Effect of nitrogen limitation on growth of Ankistrodesmus falcatus and Scenedesmus obliquus

Arch. Protistenkd. 139 (1991): 261- 273 Gustav Fischer Verlag lena

Botany Department, Faculty of Science, Assiut University, Assiut, Egypt

Effect of Nitrogen Limitation on Growth of Ankistrodesmus falcatus and Scenedesmus obliquus By A. A, MOHAMMED, A. M. AHMED & H. M. SHAFIK With 6 Figures Key words: Nitrogen limitation; Continuous culture; Green algae; Growth

Summary Ankistrodesmusfa/c(ltus and Scenedesmu.l' obliquus isolated from the Nile water were grown under N-Iimitation over a wide range of dilution rates. This study was performed in two identical chemostat devices with working volume of 1.61 I for A. fa/catus and 1.53 I for S. obliquus. The results showed that the growth rate of each algal species depends on the N-concentration. This was described by a saturation curve after the type of Monod equation. The half-maximum growth rate for A. fa/cat!ls and S. obliquus needed 210.7 flg N I I and IOS.2 flg N . 1 I, respectively. This shows that the patterns of yield coefficient and production rate for each algal species are opposite each other with respect to dilution rates. The growth of the two algal species in Nile water system under low concentrations of nitrogen was also discussed.

Introduction Besides the effect of light as a limiting factor for algal production in natural waters, the deficiency of nutrients and trace elements plays also a significant role (MOHAMMED & MULLER 1981). Phosphorus was often recorded to be growth limiting in freshwater habitats. The effects of the other nutrients, including nitrogen, have received limited attention, probably because their importance in primary productivity is less significant (GOTHAM & RHEE 1981). However, there is evidence that nitrogen limitation, which may occur during certain times of the year, plays an important role in species succession (FUHS et al. 1972; STORCH & DIETRICH 1979: GRAN ELI 1987). In addition, nitrogen can replace phosphorus in its role as a primary growth limiting element (O'BRIEN 1974; D'ELIA et a!. 1986; ISTIAVNOVES unpub!.). Moreover, nitrogen was indicated as the primary algal growth limiting nutrient in little Connemara Dam number 3 (ROBARTS & SOUTHALL 1977) and in Lake Biwa in late summer (TEZUKA 1985). Considerable attention has been paid to find out the nutrient concentration which affects the growth rate of phytoplankton species (HARRISON et a!. 1976; TILMAN et a!. 1976; MOHAMMED 1978). Growth rate is one of the prime factors determining community composition, dominant species, as a consequence of competition, grazing and sinking (see e.g. RHEE 1973; SOMMER 1988a). In addition, the variations in chemical composition of the whole phytoplanktonic algal population are always tightly coupled to the changes in algal growth rates (RHEE 1980). There is a consistent trend that increasing nutrient deficiency leads to decrease in the intracellular levels of limiting nutrients, relative to other cell components (GIDDINGS 1977). Some field studies on the Nile water system (AHMED et a!. 1986; MOHAMMED et a!. 1986; ELOTIFY, unpub!. data) showed a remarkable decline in N-eoncentration at certain times of the year. The range of the minimum concentrations of nitrogen was between 2 and 29 flg . I ~ 1. These concentrations may limit the growth rates of some phytoplankton algal species in Nile water and consequently influence the qualitative and quantitative composition of algal populations.

262

A. A. MOHAMMED et al.

However, here is still a real shortage of infonnation about N-limitation of Nile phytoplankton. Thus, a preliminary trial to find out how far such relatively low N-concentrations in Nile water could limit the growth of two dominant species of Nile algae was carried out in this investigation.

Materials and Methods Ankistrodesmus .!illcatus (CORDA) RALFS and Scenedesmus obliquus (TURP.) KUETZING were grown in batch and in nitrogen limited continuous cultures.

Batch cultures The vessels used in these experiments were previously described by AHMED et al. (1985), and the medium was a light modified Chu's 12 medium (CHU 1942). The modified medium includes only the raising of N- and Pconcentrations from 5.1 to 7.5 mg' I-I and from 0.89 to 1.0 mg' I-I, respectively. In addition, the concentrations of sodium bicarbonate and the micronutrients given by PROV ASOLI & PINTNER (1960) were doubled. The pH of the modified medium was 7.0 ± 0.2. Algae were incubated at 25± I DC under continuous illumination (cool-white fluorescent tubes, about 5,000 lux). Aeration was performed by bubbling with sterilized air. The daily cell number was used as a parameter for algal growth. The growth rate f.l (h - I) was determined according to the following formula: f.l=

In N I

-

In No

tl

-

to

where: N I and No of cell number.

= number of cells at time t l and to; t l

-

to

= the time elapsed in hours between two determinations

Continuous cultures The chemostat used in this investigation is shown in Fig. I, which is in principle similar to that described by MULLER (1972). The culture volume amounted to 1,610 ml for chemostat I (A. falcatus) and 1,530 ml for chemostat II (S. obliquus). A micro-pump (PM I, Fa. Buhler, Tiibingen, BRD) was used to maintain the flow rate of the culture medium from the reservoir to the culture vessel. Mixing and aeration of the culture were accomplished by pumping sterile air through the deeply inserted tube. The whole apparatus was sterilized in an autoclave before use. Samples were taken by a special tube inserted in the culture vessel. All nutrient concentrations were present in surplus except the limiting nutrient (nitrogen). The cultures, containing approximately the same number of cells of each test alga, were allowed to grow. Cell number was determined daily to estimate the maximum growth rate for each species before the operation of continuous supply with the nutrient medium. The nutrient pump was adjusted at the desired dilution rate. When steady state was achieved, biomass and nutrient concentrations were analysed. A steady state was assumed when the ambient Nconcentration and the cell numbcr showed no significant changes over a period of at least 3 days. At the steady state, growth rate (f.l) equals dilution rate (D). Samples were harvested to be further processed for the determination of certain parameters. Then. the dilution rate was set at the next higher value and the population was again allowed to reach the steady state. According to the theoretical basis of continuous culture given by FENCL (1966), TEMPEST (1970), PIRI (1975), GOLDMAN (1977), and MOLER ( 1987), the constants flmax and K, were calculated by transforming Monod equation SI

fl = flmax K,

+ SI

to a linear equation, where: f.l = growth rate at substrate limitation; flmax = the maximum value of fl at saturation level of substrate; K, = half saturation constant numerically equal to the substrate at which f.l = !!, flmax; S I = limited substrate concentration in culture vessel. The threshold value was calculated with the help of an iterative program, which was in the computer center at Cairo university. Cell counts were made with a haemocytometer, 0.1 mm deep, having improved Neubauer ruling (A. O. Spencer "Brighlfine"). The mean counts are given as cells' I-I algal suspension. To determine the dry weight, a definite volume (50 ml) of algal suspension was filtered through a dry weighted membrane filter (Sartorius, S. M. type 11306, porc sizc 0.45 ,1m). Data are given as mg . I ' algal suspension. Chlorophyll-a was determined spectrocolorimetrically as recommended by STRICKLAND & PARSONS (1968), and its concentration was calculated

Effect of Nitrogen Limitation on Growth

263

2

1

3

§

14§

6

I

,,- -_ -, i

i

15

I

8

Fig. ). Schematic representation of the chemostat device. I - Medium reservoir; 2 - Measuring tube; 3 - Micro pump; 4 - Chemostat culture vessel; 5 - Overflow tube; 6 - Inflow medium tube; 7 - Air inlet tube; 8 - Overflow vessel; 9 - Sampling device; 10 - Withdrawal tube; II - Sterile glass wool filter; 12 - Prefilter; 13 - Air inlet; 14 - Fluorescent lamps.

using the equation of Scor/Unesco (1966). The nitrate - nitrogen was determined using the sodium salicylate method (MULLER & WIDEMANN 1955). Phosphate-phosphorus was determined by the molybdate blue method with ascorbic acid as a reducing agent (VOGLER 1965).

Results Batch culture We used the basic nutrient medium number 12 after CHU, the maximum growth rate of A. falcatus and S. obliquus did not exceed 0.037 . h -I and 0.041 . h -I, respectively. The application of the modified nutrient solution resulted a maximum growth rate of 0.061 . h- 1 for A. falcatus and 0.060· h- I for S. obliquus (Table I).

264

A. A.

MOHAMMED

et aL

Table I. Effect of variously modified Chu 12 medium on the maximum growth rate f-lmax (calculated on the basis of cell number) and the generation time (G) of A. falcatus and S. obliquus grown at a temperature of 25 ± 1°C, and under continuous light of about 5000 Lux

Original Chu 12 medium = A A+2.5mgN·!-I=B B+O.llmgP-r ' B + Mg doubled B + HCO, doubled B + K doubled B + Micronutrient clements doubled A, doubled concentrated Final modified Chu 12 medium (B+O.II mgP·j '+HCO~, micronutrient clements doubled)

% of improvement of f-lmax in comparison with that of the original nutrient solution

Maximum growth rate (h ')

G (h)

Generation time

A jcJleatus

S.obliquus

Afaleatus

S.obliquus

Afalcatus

S.obliquus

0.037 0.057 0.048 0.051 0.052 0.049 0.057

0.041 0.059 0.056 0.051 0.048 0.054 0.056

18.73 12.16 14.44 13.59 13.33 14.15 12.16

16.91 IU5 12.38 13.59 14.44 12.84 12.38

100.0 154.1 129.7 137.8 140.5 132.4 154.1

100.0 143.9 136.6 124.4 117.1 131.7 136.6

0038 0.061

0.048 0.060

18.24 11.36

14.44 11.55

102.7 164.9

117.1 146.3

Table 2. Effect of nitrogen-concentration in inflow medium on the growth, yield coefficient and production rate of A. jillcatus and S. ohliqllus at a constant dilution rate A. fidel/tIIs

Dilution rate D (h - ') N-concentration (mg .1- 1 ) Cell number I-I . lOR Dry weight (mg' 1-- 1 ) NIiO x cells (mg) Chlorophyll a (fIg' I ') pH

S. ohliqllus

0.012515 ± 0.000388 10

5

32.650 89.000 O. 153 39.430 8.700

0.013137 ± 0.000378

± ± ± ±

1.3400 18.4000 (1.0060 0.3820

57.080 167300 0.175 99.100 8.960

10

5

± ± ± ±

1.9200 11.3700 0.0058 5.2700

40.200 ± 147.000 ± 0.124± 30.500 ± 9.440

7.8000 74.950 ± 4.2100 38.1000 256.000 ± 22.6200 0.0244 O.133± 0.0075 2.8600 73.510 ± 6.8000 9.680

Y (Yield coefficient) mg dry weightlmg app. cons. N') 17.910 ± 3.6900 Cell number· 10'/mg app. cons. N 6.570 ± 0.2300 [!g chlorophyll/mg app. cons. N 7.940 ± 0.0800

16.780 ± 1.1400 5nO± 0.1900 9.930± 0.5300

29.550 ± 7.6800 25.620 ± 2.2600 8.080 ± 1.5600 7.500 ± 0.4200 6.130 ± 0.5700 7.360 ± 0.3800

Production rate mg dry weight· I-I. hi Cell number· lOx. I-I . h- I [!g chlorophyll a . \-1 . h- I

1.110± 0.2300 0.410 ± 0.0200 0.490 ± 0.0050

2.090 ± 0.1400 0.710± 0.0240 1240 ± 0.0700

1.930 ± 0.5000 0.530 ± 0.1000 0.400 ± 0.0380

3.360 ± 0.3000 0.980 ± 0.0600 0.970 ± 0.0900

Effect of Nitrogen Limitation on Growth

265

Continuous culture Effect of N-concentration on the algal growth at a constant dilution rate: From Table 2 it can be seen that the rate of dry weight production and cell concentration more or less doubled with the doubling of the N-concentration in the inflow medium. Moreover, the amount of chlorophyll-a obtained under the N-concentration of 10 mg . l~l was more than the doubled amount obtained at the concentration of 5 mg . I~ I. The yield coefficient, calculated on the basis of dry weight, cell concentration and chlorophyll-a, in relation to the limiting substrate, remained almost unchanged for both algal species. It can be generally concluded that nitrogen at the concentration used (l0 mg . I-I) is the only limiting factor for the algal growth in the experimental medium. Correlation between the N-concentration and the growth rate at various dilution rates: Fig. 2 shows that the growth rates of A. falcatus and S. obliquus depend on the external Nconcentration in the chemostat culture vessel. This dependence was obviously clear at Nconcentrations ranging between 0 and 125 ~g . I-I for A. falcatus and between 0 and 75 ~g . I-I for S. obliquus. The threshold values below which A. falcatus and S. obliquus in the long run can not exist or their growth rate will be of negative value (i.e. death rate is greater than growth rate) are 19.5 ~g . I-I and 0.4 ~g . I -I, respectively. Taking into consideration these threshold values, the growth rate as a function of external N-concentration could be described by a saturation curve after the type of Monod equation. The maximum growth rates, calculated from chemostat data, reached 0.0366 . h -I and 0.04025 . h- I for A. falcatus and S. obliquus, respectively. For the half maximum growth rate (Ksl Ankistrodesmus needed 210.7 ~g N . I-I while Scenedesmus must be supplemented with 105.2 ~g N . I-I.

The biomass and N -concentration Fig. 3 clearly shows that in the case of A. falcatus, the cell number was, to some extent, stable until a dilution rate of 0.02 . h -I. while in the case of S. obliquus the stability of cell number continued until a dilution rate of 0.03 . h- I. Thereabove, the cell number decreased gradually with the increasing dilution rate for both algal species. The pattern of changes in external Nconcentration showed nearly an opposite trend to those of cell number and dry weight for both algal species, where the N-concentration slightly increased at dilution rate less than 0.03 . h -I, then sharply increased.

Chlorophyll-a content From the results presented in Fig. 4, it can be seen that, in the case of A. falcatus (Fig. 4A), the contents of chlorophyll-a (calculated on the basis of dry weight or apparent consumed nitrogen) were more or less affected under the various dilution rates. On the basis of cell number, a pronounced increease in chlorophyll-a content at the dilution rate of 0.0124 . h- 1 was observed. Thereabove, a gradual decrease was observed until the dilution rate of 0.02 . h -I. With further rise of dilution rate, the content of chlorophyll-a was again raised and reached its maximum value at a dilution rate of 0.0361 . h~l. On the basis of culture volume, chlorophyll-a exhibited a maximum level at a dilution rate of 0.0151 . h- I , then decreased gradually with the increase in the dilution rate with a slight increase at the highest used dilution rate. In the case of S. obliquus (Fig. 4B) the chlorophyll-a content (calculated on the basis of dry weight, cell number or apparent consumed nitrogen) exhibited the same trend with a slight increase with the rise of dilution rate. However. at the highest dilution rate (0.0397 . h- I ) the values of contents were decreased whatever the basis of calculation used. On the basis of culture volume, chlorophyll-a contents increased gradually and reached its maximum value at a dilution rate of 0.0361 . h~', then dropped sharply with the further increase in the dilution rate.

266

A. A.

MOHAMMED

et al.

~

~

a

~~-------=============-= .---/

Oll/~

0,03

0,02

I

.

1I

I

I 0,01

1000

2000

;'000

3000

5000 1JgN,(f

Fig. 2. Effect of nitrogen concentration on the growth rate of A. falcatus (X--X) and S. obliquus (0- - -0) at various dilution rates.

A

..

'I

~250

5

~

~

-':i

.

1.200

t

1SO

f-,J--L. 'i-

'"s 100 50~

I

I

,"---_

t

0,01

...

0.02

~t

- '1-,

'1- .,

-----. 0.03

.

~250

~

3

..:

2

-5 '"s 100

0.040(h-l )

II j!

300

4

I

B

..- '200

kf-..

~150

50 0

t-.,

,

~"

I-

I

13

'~ I

~2

.'+.-.- "

y\

-~

0.01

0.02

0.03

OfJ/.O(Hi)

Fig. 3. Effect of N-conccntration (=) on the biomass Icell number (--) and dry weight (-.-._)J of A A. falcatus (X) and B - S. obliquus (0) at various dilution rates.

Yield coefficient The yield coefficient refers to the degree of uptake of the substrate (here nitrogen) by organisms to build up their biomass. Due to the fluctuations in nitrogen consumption by the two test algae, the yield coefficient for dry weight, cell number and chlorophyll-a (calculated on the basis of apparent consumed nitrogen) exhibited mostly variable values. In the case of A. falcatus (Fig. SA), the yield coefficient for the dry weight showed its maximum value at the dilution rate of 0.0151' h -I. Thereabove, a continuous decrease was recorded. Concerning the yield coefficient for cell number, a slight decrease was recorded with

267

Effect of Nitrogen Limitation on Growth 0.1'

A

0,12

A 0,10

';"z 0.08

~

Cc

E

~ J

~0.06

~O.O'

~

I /1'i-_ _ y~ A'

~

~

~

o~

== en ~E

0.01

0.02

~.

0.03

~'

~

...

,

~

l'I

'52

en E

,/

~1

/

.2! .~ 0.0' Dfh-/ J

e~

u

-_. -1',.. . . . . . . .

,/ ,,/r~

~I> 2

~ ,~.::.:.~.:-:-~.~-~-~.~.:::-:.:r:.='~

'00.02

L_.r\1 ',1

---T /(1

e

c o

~

~

0.01

"

o.OJ

01)1

0.0' OflP)

~ ~

0,1'

e~.c· a. ":"..:.

B

7


J6

0,12

~

~ 0,10

B

5

~

0.08

0.0'

0.02

A'

I'.J.""-"-~-

O.CS

~_... _l---I-- +- __ .1--+ ~::'-:-:-~ _:·:t·::-:-:-·L --:.~ - ::-+-~~ 0.01

0.02

O.OJ

O.Ol Gail)

/

,,/

",

""'!

/'t-:~\

\\\\

~ ......r--

0,01

0.02

\\\\

O.OJ

0.0' Offi1)

Fig. 4. Chlorophyll - a contents of A- A. fa/calus (X) and B- S. ob/iquus (0) at various dilution rates. (--) chla.I- I ; (- -) chI-a. cells-I; (. . . ) chi-a. N- I ; (_._._) chl-a dry wt.- I . Fig. 5. Yield coefficient of A- A. fa/calus (X) and B- S. obliquus (0) at various dilution rates. (_._._) dry weight; (- -) cell number; (--) chlorophyll-a.

the rise of the rate of dilution. The yield coeffcient for chlorophyll-a biosynthesis increased gradually till it reached its maximum value at the dilution rate of 0.0151' h- I . Thereabove, a gradual decrease with the rise of dilution rate was recorded. In the case of S. ohliquus (Fig. 5B), the yield coefficient for dry weight decreased considerably with the rise of the dilution rate. As regards the cell number, the yield coefficient decreased slightly with the rise of dilution rate. With respect to chlorophyll-a biosynthesis, the yield coefficient increased slightly till it reached its maximum at the dilution rate of 0.0301 . h- 1 , above which it decreased again. Production rate The production rate refers to the biomass which overflows the culture vessel per unit time. In the case of A. falcatus (Fig. 6A), a considerable increase was recorded in the production rate of dry weight and chlorophyll-a concentration till they reached their maximum values at the dilution rate of 0.0283 . h-- 1 As regards the cell number, the rate of production increased gradually until it reached its maximum at a dilution rate of 0.02 . h- I . At relatively higher dilution rates, the production rate exhibited lower values whatever basis of calculation was used. In the case of S. ohliquus (Fig. 6B) the production rate of the three mentioned parameters (dry weight, cell number and chlorophyll-a level) increased regularly till they reached their maximum at a dilution rate of 0.030 I . h --I. Above that, they decreased considerably with the rise of the dilution rate.

268

A. A.

MOHAMMED

ct al.

35

A 30

I

25

"'f'

20

r

I

..-t--

I ~z

J... ' j1.••

15

r

g'

I

'1-.

~

-

"'1_

-..- ---

/t

!:!J 10

~ ."

h......-t---h......

"='z

g' 0,01

~ ~

0.0( [}(h1 J

0.03

0.02

~~ o 0_ ~

u '0

~

51

g

.t:

35

"k"

30

B

g' ~2S .~

Lt ."'.....

~ 20

+ ....... 1"....

g' -,

/

'0

••••.

1 __ ~\ /1 "\ '

-+--+-~

" 1----.-~ 0.01

l

0.01

0.02

, \)

Fig. 6. Production rate of A - A.falcatus (X) and BS. obliquus (0) at various dilution rates. (-. -. -) dry weight; (- -) cell number; (--) chlorophyll-a.

I---r---O.O/, O(Jil)

Table 3. Nitrogen and phosphorus levels in A. falcaws and S. obliquus and the dissolved nitrogen in the chemostat culture vessel under N-Iimitation at various dilution rates Dilution rate D (h- I )

N. dissolved

f.lg Nil 06 cells

f.lg PII 06 cells

x

x

x

s

s

I. Ankistrodesl11l1s fa/catll.1

0.0095 0.0124 0.0151 0.0200 0.0245 0.0283 0.0311 0.0361

00949 01073 01108 0289.0 04363 07088 2043.5 26475

± 000.0 ± ± ± ±

007.8 004.0 009.2 000.0 ± 017.1 :~ 063.3 ± 211.4

145 1.71 172 160 2.28 2.96 3.43 4.14

± ± ± ± ±

0.029 0.093 0.241 0.142 0.137 ± 0.079 ± 0.080 ± 0.522

0.185 0.187 0.220 0.211 0.207 0.209 0.194 0.167

± ± ± ± ±

0.009 0.010 0.033 0.026 0.017 ± 0.024 ± 0.011 ± 0.001

2. Scenedesl11l1s ob/iqlllls

0.0100 0.0120 0.0148 0.0200 0.0250 0.0301 0.0352 0.0397

0043.7 ± 000.0 0042.1 ± 004.0 0054.6 ± 013.2 0064.8 ± 002.2 0142.2 ± 000.0 0231.6±013.2 15310±408.7 48155 + 2690

1.34 ± 0.058 129 ± 0.010 1.55 ± 0.360 1.82±0.146 165±O.135 1.55 ± 0.320 1.97 ± 0521 1.79 ± 0.034

0.172±0.011 0.170 ± 0.001 0.202 ± 0.050 0.230 ± 0.019 0.201 ± 0.025 0.169 ± 0.035 0.166 ± 0.021 0.165 ± 0.006

Effect of Nitrogen Limitation on Growth

269

The apparent consumed nitrogen and phosphorus by algal cells at various dilution rates A steady-state, the difference between nitrogen concentration in the inflow medium and that in chemostat culture vessel, was used to determine the particulate nitrogen in algal cells. The results in Table 3 show that the nitrogen content can contribute to the biomass production for both algal species by increasing the dilution rate. Generally, the apparent consumed nitrogen (calculated per cell) was vigorously changed especially in the case of S. obliquus; however, that of phosphorus in both algal species showed an almost independent relation under various dilution rates.

Discussion Phytoplankton limitation could be attributed to some physicochemical factors in addition to the interaction between the various groups of the phytoplankton community. In open water areas the determination of the factors limiting phytoplankton growth could be achieved by the determination of various parameters (PORCELLA & BISHOP 1970). It has been ascertained that, at least during a certain period of the year, some inorganic nutrients, such as P, N, Si, are limiting for the primary production not only in freshwaters (LUND 1965; ISTVANOVICS et al. 1986; KAGAWA et al. 1988) but also in marine waters (HARRISON 1974; GRANELI 1987). Moreover, nutrient limitation was considered as the most critical factor in determining phytoplankton growth rate in the epilimnion of thermally stratified lakes and oceans (O'BRIEN 1974). The growth limitation of phytoplankton by nutrient deficiency was studied using phosphorus (HUTCHINSON 1957; ELSTER 1964; VOLLENWEIDER 1968; Moss 1969; BROWN & BUTTON 1979; LANG & BROWN 1981; LEAN et al. 1987), nitrogen (BRINGMANN & KUHN 1965; TALLING 1966; DUGDALE 1967; GACHTER 1968; STADELMANN 1971; CARACO et al. 1987), silicon (LUND 1950,1954; KILHAM 1971; SCHELSKE & STOERMER 1971,1972; PAASCHE 1973; HARRISON 1974; HARRISON et al. 1976, 1977; MOHAMMED & MOLLER 1981), micronutrients (RODHE 1948; GOLDMAN 1960, 1964; FUSE 1987) and organic materials (DROOP 1966, 1968, 1970; TOULIBAH, in preparation). In natural freshwaters the nitrate nitrogen concentration was repeatedly recorded to exhibit very low values at least during certain periods of the year (e.g. 55 I-lg . 1-1 in Titisee, W. Germany, SZYMANSKI-BuCARY 1974; 63-821-lg . 1-1 in Uberlingersee, W. Germany, MOHAMMED & MOLLER 1981; 33.6 I-lg . I-I in Lake Biwa, Japan, ISHIDA & MITAMURA 1988). In Lake Kariba, Rhodesia, Africa, the nitrate-nitrogen concentration rarely exceeds 20-25 I-lg . I-I (COCHE 1974). Such low N-concentration could be considered as limiting for the growth of some algal species. TOERIEN (1974) pointed out that a nitrate-nitrogen concentration of 53.3 I-lg . 1~1 permits only half the maximum growth rate for green alga Selenastrum capricomutum. In Nile water a minimum N-concentration of 14 I-lg . I-I was recorded at Assiut, Egypt in April 1981 (AHMED et al. 1986),29 I-lg . 1-1 at Sohag in March 1982 (MOHAMMED et al. 1986) and 2.35 I-lg . I-I in Aswan High Dam Lake in September 1982 (EL-OTIFY, unpubl. data). These values are lower than those mentioned above. Therefore, the growth of the phytoplankton in the Nile water may be controlled by these low ambient nitrogen concentrations. The possibility of Nlimitation in River Nile, at least at definite periods of the year, is in accordance with the results obtained by some authors. TALLING (1966) postulated some ecological evidence nitrogen was seasonally limiting in Lake Victoria. PROWSE & TALLlNG (1958) also found correlations between phytoplankton growth and organic nitrogen in White Nile. Moss (1969) found that nitrate or nitrate and phosphate were potentially limiting to algal growth in nine Malawi lakes (Africa). Also, nitrogen was found to be the primary algal limiting nutrient in an oligotrophic lake, little Connemara Dam, Rhodesia (ROBARTS & SOUTHALL 1977). The minimum N-concentration may sometimes limit the growth of only some phytoplanktonic algae, consequently influence the qualitative and quantitativc composition of algal population and could be a reason for competitive exclusion in the species spectrum.

270

A. A. MOHAMMED et al.

As regards the effect of N-limitation on Nile phytoplankton, there are no quite satisfactory informations. To clarify how far these minimal N-concentrations are limiting for growth of Nile algae, some experiments were conducted using both batch and continuous culture techniques. The continuous culture was used to maintain a steady-state growth in constantly controlled conditions. This technique was repeatedly used to study the limitation of nitrogen (MADDUX & JONES 1964; CAPERON 1968; CAPERON & MEYER 1972; THOMAS & DODSON 1972; OSBORNE & GElDER 1986), phosphorus (FUHS 1969; MOLLER 1972; GOLDMAN 1977; SHAFIK & HERODEK 1989), silicon (DAVIS 1973; HARRISON et a!. 1976; MOHAMMED 1978; SOMMER 1988b), carbon: nitrogen: phosphate ratio (GOLDMAN 1986), and phosphate and nitrate (ELRIFI & TURPIN 1985). Comparing the growth of the two algal species, it has been shown that A. falcatus requires relatively higher amounts of nitrogen than S. obliquus. This gives Scenedesmus obliquus a considerable advantage of growth under low N-concentration and can compete successfully than Ankistrodesmus falcatus. At higher dilution rates, the N-limitation diminishes and the cellular N-content comes closer to the contents of exponentially growing cells. This correlation between the growth rate and the concentration of the limiting nitrogen was illustrated by a saturation curve similar to that of Michaelis-Menten. It was noticed that the threshold value for S. obliquus was 0.4 flg . I-I, which is very low in comparison with that of A. falcatus (19.5 flg . 1-1). In addition, the N-concentration for the half maximum growth rate. K s , was found to be 105.2 flg . I-I for S. obliquus and 210.7 lAg . I-I for A. fa/catus. In nutrient limitation experiments it was repeatedly found that the concentration of the limiting substrate in the medium plays an important role not only for the growth rate, but also for the chemical composition of the cells. One consistent trend is the decrease in intracellular levels of the limiting nutrients with increasing nutrient deficiency. This pattern has been found after limitation of many nutrients (e.g. CAPERON & MEYER 1972; PAASCHE 1973; GOLDMAN 1977; MOHAMMED 1978; DROOP 1983; SHAFIK & HERODEK 1989). Also, it has been found that variations in chemical composition of phytoplankton were tightly coupled with changes in growth rate (GOLDMAN et a!. 1979; RHEE 1980). The patterns of these changes depend on the nutritional status of a cell population in response to the degree of nutrient limitation (RHEE 1973). In the case of nitrogen depletion the newly formed cells were characterized by deficiency of protein and chlorophyll contents and by high lipid and carbohydrate contents (IWAMOTO & SUGIMOTO 1958). MOHAMMED (1978) found that Si, P and N contents of Si-limited cells of Asterionella formosa and Stephanodiscus hantzschii increased with increasing dilution rate. THOMAS & DODSON (1972) found that cells of the marine diatom Chaetoceros gracilis which were given in chemostat under N-limitation. exhibited a decrease in the C: N ratio and in the cellular C: chlorophyll ratio. with the increase in growth rate. The above results ascertained that the low concentrations of nitrogen in Nile water and in Aswan High Dam Lake at certain periods of the year could be regarded as a limiting growth factor for some algal species. The data of the present experiments showed that A. falcatus at a nitrogen concentration of 210.7 flg . I-I and S. obliquus at a nitrogen concentration of 105.2 flg . I-I reached only half of their maximum growth rate. Thus, the minimum N-concentrations that were recorded in the Nile water in the previous studies could be considered as limiting levels for the growth of the test algae. Moreover, it can be said that, in general, the relatively low Nconcentrations recorded in Nile water (AHMED et a!. 1986; MOHAMMED et a!. 1986) and in Aswan High Dam Lake (EL-OTIFY, un pub!. data) were also limiting for algal growth throughout the years of study.

Literature AHMED, A. M., MOHAMMED, A. A.. HEIKAL, M. D., & MOHAMMED, R. A. (1985): Physiology of some Nile algae. I. Effect of increased NaCI concentration in the medium. Acta HydrobioI. 27: 25-32.

Effect of Nitrogen Limitation on Growth

271

AHMED, A. M., HEIKAL, M. D., MOHAMMED, A. A., & ZIDAN, M. A. (1986): Field and laboratory studies on Nile phytoplankton in Egypt. I. Some physical and chemical characteristics. Int. Rev. ges. Hydrobiol. 71: 127-138. BROWN, E. J., & BUTTON, D. K. (1979): Phosphate-limited growth kinetics of Selenastrum capricornutum (Chlorophyceae). J. Physio!. 15: 305 - 311 . BRINGMANN, G., & KUHN, R. (1965): Nitrat oder Phosphat als Begrenzungsfaktor des Algenwachstums. Gesundheits-Ingenieur 86: 210~214. CAPERON, J. (1968): Population growth response of lsochrysis galbana to nitrate variations at limiting concentrations. Ecology 49: 866-872. - & MEYER, J. (1972): Nitrogen-limited growth of marine phytoplankton. I. Changes in population characteristics with steady state growth rate. Deep-Sea Res. 19: 601-618. CARACO, N., TAMASE, A., BouTRous, 0., & VALtELA, I. (1987): Nutrient limitation of phytoplankton growth in brackish costal ponds. Can. J. Fish. Aquat. Sci. 44: 473-476. CHU, S. P. (1942): The innuence of mineral composition of the medium on the growth of planktonic algae. I. Methods and cultural media. 1. Eco!. 30: 284- 325. COCHE, A. G. (1974): Limnological study of tropical reservoir. In: E. K. BALON & A. G. COCHE (eds.), Lake Kariba: A Man-Made Tropical Ecosystem in Central Africa. The Hague. DAVIS, C. O. (1973): Effects of changes in light intensity and photoperiod on the silicate-limited continuous culture of the marine diatom Skeletonema costatum (GREV.) CLEVE. Ph. D. Diss., Univ. Washington, Seattle. D'ELlA, C. F., SANDERS, J. G., & BOYNTON, W. R. (1986): Nutrient enrichment studies in a coastal plain estuary: phytoplankton growth in largescale, continuous cultures. Care J. Fish. Sci. 43: 397-406. DROOP, M. R. (\966): Vitamin B 12 and marine ecology. III. An experiment with a chemostat. J. Mar. BioI. Ass. U.K. 46: 659-671. (1968): Vitamin B I2 and marine ecology. IV. The kinetics of uptake, growth and inhibition in Monochrysis lutheri. J. Mar. BioI. Ass. U.K. 48: 689-733. (1970): Vitamin B 12 and marine ecology. V. Continuous culture as an approach to nutritional kinetics. Helgoland. wiss. Meeresunters. 20: 629-636. (l9~3): 25 years of algal growth kinetics. Bot. Marina 26: 99-112. DUGDALE, R. C. (1967): Nutrient limitation in the sea. Dynamics, identification and significance. Limno!. Oceanogr. 12: 685-695. ELRIFI, I. R., & TURPIN, D. H. (1985): Steady state luxury consumption and the concept of optimum nutrient ratios: A study with phosphate and nitrate limited Selenastrum minutum (Chlorophyta) 1. Phyco!. 21: 592-602. ELSTER, H. J. (1964): Die Stoffwechseldynamik der Binnengewasser. Zool. Anz., Supp!. 27: 335-387. FENCL, Z. (\966): A theoretical analysis of continuous culture systems. In: I. MALEK & Z. FENCL (ed.), Theoretical and methodological basis of continuous culture of microorganisms, pp. 67-153. New York, London. FUHS, G. W. (1969): Phosphorus-limited plankton diatoms. Verh. Int. Verein. Limno!. 17: 784-786. DEMMERLE, S. D., CANELL!, E., & CHEN, M. (1972): Characterization of phosphorus-limited plankton algae (with renections on the limiting nutrient concept). In: G. E. LIKENS (ed.), Nutrients and Eutrophication, Special Symposia, pp. 113-132. The American Society of Limnology and Oceanography, Vol. I. Laurence, Kansas. FUSE, H. (\987): Effects of trace metals on growth of Red Tide phytoplankton and their accumulation of metals. Agric. BioI. Chem. 51: 987-992. Gi\.CHTER, R. (1968): Phosphorhaushalt und planktische Primarproduktion im Vierwaldstattersee (Horwer Bucht). Schweiz. Z. Hydrol. 30: 1-66. GIDDINGS, J. M. (1977): Chemical composition and productivity of Scenedesmus abundans in nitrogen limited chemostat culture. Limno!. Oceanogr. 22: 911-918. GOLDMAN, C. R. (1960): Molybdenum as a factor limiting primary productivity in Castle Lake. Hydrobiologia 42:

258-294. GOLDMAN, J. C. (1977): Steady-state growth of phytoplankton in continuous culture: comparison of internal and external nutrient equations. J. Phyco!. 13: 251- 258. (1986): On phytoplankton growth rates and particulate C: N: P ratios at low light. Limno!. Oceanogr. 31:

1358-1363. MCCARTHY, J. J., & PEAVEY, D. G. (1979): Growth rate innuence on the chemical composition of phytoplankton in oceanic waters. Nature (London) 279: 210-215. GOTHAM, I. J., & RHEE, G. Y. (1981): Comparative kinetic studies of nitrate-limited growth and nitrate uptake in phytoplankton in continuous culture. 1. Phyco!. 17: 309-314. GRANELI, E. (1987): Nutrient limitation of phytoplankton biomass in a brackish water bay highly innuenced by river discharge. Estuarine, Coastal and Shelf Science 25: 555-565.

272

A. A. MOHAMMED ct al.

HARRISON, P. J. (1974): Continuous culture of the marine diatom Skeletonema costatum (GREV.) CLEVE, under silicate limitation. Ph. D. Diss., Univ. Washington, Seattle. CONWAV, H. L., & DUGDALE, R. C. (1976): Marine diatoms grown in chemostat under silicate or ammonium limitation. I. Cellular chemical composition and steady state growth kinetics of Skeletonema costatum. Mar. BioI. 35: 177-186. - HOLMES, R. W., & DAVIS, C. O. (1977): Marine diatoms grown in chemostats under silicate or ammonium limitation. III. Cellular chemical composition and morphology of Chaetoceros deb/lis, Skeletonema costatum and Thalass/osira gra\'ida. Mar. BioI. 43: 19-31. HUTCHINSON, G. E. (1957): A treatise on limnology. Vol. I. Geography, physics, chemistry. New York, London. - (1967): A treatise on limnology. Vol. II. Introduction to lake biology and the limnoplankton. New York, London. ISHIDA, N., & MITAMURA. O. (1988): Effect of nitrogen and phosphorus addition on the phytoplankton growth in water collected from different trophic regions of Lake Biwa. Physiol. Ecol. Japan. 25: 9-17. ISTVANOVICS, V., VOROS, L., HERODEK. S., T6TH. G. L., & TATRAI, I. (1986): Changes of phosphorus and nitrogen concentration and of phytoplankton in enriched lake enclosures. Limnol. Oceanogr. 31: 798-811. IWAMOTO, H., & SUGIMOTO, H. (1958): Fat synthesis in unicellular algae. III. Absorption of nitrogen by nitrogendeficient Chlorella cells and its effects on the continuous cultivation of fatty cells. Bull. Agr. Chem. Soc. Japan 22: 410-419. KAGAWA, H., TOGASHI, M., & KAGABATA, Z. (1988): Comparison of P, Ca and Mg contents of phytoplankton between the heads of two river reservoirs with different phosphorus loading. Verh. Int. Verein. Limnol. 23: 1022-1027. KILHAM, P. (1971): A hypothesis concerning silica and the freshwater planktonic diatoms. Limnol. Oceanogr. 16: 10-18. LANG, D. S., & BROWN. E. J. (1981): Phosphorus-limited growth of a green alga and a blue-green alga. Appl. Environm. Microbiol. 1002-1009. LEAN, D. R. S., BOTT, A. A., & PICK. F. R. (1987): Phosphorus deficiency of Lake Ontario plankton. Can. J. Fish. Aquat. Sci. 44: 2069-2076 LUND, J. W. G. (1950): Studies on Asterionellaformosa HASS. II. Nutrient depletion and the spring maximum. J. Ecol. 38: 1-35. (1954): The seasonal cycle of the plankton diatom Melosira italica (EHR.) KUTz, subsp. subarctica O. MULL. J. Ecol. 42: 151-179. (1965): The ecology of freshwater phytoplankton. BioI. Rev. 40: 231-293. MADDUX, W. S., & JONES, R. F. (1964): Some interactions of temperature, light intensity and nutrient concentration during the continuous culture of Nitzschia e/osterium and Tetraselmis sp. Limnol. Oceanogr. 9: 79-86. MOHAMMED, A. A (1978): Freilandmessungen und Experimente zur Frage der Nahrstofflimitierung von Planktonalgen im Bodensee. Diss .. Fak. fUr Biologie, Albert-Ludwigs-Universitat, Freiburg. & MULLER, H. (1981): Zur Nahrstofflimitierung des Phytoplanktons im Bodensee. I. Der Zustand im Seeteil .,Oberlinger See" 1974-1975. Arch. Hydrobiol., Suppl. 59: 151-191. AHMED, A. M., & AHMED, Z. A. (1986): Studies on phytoplankton of the Nile system in upper Egypt. Limnologica (Berlin) 17: 99-117. MOLER, F. M. M. (1987): Kinetics of nutrient uptake and growth in phytoplankton J. Phycol. 23: 137-150. MONOD, J. (1942): Recherches sur la croissance des cultures bacteriennes. Paris. Moss, B. (1969): Limitation of algal growth in some central african waters. Limnol. Oceanogr. 14: 591-601. MULLER, H. (1972): Wachstum und Phosphatbedarf von Nitzschia actinastoides (LEMM.) V. GOOR in statischer und homokontinuierlicher Kultur unter Phosphatlimitierung. Arch. Hydrobiol., Supp!. 38: 399-484. MULLER, R., & WIDEMANN. O. (1955): Die Bestimmung des Nitrat-Ions im Wasser. Jb. v. Wasser 22: 247-271. O'BRIEN, W . .I. (1974): Thc dynamics of nutrient limitation of phytoplankton algae: A model reconsidered. Ecology 55: 135-141. OSBORNE, B. A., & GElDER, R. .I. (1986): Effect of nitrate-nitrogen on photosynthesis of the diatom Phaeodictylum tricomutum BOHLIN (Bacillariophyceae). Plant, Cell and Environment 9: 617-625. PAASCHE, E. (1973): Silicon and ecology of marine plankton diatoms. I. Thalassiosira pseudonana (Cye/otella nana) grown in a chemostat with silicate as limiting nutrient. Mar. BioI. 19: 117 -126. PIRT, S. J. (1975): Principles of microbe and cell cultivation. Oxford. PORCELLA, D. B., & BISHOP. A. B. (1970): Comprehensive management of phosphorus water pollution. Michigan. PROVASOLI, L.. & PINTNER. I. J (1960): Artificial media for freshwater algae. Problems and suggestion. Dymatuning symposia in ecology. Spec. Pub!. No.2., Pittsburgh.

Effect of Nitrogen Limitation on Growth

273

PROWSE, G. A., & TALLlNG, J F. (1958): The seasonal growth and succession of plankton algae in the white Nile. Limnol. Oceanogr. 3: 223- 238. RHEE, G. Y. (1973): A continuous culture study of phosphate uptake, growth rate and polyphosphate in Scenedesmus sp. J. Phycol. 9: 495-506. - (1980): Continuous culture in phytoplankton ecology. In: M. R. DROOP & H. W. JANNASCH (ed.), Advances in aquatic microbiology. Vol. 2. New York. ROBARTS, R. D., & SOUTHALL, G. C (1977): Nutrient limitation of phytoplankton growth in seven tropical manmade Lakes, with special reference to Lake Mcllwaine Rhodesia. Arch. Hydrobiol. 79: 1-35. RODHE, W. (1948): Environmental requirements of freshwater plankton algae. Symb. Bot. Upsal. 10: 1-149. SCHELSKE, C. L., & STOERMER. E. F. (1971): Eutrophication, silica-depletion and predicted changes in algal quality in Lake Michigan. Science 173: 423-424. - - (1972): Phosphorus, silica and eutrophication of Lake Michigan. Nutrient and eutrophication. Spec. Symp. 1: 157-171. SCOR/UNESCO Working group on photosynthetic pigments (1966): Monographs on oceanographic methodology. Publ. UNESCO In: 1. D. H. STRICKLAND & T. R. PARSONS, Practical handbook of seawater analysis. Fish Res. Bd. Can. Bull. 1967. Ottawa 1968 SHAFIK, H. M., & HERODEK. S. (1989): A continuous culture study into growth rate and phosphate uptake of Scenedesmus spinosus. XXIV. Congress of the [nternational Association of Limnology, SIL Miinehen. SOMMER, U. (l988a): Phytoplankton succession in microcosm experiments under simultaneous grazing pressure and resource limitation. Limnol. Oceanogr. 33: 1037-1054. - (l988b): Growth and survival strategies of planktonic diatoms. In: C. D. SANDGREN (ed.), Growth and reproductive strategies of freshwater phytoplankton, pp. 227-260. Cambridge. STADELMANN, P. (1971): Stickstoffkreislauf und Primiirproduktion im mesotrophen Vierwaldstiittersee (Horwer Bucht) und im eutrophen Rotsee. mit bcsondcrer Berticksichtigung des Nitrats als limitierender Faktor. Schweiz. Z. Hydrol. 33: 1-65 STORCH, T. A., & DIETRICH, G. A. (1979): Seasonal cycling of algal nutrient limitation in Chautauqua Lake. New York. J. Phycol. 15: 399-405. STRICKLAND, J. D. H., & PARSONS. T. R. (1968): A practical handbook of seawater analysis. Fish. Res. Bd. Can. Bull. 1967. Ottawa. SZYMANSKI-BuCAREY, E. (1974): Untersuchungen tiber die Eutrophierung des Titisees und ihre Auswirkung auf die Populationsdynamik des Zooplanktons .. Arch. Hydrobiol., Suppl. 47: 119-166. TALLING, J. F. (1966): The annual cycle of stratification and phytoplankton growth in Lake Victoria (East Africa). Int. Rev. ges. Hydrobiol. 51 542-621. TEMPEST, D. W. (1970): The continuous cultivation of microorganisms. I. Theory of chemostat. In: J. R. NORRIS & D. W. RIBBONS (ed.). Methods in microbiology Vol. 2. New York, London. TEZUKA, Y. (1985): C:N:P ratios of seston in Lake Biwa as indicator of nutrient deficiency in phytoplankton and decomposition process of hypolimnetic particulate matter. Jpn. 1. Limnol. 46: 239-246. THOMAS, W. H., & DODSON, A. N. (1972): On nitrogen deficiency in tropical Pacific oceanic phytoplankton. 2. Photosynthetic cellular characteristics of a chemostat grown diatom. Limnol. Oceanogr. 17: 515-523. TILMAN, D., KILHAM, S. S .. & KILHAM. P. (1976): Morphometric changes in Asterionellaformosa colonies under phosphate and silicatc limitation. Limnol. Oceanogr. 21: 883-886. TOERIEN, D. F. (1974): Half-saturation constants for nitrogen limitated growth of the green alga Selenastrum capricornutum PRINTZ. South African 1. Sci. 70: 75-76. VOGLER, P. (1965): Problcme der Phosphatanalytik der Limnologie und ein neues Verfahren zur Bestimmung von gelostem Orthophosphat neben kondensierten Phosphaten und organischen Phosphorsiiureestern. Int. Rev. ges. Hydrobiol. 50: 33-48. VOLLENWEIDER, R. A. (1968): Die wissenschaftlichen Grundlagen der Seen- und FlieBgewiissereutrophierung unter besonderer Berlicksichtigung des Phosphors und des Sticks(offs als Eutrophierungsfaktoren. Organisation for Econ., Cooper. and Developm. DAS/CSI 68: 27. Authors' addresses: Prof. Dr. A. M. AHMED and Dr. A. A. MOHAMMED, Botany Department, Faculty of Science, Assiut University, Egypt: H. M. SHAFIK, Balaton Limnological Research Institute of the Hungarian Academy of Sciences, Tihany, 8237 - H, Hungary.

18

Arch. Protistenkd. Bd. 139, 1-4