THE REGULATION OF PHYTOPLANKTON STRUCTURE IN VITRO BY N:P RATIOS

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

ratio.

 Introduction

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

technologies.

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.

 Results

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

the rest).

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

groups.

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

N:P.

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.

 Discussion

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

1982).

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

dominant position.

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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culture. 2. Role des nutriments: assimilation et stockage

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environmental resources and the ways of algocoenosis

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