FEMS Microbiology Ecology 46 (2003) 307^316
www.fems-microbiology.org
Is phosphorus limitation of planktonic heterotrophic bacteria and
accumulation of degradable DOC a normal phenomenon in
phosphorus-limited systems? A microcosm study
Olav Vadstein
a
a;
, Lasse M. Olsen a , Arild Busch b , Tom Andersen c ,
Helge R. Reinertsen b
Trondhjem Biological Station, Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
b
Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway
c
Norwegian Institute for Water Research (NIVA), P.O. Box 173 Kjelsafis, 0411 Oslo, Norway
Received 29 November 2002; received in revised form 2 May 2003 ; accepted 21 May 2003
First published online 27 August 2003
Abstract
A dual isotope labelling technique was used to follow the distribution of carbon and phosphorus in plankton microcosms containing
autotrophs (Tetraselmis sp.), heterotrophic bacteria and herbivores (Brachionus plicatilis) at eight different total-P concentrations. P:C
ratios of algae, bacteria and dissolved matter, as well as the general accumulation of degradable dissolved organic carbon, indicated that
both the autotrophs and heterotrophic bacteria were P-limited in all microcosms. According to the theory, such coexistence should only
be possible if bacteria have higher predation losses than algae, which was definitely not the case in our experiment. However, data are
consistent with the assumption that bacteria are superior in P uptake but have a poor ability to retain acquired P, which would promote
coexistence in a patchy P-supply environment resulting from nutrient regeneration by metazoan grazers.
7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : Heterotrophic bacteria; Alga; Coexistence; Resource competition ; Patchiness; Dissolved organic carbon
1. Introduction
In contrast to the classical assumption that heterotrophic bacteria are carbon/energy-limited, it is now well established that nitrogen and in particular phosphorus may
limit growth of heterotrophic bacteria in aquatic systems.
A review of data from limnetic systems concluded that
P limitation is a normal phenomenon, as it was detected
in 86% of the cases [1]. Nitrogen or carbon limitation was
observed in only 15% and 20% of the tested cases, respectively (the percentages add up to more than 100% due to
methodological aspects, cf. [1]). Data from marine systems
are more limited, but P limitation of heterotrophic bacteria is now documented in both brackish [2,3] and marine
waters [4^7]. Moreover, it has been observed that dissolved organic carbon (DOC) accumulates in euphotic
waters (reviewed by Thingstad et al. [8]), and that on
* Corresponding author. Tel. : +47 7359 0204 ; Fax: +47 7359 1597.
E-mail address : [email protected] (O. Vadstein).
average 19% of marine DOC (and 14% of the DOC in
lakes) is easily available for bacteria [9]. These ¢ndings
suggest that C limitation of heterotrophic bacteria is not
a normal phenomenon in aquatic systems. As planktonic
algae also are P-limited in many of these systems, an apparent paradox arises: how can these two main functional
groups of plankton organisms coexist on a single limiting
resource (Fig. 1)? The paradox is even more remarkable in
view of the fact that heterotrophic bacteria have much
higher a⁄nity for phosphate uptake than algae [1,10,11],
and are therefore considered superior competitors for P in
natural ecosystems. Two di¡erent mechanisms (models)
have been suggested for resolving the paradox.
Frede Thingstad and co-workers have elegantly resolved
the paradox in a series of theoretical and experimental
studies [12,13]. The key aspect in their model is that bacteria have to experience higher mortality than algae due to
grazing, to compensate for their competitive advantage.
Although viruses may impose high mortality rates on heterotrophic bacteria this does not obstruct the conclusion,
since viruses primarily a¡ect the diversity and not biomass
0168-6496 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0168-6496(03)00195-8
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O. Vadstein et al. / FEMS Microbiology Ecology 46 (2003) 307^316
diction we used microcosms (Fig. 1) with the algae Tetraselmis sp. and a mixed community of heterotrophic bacteria competing for P, and with the rotifer Brachionus
plicatilis as a grazer. B. plicatilis is size-selective and feeds
on bacteria with a much lower e⁄ciency than when it is
feeding on Tetraselmis-sized particles [22]. In this system
algae and rotifers produce DOC that can be used for bacterial growth (Fig. 1), and the presence of labile (easily
utilisable) DOC was used as a criterion for absence of
C limitation. The cultures were run at eight di¡erent total
P concentrations for almost 3 months. In a previous study
[23] we investigated the e¡ect of food web structure on
limiting factors and DOC accumulation. Here we explore
the dose^response relationship in a P gradient for one of
those food web structures, and go into more detail in
evaluating possible controlling mechanisms.
2. Materials and methods
Fig. 1. Schematic representation of the £ow of P and organic C in our
aquatic microbial food web. A: algae, B: heterotrophic bacteria, R: rotifers (grazers). Solid lines represent P £ow and dotted lines C £ow.
of bacteria [14]. Yngvar Olsen and co-workers proposed
another mechanism that may promote coexistence of
P-limited species. They demonstrated that there is a di¡erence between gross and net uptake of P due to release of P
from healthy cells [15,16], i.e. the net uptake curve for P
does not go through the origin. They further demonstrated
by both experimental data and numerical modelling that
this physiological phenomenon promotes coexistence [17].
This mechanism requires patchiness of P in either space or
time to promote net loss from a superior competitor when
it experiences P concentrations below a critical limit.
Although it is di⁄cult to verify experimentally, several
investigators have presented data that support this phenomenon in natural systems [18^21].
It is important to note that the two mechanisms outlined above are not mutually exclusive. The simultaneous
operation of both mechanisms would actually increase the
probability of heterotrophic bacteria being P-limited. The
mechanism related to patchiness is di⁄cult to test experimentally. However, from the selective predation mechanism we can make the following prediction, which is easier
to test experimentally : in systems where predation on algae is higher than that on bacteria, bacteria will become
C-limited and no accumulation of DOC will occur.
The aim of this study was to test this prediction. A
rejection entails that mechanisms other than selective predation must be involved in promoting coexistence of
P-limited algae and heterotrophic bacteria. A consequence
of this would be that coexistence of P-limited algae and
heterotrophic bacteria in P-limited systems is a more likely
event as several mechanisms promote it. To test the pre-
FEMSEC 1556 24-11-03
2.1. Organisms
We used a large strain (length V250 Wm) of the rotifer
B. plicatilis (SINTEF strain) in the experiments. The rotifers were pre-cultured for 3^4 weeks on the same prey as
the one used in the experiment, i.e. the prasinophyceae
Tetraselmis sp. Since none of the cultures were axenic,
mixed cultures of heterotrophic bacteria were introduced
in the microcosms together with the inocula of algae and
rotifers. Microscopic examination at the end of the experiment revealed no contamination in any cultures by unwanted eukaryotic species.
2.2. Experimental design
The experiment was performed in 10 microcosms at
eight di¡erent P concentrations in the 7.8^62 Wg P l31
range (0.25^2.00 WM). The microcosms consisted of 10-l
polyethylene vessels (Nalgene), where magnetic stirrers
provided gentle mixing of the water. The temperature
was 20 O 1‡C, and the light/dark cycle was 18/6 h. Fluorescent tubes (Philips TLD 30W/96 and F 30W/29) provided an irradiance of approximately 90 Wmol quanta m32
s31 (PAR) outside the vessels.
The culture medium was GuillardPs h/2 mineral nutrient
medium (containing macro- and micromineral nutrients
and vitamins) with reduced P content [24], based on
aged and ¢ltered seawater diluted with distilled water to
20 psu. Before use the water was autoclaved and aerated
with sterile air. The orthophosphate concentration was
6 1 Wg P l31 in the seawater before dilution. Phosphorus
was added separately to each culture as K2 HPO4 . The
N:P ratios by atoms ranged from 3530 :1 to 442:1.
H14 CO3 and 33 PO4 were added to the stock h/2 medium
before inoculation. To maintain the same speci¢c activity
throughout the whole experiment the same stock medium
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containing radioisotopes was used during dilution. In six
of the microcosms both radioactive tracers were added.
The 23 and 54 Wg P l31 total P treatments (0.75 and
1.75 WM) were replicated and only 33 P or 14 C alone was
added in each replicate, respectively. Total activity in the
microcosms at the beginning of the experiment was 20 000
cpm ml31 .
The microcosms were diluted at a rate of 0.01 day31
(100 ml day31 ). The cultures were not diluted on Saturdays and Sundays. The harvested volume was instead
doubled on Fridays and Mondays. Removed water (10ml samples) was used for fractionation by successive ¢ltering on decreasing pore sizes, rotifers were collected on 30Wm nylon net, algae and bacteria on 3- and 0.2-Wm polycarbonate membrane ¢lters (Poretics), respectively. Material passing a 0.2-Wm ¢lter was considered dissolved. Filtration pressure di¡erential varied with pore size, but
never exceeded 200 mm Hg. This size fractionation e⁄ciently separates the three groups of organisms [23].
2.3. Determination of carbon and phosphorus pools
All ¢lters were placed in 6-ml plastic scintillation vials
and stored uncapped for 18^20 h to evaporate water and
14
CO2 , before addition of scintillation cocktail. The ¢ltrate
was divided into two 5-ml aliquots in 20-ml scintillation
vials. One of the vials was used to measure dissolved organic C and total dissolved P, and one for measuring
dissolved inorganic phosphorus (DIP). Both samples
were acidi¢ed with H2 SO4 to pHW2.5 to remove dissolved
inorganic carbon. To measure DIP, dissolved organic matter was removed by adding activated charcoal [25]. The
total activity of 33 P+14 C in all sample types was measured
in a Packard Tri-Carb 1900 scintillation counter.
The L energy distributions of 14 C and 33 P are almost
identical. However, it is possible to discriminate between
the two isotopes by exploiting their widely di¡erent halflives (5730 years for 14 C and 25.4 days for 33 P). All samples are counted two times : once just after sample preparation and again after some half-lives of 33 P have elapsed.
Assuming the activity of 14 C to be identical during the two
counts, the counts attributable to 14 C and 33 P can easily be
309
calculated. For further details in these calculations the
reader is referred to Olsen et al. [23].
To convert cpm values per litre into units of C and P of
both particulate and dissolved fractions in the ¢ve sets of
samples taken per week, we divided cpm values with speci¢c activities of 33 P and 14 C (cpm [mg C or Wg P]31 ).
Speci¢c activity of P was based on added 33 P (cpm l31 )
and total P of the microcosms. We used data from the
previous study for speci¢c activity of C (determined
from the ratio between 14 C and total C of particulate
matter collected on Whatman GF/F ¢bre glass ¢lters
[23]). The counting e⁄ciency was assumed to be equal in
particulate and dissolved fractions. Stable speci¢c activities were obtained after approximately 10 days [23].
2.4. Bacterial re-growth experiment
At the end (day 60) of another experiment where three
of our cultures were replicated [23] water samples from
cultures with 7.8, 31 and 62 Wg P l31 were ¢ltered through
glass ¢bre ¢lters (Whatman, GF/F) and stored frozen
(320‡C). After termination of the main experiment the
degradability of 14 C-labelled DOC in these samples was
measured in a bacterial re-growth experiment. Bacterial
samples for inoculation were mixed from cultures of
B. plicatilis, Tetraselmis sp. and from natural seawater,
and ¢ltered through 1-Wm polycarbonate membranes to
remove eukaryotes. Inorganic macro- and micronutrients
were added in amounts corresponding to complete h/2
medium in 50-ml samples of the GF/F ¢ltrate from the
microcosms, and bacteria were inoculated to a ¢nal concentration of 2^3U103 ml31 . For methodological details
see Olsen et al. [23].
3. Results
The concentration of DIP was reduced to concentrations 6 1 Wg P l31 in all cultures, and stayed low for the
rest of the experiment. The biomass development was
fairly similar in all cultures, except for di¡erences in carrying capacity due to varying total P concentration (Fig. 2).
Table 1
Net speci¢c increase or decrease in organic carbon fractions during the post-bloom period assuming an exponential model (day no. v 40, (dX/dt) X31 ,
dimension day31 )
Total P (Wg P l31 )
Heterotrophic bacteria
Algae
Rotifers
DOC
8
16
23
31
39
47
54
62
30.026 O 0.003
30.026 O 0.003
30.023 O 0.003
30.020 O 0.002
30.028 O 0.003
30.026 O 0.003
30.025 O 0.003
30.011 O 0.003
(30.001 O 0.002)
30.018 O 0.003
0.009 O 0.003
0.008 O 0.002
(0.006 O 0.004)
0.012 O 0.003
(0.001 O 0.002)
0.009 O 0.002
0.011 O 0.003
0.005 O 0.002
0.015 O 0.002
0.013 O 0.002
0.008 O 0.003
0.012 O 0.002
0.006 O 0.002
(0.003 O 0.002)
30.0024 O 0.0006
30.0031 O 0.0006
30.0042 O 0.0005
30.0045 O 0.0003
30.0039 O 0.0005
30.0041 O 0.0008
(30.0005 O 0.0006)
30.0046 O 0.0004
Rates are given with S.E.M. Slopes not signi¢cantly di¡erent from zero (P s 0.05) are in parentheses.
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Fig. 2. Development of biotic carbon compartments (mg C l31 ) for each total P concentration in a time course. Total P concentrations are shown in
each panel.
The algae grew exponentially without a lag phase, leading
to an algal bloom and a subsequent decline due to grazing.
Both the peak level and the duration of the bloom depended on total P concentration. The termination of the
bloom involved a 90^97% reduction in algal biomass,
which remained fairly stable thereafter (Table 1) with an
average rate of change ( O S.D.) of 0.004 O 0.010 day31 . A
signi¢cant increase in algal biomass was seen in four of the
cultures, whereas in one case a signi¢cant decrease was
observed (Table 1). However, the rates were so low
( 6 0.01 day31 ) that the algae could be considered as being
in a steady state during the post-bloom period (day no.
s 40).
Also the rotifers initially grew exponentially (Fig. 2)
with an overall average rate of 0.34 O 0.09 day31
( O S.D.). However, there was a statistically signi¢cant re-
FEMSEC 1556 24-11-03
duction in initial speci¢c growth rate as the total P increased (30.116 O 0.04 day31 (WM P)31 , P = 0.024). The
time span of the exponential growth depended indirectly
on total P concentration, and the termination of the exponential growth occurred simultaneously with the termination of the algal bloom. Thus, growth of the rotifer was
controlled by food availability. In all cultures the rotifer
biomass increased slightly during the post-bloom period
(Table 1). Except for the culture with 62 Wg P l31 (2 WM)
the increase was statistically signi¢cant with an average
net speci¢c rate of 0.009 O 0.004 day31 , and with a tendency for reduced rates at the higher total P concentrations. Although statistically signi¢cant, these rates are of
limited biological signi¢cance and steady state may be
assumed. There were no tendencies for prey^predator oscillations in any of the cultures. Due to natural mortality
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O. Vadstein et al. / FEMS Microbiology Ecology 46 (2003) 307^316
311
of the rotifers (average life span 10 days), the carbon in
the rotifer fraction ( s 30 Wm) consisted partly of detritus.
However, in this study this is not treated further as it has
no implications for testing of the prediction.
Changes in bacterial biomass had three distinct phases
(Fig. 2). An initial phase was characterised by a rapid
constant exponential growth (0.37^0.81 day31 , average
0.53 O 0.15 day31 ) that lasted for 5^6 days. This was followed by a shift-down situation where growth was still
exponential, but at rates reduced by almost an order of
magnitude (0.04^0.16 day31 , average 0.08 O 0.04 day31 ).
After this second phase all but the two cultures with the
lowest total P were characterised by a fall in bacterial
biomass. The two cultures with the lowest total P showed
a decline of biomass at a low but statistically signi¢cant
rate (Table 1) with an average of 30.023 O 0.005 day31
( O S.D.). The ¢rst phase lasted while the algae grew exponentially. The second phase occurred during the stationary phase of the algae and lasted until the algal bloom was
terminated (Fig. 2), which coincided with maximum rotifer
biomass. The fact that no collapse in bacterial biomass
was observed after the biomass maximum suggests that
viruses did not play an active role in controlling bacterial
biomass dynamics.
The changes in DOC and total P concentrations were
also related (Fig. 3). The DOC concentration increased
during 3^5 weeks. The maximum DOC concentration
was generally reached at the same time as the rotifer biomass peaked. Thereafter it levelled o¡ in the two cultures
with the lowest total P and decreased by 3^30% before
levelling o¡ in the rest of the cultures. During the phase
after this reduction, the DOC concentration decreased at a
very low but in most cases statistically signi¢cant rate (on
average 30.004 O 0.001 day31 , when excluding one nonsigni¢cant value, Table 1).
The averages of all carbon pools of the post-bloom
period, on which the testing of our prediction is mainly
focussing, are shown in Fig. 4. Generally we observed a
nearly linear increase in all carbon pools with increased
total P concentration. The response was stronger for rotifer biomass (predator) than for algae and bacterial biomass (prey). However, the response in DOC concentration
was as strong as for the rotifers. The slopes were
18.9 O 1.6, 4.7 O 0.9, 33.5 O 1.6 and 34.4 O 3.4 ( O S.E.M.,
dimension mg C (Wg P)31 ) for algae, bacteria, rotifers
and DOC, respectively. It must be noted that a portion
of the particulate carbon in the rotifer fraction was detri-
Fig. 4. Average size of biotic carbon pools and DOC during the postbloom period as a function of total P concentration. Error bars are
S.D.
Fig. 5. Percent distribution of organic carbon pools during the postbloom period with total P (Wg P l31 ) indicated, and average distribution
of organic C and P for all total P concentrations.
Fig. 3. Development of DOC in each of the eight microcosms as a function of time. Legend indicates total P concentration in Wg P l31 .
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O. Vadstein et al. / FEMS Microbiology Ecology 46 (2003) 307^316
Fig. 6. Utilisation of DOC by heterotrophic bacteria during the regrowth experiments. The concentration is given as a fraction of initial
concentration. DOC was sampled at the end of the post-bloom period.
tus particles. As a consequence DOC was the strongestresponding carbon pool in the P gradient, and most of the
carbon ¢xed by algae ended up as detritus sensu Wetzel
[26], i.e. DOC+dead particulate matter.
The relative distribution of organic C pools was remarkably stable throughout the P gradient during the postbloom period (Fig. 5). The rotifers increased their contribution by 10% within the P gradient, whereas the algal
fraction had a similar gradual reduction at low to intermediate level of total P. In contrast to this the contribution of DOC was 5% higher at intermediate level of total P
Fig. 7. P:C ratio of algae and heterotrophic bacteria during the postbloom period. Error bars are S.D.
FEMSEC 1556 24-11-03
Fig. 8. P:C ratio of dissolved organic matter during the post-bloom period. Error bars are S.D.
than at high or low concentrations, and in heterotrophic
bacteria there was no trend within the P gradient. DOC
was the dominant C pool at all P concentrations and constituted 42 O 3%. The average contributions of heterotrophic bacteria, algae and rotifers to carbon were 6 O 1%,
18 O 4% and 31 O 4%, respectively. Again it must be noted
that detritus contributed signi¢cantly to carbon in the rotifer fraction. The same calculations on a P basis were
even more stable within the P gradient. However, there
was a shift in the distribution of P as compared to C.
The major di¡erence was that dissolved organic matter
contributed less to organic P, and heterotrophic bacteria
and rotifers increased their relative contributions by a factor of 2.3 and 1.3, respectively.
Whether heterotrophic bacteria are C-limited or not will
depend on to what extent the accumulated DOC is easily
available for bacterial growth. The availability of accumulated DOC was tested in re-growth experiments with DOC
from the post-bloom period. On average 20 O 1% of the
DOC was rapidly used by heterotrophic bacteria (Fig. 6),
with a growth yield of 0.3^0.5. This labile DOC corresponded to 0.13, 0.37 and 0.61 mg C l31 for total P concentrations of 7.8, 31, and 62 Wg l31 (0.25, 1, 2 WM),
respectively. This amount of labile DOC is su⁄cient to
increase the average post-bloom bacterial biomass by
50%. It must, however, be kept in mind that the species
composition of the heterotrophic bacteria may have differed during the post-bloom period and the re-growth experiment.
P:C ratios of algae and heterotrophic bacteria are considered to be good indicators of the degree of P limitation
[27]. Fig. 7 shows a statistically signi¢cant increase in the
P:C ratios of both groups with increasing total P concentration during the post-bloom period (P 6 0.01). Also the
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O. Vadstein et al. / FEMS Microbiology Ecology 46 (2003) 307^316
P :C ratio of dissolved organic matter increased signi¢cantly with increasing total P (P 6 0.001), but the P content of dissolved organic matter was very low compared to
other system components (Fig. 8).
4. Discussion
4.1. Development of organic carbon pools
Generally all biomass pools developed as normal prey^
predator interactions (Fig. 2). It is, however, notable that
no prey^predator oscillations were observed. In accordance with prey^predator theory, the rotifer biomass increased with increasing total P in the post-bloom period
(Fig. 4). However, although the responses were much lower, also the prey biomasses (algae and bacteria) increased
with increasing total P ^ an observation that is not in
accordance with the classical theory.
The DOC concentrations during the post-bloom period
(0.64^3.04 mg C l31 ; Fig. 4) were in agreement with those
recorded in marine surface waters of varying trophic status (range 0.7^3.6 mg C l31 ; reviewed by Thingstad et al.
[8]). Daily surplus production of DOC during the postbloom period may be calculated as the sum of speci¢c
net rate of change for DOC (Table 1) and the dilution
rate (0.01 day31 ), multiplied by the average DOC concentration. These calculations indicate that DOC accumulated
at a rate of 4.8^22.6 Wg C l31 day31 (Table 2). These rates
are considerably lower than those emerging from a numerical model [8] but the model prediction of a linear increase
in DOC accumulation with increasing total P of the system is supported by our data. The quality of the DOC
produced in our experiments seems to be comparable to
DOC in natural ecosystems, as the percentages of easily
available DOC were very similar to those determined from
natural ecosystems [9].
Traditionally it has been assumed that algal exudates
are the primary DOC source that supports growth of heterotrophic bacteria in pelagic systems. The present study
strongly indicates that the grazers were the main source of
DOC. This is supported by the fact that the concentration
of DOC increased continuously (Fig. 3) after the algal
bloom had reached its maximum, and continued to do
so as long as the biomass of the rotifer increased (Fig.
2). In some previous experiments with variable food web
structure we also concluded that DOC production in the
systems increased dramatically when we introduced a grazer to the system [23]. The assimilation e⁄ciency of herbivores is typically 60^80% [28], which indicates that 20^
313
40% of ingested C is egested. Only a part of egested carbon is released directly as DOC, but hydrolysis may convert particulate detritus to DOC at high rates. Previous
work on the freshwater cladocera genus Daphnia showed
that 30% of egested carbon was released directly as DOC,
which was rapidly taken up by heterotrophic bacteria [29].
A review suggests that average exudation by algae is 13%
of primary production (normal range 5^30% [30]). Both of
these results support our conclusion that herbivores are
the most signi¢cant source of DOC.
4.2. Phosphorus status and limiting factor for algae and
heterotrophic bacteria
Contrary to the rotifers, algae and bacteria cannot be
assumed to be in the same physiological state for the
whole post-bloom period and throughout the P gradient.
Because the rotifer biomass increased with increasing total
P, the grazing pressure also increased. As food biomass
was below the incipient limiting concentration for B. plicatilis [31], an individual rotifer would ¢lter water at approximately the same rate in all cultures. As a consequence the population grazing pressure on algae and
bacteria must have increased linearly within the P gradient. Because the speci¢c rates of change in algal and
bacterial biomass were very low during the post-bloom
period ( 6 0.03 day31 , Table 1), the speci¢c growth rate
(W) of algae and bacteria must have equalled the predation
rate and therefore also increased linearly with biomass
(and thus also with total P). Consequently, the degree of
limitation, i.e. the suppression below the maximum speci¢c
growth rate (W : Wmax ), of algae and bacteria decreased
along the total P gradient. Assuming a speci¢c growth
rate of 0.1 day31 (unpublished), a respiration rate of 0.3
day31 [23], and an assimilation e⁄ciency of 0.7 [28], the
speci¢c ingestion rate of carbon by B. plicatilis will be
(0.1+0.3)/0.7 = 0.57 day31 . The community clearance rates
for each microcosm during the post-bloom period may
then be calculated as this speci¢c ingestion rate of carbon
multiplied by rotifer biomass and divided by food concentration. Such calculations give community clearance rates
and hence speci¢c growth rates on algae, in the 0.59^1.32
day31 range. Assuming a Wmax of 1.7 day31 for Tetraselmis
sp. [32], the relative speci¢c growth rate (W : Wmax ) of algae
was 0.35^0.78, and increased linearly with total P
(R2 = 0.780). Thus, the physiological state of the algae
ranged from strong limitation at low total P to only moderate limitation at high total P. If we assume a size selectivity coe⁄cient of 0.2 for bacteria [22] and a Wmax = 2.2
day31 for bacteria [33], the relative speci¢c growth rate of
Table 2
Surplus DOC production in the microcosms during the post-bloom period determined as the sum of net speci¢c rate of change and dilution rate multiplied by average DOC concentration
Total P (Wg P l31 )
Surplus DOC (Wg C l31 day31 )
8
4.8
16
6.0
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23
7.4
31
11.2
39
13.7
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47
14.7
54
22.6
62
16.4
314
O. Vadstein et al. / FEMS Microbiology Ecology 46 (2003) 307^316
heterotrophic bacteria increased linearly in the same manner as for algae, but the rates were always 6 0.07 day31 .
The bacteria therefore experienced much stronger limitation than algae at all total P concentrations. Although
these calculations are coupled to some uncertainty, our
general conclusions regarding limiting factor and changes
in degree of limitation with total P concentration are fairly
robust.
The growth medium was designed to give P limitation
of the algae. The measured P:C ratios of the algae, with
averages of 5^13 Wg P (mg C)31 , indicate that this was the
case. Previous studies with the same Tetraselmis strain
revealed a P content of 6 Wg (mg C)31 for severely P-limited cells [32]. It is well documented that the P content of
algae increases with increasing speci¢c growth rate [27].
Therefore, the fact that the P:C ratios increased with increasing total P supports the calculation above that the
degree of limitation decreased with total P concentration
in the medium.
The prediction from the ‘selective predation’ mechanism
stated that the heterotrophic bacteria in our system would
be C-limited. The evaluation above regarding predation
and relative speci¢c growth rate indicated strong limitation of bacteria at all total P concentrations. As 20% of
the DOC in the cultures was easily available for heterotrophic bacteria (Fig. 6), carbon must be ruled out as a
limiting factor in our experiment [14]. We believe that this
conclusion is fairly robust, even though it can be argued
that the composition of the bacterial community was
di¡erent during the re-growth experiment. Subsistence
P quotas for P-limited heterotrophic bacteria are typically
32 Wg P (mg C)31 , but with considerable species di¡erences
as indicated by an interquartile range of published values
of 15^55 Wg P (mg C)31 [1]. In the present study the
P content of bacteria was well below the typical subsistence
quota of P-limited bacteria, and even averages plus standard deviations were below this limit. As large amounts of
labile DOC and all minerals except P were present in excess, we conclude that the heterotrophic bacteria were
strongly P-limited in the cultures at all total P concentrations. We therefore have to reject the proposed hypothesis.
As a consequence, other mechanisms than selective predation of bacteria promoted coexistence of P-limited algae
and heterotrophic bacteria in our experimental system.
4.3. Can patchiness explain the coexistence of P-limited
algae and heterotrophic bacteria?
The ‘release from healthy cells’ mechanism provides a
physiological explanation for coexistence in our experiments, and covers patchiness in time and space. In our
experiments, the daily 1% dilution provides a pulse of
new nutrients, and it has been shown that a discontinuous
nutrient addition regime may provide coexistence [17] and
increased diversity [21]. However, as the daily dilution was
so low in our experiments, it is not likely that this tempo-
FEMSEC 1556 24-11-03
ral patchiness was the main factor creating coexistence. As
addition of new nutrients occurred at such low rates (0.01
day31 ), regenerative processes must have provided most of
the nutrients.
Rotifers must have been the principal source of regenerated P, and they regenerate P by both egestion and excretion. Inorganic P seems to be the principal constituent
of P regenerated by grazers (cf. [1]). As rotifers act as
point sources of P, they may have created a spatial patchiness. Strongly starved B. plicatilis have a swimming speed
of 25^30 mm min31 , leaving behind them a path of regenerated P while swimming. Assuming a regeneration rate of
1 Wg P (mg C)31 h31 [34], a swimming path width of 150
Wm, and a swimming speed of 30 mm min31 , a rough
estimate of the concentration increase in the path behind
a rotifer is 100 Wg P l31 . This concentration is two to three
orders of magnitude higher than that of the cultures with
P-limited algae and bacteria. The P regeneration rate per
unit of biomass is linearly related to the P content of the
food [34]. As the P:C ratio of algae and bacteria in this
study increased with increasing total P (Fig. 7), also the
regeneration rate and hence the concentration increase
that it creates will increase with total P. Also the frequency
of these paths with elevated P concentration will increase
with total P, as the rotifer density increased with total
P concentration (Fig. 4). The P-limited algae and bacteria
in our microcosms therefore experienced a spatial feast
and famine environments, which could have enabled coexistence due to the ‘release from healthy cells’ mechanism.
Patchiness in P created by grazers has been shown to
in£uence the outcome of P competition between algae
and bacteria [20]. Our data do not contain information
that would evaluate if the ‘release from healthy cells’
mechanism was the physiological reason for the observed
coexistence in our experiments, but the calculation and
reasoning above indicate that the necessary assumptions
are ful¢lled. Also physiological data suggest that heterotrophic bacteria are not superior competitors for P in
patchy environments [1].
4.4. Coexistence of P-limited algae and heterotrophic
bacteria as a phenomenon in plankton communities
Our data are in accordance with the observation of
coexisting P-limited algae and heterotrophic bacteria as a
normal phenomenon in lakes [1]. Moreover, the patchiness-related mechanisms and the ‘selective grazing’ mechanism may both serve as physiological mechanisms that
together make P co-limitation very likely in these environments where high N:P ratios are the normal phenomenon.
The cladoceran genus Daphnia, which often dominates the
metazoan zooplankton in lakes, contains many species
that are e⁄cient grazers of bacteria. Thus, it is likely
that Daphnia, possibly in combination with protozoa,
may ful¢l the requirement of the ‘selective grazing’ mechanism in lakes where they dominate.
Cyaan Magenta Geel Zwart
O. Vadstein et al. / FEMS Microbiology Ecology 46 (2003) 307^316
Growth-limiting factors of heterotrophic bacteria are
much less studied in marine systems, but as commented
above, recent studies suggest that this is not a rare phenomenon. Marine o¡-shore waters are characterised by
N:P ratios that are close to the typical optimal N:P ratio
of algae, and are therefore a fairly balanced medium for
algal growth. However, heterotrophic bacteria have P requirements that are typically 10 times higher than those of
algae, whereas their N requirements are not so di¡erent
[1,35]. Thus, a balanced medium for algal growth may
turn into a medium that is P-limited for both algae and
bacteria when the two groups of organisms are grown
together. The question whether this is a real phenomenon
in natural ecosystems is still to be answered. In many
coastal marine areas the freshwater input entails an increase in the N:P ratio of the system, and hence, the
probability of P limitation increases. In fact several of
the studies where P limitation of bacteria has been documented in marine systems are from brackish/coastal areas
[2^4].
Conditions for satisfying the requirements for the patchiness hypothesis are comparable in freshwater and marine
systems. The requirement for the ‘selective grazing’ mechanism is more di⁄cult to evaluate due to higher diversity
of metazoans in marine systems and the fact that generalist ¢lter feeders are normally not present in high densities.
Thus, the dual predation pressure on heterotrophic bacteria in freshwater systems due to grazing by both Daphnia
and heterotrophic £agellates is unlikely under normal conditions in marine systems. An exception may be in cases
where appendicularians form a signi¢cant fraction of the
metazooplankton. We therefore claim that coexistence of
P-limited algae and bacteria is less likely to be a usual
phenomenon in marine systems, but we do not anticipate
that it is a rare phenomenon.
We conclude that, as several mechanisms can promote
coexistence of simultaneously P-limited algae and heterotrophic bacteria, this may be a normal phenomenon in the
environments where P-limited algae are common, such as
lakes and coastal areas with skewed N:P ratios. In the
environments where P limits heterotrophic bacteria labile
DOC will accumulate.
Acknowledgements
We thank Y. Olsen for comments on the manuscript.
This work was supported by the Norwegian Research
Council (Contract 127176/120) and EC-FP5 contract
EVK3-2001-0023 (DANLIM).
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