Photosynthetic characteristics of algae grown under constant illumination and light-dark regimes

J. e-up. mur. Biol. Ecol., 1979, Vol. 40, pp. 63-70 0 Elsevier/North-Holland Biomedical Press

PHOTOSYNTHETIC

CHARACTERISTICS OF ALGAE GROWN UNDER

CONSTANT ILLUMINATION AND LIGHT-DARK REGIMES

G.

F. HUMPHREY

CSIRO. School of’ Biological

Sciences, Sydney University, Australia

Abstract: When grown under a 12-12 light-dark regime, Amphidinium, Biddulphia, Chaetoceros, Chroomonas, Qlindrotheca, Dunaliek, Pavlova, and Phaeodactylum had a higher photosynthetic rate

and photosynthesis : respiration ratio than when grown under constant illumination. The chlorophyll content was also higher (except for Biddulphia and Chaetoceros), the assimilation number was higher (except for Pavlova), but growth was less (except for Biddulphia which showedno differenceand for Amphidinium which grew faster).

Under natural conditions most algae live in alternating periods of several hours darkness and several hours light. Under polar conditions the periods increase to weeks at certain times of the year. Although most laboratory cultivation of algae is in continuous light (LL) i.e. conditions which are natural only at the poles, often light-dark (LD) regimes of illumination are used in order to simulate the more usual natural condition and to obtain some synchrony in division. Growth in LD produces die1 rhythms in biochemical and other properties (Sournia, 1974) and, therefore, it is often unjustified to pool results obtained from algae grown in LL with results from algae grown in LD. Although chlorophyll content and photosynthetic rate are the biochemical properties most studied, even with a basic property such as chlorophyll content there was not sufficient information available to allow Sournia (1974) to decide whether there is a general type of periodicity or whether different classes of algae behave differently; the position might even be that there are differences between members of the same class. If, however, a conclusion has to be drawn from the results already in the literature, it could be that there seems to be a periodicity in the photosynthetic rate (peak and subsequent decrease during L) but none generally valid for other properties such as cell division and chlorophyll content. Furthermore, there have been no studies in which, except for a variation in illumination regime, several algae have been examined under identical conditions of growth and sampling. Finally, some studies suffer from the disadvantage that the observation period for determination of photosynthetic rate (several hours) was comparable with the period of the regime. 63

G.F.HUMPHREY

64

In the present

study, eight species of marine

and in a 12-12 h LD regime.

Samples

were used for determinations synthetic

of cell number,

rate. From these the assimilation

of any rhythm

detected

unicellular

taken at comparable

algae were grown

chlorophyll

number

in LL

times in the light periods content,

was calculated

and

photo-

and the presence

graphically.

METHODS

Most details are given by Humphrey (1975). Following Green (1975), Monochrysis lutheri Droop is referred to as Pavlova lutheri (Droop) Green. LD cultures were inoculated at the beginning of a light period and LL cultures were inoculated at the same local time. After 48 h growth under conditions which ensured that nutrients were not limiting (Humphrey, 1974, 1975) and at a light intensity of 80 ,uEinsteins Co.) samples were taken at mm2 s-’ (LI 185 q uantum sensor, Lambda Instrument z 2-h intervals throughout the next 12 h, i.e. an L period. When it was not possible to work during the whole 12 h, samples were taken during the first 10 h or the last 10 h, each separate experiment including a sample at 8.5 h. To combine these separate sets of results, values were calculated as percentages of the 8.5-h value. Photosynthesis was measured for 10 min with an oxygen-electrode (Humphrey, 1975) at 800 ~Einsteins m-? s-’ which was slightly greater than saturating. Chlorophylls a and c were measured in 90% acetone and calculated by the equations of Jeffrey & Humphrey (1975) but since these authors did not give equations for 907” acetone extracts of algae containing chlorophylls a and c, (Amphidinium and Chroomonas)

the following

new equations

were calculated.

Chlorophyll

a = 11.43 E,,, - 0.40 I&

Chlorophyll

c? = 24.88 EhjO - 380 EGh4.

where chlorophyll is in pg. ml-’ in the extract and E is the extinction for a light path. On some occasions the chlorophyll was not completely extracted Biddulphia, Cylindrotheca, and Phaeodactylum. In these cases the residue acetone extraction was stirred with 0.2 ml methanol, kept in the dark for 5 diluted with 0.8 ml 100% acetone, and centrifuged. The methanol-acetone was added to the 90% acetone extract before determining the extinction. sitions of the spectral peaks were not altered by the methanol. Cell numbers were determined by haemocytometer counts and growth culated as the number of doublings in 24 h.

l-cm from after min,

extract The porates cal-

RESULTS

Only Amphidinium (Table I) grew faster in LD than in LL. Rates were similar in LL and LD for Biddulphia and the remaining six species showed faster rates in LL.

PHOTOSYNTHETIC

CHARACTERISTICS

There are no other such direct comparisons worth

that Dunaliella

(1966) calculated

for marine

OF ALGAE

65

species but Eppley & Coats-

had 1.5 divisions

a day in LL and 0.8 in a

I2 : 12 LD regime. TABLE I

Mean growth

rates as doublings

per day; LL = continuous Growth

Organism Amphidinium

Hulburt

carterae

rate

LL

LD

0.6

0.8

0.3 Biddulphia

aurita

(Lyngbye)

light; LD = light- dark

de

0.6

0.6

Ehrenberg

0.6

0.4

0.5

0.3

I.1

0.9

1.1

0.8

1.5

0.8

Reference This paper Jitts et al. (1964) This paper

Brebisson Chaetoceros

di&mum

Chroomonas

sp.

Cylindrotheca

Reimann Dunaliella

(Ehrenberg)

closterium

& Lewin tertiolecta

Butcher

0.8 Pavlova

This paper This paper This paper

lutheri

(Droop)

Green

0.9

0.6 0.6

Phrreodactyium

tricornutum

Bohlin

1.7

1.2

This paper Eppley & Coatsworth Jitts rt al. (I 964) This paper Jitts et ~11.(1964) This paper

(I 966)

In LD, Phaeodactylum was the only alga to divide in L, beginning its division in the second half. This differs from the finding of Palmer et al. (1964) that this alga divided during the second half of D and the first half of L. Both differ from the continuous culture result of Uno (1971) that Phaeodactylum divided only in L. Although no samples were taken during D, it is presumed that the other algae divided in D since no increase was observed in L. This has already been shown for Amphidinium

(Galleron, 1976; Hersey & Swift, 1976), Biddulphia (Subrahmanyan, 1945). and Dunaliella (Paasche, 1971; Whitney, 1973) although Rieth (1939) found that Biddulphia divided in L and Eppley & Coatsworth (1966) found that Dunaliellu began to divide in the second half of L. The data which are available in the literature for other marine phytoplankton do not allow any generalization as to the timing of cell division, i.e. cells appear to divide at various times in L or D according to species or to experimental conditions. Most of the relevant data are summarized in Sournia (1974).

Fig. 1 shows the variations in photosynthetic rate, chlorophyll a concentration, and assimilation number during L. For photosynthetic rate, Amphidinium, Biddulphia, Chaetoceros, and Cylindrotheca show a clear peak, Phaeoductylum a plateau, and Chroomonas, Dunaliella, and Pavlova a continuous increase. Previously Paasche (1971) and Whitney’(1973) have shown the continuous rise for Dunaliella. In contrast to the present results, Uno (1971) found that Phaeoducrylum gave a clear

G. F. HUMPHREY

I__ 0

”

0 0

0 (0

0 ID

PHOTOSYNTHETIC

maximum. though

For chlorophylls

CHARACTERISTICS

a and c all algae

this was small in Amphidinium.

Increases

showed

OF ALGAE

61

a continuous

in chlorophyll

increase

al-

a have previously

been shown by Eppley & Coatsworth (1966) in Dunaliellu and by Palmer rt al. (1964) in Phaeodactylum. The assimilation number shows a peak during L for all algae except Pavlova (constant) and perhaps Biddulphia (plateau, then a decrease). As previously shown by Sournia (1967) for phytoplankton, the “midday depression” of metabolism is, therefore, more evident phyll is used rather than photosynthetic

when photosynthetic rate alone.

rate per unit chloro-

TABLE II Variation

in photosynthetic

rate, chlorophyll c1concentration, and assimilation figures are ratios of highest to lowest values. Photosynthetic rate

Chlorophyll concentration

number

in LL and LD:

Assimilation number

Organism

LL

LD

LL

LD

LL

LD

Amphidinium Biddulphia Chaetoceros Chroomonas Cylindrotheca Dunaliella

1.1 1.4 1.7 1.5 1.2 1.1

1.3 1.5 2.4 2.9 1.6 1.6

1.2 1.4 1.1 1.2 1.1 1.1

1.3 2.0 1.8 1.8 1.7 1.7 1.6

I.1 1.4 1.7 1.3 1.1 1.1

1.2 1.5 3.5 1.8 1.4 1.2 1.2

Pavlova Phaeodactylum Ditylum Gon)aulax polyedra Skeletonema

1.1 1.2

1.9 1.5 1.4 1.7 2.5 1.5 1.8 13

* Depending

1.2 1.2 1.2

1.2 1.4 2.1 1.4 1.7 1.6/3.6*

1.1 I.1

1.3 1.6 1.2 1.5 I .8/3.6*

Reference This This This This This This

paper paper paper paper paper paper

Epplcy & Coatsworth (1966) Pdasche (1971) Whitney (1973) This paper This paper Eppley e/ al. (1967) Hastings et al. ( I96 1) Jorgensen (1966) Honjo & Hanaoka (I 969) Uno (1971)

on cell-type.

The extent of the variations in the above properties is shown in Table II as ratios of the highest to the lowest point on the curves. For comparison, ratios were calculated for variations similarly found with algae grown in LL. When there is no biological variation in continuous light cultures, these latter values show the experimental error. With algae easy to sample and count, the ratios do not exceed 1.2. With Biddulphia (gum-producing), Chaetoceros (chain-forming), and Chroomonas (aggregating during growth), the ratios reached 1.7. The ratios for LD regimes were, however, always greater than those for LL. In general, Chaetoceros and

68

G. F. HUMPHREY

Chroomonas showed the largest ratios with Biddulphia and Phaeodact_vlum following. Previous

work (Table II) has shown for Dunaliefla a ratio of 1.5 for photosynthetic

rate and 1.2 for chlorophyll a (Whitney, 1973) 1.6 for chlorophyll a and 1.2 for assimilation number (Eppley & Coatsworth, 1966) and 1.9 for photosynthetic rate (Paasche,

1971).

With all algae (Table

III) growth

in LD produced

cells which

higher photosynthetic rates and higher photosynthesis : respiration grown in LL. In all cases except Pavlova the assimilation numbers

could

exhibit

ratios than cells were also higher.

TABLE III

Maximum values of photosynthetic rate, photosynthesis : respiration ratio, chlorophyll concentration. assimilation number, and growth: rates are ~1 0,. 10m6 cells. hh’; concentrations are pg. IO-’ cells; numbers are mol 02. mol-’ chl (I. h-’ : growth is doublings per day.

Photosynthetic rate

Photosynthesis respiration ratio

:

Chloro-

Chlorophylf h or c

phyll ‘I

Assimilation number

Growth

Organism

LL

LD

LL

LD

LL

LD

LL

LD

LL

LD

LL

LD

Amphidinium Biddulphiu Chaeroceros Chroomonas Cylindrothecu Dunaliellu Pavlova Phaeodactylum

12.6 33.0 16.1 5.9 7.1 11.3 2.7 2.7

25.7 56.0 23.5 11.9 IO.8 13.9 3.4 3.8

3.8 8.7 3.4 4.3 Il.1 7.2 5.7 5.6

7.3 17.7 5.1 6.8 15.7 10.1 8.2 13.6

1.1 5.9 3.0 1.o 1.3 1.3 0.29 0.32

2.7 5.1 2.1 1.9 1.5 1.8 0.43 0.47

0.39 1.10 0.58 0.16 0.17 0.26 0.02 0.05

1.OS 1.01 0.49 0.27 0.21 0.35 0.06 0.09

367 352 246 258 320 359 388 350

454 630 482 285 383 387 320 437

0.7 0.7 0.7 0.6 1.2 1.3 1.0 7.2

0.9 0.X 0.6 0.4 1.1 0.9 0.7 I .4

The chlorophyll a (and also b or c) concentrations were higher with all species except for the centric diatoms Biddzdphia and Chaetoceros. Previously Dunaliella has been shown to have a higher chlorophyll a content (2.0 ,ug chl. a. lO-h cells compared

with 1.4, Eppley

& Coatsworth,

1966; 0.7 compared

with 0.5, Whitney,

1973). Contrary to the present work, Eppley & Coatsworth (1966) found that Dunaliella had a lower assimilation number in LD (80 compared with 120) but these numbers are so low that probably the cells were not in good biochemical condition.

DISCUSSION

Although the reactions of the algae to the different illumination regimes cannot be strictly related to taxonomic position there are important variations between species. For the dinoflagellates, Sweeney (1959) and Hastings et al. (1961) have shown that Gonyaulax polyedra, Gymnodinium, and Prorocentrum could divide in L, but Gonyaulax sphaeroidea did not; also Amphidinium did not divide in L

PHOTOSYNTHETIC

CHARACTERISTICS

(Table

I). The centric

diatoms

Biddulphia

(Table

I) but Ditylum

(Eppley

et al., 1967) and

Uno, 1971) did. The pennate tylum divided Phaeodactylum

diatom

and

Cylindrotheca

69

OF ALGAE

Chaetoceros Skeletonema

did not

divide

(Jorgensen,

in L 1966;

did not divide in L but Phaeodac-

in the second half of L. Previously Palmer et al. (1964) found that divided in the first half of L and Uno (1971) showed division oc-

curred throughout L. The discrepancy in information for this organism, and to a smaller extent for other organisms, is probably due to differences in culture conditions. Of all the algae so far examined, Chaetoceros, Chroomonas, Gonyaulax, and Phaeodactylum show the greatest extent of variation in metabolic properties (Table II). The high ratio of 13 obtained by Uno (1971) for photosynthetic rate of Skeletonema may be due to the use of continuous cultures or may be due to determining photosynthetic rate at only 120 ft-c, a value almost certainly below saturating. There is still not sufficient information, however, to show within the various classes of algae the extent of variations in metabolic properties during L. Fig. 1 shows that centric diatoms (Biddulphia and Chaetoceros) give the most pronounced peaks in photosynthetic rate whereas pennate diatoms (C?‘lindrotheca and Phaeodactylum) give flatter curves. The dinoflagellate Amphidinium shows the greatest stability. With only few exceptions, growth in LD increases the intensity of the metabolic characteristics of all classes of algae (Table III). This is always found for the photosynthetic rate and the photosynthesis : respiration ratio. For chlorophyll content the only exceptions are the centric diatoms, Biddulphia and Chaetoceros. For assimilation number, Pavlova and perhaps Dunaliella are exceptions. Although working with cells grown in LD gives the advantage of a closer approach to most natural conditions, it brings the need for a consistency in sampling time to offset variation in biochemical response caused by rhythmic changes in metabolic characteristics. Endogenous rhythms would, however, still be present. The higher maximum photosynthetic rates, photosynthesis and assimilation numbers but lower maximum growth rates,

: respiration ratios, suggest that meta-

bolism is increased in intensity to compensate for the reduced total illumination. The increases do not, however, allow the higher growth rates found under constant illumination.

REFERENCES R. W. & J. L. COATSWORTH, 1966. Culture of the marine phytoplankter. Dunuliellu tc~rtrolt~~u. with light--dark cycles. Arch. Mikrobiol., Vol. 55. pp. 6680. EPPLEY. R. W.. R. W. HOLMES & E. PAASCHE, 1967. Periodicity in cell divtsion and physiological behaviour of Ditylum brigh/d/ii. a marine planktonic diatom, during growth in light-dark cycles. Arch. Mikrobid., Vol. 56, pp. 305-323. GALLEROY, C.. 1976. Synchronization of the marine dinoflagellate Amphidinium cartrri in dense cultures. J. Phwol., Vol. 12, pp. 69-73. GREEN, J.C., 1975. The tine-structure and taxonomy of the haptophycean flagellate Pdovu hrrhrr/ (Droop) Comb. Nov. (= Monochrysis lutheri Droop). J. mar. hiol. Ass. U.K., Vol. 55, pp. 785-793. EPPLEY.

70

G. F. HUMPHREY

HASTINGS, J. W., L. ASTRACHAN & B. M. SWEENEY, 1961. A persistent daily rhythm in photosynthesis. .I. gen. Physiol., Vol. 45, pp. 69-76. HERSEY, R. L. & E. SWIFT, 1976. Nitrate reductase activity of Amphidinium carteri and Cachonina nisi (Dinophyceae) in batch culture: die1 periodicity and effects of light intensity and ammonia. J. Phycol., Vol. 12, pp. 36-44. HONJO, T. & T. HANOAKA, 1969. Diurnal fluctuations of photosynthetic rate and pigment contents in marine phytoplankton. J. oceanogr. Sot. Japan, Vol. 25. pp. 182-190. HUMPHREY, G.F., 1974. Effects of carbon dioxide and phosphate supplied during growth, on phosphorus content and photosynthetic rates of some unicellular marine algae. J. mar. biol. Ass. India, Vol. 16, pp. 358-366. HUMPHREY, G. F., 1975. The photosynthesis: respiration ratio of some unicellular marine algae. J. exp. mar. Biol. Ecol., Vol. 18, pp. 111-119. JEFFREY, S. W. & G. F. HUMPHREY, 1975. New spectrophotometric equations for determining chlorophylls a, h. c, and cz in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanzen. Vol. 167. pp. 191~194. JITTS, H. R., C. D. MCALLISTER, K. STEPHENS & J. D. H. STRICKLAND, 1964. The cell division rates of some marine phytoplankters as a function of light and temperature. J. Fish. Res. Bd Can., Vol. 21, pp. 139-157. JORGENSEN, E.G., 1966. Photosynthetic activity during the life cycle of synchronous Skeleronema cells. Physiologiu PI., Vol. 19, pp. 789-799. PAASCHE, E., 1971. Effect of ammonia and nitrate on growth, photosynthesis and ribulose diphosphate carboxylase content of Dunaliella tertiolecta. Physiologia PI., Vol. 25, pp. 294-299. PALMER. J.D., L. LIVIN~;S~ON & F.D. ZUSY, 1964. A persistent diurnal rhythm in photosynthetic capacity. Nature, Lond., Vol. 203, pp. 1087-1088. RIETH, A., 1939. Photoperiodizittit bei zentrischen Diatomeen. Planta, Vol. 30, pp. 294-296. SOLJRNIA. A.. 1967. Rythme nycthCmtral du rapport “intensitC photosynthitique/chlorophylle” dans le plancton marin. C.r. hebd. SPanc. Acad. Sci., Paris, Str D, Vol. 265, pp. 1000-1003. SOURNIA, A., 1974. Circadian periodicities in natural populations of marine phytoplankton. Adv. mar. Biol., Vol. 12, pp. 325-389. SUBRAHMANYAN, R., 1945. On the cell-division and mitosis in some south-Indian diatoms. Proc. Indian Acad. Sri.. Vol. 22B, pp. 331-354. SWEENEY, B. M.. 1959. Endogenous diurnal rhythms in marine dinoflagellates. International Oceanographic Congress Reprints, New York 1959. edited by M. Sears, Am. Ass. advmt Sci., Washington, D.C., pp. 204-207. UNO, S., 1971. Turbidometric continuous culture of phytoplankton. Constructions of the apparatus and experiments on the daily periodicity in photosynthetic activity of Phaeodactylum tricornutum and Skeletonema costatum. Bull. Plankton Sot. Japan, Vol. 18, pp. 1427. WHITNEY, D., 1973. The effects of temperature, light intensity, and day length upon growth and the release of newly assimilated carbon during photosynthesis in the marine alga, Dunaliella terriolecta Butcher. Ph.D. thesis, University of Delaware, 1973 Biology, University Microfilms, Ann Arbor, Michigan.