptake activity of the entire population.
The reaction time of organisms regulates how fast the
outcome of the antecedent adaptation guides the subsequent adapted state in a distinct manner. This is the time at
which a stable state, originating from an antecedent pulse, is
disturbed in a subsequent pulse and a new adapted state is
established. In this respect, the reaction time appears to be
an index of the sensitivity of microorganisms to an environmental alteration, determining the transfer of information
from one pulse to the next.
The dependence of possible information processing on
the relation between the reaction time and the exposure time
is revealed in the pattern of phosphate pulses. Several
frequently found examples are depicted in Fig. 1. The first
example (Fig. 1a) refers to a situation in which the external
concentration fluctuates in the vicinity of the threshold
value so that the uptake system of the community is only at
the threshold value in a stable adapted state. In such a case,
during a transient increase of the external concentration
FEMS Microbiol Ecol 77 (2011) 1–16
5
Adaptive phosphate uptake behaviour of phytoplankton
(a)
(b)
T
(c)
T
(d)
T
T
Fig. 1. Four theoretical situations about information processing by phytoplankton during different pulse patterns with respect to the relation between
the reaction time (tR) and the exposure time (tE). (a) Phosphate uptake during a sequence of low-concentration pulses, fitted by nonlinear flow–force
relations. (b) Phosphate uptake of higher concentration sequence of pulses, in which the higher and lower part of the first pulse could be fitted by a
linear flow–force relation that determined the uptake behaviour of the following pulses. (c) Sequence of pulses, fitted by linear flow–force relations. (d)
Sequence of pulses, in which an antecedent phosphate pulse is fitted by a linear flow–force relation, but not the subsequent pulse. [Pe], external
phosphate concentration; JP, uptake rate; new [Pe]A, new threshold value; Pre [Pe]A, previous threshold value.
above the threshold value, if the reaction time is longer than
the exposure time, the phosphate flow into the cell does not
perturb the previously attained stable state. As a result, the
time course of the decrease in the external concentration is
similar after repeated phosphate additions, because a new
stable adapted state with new kinetic properties is never
established. Naturally, in this case, no information about
adaptation to an antecedent pulse is transferred to the
subsequent pulse and the change in the external phosphate
concentration does not follow a linear flow–force relationship.
A different situation arises when the reaction time is
shorter than the exposure time (Fig. 1b–d). Figure 1b depicts
a case in which a new adapted state is rapidly reached by a
phosphate-deficient community after a transient increase in
the external concentration. Therefore, the time course of the
decrease in the external concentration is likely to follow the
nonlinear function only after the onset of the uptake process.
It can, however, be fitted by the linear flow–force relationship
after an exposure time that reflects the duration needed for
the attainment of a new stable adapted state. Because of its
stability, this state potentially determines the uptake behaviour in the following pulse(s): the time course of the decrease
in the external concentration in subsequent pulses then obeys
the linear flow–force relationship with similar conductivity
coefficients, ‘inherited’ from an antecedent pulse, as long as
no adaptive reconstruction of the uptake system takes place.
Curve fitting of the time courses of the decrease in the external
concentration in these pulses should therefore result in similar
conductivity coefficients of the semi-logarithmic function.
If, however, in a sequence of pulses, all time courses can
be fitted by linear semi-logarithmic functions, but with
different conductivity coefficients, new stable states are
created, when the external concentration approaches the
threshold value; the kinetics of subsequent pulses then
reveals to what extent the uptake system has been modified
at the end of former pulses (Fig. 1c). Theoretically, it is also
FEMS Microbiol Ecol 77 (2011) 1–16
possible that in a sequence of pulses, the time course of an
antecedent pulse follows a linear semi-logarithmic function,
but not in the subsequent one. This refers to a situation in
which an adapted state, originating from the former pulse,
has been disturbed before the external concentration has
declined to the threshold value (Fig. 1d).
The line of arguments presented above was used for an
analysis of information processing by a phytoplankton
community of Lago Rodó.
Materials and methods
Study area
The physiological response of phytoplankton to phosphate
fluctuations was studied during the growing season on five
occasions in 2003 and 2007. The phytoplankton samples
were taken from Lago Rodó, an urban lake in Montevideo,
Uruguay. Lago Rodó is a shallow, polymictic and hypereutrophic lake, with a mean depth of 1.5 m (maximum
depth: 2 m, area: 1.3 ha). Water is mainly supplied by
inflowing groundwater that contains high concentrations
of nutrients (Aubriot et al., 2000). During the study period,
the content of dissolved oxygen in the water column ranged
between 2.5 and 20.0 mg L1 (at the surface) and 1.0 and
16.0 mg L1 (at the bottom) at temperatures between 15.5
and 27.5 1C. In the surface layer of the lake, the mean total
phosphorus (TP) content was 6.0 1.6 mM, the content of
total nitrogen (TN) varied between 56.5 and 497.0 mM and
the concentration of dissolved inorganic nitrogen (DIN)
varied between o 5.0 and 175.0 mM, and consisted of up
to 80% nitrate. The concentration of orthophosphate
was usually below the limit of detection by conventional
analytical methods, suggesting that the phytoplankton
community, mainly dominated by cyanobacteria, was in a
phosphate-deficient state. Bacterioplankton biomass of
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
6
Lago Rodó is less than expected for hypertrophic lakes
(3.6% of the total biovolume of phytoplankton and heterotrophic bacterial community, Sommaruga, 1995; Sommaruga & Robarts, 1997). Phosphate deficiency was confirmed
by the corresponding phosphate uptake characteristics of
the phytoplankton community (activated phosphate influx
rates, threshold values in the nanomolar range).
Sampling and in situ measurements
Lake water samples of 2 L were taken from the surface with
dark bottles and immediately transported to the laboratory,
before uptake experiments were started, which took about
30 min. In order to characterize the water column, in situ
measurements of temperature, dissolved oxygen and pH
were performed every 20 cm from the surface to the bottom,
using Horibar OM–14 and D–24 sensors.
Spectrophotometric determinations
After filtration of the original lake sample through GF/F
Whatman glass fibre filters, presoaked in ultrapure water,
the concentrations of orthophosphate and DIN were analysed in subsamples of 100 mL, using the colorimetric
methods described by Strickland & Parsons (1972). The
filters with the particulate material were stored at 20 1C
for extraction of Chl a in triplicate samples, which was
performed later using hot ethanol and quantified according
to Nusch (1980). The amount of TP and TN was determined
following the method given by Valderrama (1981).
Determination of phytoplankton composition
Phytoplankton samples (450 mL) were fixed with acid
Lugol’s solution for qualitative and quantitative analysis
and kept in the dark until processing within 3 months.
Taxonomic identifications were performed with an Olympus optic microscope, using magnifications from 400 to
1000. Organisms larger than 5 mm diameter were identified to the genus or the species level, when possible,
according to Sant’Anna (1984), Wehr & Sheath (2003) and
Komárek & Anagnostidis (2005). Although picophytoplankton (0.2–2 mm) was not evaluated in this study, it has been
shown that larger organisms dominate the phytoplankton
community in Lago Rodó (Sommaruga, 1995; Scasso et al.,
2001). The organisms were counted in a 1-mL Sedgwick–Rafter chamber at 400 magnifications from random
fields according to Guillard (1978). Cell biovolume calculations were based on geometric shapes and the microscopically determined average size of 10–30 organisms per taxon
(Hillebrand et al., 1999). In order to calculate the biovolume
of each taxon (BT) in the community (mm3 L1), counts
were multiplied by the specific biovolume (mm3). The
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
L. Aubriot et al.
biovolume contribution of each taxon was expressed as a
percentage of the total biovolume.
Measurement of phosphate uptake
Phosphate uptake by the phytoplankton community was
measured in two identical subsamples of 50 mL (termed S1
and S2), obtained from the same original lake sample
that was taken immediately before each experiment (A to E,
Table 1). Before each experiment, the sample was filtered
through an 80-mm mesh. This allowed the passage of
filamentous algae, but removed large-sized zooplankton.
The subsamples were then illuminated (150 mmol
photons m2 s1), gently mixed with a magnetic stirrer and
kept at the same temperature as at the sampling site in a
temperature-controlled chamber (20–26 1C). These incubation conditions were maintained for 30 min before phosphate
uptake measurements began.
Naturally, information processing about phosphate fluctuations could only be studied when the growth of the
phytoplankton in Lago Rodó was limited by phosphate
supply. Unfortunately, this was not always the case; there
were periods in which an increase in the external phosphate
concentration was paralleled by a decrease of DIN, indicating that nitrogen has become a growth-limiting factor.
Under these conditions, adaptive responses to experimentally used phosphate concentrations could be reestablished,
when the phytoplankton sample was preincubated with
250 mM nitrate for 41 h before starting the [32P] phosphate
uptake measurements (procedure applied to experiment E).
The nitrate preincubation was performed under the same
incubation conditions as described previously.
[32P] phosphate was applied to each subsample at different initial concentrations in the micromolar range. At these
high initial [32P] phosphate concentrations, the influx rate
of the tracer into the cell is several orders of magnitude
higher than the efflux of endogenous phosphate. Consequently, isotopic exchange can be neglected and the observed uptake essentially represents the net incorporation of
phosphate (Aubriot et al., 2000). This was confirmed by the
fact that the apparent threshold value remained constant in
several subsequent pulses during which the activity of the
uptake system was not affected (the occurrence of an
isotopic exchange between [32P] phosphate in the incubation medium and endogenous [31P] phosphate would
decrease the specific activity of the [32P] phosphate and, as
a consequence, in the calculation, misleadingly providing a
lower threshold value in the first pulse). In order to measure
the decrease in external phosphate concentrations resulting
from phytoplankton uptake activity, aliquots of 0.8–1.5 mL
were filtered through a Millipore HA filter (0.45 mm pore
size) at appropriate time intervals. No detectable interference of prokaryotic cells was found in the filtrate using
FEMS Microbiol Ecol 77 (2011) 1–16
7
Adaptive phosphate uptake behaviour of phytoplankton
0.45 mm pore size filters with water samples of Lago Rodó.
We performed eight comparisons of filtering adequacy using
0.22 and 0.45 mm pore size filters to determine [32P]
phosphate in the filtrate. No significant differences were
found between pore-size filters when filtering the same
aliquot (Mann–Whitney U-test, P = 0.7). This finding is
consistent with the size of bacterioplankton found in Lago
Rodó ( 4 0.5 mm length; Sommaruga & Conde, 1997).
We chose 0.45 mm pore-size filters because of a less filtering
pressure required to pass the volume of aliquots, with
the aim of minimizing a possible release of internal
[32P] phosphate by cell damage. Hence, the phosphate
uptake activity measured in our study integrates phosphate
removal by phytoplankton and phosphate uptake-active
bacterioplankton. The radioactivity in the filtrate was
measured as Cerenkov radiation in water using a Beckman
Liquid Scintillation Counter (LS 6000). In order to study
the uptake behaviour in a sequence of pulses, the concentrations indicated in the figures were added several times to
the same sample after the threshold had been attained
following each addition.
Experimental design
The complex phosphate uptake behaviour is demonstrated
in five experiments as summarized in Table 1. In all these
experiments, the same total [32P] phosphate was applied to
both subsamples (S1 and S2). In experiment A, two identical
samples were exposed to a succession of three phosphate
pulses, in order to check the reliability of the method. In
experiments B, C, D and E, two identical samples were
exposed first to two different patterns of ‘imprinting pulses’,
before the uptake behaviour was analysed in two final ‘test
pulses’, containing the same amount of phosphate. The
test pulses were applied when, after the last imprinting
pulse, the external concentration remained constant at the
corresponding threshold value.
Data analysis
The linear and nonlinear flow–force relationships [Eqns (8)
and (9), respectively] were fitted using the computer program MLAB (Mathematical Modelling System, Civilized
Software Inc.). Fitting of the linear function was performed
first with the linear term with data close to the threshold
value, i.e. in a region where the value for the nonlinear term
becomes negligible in comparison with the linear term (see
Theoretical considerations). The parameters of the nonlinear term were then obtained after the insertion of the
parameters of the linear function into the entire expression
(Falkner et al., 2006).
For comparison, in some cases, we constructed a semilogarithmic replot of the time course of phosphate removal
from the external medium according to Thellier (1970). For
this purpose, the measured uptake rates (corresponding to the
tangents of the computer-fitted time courses of the decrease in
the external concentration) at the time intervals at which the
uptake measurement had been performed were related per
hour and the Chl a content of the phytoplankton community.
These values were then plotted against the logarithm of the
external concentration given on the ordinate at these time
intervals. In order to obtain a dimensionless number for the
logarithmic concentration, for example 6 for 1 mM and 9
for 1 nM, the molarity of the concentrations used was related
to unit standard concentration (1 M).
The exposure time (tE) indicates how long phytoplankton
had experienced external phosphate concentrations above
the threshold value; it represents the period of time between
[32Pe] phosphate addition and the moment at which 99%
of the amount of [32Pe] phosphate that can be incorporated
into a pulse had been taken up by the phytoplankton.
The cumulative exposure time of phytoplankton to experimentally applied concentrations above the threshold value
(StE) was obtained by summing the tE-values determined in
each phosphate pulse.
Table 1. Summary of the different pulse patterns used in experiments A to E, describing the initial [32P] phosphate concentration of the imprinting and
test pulses and the cumulated exposure time of phytoplankton to phosphate concentrations above the threshold value StE
Initial [32P] phosphate (mM)
StE
Experiment
Dates
Sbs
Imprinting pulses
First and second test pulse
(min)
A
13/03/2007
B
27/03/2003
C
13/03/2003
D
08/05/2003
E
23/01/2007
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
3.0 (nonlinear) 1.5 (linear) 1.5 (linear)
3.0 (nonlinear) 1.5 (linear) 1.5 (linear)
0.2 (5 times)
1.0 (once)
1.0 (10 times)
5.0 (twice)
1.0 (4 times) then 0.5 (twice)
5.0 (once)
1.0–3.0
3.0–1.0
–
–
0.5 (nonlinear)–0.5 (nonlinear)
0.5 (nonlinear)–0.5 (nonlinear)
1.0 (nonlinear)–1.0 (nonlinear)
1.0 (linear)–1.0 (linear)
0.4 (linear)–0.4 (linear)
0.4 (linear)–0.4 (linear)
2.0 (nonlinear)–2.0 (nonlinear)
2.0 (nonlinear)–2.0 (nonlinear)
437
437
535
558
577
335
1756
1826
634
1166
Sbs, subsample designation S1 and S2; (linear) and (nonlinear) refer to the flow–force equations used for curve fitting.
FEMS Microbiol Ecol 77 (2011) 1–16
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
8
L. Aubriot et al.
For technical reasons, it was not possible to follow
simultaneously in duplicate the rapid decrease in the
external concentration in two phytoplankton subsamples.
We have therefore replicated the experiments in different
dates in order to determine whether the adaptive behaviour
of phytoplankton to phosphate fluctuations occurs under
different species composition and environmental conditions. The reliability of the method was therefore checked
by comparing the uptake kinetics of two identical subsamples, originating from the same phytoplankton assemblage.
A plot of the data from the time courses, observed after three
subsequent additions of phosphate, shows that the differences of the uptake kinetics in the two subsamples are
smaller than the symbol used in our graphs (an example is
given in Fig. 2).
The 95% confidence interval (CI95%) was calculated from
the flow–force equation fitting of each time course of
phosphate removal. Significant differences of [Pe]A between
subsamples were accepted when no overlap occurred between the upper and the lower limits of CI95%. Significant
differences between subsamples in the slope of time course
of phosphate removal were evaluated using GLM-ANCOVA
(STATISTICA 6.0, StatSoft Inc.).
Results and discussion
Concomitant kinetic and energetic alterations of
phosphate uptake by phytoplankton as an index
of antecedent phosphate supply
Figure 2 presents an analysis of the uptake behaviour in three
consecutive pulses, using the nonlinear and linear flow–force
relationship (the parameters of the corresponding functions
3.5
3.0
[Pe] (µM)
2.5
2.0
1.5
1.0
0.5
0.0
0
100
200
300
400
Time (min)
500
600
Fig. 2. Time course of [32P] phosphate removal by two identical phytoplankton subsamples (open and closed circles) from Lago Rodó during
three consecutive imprinting phosphate pulses, the first containing 3 mM
and subsequently 1.5 mM [32P] phosphate (experiment A). The curves of
the first pulse represent the best computer fit using the linear and
nonlinear equation. The corresponding kinetic parameters are shown in
Table 2. The TP content of the lake sample was 4.3 mM.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
are given in Table 2). In this experiment, the best fit for the
first pulse was obtained with the nonlinear function, whereas
the linear flow–force relationship was more appropriate for
the two subsequent pulses. Following the line of argument
given in Theoretical considerations, we may infer that the
properties of the uptake systems of the community were
modified after the abrupt increase in the external concentration in the first pulse during an exposition time tE of
120 min. In the resulting stable adapted state, the uptake
behaviour obeyed a linear flow–force relationship over a
wide range, extending from nanomolar threshold values up
to micromolar concentrations. The conductivity coefficients
(LP) showed minor modifications after the second and third
pulse (LP = 9.5 and 10.7 nM min1, respectively), indicating
that the kinetic properties of the uptake system, attained
during the (higher) first pulse, were maintained during the
subsequent (lower) pulses. This is in accordance with the
postulate that in adapted states, in which the uptake behaviour obeys the linear flow–force relationship, an uptake
system is characterized by a certain degree of stability. A
replot of the time courses of the second and third pulses
according to Thellier (Fig. 3) illustrates that the relative
invariance of the conductivity coefficients is contrasted with
a continuous increase of the threshold value from pulse to
pulse ([Pe]A = 28, 71 and 179 nM, first, second and third
phosphate pulse, respectively; Table 2). Apparently, exposure
to micromolar phosphate concentrations led to a gradual
decrease of the energy invested in the uptake process.
It is noteworthy that, in a sequence of pulses, a diversified
phytoplankton community consisting of Scenedesmus ellipticus, small flagellates, Sphaerocystis schroeteri, Planktothrix
agardhii, Oocystis sp., Scenedesmus acuminatus, Mallomonas
sp., Microcystis sp., Peridinium sp. (Table 3, experiment A)
and a bacterioplankton community (Sommaruga & Conde,
1997) showed similar uptake behaviour as a phosphatedeficient monospecific cyanobacterial culture. For example,
when Anabaena variabilis was exposed to a sequence of
pulses, the first pulse could not be fitted by a linear
flow–force relationship and a new stable state, representing
an attractor for adaptation to higher concentrations, was
only attained after repeated stimulations by elevated external concentrations (Falkner et al., 2006). Our results suggest
that adaptation of the different organisms of the phytoplankton community and the portion of phosphate uptakeactive bacterioplankton follows similar energetic constraints
as observed with a monospecific cyanobacteria population.
Previous observations have shown that a semi-logarithmic
plot of the concentration dependence of uptake rates exhibited a wide linear range when the phytoplankton assemblage
of Lago Rodó experienced an enhanced nutrient supply by
inflowing ground water (Aubriot et al., 2000). The fact that
the first pulse could not be fitted by such a linear relationship
indicates that the phytoplankton assemblage used for this
FEMS Microbiol Ecol 77 (2011) 1–16
9
Adaptive phosphate uptake behaviour of phytoplankton
Table 2. Kinetic parameters of time courses of phosphate removal by phytoplankton sampled from Lago Rodó
Exp.
A
B
Pulse type
First imprinting
Second imprinting
Third imprinting
Imprinting
First test
Second test
C
Imprinting
First test
Second test
D
Imprinting
First test
Second test
E
First imprinting
Second imprinting
First test
Second test
Sbs
S1 and S2
S1 and S2
S1 and S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
S1
S2
[32P] pulse (mM)
LP (nM min1)
3.0
1.5
1.5
0.2 (5 times)
1.0 (once)
0.5
0.5
0.5
0.5
1.0 (10 times)
5.0 (twice)
1.0
1.0
1.0
1.0
1.0 (4 times)10.5 (twice)
5.0 (once)
0.4
0.4
0.4
0.4
1.0
3.0
3.0
1.0
2.0
2.0
2.0
2.0
L (nM min1)
7.1
9.5
10.7
–
–
1.9
2.1
1.3
2.3
–
–
6.9x
14.2x
14.0x
10.8x
–
–
2.2
2.4
1.7x
11.4x
21.2
18.8
18.9
19.1
10.8x
9.6x
2.1x
0.8x
0.01
–
–
–
–
0.03
0.03
0.06
0.25
–
–
0.05
–
0.10
–
–
–
–
–
–
–
–
–
–
0.2
0.5
0.5
1.0
1.0
[Pe]A (nM)
+
28 51
71 66
179 93+
–
–
14 12
15 10+
33 31
66 6,+
–
–
71 41+
66 52
271 51,+
92 43
–
–
201 15,+
308 27,+
280 29,+
735 25,+
14 3+
35 50+
24 12+
49 9+
48 45
80 43
103 66+
184 86+
R2
0.9996
0.9975
0.9915
–
–
0.9995
0.9995
0.9993
0.9997
–
–
0.9969
0.9941
0.9902
0.9969
–
–
0.9981
0.9887
0.9786
0.8617
0.9999
0.9995
0.9977
0.9999
0.9992
0.9992
0.9986
0.9969
Significant differences between subsamples S and S by no overlap of CI
1
2
95%.
+
Significant differences between pulses within the same subsample by no overlap of CI95%.
Significant differences between subsamples S1 and S2 (GLM-ANCOVA, P o 0.01).
Experiment identification (Exp), type of phosphate pulse applied, subsample designation (Sbs) as S1 and S2; initial [32P] phosphate concentration ([32P]
pulse). Parameters were obtained with curve fitting of the linear and nonlinear equations of the flow–force relationship (m = 5 for experiments A, B, C
and m = 3 for experiment E). LP and L, conductivity coefficients of the flow–force equation; [Pe]A, threshold value CI95%; R2, determination coefficient.
x
experiment had not been exposed in the lake to micromolar
phosphate concentrations before the experiment.
The effect of the pattern of antecedent pulses on
the subsequent adaptive response
The experiment presented above shows that in a sequence of
pulses, subsequent adaptations of a natural phytoplankton
community may be influenced by antecedent adaptations. A
difference in the pattern of experienced phosphate fluctuations can therefore be expected to lead to a corresponding
difference in the subsequent adaptive behaviour, even when
the same amount of phosphate has been incorporated
during those fluctuations. In order to investigate the conditions under which information about the pattern of phosphate fluctuations in a lake is transcribed into a distinct
subsequent adaptive response of a phytoplankton community, the same amount of [32Pe] phosphate was supplied in
FEMS Microbiol Ecol 77 (2011) 1–16
two different modes to two identical subsamples (termed S1
and S2) from Lago Rodó, before the adaptive behaviour
was analysed in subsequent test pulses. In the experiments described below, externally supplied phosphate was
added at different concentrations in order to investigate the
community’s sensitivity during an adaptive response to
environmental phosphate fluctuations. The phytoplankton
community (containing between 175.6 3.4 and 277.3 3.4 mg Chl a L1) was mainly dominated by two cyanobacteria: P. agardhii and Raphidiopsis mediterranea (Table 3,
experiments B to E).
In order to study the effect of lower phosphate concentrations, we exposed one subsample (S1) to five imprinting
pulses of [32P] phosphate, 0.2 mM each, and another subsample (S2) to the same total phosphate concentration, but
applied in one pulse of 1.0 mM (Fig. 4a). The threshold
values, attained at the end of this pretreatment, were
identical in both subsamples, in accordance with previous
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
10
L. Aubriot et al.
observations that the threshold value was also not affected
by pulses in the nanomolar range (Aubriot et al., 2000).
However, the exposition times tE resulting from this pretreatment were slightly different in the two subsamples
(tE = 135 and 160 min, S1 and S2, respectively). After this
pretreatment, 280 min after the first addition of phosphate,
we exposed the two subsamples to two more test pulses of
0.5 mM, in order to investigate possible differences in the
time course of [32P] phosphate removal by the phytoplank10
8
JP
6
4
2
0
–7.5
–7.0
–6.5
log [Pe]
–6.0
–5.5
–5.0
Fig. 3. Thellier plot of the concentration dependence of incorporation
rates obtained from the time course of phosphate removal in the second
and third pulse, shown in Fig. 2. The uptake rate JP is given in
mmol Pi mg1 Chl a h1; the logarithm of the external phosphate concentration is expressed relative to the unit standard concentration (1 M).
ton. However, this was not the case; the time course of
phosphate removal in both subsamples was practically
identical, with the exception of a more pronounced increase
in the threshold values in subsample S2 after the second test
pulse (Fig. 4b). None of the four test pulses could be fitted
by a linear flow–force relationship, indicating that no stable
adapted state had been established during the pretreatment,
which is a precondition for storage and transfer of information from one pulse to the next (see Table 2, experiment B).
A totally different situation arose after exposure to higher
imprinting pulses in experiment C (Fig. 5a), in which the
phytoplankton of subsample S1 experienced 10 phosphate
increments of 1 mM, while in S2, it was subjected to two
pulses of 5 mM [32P] phosphate (Fig. 5a); thereby, the
phytoplankton of subsample S1 was exposed to an elevated
phosphate concentration twice as long as the phytoplankton
of the reference sample S2 (tE = 320 and 180 min for S1 and
S2, respectively). An analysis of the uptake behaviour
performed after this pretreatment with two more test pulses
of 1 mM (Fig. 5b) revealed remarkable differences in the
kinetics of phosphate removal from the external medium in
the two subsamples (the parameters of the fitted functions
are given in Table 2). The phytoplankton of subsample S1
incorporated phosphate into the first test pulse at half the
rate of subsample S2. Both phytoplankton subsamples
nevertheless established essentially the same threshold value.
In the second pulse, however, the threshold value of
subsample S1 was significantly increased so that the phytoplankton incorporated less phosphate at a lower rate than
Table 3. Main phytoplankton taxa, chlorophyll a concentration (Chl a), total biovolume and the relative biovolume of each taxa in independent
phytoplankton samples taken from Lago Rodó
Phosphate uptake experiment and sampling dates
1
Chl a (mg L )
Total biovolume (mm3 L1)
Main taxa (% of total biovolume)
Anabaena sp.
Microcystis sp.
Raphidiopsis mediterranea
cf. Planktolyngbya
Planktothrix agardhii
Synedra ulna
Oocystis sp.
Scenedesmus acuminatus
Scenedesmus ellipticus
Sphaerocystis sp.
Cryptomonas sp.
Rhodomonas sp.
Mallomonas sp.
Peridinium sp.
Flagellates ( o 5 mm)
Ind. volume
A 13.03.07
B 27.03.03
C 13.03.03
D 08.05.03
E 23.01.07
204.0 41.6
32.59
211.1 3.4
81.69
175.6 3.4
59.54
196.5 2.0
85.98
106.2 19.0
20.82
0.0
3.0
0.0
0.0
11.4
0.0
5.6
4.8
23.5
12.8
0.0
0.0
3.0
2.3
19.7
0.0
0.0
12.4
0.2
83.5
0.4
0.0
0.0
0.0
0.0
0.2
0.1
0.0
2.6
0.0
0.0
0.0
50.4
2.8
42.2
0.0
0.0
0.0
0.0
0.0
1.3
2.8
0.0
0.0
0.0
0.0
0.0
18.9
0.0
80.4
0.0
0.0
0.0
0.0
0.0
0.5
0.2
0.0
0.0
0.0
3.9
0.1
4.6
0.0
85.5
2.5
0.0
0.6
0.0
0.0
0.0
0.0
0.0
3.0
0.0
(mm3)
1096
493
460
208
2091
2244
380
254
53
144
159
135
800
7381
14
The letters A, B, C, D and E refer to the experiments performed with these phytoplankton samples shown in Figs 2, 4, 5, 6 and 7, respectively. Ind.
volume, specific volume of the main taxonomic groups.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
FEMS Microbiol Ecol 77 (2011) 1–16
11
Adaptive phosphate uptake behaviour of phytoplankton
(a)
5
0.8
4
0.6
0.4
3
2
0.2
1
0.0
0
50
100
150
200
0
250
(b) 0.6
0
100
200
300
0
50
100
150
Time (min)
400
500
200
720
(b) 1.2
0.5
1.0
0.4
[Pe] (µM)
[Pe] (µM)
6
1.0
[Pe] (µM)
[Pe] (µM)
(a) 1.2
0.3
0.2
0.8
0.6
0.4
0.1
0.2
0.0
0
50
100
150 200 250
Time (min)
300
350
400
0.0
32
Fig. 4. Time course of [ P] phosphate removal by two identical phytoplankton subsamples of Lago Rodó, containing 7.8 mM TP. (a) Subsample
S1 (closed symbols) was exposed to five imprinting pulses of 0.2 mM each,
while subsample S2 (open symbols) was exposed to one pulse of 1 mM.
(b) After this pretreatment, 280 min after the first addition of phosphate,
both subsamples were subjected to two more phosphate pulses of
0.5 mM each and the time course of [32P] phosphate removal by the
phytoplankton in the two subsamples was followed. The solid curves
represent the best computer fit for the two test pulses, using the
nonlinear equation. The corresponding kinetic parameters are shown in
Table 2 (experiment B).
the plankton of subsample S2 ([Pe]A = 271 68 and
92 56 nM, S1 and S2, respectively). In these two test pulses,
both the threshold values and the conductivity coefficients,
reflecting the energetic and kinetic properties of the uptake
systems of the phytoplankton community, respectively, were
altered independent of each other. Furthermore, it is notable
that only the two test pulses in the subsample, in which the
community had been subjected to higher concentrations of
5 mM, could be fitted by the linear semi-logarithmic function. In the other subsamples that were repeatedly exposed to
lower concentrations, the nonlinear function was more
appropriate. In this experiment, fitting with the linear
function was not possible before exposure to elevated concentrations, similar to experiment A (Fig. 2). Hence, this
experiment represents a situation in which a difference in the
environment of the community of two subsamples (i.e. a
longer exposure time tE due to a persistent stimulation by 10
imprinting pulses of 1 mM phosphate vs. a shorter exposure
FEMS Microbiol Ecol 77 (2011) 1–16
Fig. 5. Time course of [32P] phosphate removal by two identical phytoplankton subsamples of Lago Rodó, containing 7.5 mM TP. (a) Subsample
S1 (closed symbols) was exposed to 10 imprinting pulses of 1.0 mM each,
and subsample S2 (open symbols) to two pulses of 5 mM each. Five
hundred and fifty minutes after the first addition of phosphate, both
subsamples were subjected to two test phosphate pulses of 1.0 mM each
and the time course of [32P] phosphate removal by the phytoplankton in
the two subsamples was followed (b). The solid curves represent the best
computer fit of the time course of the two test pulses, using the
nonlinear equation (m = 5) for subsample S1 and the linear equation for
subsample S2. The corresponding kinetic parameters are shown in Table
2 (experiment C).
time tE during two imprinting pulses of 5 mM) led to a
difference in the adaptive behaviour in the two consecutive
test pulses. Apparently, under these conditions, the phytoplankton community was capable of some sort of information processing about environmental phosphate fluctuations.
Nutrient supply by the groundwater pumping system that
was occasionally switched on led to a decrease in the uptake
activity of the phytoplankton assemblage. When an experiment is performed with such an assemblage, the cells need
more time for removal of the added phosphate and are
therefore exposed to elevated phosphate concentrations in
the individual pulses for a longer period of time. In this case,
greater differences in the adaptive behaviour can be observed after antecedent exposures to two different pulse
patterns. In experiment D, presented in Fig. 6a, phosphate
was provided in two different supply modes to the two
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
12
(a)
L. Aubriot et al.
6
[Pe] (µM)
5
4
3
2
1
0
0
4
8
12
16
20
24
(b)
[Pe] (µM)
0.8
0.6
0.4
0.2
0.0
24
26
28
30
Time (h)
32
34
Fig. 6. Time course of [32P] phosphate removal by two identical phytoplankton subsamples of Lago Rodó, containing 7.9 mM TP and a Chl a
content of 196.5 2.0 mg L1. (a) The phytoplankton of subsample S1
(closed symbols) was exposed to four imprinting pulses of 1.0 mM,
followed by two more pulses of 0.5 mM each, and the phytoplankton of
subsample S2 (open symbols) to one imprinting pulse of 5 mM. The dark
period is shown by hatching. Twenty-four hours after the first addition of
phosphate, both subsamples were subjected to two test phosphate
pulses of 0.4 mM each and the time course of [32P] phosphate removal
by the phytoplankton in the two subsamples was followed [(b) note that
the initial phosphate concentration in the final pulses corresponds to the
external concentration at the time of phosphate addition plus the
phosphate concentration added]. The solid curves represent the best
computer fit for the two final pulses, using the linear equation with the
corresponding kinetic parameters shown in Table 2 (experiment D).
reference subsamples. In one subsample, this was applied in a
single dose (S2), while in the other subsample, the phosphate
was added in six stages (S1) (Table 2). Because of the lower
uptake activity, the external concentration decreased slowly
to the threshold values (of 322 and 313 nM) within 24 h. As a
result, only the phytoplankton of subsample S1 was subjected
to an intermittent phosphate supply during the imprinting
pulses, whereas in subsample S2, it was permanently exposed
to an elevated phosphate concentration (Fig. 6a). The
following day, the community in both subsamples showed
significant differences in uptake behaviour (Fig. 6b). When
exposed twice to 0.4 mM phosphate, the phytoplankton that
had experienced lower pulses the day before continued to
incorporate phosphate after the second addition, thereby
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
approaching a threshold value of the same order of magnitude as in the former pulse. In contrast, the other phytoplankton assemblage that had been exposed to an elevated
phosphate level the day before stopped the uptake process
after the second addition of phosphate. Further studies will
show to what extent the ‘memory’ of former phosphate
fluctuations is a function of the nutrient status and possibly
of the growth activity of the cells.
After a prolonged nutrient supply to Lago Rodó, the
phytoplankton assemblage adapts to a permanent increase
of the external concentrations above the threshold value.
The uptake rate is then reduced to a level that covers the
phosphorus demand of growing organisms and the capacity
of excessive phosphate storage during transient increases of
the external phosphate concentration is lost. The growth
rate can then be limited by other nutrients or light.
A phytoplankton assemblage of Lago Rodó then renews
the ability for phosphate storage and information processing
about phosphate fluctuations, when incubated for several
hours with nitrate before the experiment, indicating that in
this situation the availability of inorganic nitrogen compounds was the growth-limiting factor. In the following
example, preincubation with nitrate restored the discriminatory potentiality of a phytoplankton assemblage. This
could be demonstrated in a simple way by exposing two
identical subsamples to one small pulse of 1 mM and one
greater pulse of 3 mM, but in reverse order: to one subsample
(S1), first, the smaller and then the greater amount was
added, and to the other subsample (S2), phosphate was
given the other way round (experiment E, Fig. 7, Table 2). A
comparison of the uptake kinetics in two subsequent test
pulses of 2 mM revealed significant differences with respect
to the successive concatenation of adaptive events in the two
samples. Thereby, the sample that had been exposed to the
smaller pulse after the greater one was less active, indicating
that a gradual decrease in the dose rate had a greater effect
than a gradual increase. Apparently, the small difference in
the pattern of the imprinting pulses established different
adapted states, representing two distinct initial conditions
for unequal adaptive operation modes in the two subsequent test pulses. Furthermore, the experiment shows that
information processing about phosphate fluctuations does
not require a contrast of either low or high pulses, as has
been the case in the previous experiments. Finally, it should
be noted that the experimental design, presented in this
example, allows obtaining information about possible
growth-limiting compounds other than phosphate.
Conclusions for further investigations
The considerable variability of the uptake behaviour, exhibited in the response of phytoplankton and bacterioplankton to different phosphate supply modes, renders difficult a
FEMS Microbiol Ecol 77 (2011) 1–16
13
Adaptive phosphate uptake behaviour of phytoplankton
3.5
3.0
[Pe] (µM)
2.5
2.0
1.5
1.0
0.5
0.0
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (h)
32
Fig. 7. Time course of [ P] phosphate removal by two identical phytoplankton subsamples of Lago Rodó, containing 6.3 mM TP and a Chl a content of
106.2 19.0 mg L1. The preincubation was performed by adding 250 mM nitrate 41 h before the first pulse of [32P] phosphate. Closed symbols, the
phytoplankton subsample (S1) was exposed to one pulse of 1 mM, followed by one pulse of 3 mM. Open symbols, application of one pulse of 3 mM followed by
one pulse of 1 mM to the parallel subsample (S2). Four hours after the first addition of phosphate, both subsamples were subjected to two test phosphate
pulses of 2 mM each and the time course of [32P] phosphate removal by the phytoplankton in the two subsamples was followed. The solid curves represent the
best computer fit of the time course using the linear and nonlinear equations, with the corresponding kinetic parameters shown in Table 2 (experiment E).
functional attribution of defined uptake characteristics in
terms of Michaelis constants and maximum uptake velocities to individual species. This is in accordance with other
physiological and biochemical studies, performed with
phytoplankton and cyanobacterial species (Falkner et al.,
1974, 1995; Smith et al., 2009; Pitt et al., 2010). For example,
the values for Vmax and KM, obtained from the initial velocities
of 32P-phosphate influx of phosphate-deficient cells of the
unicellular Synechococcus leopoliensis were 600 mmol mg1
Chl a h1 and 130 nM at 37 1C (Wagner et al., 1995), whereas
for nondeficient cells, the values for Vmax and KM, measured at
22 1C, were 75 nmol mg1 Chl a h1 and 6 mM, respectively
(Falkner et al., 1974). A huge variation range of the uptake
activity can also be observed with the slower-growing
A. variabilis (Falkner et al., 2006). For this reason, a characterization of the uptake behaviour using equations with fixed
parameters does not appear to be meaningful for ecological
conditions, under which these parameters are altered, apart
from the fact that no physicist would use the term ‘constant’
for parameters, which are affected by the way in which
the investigation is performed. To overcome these difficulties,
we analysed the adaptive properties of the uptake system
by means of flow–force relationships, including only variable parameters that are potentially altered in response to the
measurement conditions: threshold values and concentrationdependent phenomenological coefficients. A characterization
of information processing by the flow–force relationship is
justified for another reason: the regulation of the uptake
system is very complex because its activation, via an increase
in the degree of coupling between the transport process and
the activity of a membrane ATPase, also occurs in the presence
of an inhibitor of protein biosynthesis (Wagner & Falkner,
FEMS Microbiol Ecol 77 (2011) 1–16
1992). The energetic analysis of the uptake behaviour, performed in this study, led to several interesting ecophysiological
problems. In particular, the influence of the pattern of
phosphate fluctuations on the reaction time as an index of
cellular information processing, the common threshold value
of a community composed by autotrophic and heterotrophic
planktonic organisms and the connectivity of adaptive events
deserve further studies.
The role of the reaction time in information
processing about the pattern of phosphate
pulses
The reaction time at which a stable state, originating from
an antecedent pulse, is disturbed in a subsequent pulse and a
new adapted state is established depends on the pattern of
antecedent phosphate pulses. For this reason, the adaptive
response of phytoplankton organisms to an experimentally
used pulse can also be expected to depend on the nature of
phosphate exposures experienced by the organisms during
its previous growth. In a monospecific culture, this was the
case: a kinetic analysis of the concentration dependence of
the time course of [32P] phosphate influx into the cells of A.
variabilis has shown that lower pulses in the growth medium
resulted in a decrease of the reaction time and in an onset of
adaptive processes at lower concentrations (Falkner et al.,
2006). It remains to be established to what extent the
reaction time of phytoplankton can be used to assess the
prehistory of phosphate supply to a lake.
Another problem that deserves further studies is the
seemingly coherent behaviour of a phytoplankton community with regard to a common threshold value. A sequence
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
14
of pulses occasionally resulted in an increase of the threshold
value that was then maintained by phytoplankton and
bacterioplankton of Lago Rodó for a certain period of time.
This remarkable behaviour can be explained by the interdependence of adaptive events among the organisms of a
community, by which the adaptive responses of the individual organisms influence each other. Naturally, this only
takes place when every organism is capable of adapting to
the concentration changes that are caused by the uptake
activity of other organisms of the community in question.
Phosphate-deficient bacterioplankton is expected to utilize inorganic phosphate when available (Bañeras et al.,
2010). The contribution of bacterioplankton in the overall
uptake adaptive behaviour may be complex due to their
flexibility to exploit inorganic and organic phosphate esters
(del Giorgio & Cole, 1998; Jansson et al., 2006). Our results
did not show the expected supremacy of bacterioplankton
on phytoplankton for the uptake of inorganic phosphate
(Løvdal et al., 2007); otherwise, the higher threshold values
achieved will be immediately lowered by the heterotrophic
bacterial assemblage. Therefore, the high and stable threshold values found in our study indicate that bacterioplankton
that participates in the active uptake should also be implicated in the entire adaptive process.
It is conceivable that as a result of such a mutual
interference the threshold values of different organisms can
potentially converge to the same level, possibly for the
following reason: during a new phosphate pulse, the organisms with shorter reaction times will begin to adapt to the
transient increase of the external concentration, by diminishing the uptake activity and increasing their threshold
value. Because this nutrient is then not so rapidly removed
by phytoplankton, cells with a longer reaction time can also
respond to elevated phosphate concentrations by increasing
their threshold values, even though to a correspondingly
lower level. This will further reduce the rate of the decrease
in the external concentration and allow the organisms with
shorter reaction times to adapt a second time to the external
concentration, thereby establishing a new threshold value
that approaches the threshold value of organisms of a longer
reaction time. By a repetition of this interdependent sequence of adaptive event, finally, a stable state of minimum
entropy production can be attained at a common threshold
value for all organisms present.
This hypothesis offers a possible solution for the apparent
paradoxical coexistence of many phytoplankton species
(Hutchinson, 1961), but requires further experimental verification by laboratory studies, in which the uptake behaviour of mixed populations in a sequence of pulses can be
analysed. The populations may either be composed of cells
of different species or the same species, but they must differ
in their reaction times and threshold values (as a result of
different growth conditions before the experiment). These
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
L. Aubriot et al.
experiments can be supplemented by computer simulations
of adaptive responses to the changing external concentration, in which the program alters its properties in response
to its own simulation at different, but interdependent
reaction times. An example for this type of simulation has
been provided previously (Plaetzer et al., 2005).
Most present-day hypotheses for explaining the paradoxical diversity of phytoplankton under limiting growth conditions have serious limitations and no theory has hitherto
gained universal acceptance (Reynolds, 1998; Roy & Chattopadhyay, 2007). A recent general ecological discussion
poses that species coexistence is achieved by the co-occurrence of originally ‘similar’ (Chesson, 2000; Barton et al.,
2010) or niche differentiated species (Huisman, 2010).
Nearly all hypotheses assume that the main force governing
the biology of phytoplankton populations is competition.
This is an obstacle for a solution to the paradox of the
plankton because hypotheses of competition presume a
mechanistic operation of the uptake systems of organisms.
Phytoplankton growth is thus almost unvaryingly described
by equations that define the nutrient uptake behaviour in
terms of constants, such as a Michaelis constant and a
maximum uptake velocity, determining the fate of the
organisms under ever-changing environmental conditions.
However, an increase in diversity is predicted when changeable physiological properties of species are included in
model simulations of coexistence (Huisman et al., 2001).
The capacity of several species to attain a common threshold
value as a result of an interdependent sequence of adaptive
events provides a new perspective for explaining the coexistence of many phytoplankton species. Future studies may
show which phytoplankton assemblages are able to develop
a coherent behaviour with respect to a common threshold
value and which assemblages do not have this capacity.
In the interaction between organisms and environment,
the adaptive response to changes in the external phosphate
concentration leads to new uptake properties, which, in
turn, determine further changes in the external concentration. This mutual interdependence results in a stationary
state, in which energy conversion is optimized (see Theoretical considerations). An investigation of such a concomitant alteration of organisms and environment is opposed to
any experimental design in which the organisms are kept
under constant external conditions like in a chemostat.
Whereas in the latter case the interaction of different species
may be adequately described by ‘resource competition
theories’, competition provides an insufficient explanation
for a process, in which an entanglement of physiological and
environmental alteration is directed towards stationary
states of minimum entropy production. It is obvious that a
competitive faculty of some organisms at other organism’s
expense plays a minor role in the experimental and theoretical framework presented here. Instead, we hypothesize that
FEMS Microbiol Ecol 77 (2011) 1–16
15
Adaptive phosphate uptake behaviour of phytoplankton
variations in the number of the different species would
rather reflect differences in the capacity of individual cells
to exploit transient increases of the external concentration
for the subsequent growth process, relying on stored information about former nutrient fluctuations.
In conclusion, our results offer a new perspective for
explaining the coexistence of phytoplankton species under a
single limiting nutrient. We propose that species with
different nutrient uptake features are able to ‘resemble’ each
other due to their capacity of mutual adjustment of individual physiologies. Thus, the adaptive behaviour of phytoplankton can be proposed as an equalizing ecophysiological
process that allows the stable coexistence of species.
Acknowledgements
We wish to thank Renate Falkner, Luis Acerenza, Daniel
Conde, Carla Kruk and Malvina Masdeu for helpful discussions. We also thank Anamar Britos, Leticia Vidal and
Mauricio González for technical assistance, and Dermot
Antoniades for valuable linguistic corrections. We are grateful for the financial help of CSIC-Universidad de la República, PEDECIBA-Basic Science Development Program of
Uruguay, DINACYT-Uruguay (No. 7026), SNI-ANII and
the Austrian Research Fund.
References
Aubriot L, Wagner F & Falkner G (2000) The phosphate uptake
behaviour of phytoplankton communities in eutrophic lakes
reflects alterations in the phosphate supply. Eur J Phycol 35:
255–262.
Bañeras L, Ros-Ponsatı́ M, Cristina XP, Garcia-Gil JL & Borrego
CM (2010) Phosphorus deficiency and kinetics of alkaline
phosphatase in isolates and natural populations of
phototrophic sulphur bacteria. FEMS Microbiol Ecol 73:
243–253.
Barton AD, Dutkiewicz S, Flierl G, Bragg J & Follows MJ (2010)
Patterns of diversity in marine phytoplankton. Science 327:
1509–1511.
Bateson G (2000) Steps to an Ecology of Mind. The University of
Chicago Press, Chicago.
Chesson P (2000) Mechanisms of maintenance of species
diversity. Annu Rev Ecol Syst 31: 343–366.
del Giorgio PA & Cole JJ (1998) Bacterial growth efficiency in
natural aquatic systems. Annu Rev Ecol Syst 29: 503–541.
Droop MR (1974) Nutrient status of algal cells in continuous
culture. J Mar Biol Assoc UK 54: 825–855.
Falkner G, Werdan K, Horner F & Heldt HW (1974)
Energieabhängige Phosphataufnahme der Blaualge Anacystis
nidulans. Ber Deutsch Bot Ges 87: 263–269.
Falkner G, Falkner R & Schwab A (1989) Bioenergetic
characterization of transient state phosphate uptake by the
FEMS Microbiol Ecol 77 (2011) 1–16
cyanobacterium Anacystis nidulans. Arch Microbiol 152:
353–361.
Falkner G, Falkner R & Wagner W (1993) Adaptive phosphate
uptake behaviour of the cyanobacterium Anacystis nidulans:
analysis by a proportional flow-force relation. CR Acad Sci III
Vie 316: 784–787.
Falkner G, Wagner F & Falkner R (1994) On the relation between
phosphate uptake and growth of the cyanobacterium Anacystis
nidulans. CR Acad Sci III Vie 317: 535–541.
Falkner G, Wagner F, Small JV & Falkner R (1995) Influence of
fluctuating phosphate supply on the regulation of phosphate
uptake by the blue-green alga Anacystis nidulans. J Phycol 31:
745–753.
Falkner R & Falkner G (2003) Distinct adaptivity during
phosphate uptake by the cyanobacterium Anabaena variabilis
reflects information processing about preceding phosphate
supply. J Trace Microprobe T 21: 363–375.
Falkner R, Priewasser M & Falkner G (2006) Information
processing by cyanobacteria during adaptation to
environmental phosphate fluctuations. Plant Sign Behav 1:
212–220.
Guillard R (1978) Counting slides. Phytoplankton Manual
(Sournia A, ed), pp. 182–190. Unesco, Paris.
Hillebrand H, Dürselen C, Kirschtel D, Zohary T & Pollingher U
(1999) Biovolume calculation for pelagic and benthic
microalgae. J Phycol 35: 403–424.
Hudson JJ, Taylor WD & Schindler DW (2000) Phosphate
concentrations in lakes. Nature 406: 54–56.
Huisman J (2010) Comment on ‘Patterns of Diversity in Marine
Phytoplankton’. Science 329: 512.
Huisman J, Johansson A, Folmer E & Weissing F (2001) Towards
a solution of the plankton paradox: the importance of
physiology and life history. Ecol Lett 4: 408–411.
Hutchinson GE (1961) The paradox of the plankton. Am Nat 882:
137–145.
Jansson M, Bergström A-K, Lymer D, Vrede K & Karlsson J
(2006) Bacterioplankton growth and nutrient use efficiencies
under variable organic carbon and inorganic phosphorus
ratios. Microb Ecol 52: 358–364.
Katchalsky A & Curran P (1975) Nonequilibrium
Thermodynamics. Harvard University Press, Cambridge.
Kedem O & Caplan SR (1965) Degree of coupling and its
relationship to efficiency of energy conversion. T Faraday Soc
61: 1897–1911.
Komárek J & Anagnostidis K (2005) Cyanoprokaryota II. Teil
Oscillatoriales. Spektrum Akademischer Verlag, München.
Kulaev I & Vagabov V (1983) Polyphosphate metabolism in
microorganisms. Adv Microb Physiol 24: 88–171.
Løvdal T, Tanaka T & Thingstad TF (2007) Algal–bacterial
competition for phosphorus from dissolved DNA, ATP, and
orthophosphate in a mesocosm experiment. Limnol Oceanogr
52: 1407–1419.
Nusch E (1980) Comparisons of different methods for
chlorophyll and phaeopigments determination. Arch
Hydrobiol Beih Ergebn Limnol 14: 14–36.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
16
Pitt FD, Mazard S, Humphreys L & Scanlan DJ (2010) Functional
characterization of Synechocystis sp. strain PCC 6803 pst1 and
pst2 gene clusters reveals a novel strategy for phosphate uptake
in a freshwater cyanobacterium. J Bacteriol 192: 3512–3523.
Plaetzer K, Thomas SR, Falkner R & Falkner G (2005) The
microbial experience of environmental phosphate
fluctuations. An essay on the possibility of putting intentions
into cell biochemistry. J Theor Biol 235: 540–554.
Reynolds CS (1998) The state of freshwater ecology. Freshwater
Biol 39: 741–753.
Rigler FH (1956) A tracer study of the phosphorus cycle in lake
water. Ecology 37: 550–562.
Rosenberg H (1987) Phosphate transport in prokaryotes. Ion
Transport in Prokaryotes (Rosen BP & Silver S, eds), pp.
205–248. Academic Press, New York.
Rottenberg H (1979) Non-equilibrium thermodynamics of
energy conversion in bioenergetics. Biochim Biophys Acta 549:
225–253.
Roy S & Chattopadhyay J (2007) Towards a resolution of ‘the
paradox of plankton’: a brief overview of the proposed
mechanisms. Ecol Complex 4: 26–33.
Sant’Anna CL (1984) Chlorococcales (Chlorophyceae) do Estado de
Sao Paulo, Brasil. J. Cramer, Vaduz.
Scasso F, Mazzeo N, Gorga J, Kruk C, Lacerot G, Clemente J &
Bonilla S (2001) Limnological changes in a sub-tropical
shallow hypertrophic lake during its resoration: two years of a
whole-lake experiment. Aquat Conserv 11: 31–44.
Schindler D (1977) The evolution of phosphorous limitation in
lakes. Science 195: 260–262.
Smith SL, Yamanaka Y, Pahlow M & Oschlies A (2009) Optimal
uptake kinetics: physiological acclimation explains the pattern
of nitrate uptake by phytoplankton in the ocean. MEPS 384:
1–12.
2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
L. Aubriot et al.
Sommaruga R (1995) Microbial and classical food webs: a visit to
hypertrophic lake. FEMS Microbiol Ecol 17: 257–270.
Sommaruga R & Conde D (1997) Seasonal variability of
metabolically active bacterioplankton in the euphotic zone of a
hypertrophic lake. Aquat Microb Ecol 13: 241–248.
Sommaruga R & Robarts RD (1997) The significance of
autotrophic and heterotrophic picoplankton in hypertrophic
ecosystems. FEMS Microbiol Ecol 24: 187–200.
Strickland JDH & Parsons TR (1972) A practical handbook of
seawater analysis. Fisheries Research Board, Ottawa.
Thellier M (1970) An electrokinetic interpretation of the
functioning of biological systems and its application to the
study of mineral salt absorption. Ann Bot 34: 983–1009.
Thellier M, Ripoll C, Vincent JC & Mikulecki D (1993)
Interpretation of the fluxes of substances exchanged by cellular
systems with their external medium. Some Physicochemical and
Mathematical Tools for Understanding of Living Systems
(Greppin H, Bonzon M, Degli Agosti R & Rapala J, eds), pp.
221–277. University of Geneva, Geneva.
Valderrama JC (1981) The simultaneous analysis of total nitrogen
and total phosphorus in natural waters. Mar Chem 10:
109–122.
Wagner F & Falkner G (1992) Concomitant changes in phosphate
uptake and photophosphorylation in the blue-green alga
Anacystis nidulans during adaptation to phosphate deficiency.
J Plant Physiol 140: 163–167.
Wagner F, Falkner R & Falkner G (1995) Information about
previous phosphate fluctuations is stored via an adaptive
response of the high-affinity phosphate uptake system of the
cyanobacterium Anacystis nidulans. Planta 197: 147–155.
Wehr JD & Sheath RG (2003) Freshwater algae of North America.
Ecology and classification. Academic Press, Amsterdam.
FEMS Microbiol Ecol 77 (2011) 1–16