372 IAN MORRIS AND K. FARRELL Physiol. Plant., 25, 372-377, 1971

Photosynthetic Rates, Gross Patterns of Carbon Dioxide Assimila-tion and Activities of Ribulose Diphosphate Carhoxylase in

Marine Algae Grown at Different Temperatures

By

IAN MORRIS and K. FARRELL

Department of Botany and Microbiology, University College LondonGower Street, London W.C.I.

(Received May 24, 1971)

AbstractStudies of the marine green flagellate Dunaliella tertiolecta

have confirmed and extended previous observations of Stee-mann Nielsen and his colleagues. Algae, grown at 12°C,assimilated carbon dioxide under light-saturated conditionsmore rapidly than did those grown at 20°C; for both, theassimilation rate being higher at 20°C than at 12°C. Cellsgrown at the lower temperature contained higher concentra-tions of soluble protein, higher activities of ribulose diphos-phate carboxylase and showed an enhanced relative rate ofprotein synthesis during the photosynthetic assimilation ofcarbon dioxide. This appears to represent true adaptation sinceit allowed the growth rate at 12°C to be almost the same asthat at 20°C.

Studies of the marine diatom Phacodactylum tricornutumhave not revealed the same picture of temperature adaptation.Cultures grown at 5°C had significantly higher rates ofphotosynthesis than did those grown at 10°C, but the samewas not true when algae grown at 10°C were compared withthose grown at 20°C. In this organism, growth at the lowertemperatures reduced its ability to photosynthesize at 2O'-'C.Cells grown at the lower temperatures contained more pro-tein than did those grown at 20°C; this was particularlymarked in cells growing at 5°C, a temperature which reducedthe growth rate. The relative rate of protein synthesis washigher in Phaeodactylum grown at lower temperatures; butthis difference was most marked when the measurements weremade at 20°C.

IntroductionFor some time there has been considerable interest in

the adaptation of micro-algae to various light intensitiesand temperatures (e.g. Steemann Nielsen and Hansen1959, Jorgensen 1964, Aruga 1965, Jorgensen and Stee-mann Nielsen 1965, Yentsch and Lee 1966, SteemannNielsen and Jergensen 1968 a, b, Jergensen 1968). De-

spite this interest, the biochemical mechanisms whichpermit an alga to adapt to changing temperatures andlight intensities remain unknown. In this laboratory, weare interested in understanding further the precise bio-chemical changes accompanying such adaptations.

The experiments described in this present paper areattempts to test the generalizations of Jorgensen andSteemann Nielsen (1965) and Steemann Nielsen andJorgensen (1968 a), and to extend the specific observa-tions of Jergensen (1968) on temperature adaptation ofthe algae. These workers have proposed that adaptationto sub-optimal temperatures involves an increase in theconcentration of enzymes concerned in carbon dioxideassimilation (thus allowing the rate of photosynthesis atthe lower temperature to be comparable to that at thehigher). Indirect support for their hypothesis came fromthe observations that cells of Skeletonema costatumgrown at 7°C contained greater amounts of protein thandid those grown at 20°C (Jargensen 1968).

Using two marine algae, the green flagellate Dunaliellatertioleeta and the diatom Phaeodactylum tricornutum,we have attempted to extend the observation of Stee-mann Nielsen and his co-workers in three ways. Firstly,we ask whether these algae resemble Skeletonema costa-tum in showing higher rates of photosynthesis (at satu-rating light intensities) at lower temperatures when pre-viously grown at those lower temperatures. That is, whengrown at 20°C, Skeletonema assimilated carbon dioxideat 7°C more slowly than it did at 20°C; when grown at8°C, however, the rate of photosynthesis at the lowertemperature was comparable to that at 20°C (SteemannNielsen and Jorgensen 1968 a). Is the same true forDunaliella and Phaeodactylum}

The second question we ask is whether Phaeodactylumand Dunaliella, when grown at lower temperatures, also

Physiol. Plant., 25, 1971 PHOTOSYNTHESIS IN MARINE ALGAE AT DIFFERENT TEMPERATURES 373

contain greater amounts of protein; and also whetherthey possess higher activities ribulose-l,5-diphosphate(RUDP) carboxylase than do cells grown at higher tem-peratures. RUDP carboxylase is a key enzyme of thereductive pentose phosphate cycle (Calvin cycle) ofphotosynthetic carbon dioxide assimilation and measure-ment of its activity in organisms grown at different tem-peratures might be a more sensitive test of SteemannNielsen and Jorgensen's hypothesis than measurementsof bulk protein content.

Thirdly, we ask whether growth at lower temperaturesis accompanied by a change in the pattern of carbon di-oxide assimilation towards a greater relative rate ofprotein synthesis.

Methods

Growth of organisms

The marine algae Dunaliella tertiolecta (kindly sup-plied by Prof. C. S. Yentsch) and Phaeodactylum tri-cornutum (Cambridge Culture Collection) were grownin the F^ medium of Guillard and Ryther (1962). Formost experiments both algae were grown in stationarycultures in constant-condition growth cabinets illum-inated with 'Warm White' fluorescent tubes giving alight intensity of about 8000 lux. All cabinets were sub-jected to alternation between 16 hours illumination and8 hours darkness. For Phaeodactylum day temperaturesof 5°C, 10°C and 20°C (with accompanying night tem-peratures of 5°C, 8°C, and 15°C, respectively) wereused. For Dunaliella the day temperatures were 20°Cand 12°C and the night temperatures 15°C and 6°Crespectively. (Throughout this report only the day tem-peratures will be specified.) For some experiments withDunaliella temperatures from lO'-'C to 30°C wereachieved in illuminated water baths. In these experimentsthe algae were also supplied with 16 hours of light and8 hours of darkness, but the day and night temperatureswere the same and the light intensity was only about3500 lux. The main cultures were inoculated with sus-pensions previously maintained for several weeks at thesame temperatures as subsequently used for growth.

Measurements of photosynthetic carbon dioxideassimilation

After 4-6 days (Dunaliella) or 5-8 days {Phaeodac-tylum), the algae were harvested by centrifugation, re-suspended in fresh growth medium, transferred to 50 mlor 100 ml conical flasks and these sealed with a vaccinestopper. After incubation in the light (in the same cabi-nets as used for growth) for 10-20 minutes, '""C-sodiumbicarbonate (giving a final concentration of 5.0 mM anda specific activity of 0.25 iCi/f.imol) was added. Formeasurements of rates of photosynthesis, 1.0 ml sampleswere removed at various times over four hours and

added to 2.0 ml of 5 "/o acetic acid in ethanol. Afterdrying down, scintillant (Butyl-PBD) was added and theradioactivity counted with a Packard Tricarb liquidscintillation counter. For fractionation studies, 4.0 mlsamples were removed at various times up to five hours,centrifuged and the pellet resuspended in 2.0 ml of abso-lute ethanol. Fractionation into the alcohol-soluble, hot-trichloroacetic acid (TCA)-soluble and TCA-insolublefractions was then achieved by the method described byMorris (1969); radioactivity in each fraction beingcounted with the Packard Liquid Scintillation Counter.From work with other algae (Morris 1967) it seemedreasonable to assume that the hot TCA-soluble fractionwas mainly polysaccharide and the TCA-insoluble, pro-tein.

Preparation of cell-free extracts

Suspensions of the algae were harvested by centrifuga-tion, washed once with ice-cold 0.01 M Tris pH 8.0 andresuspended in this. The suspension was broken with asmall homogenizer (after including a glass fibre disc)running for 2 to 3 minutes. Comparison of cell countsbefore and after treatment confirmed a 90-100 "/o break-age. After breakage, the suspensions were spun at about60,000 g for 1 hour and the supernatant used as thecrude cell-free extract for measurements of soluble pro-tein, and for enzyme assays. In other experiments,Phaeodactylum was broken by two passages through aFrench Pressure Cell at 835-1115 kg/cm^ (12-16,000 lbs.per sq. in.). In these experiments, the broken suspensionwas centrifuged at about 20,000 g for 30 minutes andthe supernatant from this used for protein estimations.

Assay of ribulose-l,5-diphosphate (RUDP)carboxylase

The reaction mixture contained in 1.0 ml: 50 |,imol'Tris' buffer pH 8.2, 10 |.tmol MgCl,, 5 jxmol reducedglutathione, 0.5 [xmol ribulose-l,5-diphosphate, 25 |xmol'••C-sodium bicarbonate (0.8 jxCi per |j.mol). After equil-ibration for 10-20 minutes, 0.1-0.3 ml extract (contain-ing about 0.5 mg protein per ml) was added to start thereaction, incubation temperature was 22-25°C. At fiveminute intervals 0.1 ml samples were removed from thereaction mixture and added to 0.2 ml of 5 "/o acetic acidin ethanol. After drying down, scintillant was added andthe radioactivity measured as above. The amount ofacid-stable radioactivity increased linearly during thefirst 30 minutes of incubation, and the rate of reactionwas determined from this linear period. The slight fixa-tion of '""C-bicarbonate in the absence of RUDP wassubtracted from that in the presence of the substrate incalculation of RUDP carboxylase activities.

Cell counts were made with a haemacytometer; pro-tein in the cell-free extracts was determined by themethod of Lowry et al. (1951). "C-sodium bicarbonate

374 IAN MORRIS AND K. FARRELL Physiol. Plant., 25, 1971

100

Figure 1. Growth of Dunaliella tertio-lecta (A) and Phaeodactylum tricornu-tum (B) at various temperatures. ForDunaliella the temperatures were 20°C(•) and 12°C (^,A); for Phaeodacty-lum temperatures were 20°C (•), 10°C(A, A) and 5°C (•, n). For both algaethe solid symbols represent culturespreviously maintained at the same tem-perature as used for growth; opensymbols represent cultures transferredfrom 20°C to the growth temperatures.

was obtained from the Radiochemical Centre, Amers-ham; ribulose-l,5-diphosphate from Sigma, reduced glu-tathione from Koch-Light Co.

Results

1. Growth rates of Dunaliella and Phaeodactylum atdifferent temperatures

At the start of this investigation, it seemed desirableto measure growth of the algae at the various tempera-tures to be used in later experiments. Such observationswould allow us to know the extent to which we wereexamining the biochemistry of organisms growing atwidely different rates; also we could investigate the pos-sibility that transfer from one temperature to anotherwas followed by a period of adaptation.

Figure 1 gives some examples of growth of the algaeat different temperatures. Although Dunaliella grewniore rapidly at 20°C than at 12°C (Figure 1 A), thedifference was not very marked. From several experi-ments, generation times at 20°C were 14.0, 22.5, 21.0,and 19.5 (mean, 19.25 hours); those at 12°C being 22.0,18.0, 22.5 and 21.0 hours (mean, 20.9 hours). This dif-fers from the observations of Eppley and Sloan (1966),who report the growth rate of Dunaliella tertiolecta at20°C to be three times as fast as at 10°C. However,these authors were using continuous light and constanttemperature (i.e. not lower night temperature). Possibly,the differences between our conditions and those ofEppley and Sloan (1966) explain the differences betweenthe two sets of results. No significant lag could be de-tected following transfer from 20 °C to 12°C (an adapta-

tion period of less than 8-12 hours would of course bedifficult to detect by following changes in cell numbers).Other experiments performed in illuminated water baths(at lower light intensities than those in the growth cabi-nets) also showed rates of growth to be similar at 30^,25°, 20° and 14°C.

For Phaeodactylum initial growth rates at 20 °C weresignificantly higher than those at 10°C (doubling timesof 7 and 20 hours respectively), but after about 30 hours,the growth at 20°C began to decline (presumably dueto self-shading at higher cell densities). Thus, at the timeof harvesting, Phaeodactylum was growing approx-imately at the same rates at both 20" and "10°C. Fromseveral experiments, there was no indication of a lagfollowing transfer from 20° to 10°C. Growth at 5°Cwas significantly slower than at either 10° or 20°C(doubling times of about 25 hours; although long lagperiods made accurate measurement of growth rates dif-ficult), and growth ceased at cell densities below thoseat 20° and 10°C.

At this stage in the investigation, we have not beenconcerned with studying growth curves of these algae atdifferent temperatures in any detail. We present themmerely to point out that, under our conditions, the algaewere growing at similar rates at different temperatures(with the exception of Phaeodactulym, at 5°C). That is,we have not been concerned with extremes of tempera-tures to which algae must adapt over prolonged periodsof time. Any adaptation was presumably 'physiological'(as opposed to 'genetic') and occurred over time periodstoo short to be detected by measuring growth of theorganism.

Physiol. Plant., 25, 1971 PHOTOSYNTHESIS IN MARINE ALGAE AT DIFFERENT TEMPERATURES 375

Table 1. Rates of photosynthesis at various temperatures byDunaliella tertiolecta and Phaeodactylum tricornutum pre-viously grown at different temperatures. Rates were calculatedfrom linear time courses of incorporation of "C-sodium bicar-bonate over two hours. Further details as in Methods.

Organism

Dunaliella tertio-lecta .

Dunaliella tertio-lecta

Phaeodactylum tri-cornutum

Phaeodactylum tri-cornutum

Phaeodactylum tri-cornutum

Temperatureof growth

°C

20

12

20

10

5

Temperatureof incubation

C

2012

2012

2010

5

20105

20105

Rate of photo-synthesis

CO2 nmol/hxlO« cells

75.051.0

105.072.5

78.062.342.0

42.052.541.5

39.067.552.5

2. Rates of photosynthetic carbon dioxide assimilationat different temperatures by algae grown at

different temperatures

We have not measured light saturation curves at dif-ferent temperatures but several measurements with in-tensities reduced by 30-50 per cent suggested that 8000lux (the intensity used in all these experiments) was nearsaturating. The rate of photosynthesis by Dunaliella washigher at 20°C than at 12°C (Table 1). This was so withboth cultures grown at 20° and those grown at 12°C.However, at each temperature the algae previouslygrown at 12°C assimilated carbon dioxide more rapidllythan did those previously grown at 20^C; the ratios ofphotosynthesis at 20° to that at 12°C were approx-imately the same for cultures grown at both tempera-tures. These results resemble those of Steemann Nielsenand Jorgensen (1968 a); that is, when the alga was pre-viously grown at 20°C, photosynthesis at 12° was about33 "/o lower than at 20°C, but organisms grown at thelower temperature showed about the same rate at 12°as did 20 -grown algae when incubated at 20°C.

However, for Phaeodactylum tricornutum, growth at10°C did not appear to increase the rates of photo-synthesis at that temperature (compared with algaegrown at 20°C, Table 1). But when Phaeodactylumwas grown at 5°C, the rate of photosynthetis (at both10° and 5°C) was 25—30 "/o higher than by cells grownat 10°C (Table 1). Reducing the growth temperature de-creased the subsequent rate of photosynthesis at 20 °C.

This temperature was only optimal for photosynthesis byorganisms previously grown at 20°C.

3. Protein content and ribulose-1,5-diphosphate carboxy-lase activities of extracts from algae grown at

different temperatures

We have confirmed higher protein contents of cellsof Phaeodactylum and Dunaliella grown at the lowertemperatures this being particularly marked in cells ofPhaeodactylum grown at 5°C (Steemann Nielsen andJergensen 1968 a). We wish to test further the idea ofSteemann Nielsen and Jorgensen (1968 a) that higherprotein contents indicated higher concentrations of en-zymes by measuring the activity of RUDP carboxylasein extracts of algae grown at different temperatures. Un-fortunately, we have so far failed to detect activity ofthis enzyme in extracts of Phaeodactylum tricornutum.Results for Dunaliella tertiolecta are presented in Table2. Extracts from algae grown at 12°C contained about50 Vo more RUDP carboxylase activity (when expressedper unit amount of protein) than did extracts from algaegrown at 20°C. This increase was greater than the in-crease in protein content; hence the increase in enzymactivity per cell was greater than 50 "/o. In the secondseries of experiments in which algae were grown in thewater baths, a greater effect of low growth temperatureon the level of RUDP carboxylase could be observed.Thus, the specific activity (expressed per unit protein)in extracts from algae grown at 17°C was nearly threetimes that from algae grown at 23°C; the difference inactivity per cell was nearly four-fold (Table 2). No fur-ther increase accompanied a further reduction in growthtemperature from 17° to 10°C; rather there was somedecrease.

Similar results have been obtained in about six inde-pendent experiments. However, there was considerablevariability in the results from one experiment to another(for example. Table 3). Those experiments showing leastincrease (0-20 Vo) in RUDP carboxylase activity whenthe algae were grown at lower temperatures were those

Table 2. Activities of RUDP carboxylase in cell free extractsfrom cultitres of Dunaliella grown at various temperatures.For experiment 1, the cultures were grown in the growthcabinets; for experiment 2 in water baths. Further detailsas in Methods

Experimentno.

RUDP carboxylase activity

xmgprotein

l-imol/hx 10"cells

2 .

2012

231710

2.13.1

0.92.61.8

1.93.3

4.717.213.0

376 IAN MORRIS AND K. FARRELL Physiol. Plant., 25, 1971

Table 3. Relative rates of incorporation of '•'C-carbon dioxideinto various fractions of Dunaliella. Experiment 1 was per-formed (and the cultures grown) in growth cabinets; experi-ment 2 in water baths (details in Methods). Total incorpora-tion was calculated from the sum of the three fractions; re-covery varied between 85 and 120 "/o. Incubation time was3 hours.

Experimentno.

Tem-perature

forgrowth

°G

Tem-perature

forexperi-ment

°G

Alcohol-soluble

Hot TGA-soluble

TGA-insol-uble

2012

231710

2020

232323

32.239.0

40.645.549.0

65.155.3

57.350.045.0

2.75.7

2.14.56.0

Table 4. Relative rates of incorporation of '''C-carbon dioxideinto various fractions of Phaeodactylum. The results of twoseparate experiments are shown; for each one the mean ofduplicate flasks is given. Total incorporation was calculatedfrom the sum of the three fractions; a comparison with mea-sured total incorporation showing a recovery of between 80and 130 "/is. Incubation time was 5 hours.

in which the absolute activities (at both temperatures)were much lower than in the experiments presented inTable 2. We assume that changes in the physiologicalstate of the cells (possibly related to stage in the growthcurve at which they were harvested) and variations inthe efficacy of extraction and assay of the enzyme fromone experiment to another were responsible for theselower activities.

4. Gross patterns of carbon dioxide assimilation by algaegrown at different temperatures

Higher protein contents of organisms adapted to lowertemperatures presumably arises from altered patterns ofmetabolism resulting in proportionally greater rates ofprotein synthesis. We have measured the proportion ofcarbon dioxide assimilated into protein, polysaccharideand alcohol-soluble fractions during photosynthesis byDunaliella and Phaeodactylum previously grown at dif-ferent temperatures.

Suspensions of Dunaliella grown at 12°C incorporatedtwice as much carbon into protein as did algae grown at20°C (Table 3). Similar increases in the proportion ofcarbon incorporated into protein were also observed ina second series of experiments in which Dunaliella wasgrown at 20°, 17° and 10°C in water baths (Table 3,Experiment 3).

However, Dunaliella incorporated only a small pro-portion of assimilated carbon into protein, so thatchanges from only 2-6 "/o were measured. It seemed ad-visable to investigate this more intensively in Phaeodac-tylum, where greater relative rates of protein synthesiscould be observed. Results from these experiments arepresented in Table 4. When photosynthesis was measuredat 20°C, there was an increase in the relative rate ofprotein synthesis with decreasing temperature used forgrowth of the algae. Thus, algae grown previously at

Tem-perature

forgrowth

°G

Tem-perature

forexperi-ment°G

Experi-mentno.

Alcohol-soluble

Hot TGA-soluble

TGA-insoluble

20

10

20

10

5

20

10

5

20

10

5

12121212

1212

12

12

12

39.552.935.032.5

37.031.7

30.929.8

30.028.7

33.332.2

33.533.0

31.031.1

34.542.6

39.030.050.659.0

52.156.6

43.044.4

57.059.0

51.057.0

33.534.0

50.051.5

50.544.4

21.517.114.48.5

10.911.7

25.125.8

13.012.3

15.710.8

33.033.0

19.017.4

15.013.0

20°C assimilated about twice as much carbon into poly-saccharide as they did into protein; whereas for thosegrown at 5°C, as much carbon was assimilated into pro-tein as into polysaccharide. Interestingly, this increase inrelative rate of protein synthesis was not so markedwhen carbon dioxide assimilation was measured at thelower temperatures of 10° and 5°C. When the experi-ment was performed at the same temperature as thatused for growth, cultures at 20 °C showed a ratio of in-corporation into polysaccKaride to that into protein ofabout 2 to 1; at 10'C the ratio was approximately 4.5to 1; and at 5°C, about 3.5 to 1 (Table 4).

DiscussionThis present contribution extends the observations of

Steemann Nielsen and Jorgensen (1968 a) and Jorgensen(1968) in two main ways. Firstly, from experiments withDunaliella tertiolecta, we have evidence supporting thehypothesis of Steemann Nielsen. But, secondly, studieswith Phaeodactylum tricornutum suggest that the phe-nomena exhibited by Dunaliella and Skeletonema mightnot be general for all micro-algae growing at differenttemperatures.

Physiol. Plant., 25, 1971 PHOTOSYNTHESIS IN MARINE ALGAE AT DIFFERENT TEMPERATURES 377

Four observations with Dunaliella support the hypo-thesis of Steemann Nielsen: the higher rates of photo-synthesis at saturating light intensities by algae grownat the lower temperatures, the higher protein content andthe higher activity of RUDP carboxylase in cells grownat the lower temperature, and the greater relative rateof protein synthesis during photosynthesis by algaegrown at the lower temperatures.

This enhanced relative rate of protein synthesis andaccompanying increased activity of RUDP carboxylaserepresent true adaptation to a lower temperature, sincethe growth rate of Dunaliella tertiolecta at 12°C isapproximately the same as that at 20°C. This is dif-ferent from Skeletonema, where there does not seem tobe adaptation to a lower temperature (7°C) in the senseof it permitting the growth rate at this lower tempera-ture to be comparable to that at 20°C.

The response of Phaeodactylum to growth at varioustemperatures appears to be different from that of Duna-liella and Skeletonema. The rate of photosynthesis byPhaeodactylum grown at 5°C is greater than by the algagrown at 10°C; there is no similar effect when 10°-grown algae are compared with those grown at 20°C.Rather, growth at the lower temperatures appears toreduce the ability of the alga to assimilate carbon di-oxide when returned to 20°C. Possibly, higher rates ofphotosynthesis by algae grown at lower temperaturescan only be observed over a certain restricted tempera-ture range (a range which depends upon the particularorganism being studied). The protein content of Phaeo-dactylum grown at lower temperatures is higher thanthat of those grown at 20°C. This is particularly so fororganisms growing at 5°C, a temperature which reducesthe growth rate markedly (this is a situation analogousto that reported by Jergensen 1968, for Skeletonema).Our failure to measure RUDP carboxylase in Phaeo-dactylum has prevented us from establishing whether in-creased protein content is accompanied by increased ac-tivity of an enzyme important in carbon dioxide assim-ilation.

Another point of similarity with Dunaliella is thegreater relative rate of protein synthesis by cultures ofPhaeodactylum grown at lower temperatures. However,an interesting feature of this was the fact that this en-hanced protein synthesis could only be observed whencarbon dioxide assimilation was measured at 20°C. Thereseems no obvious explanation for the failure to observesuch enhanced relative protein synthesis when measure-ments are made at 10°C and 5°C.

Future investigations will need to emphasize changingmetabolic patterns and changing activities of specific en-zymes accompanying growth of algae at various tem-peratures so that the biochemical basis of enhanced rela-tive rate of protein synthesis in cells grown at sub-optimal temperatures may be better understood. It ap-pears, also, that comparative studies with more than onealgae will be necessary before any generalized picture oftemperature adaptation can emerge.

We acknowledge the expert technical assistance of Miss M.Goodson and the preliminary results of Mr. G. B. Weekes onwhich later work was based. We also thank Dr. K. Taylorfor making room available in the growth cabinets.

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ter production of phytoplankton. II. Photosynthesis ofalgae in relation to light intensity and temperature. — Bot.Mag. Tokyo 78:360-365.

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— 1968. The adaptation of plankton algae. II. Aspects ofthe temperature adaptation of Skeletonema costatum. —Ibid. 21:423-427.

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Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall,R. J. 1951. Protein measurement with the Folin phenolreagent. — J. Bioi. Chem. 193: 265-275.

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Steemann Nielsen, E. & Hansen, V. K. 1959. Light adapta-tion in marine phytoplankton populations and its inter-relation with temperature. — Ibid. 12: 353-370.

— & Jorgensen, E. G. 1968 a. The adaptation of planktonalgae. I. General part. — Ibid. 21: 401-403.

— — 1968 b. The adaptation of plankton algae. III. Withspecial consideration of the importance in nature. — Ibid.21:647-654.

Yentsch, C. S. & Lee, R. W. 1966. A study of photosyntheticlight reactions, and a new interpretation of sun and shadephytoplankton. — J. Mar. Research 24: 319-337.