THE REGULATION OF PHYTOPLANKTON STRUCTURE IN VITRO BY N:P RATIOS
LEVICH A.P., CHUDOYAN A.A., BULGAKOV N.G. and ARTYUCHOVA V.I.
Subfaculty of Zoology of Vertebrates and General Ecology,
Department of Biology, Moscow State University
Vorobyovy gory, 119899 Moscow, Russia
The influence of basic nutrients nitrogen and phosphorus
ratio on taxonomic and size composition of natural phytoplankton
community in laboratory bath culture was studied. High ratios N:P
in nutrient medium (20-50) stimulate growth of Chlorophyta, while
Cyanophyta grow better at low ratios (2-5). The mean cell mass of
Chlorophyta is increasing but the one of Cyanophyta is decreasing
with the raising of N:P ratio. The analysis of experimental data
shows that it possible to manage a distribution of phytoplankton
in natural algal community by varying of nutrient resources
In the context of this study, the management of structure in
algocoenosis is understood as variation of biomass in taxonomic
or size groups of phytoplankton when the community is under
purposeful impact of the managing environmental factors. Such a
management may turn out to be useful for the optimization of
phytoplankton size, biochemical, toxicological, trophic and
production characteristics in reservoir blooming type regulation
problems, for providing feed supply for herbivorous fish and
invertebrates and also for the development of bioenergetic
Most of the above problems dictate the control strategy
basing on the domination of Chlorophyta, Bacillariophyta and
Euglenophyta cells and on diminishing the fraction of Cyanophyta,
as well as on the increased representation of large-size algal
species in the whole biomass.
In addition to the physical and chemical methods of
suppression of certain phytoplankton species, a purely ecological
approach is possible, i.e., the life of various microalgal groups
can be regulated by creating proper conditions, taking into
account the requirements of these groups in certain environmental
resource factors. In particular, the blooming type can be
drastically changed type by insertion of the basic biogenic
elements in ratios corresponding to the cellular quotas of the
phytoplankton groups to be optimized (Levich 1989). The cited
works contain model justifications of the suggested control
method and a review of empirical data on the effect of ratios of
mineral nutrition components on the phytoplankton. The best
nitrogen to phosphorus ratios (N:P) for green algae are those >
29 (Smith 1983). On the contrary, the blue-green algae dominate
in the community mostly at ratios of 5 to 10 (Schindler 1977),
while higher values inhibit their growth. It is shown that the
stimulating or inhibiting influence of biogenic element ratios
may also cause narrower effects characteristic of concrete
species. In other works (Levich and Bulgakov 1992, 1993) the
authors studied the regulation problems using as examples
artificial laboratory algocoenoses and natural phytoplankton
communities in situ.
Under laboratory bath culturing conditions, 4 species of
Chlorococcales were cultivated on two media with different
initial values of N:P (the first - 11 mg/l N and 3 mg/l P,
N:P=3.5; the second medium - 50 mg/l N and 2.5 mg/l P, N:P=20).
The species structure of the artificial community in the final
stationary condition greatly varied depending upon the value of
N:P (Levich and Bulgakov 1993, Table 1). Regarding the growth on
cellular reserves and on the medium substrates, Scenedesmus
quadricauda dominated in the community in terms of its increasing
abundance on both media. However, with N:P=3.5 this domination
was not absolute (44% of total abundance). When the value of N:P
rose to 20, S.quadricauda practically forced the three other
species out of the community. We analyzed the growth of the
species only due to N and P of the medium and found that when the
N:P ratio changes, the dominating species of algocoenosis also
changes: with N:P=3.5, it is Chlorella vulgaris, with N:P=20 it
is S. quadricauda (the growth of the other two species was zero).
To regulate the species and size structure of phytoplankton,
nitrogenous and phosphate fertilizers were applied in
fish-breeding ponds situated in the delta of Volga river (levich
and Bulgakov 1992). N:P ratios in fertilizers introduced into
experimental ponds was 25:1-50:1. Fertilizers were introduced 2
times a week from April to September. In the control ponds, N and
P were added in a 4:1 ratio every ten days. Biomass of
Chlorophyta was higher in the experiment than in the control
(Table 2), while the opposite situation was observed for
Cyanophyta. The average cell size of all phytoplankton phyla was
higher in the experiment than in the control.
The challenge of the present work is to study empirically
the influence of different values of the mineral nitrogen to
phosphorus ratio on the species and size structure of a natural
algocoenosis cultivated under controlled laboratory conditions.
Materials and methods
The experiments with natural phytoplankton have been
performed under controlled conditions. Water from a fish-breeding
pond (Astrakhan region) was placed in six 20-liter aquaria and
NH4NO3 and Ca(H2PO4)2 were added in different quantitative
combinations (Table 3). The initial biomass were the same in all
the aquaria. To prevent the effect of eating-out by zooplankton,
the water to be used in the experiment was let through a cellular
net with the corresponding cell size and left for two days in the
darkness. All aquariums were situated 50 m off the pond which was
used for water sampling; the illumination conditions in them and
in the pond were the same. One of the aquariums (No 7) did not
receive N and P, its concentration of nutrients equaled the
concentration in the pond. The aquariums with the same initial
N:P value were considered as repetitions for which the final
biomass of taxa and of size groups of phytoplankton were
averaged. In this way, in case of N:P=5 we found the mean for
aquariums 3,6 and 7; in case of N:P=11 - for aquariums 1,2 and 5.
The abundance and, simultaneously, masses of phytoplankton
cells (the latter by measuring individual size) were determined
under a microscope. The obtained biomass served as the basic
functional parameter for different systematic groups. The
observations of algal growth dynamics were conducted within 14
days. However, as early as in the middle of the experiment the
zooplankton abundance grew in the aquaria to a large extent. This
is explained by the fact that eggs and small-size forms of the
plankton had still penetrated through the net cells. As a result,
5. after about 10 days the phytoplankton mass values cannot be
regarded as true functions of nutrition and growth. In view of
this, the final biomass of phytoplankton were identified as the
mean values between the sixth and tenth days of the experiment.
The scheme of the second experiment, with a wider range of
initial biogenic element ratio values (Table 4), was not
fundamentally different, apart from the fact that the selected
portions of pond water were placed in 2-liter flasks. The
measures for zooplankton removal turned out to be more efficient
and within the eight days, while the experiment lasted, the algae
did not experience the pressure of grazing.
The biomass analysis was carried out both for large
phytoplankton taxons (Volvocales, Protococcales, Chlorophyta,
Bacillariophyta and Cyanophyta) and on the level of dominant
genera and species. To make clearer the terminology it is
necessary to indicate that all the species and genera were
partitioned into dominant ones (whose biomass amounted to no less
than 20 per cent of the total one on the 6th day of the
experiment in at least one of the aquaria), unrepresentative ones
(with biomass lower than 1 per cent) and subdominant ones (all
As seen from Figure 1,A, a stimulating influence on the
growth of Chlorococcales is exerted by the highest N:P equal to
19. For other taxa an increased ratio leads to growth inhibition.
The most pronounced growth degradation was observed for
Cyanophyta at the ratio of 19. A stimulating effect of high
ratios was observed as well for dominant Protococcales, namely,
Scenedesmus acuminatus and the Coelastrum. The representatives of
Bacillariophyta (Nitschia) and Cyanophyta (Phormidium) exhibit a
reverse dependence (Fig. 1,B,C).
The size structure analysis included a comparison of average
individual masses within phyla (this quantity is determined by
dividing the total phylum biomass by its total abundance on the
same day of the experiment). Besides, the fractions of certain
size classes of algae in the biomass were compared. For that
purpose all the species found were divided into six size groups
according to their volumes. After a recalculation from size to
mass units the following classes of cells were obtained: (1) less
than 0.1 ng; (2) 0.1 to 0.3 ng; (3) 0.3 to 1 ng; (4) 1 to 3.2 ng;
(5) 3.2 to 10 ng; (6) more than 10 ng.
An analysis of the average individual sizes (Fig. 2) shows
that an increase of the environmental N:P regularly diminishes
the sizes of Volvocales and Cyanophyta cells and slightly
increases the Protococcales cells (at a ratio equal to 16).
It should be clarified that in our case we do not consider
the changes in the absolute individual cell sizes but that in the
abundance of species with different individuals sizes. For
instance, when the N:P ratio increased among the Chlorococcales,
species with more massive cells or colonial species became
dominating (Scenedesmus quadricauda, S.acuminatus, Coelastrum
sp., Oocyctis sp., Pediastrum duplex).
An effect of high N:P on the size class representation is
pronounced only with respect to the biggest cells (> 10 ng) which
increase their biomass (Fig. 3).
As follows from the data presented, the N:P is an active
factor for phytoplankton distribution in pond water. However, the
rather narrow range of ratios (5 to 16) tested in the above
experiment, does not cover all possible combinations of factors
which regulate the phytoplankton state.
Therefore in another experiment we made an attempt to follow
the phytoplankton taxa responses to a wider range of ratios (in
magnitude and quantity).
Fig. 4 shows the final biomass for three main phytoplankton
phyla versus the initial biogenic ratio values. It can be seen
that ratios greater than 5 drastically change the algocoenosis
structure in the direction of absolute dominance of Chlorophyta.
The dependence curve for Chlorophyta has a single peak at N:P=20,
corresponding to the most rapid growth. For Bacillariophyta and
Cyanophyta the greatest biomass is achieved at lower ratios (2 to
5). Increased nitrogen addition suppresses the development of all
Chlorophyta consists mainly of Scenedesmus quadricauda (Fig. 4).
For the diatoms Stephanodiscus and Nitzschia the ratios
between 5 and 20 are optimal. Lastly, the blue-green alga
Microcystis develops best of all at ratios between 2 and 5.
Higher ratio values are an inhibiting factor for it.
The response of average individual sizes of the main phyla
to different biogenic element ratios on the eighth day of the
experiment nearly coincide with those for the biomass. The
biggest Chlorophyta cells are found in the flask with a ratio of
20 (Fig. 5). Passing to greater ratio values, one finds a
decrease of the average size, although it remains greater than at
ratios of 2 and 5. An increase of cellular volumes of diatoms at
N:P ratio of 100 should be noted. The Cyanophyta show a monotone
tendency of cellular volume decrease in response to an increased
The fractional abundances of algal size classes are shown in
Fig. 6. As the cells > 10 ng are extremely rare in the biomass,
we have excluded this class from analysis. Individuals from the
range 1 to 3.2 ng occupy a dominant position in the community at
N:P ratios of 20 and 50 while at higher and lower ratio values
their fractional biomass decreases. Cells with masses between 0.3
and 1 ng are the most abundant at the ratio equal to 5. The
representation of the two smallest classes falls down in the
transition from a ratio of 2 to 50; however, at a ratio of 100
they restore their dominant position.
Summarizing the results of the work, we conclude that the
ratio of nitrogen and phosphorus concentrations in water solution
acts as one of the regulating factors for the pond algocoenosis
structure. Placing the phytoplankton to partially controllable
conditions, we gain the possibility to directly follow the
process of consumption off nutrients and growth of cells in the
environment created by themselves. The results of Experiment 1
indicate that phosphate concentration within the studied range
and on the background of the prescribed nitrogen additions cannot
explain the microsuccession in the aquaria. As for nitrogen salt
content, its changes act in approximately the same direction as
does N:P ratios (Fig 1, 2). However, an increased initial nitrogen
concentration in the same experiment does not always lead to
evident results. Some species of Chlorococcales, both the
dominating (S. acuminatus, Coelastrum sp.) and the subdominating
ones (Actinastrum sp., Ankistrodesmus sp., Crucigenia sp.,
Dictyosphaerium sp., Micractinium quadrisetum, Nephrochlamis
subsolitaria, Tetraedron sp.) decrease their biomass when the
initial nitrogen concentration increases from 3.3 to 5.8 mg/l.
That means that their is no monotony of response if concentration
overfalls are not too great. Therefore, from our viewpoint it is
still the biogenic element ratio that determines the algal group
distribution. in the community. The point is that N:P ratio
determines that part of biomass of phytoplanctonic species,
genera, size class. In other words, the N:P value affects the
relative biomass p 4i 0 of phytoplanktonic groups. If the total
biomass of the community B is fixed, then the N:P ratio also
specifies the absolute biomass of the given groups: B 4i 0 = p 4i 0B.
As for the action of concrete values of the established
factor, the ratios near of 20 are the most favourable for the
growth of green algae, in particular, the Protococcales. It is to
be noted that higher ratios (50 and 100), although do not lead to
absolute biomass maxima for the Chlorophyta, by no means change
their dominant position in the community. Successful development
of the Cyanophyta is determined by low N:P (2 to 5). In all other
cases the growth of the phylum and its constituent dominant
species is substantially slowed down. For the Bacillariophyta the
stimulating ratio values are probably confined between 5 and 20.
Evidently the content of nutrients in the environment must
conform to the phytoplankton cells' requirements for them. This
concerns both the absolute concentrations of nitrogen and
phosphorus and their ratio. The requirements of phytoplankton
organisms in mineral nutrition components (or cellular quotas)
are not constant and vary depending on the growth stage (Levich
1989). For a given algal species a ratio of limiting factors in
the water is optimal if it equals the ratio of minimal quotas
(Rhee 1978; Levich 1989). Following Droop (Rhee and Gotham 1980)
we call a minimal quota the amount of the limiting nutrient in
the cell at zero growth rate.
A number of papers describe methods of determining the
requirements of microalgae in nitrogen and phosphorus (Levich
1989; Levich et al. 1986; Levich and Artyukhova 1991). The values
of cellular requirements for a number of species of green and
blue-green algae grown in laboratory in the bath culture from
Levich and Artyukhova 1991 are presented in Table 5.
Comparing those data with the final biomass of species in a
pond polyculture, we conclude that the requirements ratio for
green algae is at average close to their optimal combination in
the initial environment, i.e., to 20. Apparently for some species
of the Protococcales greater ratio values like 30 or 40 can be
also stimulating. For 1Scenedesmus quadricauda 0 the biogenic
element ratio which led to their maximum growth, turned out to be
one third of the cell quotas ratio (60, Table 5). In this case
most probably the minimal nitrogen quota was not achieved in the
experiment determining the requirements and the mitosis stopped
due to cells self-darkening.
The blue-green algae (Anabaena, Anacystis) can possess high
minimal quotas ratios (about 20) as well, whereas the growth of
the representatives of this phyla was restrained by just this
ratio value. However, there were no representatives of the above
species among the blue-green pond dominants which efficiently
developed at ratios of 2 to 5. Apparently, Microcystis, which
dominated among the Cyanophyta in the second experiment,
possesses another optimal ratio N:P=4, as derived by Rhee and
Gotham (1980). This value is close to the optimum in our
experiments (2-5). This example characterises the cyanobacteria
as strongly euribiotic organisms with respect to combinations of
nitrogen and phosphorus: their upper bound of the requirements
ratio reaches 100, as exemplified by Anabaena cylindrica (Dauta
The results of our experiments coincide with those of other
authors. From the already cited paper by Smith (1983) it follows
that the Cyanophyta dominate in lakes within the period when the
N:P is lower than 29. When this value is exceeded, the
Chlorophyta and Bacillariophyta begin to dominate.
Schindler (1977) found out in his experiments with small
lake fertilization that when the fertilizers are inserted with
the N:P equal to 30, the green alga 1Scenedesmus 0 dominated in the
phytoplankton community all over the experiment. When in another
lake the ratio of 11 was used, the nitrogen-fixing blue-green
algae Anabaena became dominant. Further on, when in the first
lake the ratio was lowered to a value of 5, it also changed the
blooming type: the Cyanophyta (Aphanizomenon gracile) occupied a
The size distribution of the organisms is also subject to an
influence of the biogenic element ratio. The ratio being
increased, the average individual size of the Chlorophyta grows
while the average individual size of Cyanophyta diminishes. Here
the value of 20 is also optimal for the Chlorophyta. Notably the
biggest cells of Bacillariophyta are met in the flask with the
initial ratio value equal to 100.
The results presented show that by varying the amount and
ratio of the nitrogen and phosphorus components of mineral
nutrition it is possible to regulate the taxonomic and size
composition of natural phytoplankton in vitro. The regulation is
conducted most probably on the level of algal species and may be
even on genera.
The authors thank A.A.Khudoyan and V.I.Artiukhova who have
participated in laboratory cultivation of natural phytoplankton
and in detecting of algal species in the samples as well.
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