Fredriksen and Rueness: Culture studies of Gelidium latifolium from Norway 539

Botanica MarinaYot.32, pp. 539-546, 1989

Culture Studies of Gelidium latifolium (Grev.) Born et Thur.(Rhodophyta) from Norway.Growth and Nitrogen Storage in Response to VaryingPhoton Flux Density, Temperature and Nitrogen Availability

S. Fredriksen and J. Rueness

Sectionfor Marine Botany, Department of Biology, University of Oslo, P.O. Box 1069 Blindern,03l6 Oslo 3,

Norway

(Accepted 25 June 1989)

Abstract

Gelidium latifotium from the Norwegian west coast was grown at 25 combinations of photon flux density

(PFD,range20-300pmolm's-t)andtemperature(range14-28'C)undernitrogensufficientandnitrogendeficient conditions. Growth rate under nitrogen sufficient conditions increased towards higher PFD, while

it was less allected by temperature. Higher growth rates at the higher PFD were correlated with increased

lateral branching. Pigmentation was aflected by both PFD and nitrogen availability. Phycobiliproteins and

chlorophyll a decreased with increasing PFD, with the lowest pigment content in plants grown under N-deficient conditions. Analyses of free amino acids in the algae indicated that phycobiliproteins were more

important as storage compounds for nitrogen than free amino acids. The importance of phycobiliproteins

and other nitrogen compounds as nitrogen pools under nitrogen limiting conditions is discussed. The C: Nratio was not affected by PFD as long as nitrogen was in excess, but increase with increasing PFD when

nitrogen was limiting.

Offprint

Stages of spore germination have been repeatedlyreported (e. g. Katada 1955, Boillot 1963), and a fewpapers on effects of environmental factors on growthof sporelings have been published (Correa et al. 7985,

DAntonio and Gibor 7985, Carter 1985). The devel-

opment from sporelings to mature thalli and comple-tion of life history was reported for the hrst timerecently by Macler and West (1987). They studiedGelidium coulteri Harv. from California, and Macler(1986) used cloned strains of this species to examine

the regulation of carbon flow by nitrogen and light.Most physiological studies on Gelidium have been

undertaken using field-collected thalli brought intoexperimental culture condition. Effects of light, tem-perature and water movement were examined in some

Gelidiales from Hawaii (Santelices 1978), while Bird(1976) examined nitrogen uptake rates and Hansen(1980) measured photosynthesis as a function of light

Introduction

Species of the genus Gelidium represent an importantsource of the phycocolloid agar, and are extensively

harvested from natural populations. A pre-requisite

for developing a controlled mass-cultivation system

is a better understanding of the physiological require-ments for growth and the factors controlling repro-

duction and life history. Hence, culture studies under

controlled conditions are indispensable in resolvingmany of these problems.

While extensive work has been published on species

ol the agarophyte Gracilqria in recent years (for ref-

erences see Dawes 1987), only a few culture studies

have been reported for Gelidium. Apparently, diffi-culties in growing species of Gelidium have limitedour basic knowledge on life history, physiological

requirements and species delimitation in this genus.

Botanica Marina I Yol.32 I 1989 I Fasc. 6

Copyright O 1989 Walter de Gruyter' Berlin 'New York

l-c 76

540 Fredriksen and Rueness: Culture studies ol Gelidium latfolium from Norway

and temperature in other Pacific Gelidium species.

Ecophysiological studies of four Chilean Gelidiumspecies (Oliger and Santelices 1981) demonstrated thattaxonomically confused species responded differentlyto variation in irradiance, water movement and tem-perature. It is important to note that large differencesin physiological responses may occur if diverse indi-viduals from field-collected samples are used in ex-periments. This may be due to variation in age, re-productive stage, collection site and nutrient status.To reduce plant-to-plant variation ciones of a singleindividual should be used in many experiments, andthere is a need to describe the full range of phenotypicplasticity.

The alga used in the present investigation is the same

as that used by Rueness and Tananger (1984), whoreported on growth responses under various regimesof salinity, temperature and irradiance. The taxonunder discussion was referred to as Gelidium sp. byRueness and Tananger (1984). Taxonomic and bio-geographic work (Rueness and Fredriksen 1989) has

revealed that the alga is referable to Gelidium latifol-ium (Grev.) Born. et Thur. sensu Dixon and Irvine(1977). A considerable phenotypic variation in mor-phology and pigmentation has been demonstratedboth in culture and in the field. The colour may rangefrom almost black to yellowish-green and branchesare terete or flattened depending on growth condi-tions.Differences in pigment composition of red algae mayarise in response to changes in environmental condi-tions. Ample evidence suggests that pigment concen-trations in red algae increase or decrease in response

to low and high photon flux density (PFD), and notin response to the quality of light (Ramus et al. 1916,

Dring 1981, Ramus and van der Meer 1983). It is

also well known that there is a correlation betweennitrogen dehciency and reduction of pigment contentof algal cells (DeBoer 1981). Hence, bleaching of red

algae may be attributed to both photobleaching andnitrogen starvation. Previous studies of Gracilaria spe-

cies have demonstrated a high luxury consumptionand storage of nitrogen during periods of high nitro-gen availability (Bird et al. 7982, Lapointe and Duke1984, Lignell and Peders6n 1987). Free amino acidsand phycobiliproteins appear to form the major ni-trogen storage pools (Bird et a|.1982).

Knowledge of the relationship between light intensityand nitrogen metabolism may have practical impli-cations for aquacultural mass cultivation. The abilityto store nitrogen allows pulse fertilization, which mayreduce growth of undesirable weeds in open systems.

Furthermore, the so called Neish effect, i. e. a markedincrease in the biosynthesis of cell wall polysacchar-

ides like agar and carrageenans in nitrogen starvedplants, has been demonstrated in several red algae bye.g. Bird et al. (1982) and Macler (1986).

In the present study growth rates were determined at25 combinations of PFD and temperature under ni-trogen sufficient conditions. Experiments were under-taken to determine the content of chlorophyll a, phy-coerythrin, phycocyanin, free amino acids and theC:N ratio in individuals of Gelidium latifolium grownat various PFD under both nitrogen sufficient andnitrogen limiting conditions.

Material and Methods

A tetrasporophyte of Gelidium latifolium, originallyisolated from Eggholmen, south of Bergen, Norway,was isolated into unialgal culture and maintained as

vegetative plants. Stock cultures were maintained at17 'C in an enriched seawater medium (IMR/2, Ep-pley et al. 1967) adjusted to a salinity of 30%o, andgrown under a 16 : 8 light: dark cycle at a photon fluxdensity (PFD) of 10-20 pmol m-2 s-1 given byfluorescent tubes (Philips TL 40 W 132). Clones of thisplant were used in the present study and inocula wereexcised apical portions. A cross-gradient growth tablesimilar to that described by Yarish et al. (1979) wasused for experiments. Photon flux densities and tem-peratures used were 20, 50, 100, 200, 300 pmol m 2

s 1 and 1.4, 1.8, 21., 24, 28 "C, respectively.

Ten excised apical portions (ca 7 cm) of G. latifoliumwere weighed and used as inocula in each of 25 100-

ml Ehrlenmeyer flasks containing 75 ml full strengthmedium (initial NO3-N > 500 pu, N-sufficient con-ditions). In addition 6 flasks each with a similar in-oculum, were incubated in the same medium minusnitrogen enrichment (initial NO3-N I 2 Vxt, N-defi-cient conditions). These 5 cultures were placed at a

constant temperature of 20 'C and at each of the fivedifferent PFDs on the growth table. The first 25

cultures were placed at 25 combinations of tempera-ture and PFD. Culture medium was changed everysecond day. The cultures were continuously aerated,keeping the plants free-floating in constant motion.Photon Flux Density was measured by a QSL-100Laboratory Quantum Scalar irradiance meter, with aspherical quantum sensor measuring photosyntheticactive radiation (PAR).

Morphological observations were made after 2 weeks.

The length of the main axis and the number of lateralbranches and branch initials were determined in each

of the 250 experimental plants. Plants were harvestedafter 4 weeks. The fresh weight of the algae containedin each flask was determined following a standard

Botanica Marina lYol.32 I 1989 lFasc.6

Fredriksen and Rueness: Culture studies of Gelidium latifolium lrom Norway 541

blotting procedure (giving an accuracy of -f 2.60/o,

n : 10). The growth was determined in terms ofweight increment and expressed as specific growthrate, po/o day 1 : 100 lnNt/No/t, whereNo : initialbiomass, Nt : biomass on day t, t : time interval(days).

The method for phycobiliprotein extraction followedBeer and Eshel (1985). Fresh material (0.03-0.1 g)

from each culture was ground in 5 ml 0.1 rl phosphatebuffer (pH : 6.8) and centrifuged. The absorbancewas determined at 455, 564, 592, 618 atd 645 nm andthe phycobilins quantified as mg pigment/g freshweight. Chlorophyll a was extracted with 700oh ace-tone from the solid material left after phycobiliproteinextraction, and the amount of chlorphyll a was de-termined according to Jensen (1978).

The carbon and nitrogen content was determined ina Carlo Erba Elemental CHN Analyzer. Samples fromN-sufficient and N-deficient conditions were ana-lyzed, using 2 and 3 replicates, respectively.

Free amino acids were extracted, with some modifi-cations, after a method described by Laycock andCraigie (1911). One g of alga (fresh weight) wascrushed in liquid nitrogen. Amino acids were ex-tracted with five 10-ml volumes of 1 rra NH+OH in80%o ethanol. The pooled extracts were taken to dry-ness on a rotary evapourator and redissolved in amixture of 5 ml propanol, 5 ml petroleum benzine(b.p. 60-80') and 5 ml water. The water phase wasonce again taken to dryness and redissolved in 5 mlwater. This extract was analysed in Biotronik LC 5000amino acid analyzer. Material for this experiment wasnitrogen starved plants grown in a 2l Ehrlenmeyerflask containing nitrogen free medium (< 2 pvr).

Plants were continuously aerated. Nitrogen (as NO3)was added at the beginning of the experiment to aconcentration of > 500 prra and was maintained insurplus during the experiment. Samples were takenfor analyses of free amino acids, phycobiliprotein andnitrogen content at 0, 8, 24, 48,96, 792, 360, 526hours after addition of nitrogen. The culture wasgrown at 77 "C and a PFD of 50 pmol m 2 s 1 in a16 :8 light:dark period.

Results

Growth rqtes and branching of thallus

The specific growth rates at the 25 combinations ofPFD and temperature under N-sufficient conditionsvaried from about 2.5oh day 1 to 8.87o day 1 (Fig.1). Within the temperature range tested (14-28 "C)no significant differences in growth rates were re-

Botanica Marina I Yo1.32 I 1989 I Fasc.6

28 24 21 18 14

Temperature ('C)

Fig. 1. Specihc growth rate of Gelidium latlfulium under dif-ferent combinations of photon flux density and temperature.

corded (p > 0.1, student t-test). There seemed to bea broad optimum in the range of 14-25 'C with28'C as slightly suboptimal. Significant dilferencesin growth rates were seen between the highest andlowest PFD (p < 0.05, student t-test) with a maximumgrowth rate at the highest PFD tested (300 pmol m 2

t\s').The number of lateral branches was determined after2 weeks of culturing (Table I). While there was noclear trend in the number of lateral branches withincreasing temperature, the branching increasedmarkedly with increasing PFD. There was a strongpositive correlation (r : 0.96, p < 0.001) between thenumber of lateral branches and the biomass as deter-mined after two weeks of growth.

Table I. Number of branches and branch initials per plant inG. latifolium after two weeks in culture at various photon fluxdensities and temperatures. Figures are mean values for tenplants at each treatment.

Temperature.C

Photon flux density pmol m 2 s 1

100

9

B

1

6

5

4

J

2

1

n

/ ,\\2OO :v %

I

G!

oo

3ooo

.O

oo-

CN

/ .<a\^\100 <s"

,/ .\i50 .,tr'

(rs20 ,oc-'ao'

3002005020

23 3625 5228 7328 6221 37

1418

21

2428

8693

1141t5118

72 84119 117159 221

156 300166 198

Pigment composition

The concentration of phycoerythrin, phycocyanin andchlorophyll (t were determined after 4 weeks ofgrowth. No significant differences in pigment content

r

542 Fredriksen and Rueness: Culture studies of Gelidium latifolium from Norway

Table IL Pigment content tn G. latifolium grown under nitrogen sufficient (+N, n:5, SD indicated) and nitrogen dehcient

(-N) condiiions under diiferent photon flux densities (PFD). A11 values given as mg pigment/g fresh weight.

PFDpLmol m-2S

Phycoerythrin Phycocyantn Chlorophyll a

+N +N-N -N +N -N

300200100

5020

1.78 + 0.442.5'7 + 0.453.78 + 0.505.6'.7 + 0.606.84 + 0.68

0.030.080.211.132.72

0.0050.0130.0260.0890.128

0.050.080.090.240.41

0.086 + 0.030.124 + 0.040.199 + 0.040.309 + 0.060.369 + 0.08

0.22 + 0.070.29 + 0.030.41 + 0.030.61 + 0.070.64 + 0.03

were found between the algae grown at the vanous

temperatures. Hence, the f,rve values corresponding to

different temperatures at each PFD were averaged

and given with S. D. (Table II).

Under N-deficient conditions there was a significantincrease in phycoerythrin from the highest to the

lowest PFD tested. By visual judgement this appeared

as a change in colour from yellow to pale red. Plants

grown under N-sufficient conditions showed onlyslight visual differences in colour at the different PFD.At the highest irradiances plants were still red, but

r+ NPE+PClChIao- NPE+PC/Chla

100 200 300

Photon f lux density (p mol m-2 s 1 )

turned dark red at the lowest PFD. For all pigments,

an increase in concentration with decreasing PFD was

measured. The ratio between total phycobiliproteinand chlorophyll a is plotted against PFD for the twodifferent levels of nitrogen availability in Figure 2.

Under nitrogen limiting conditions there was a

marked increase in the ratio towards lower PFD. Inother words, the light harvesting pigments decrease

more rapidly than chlorophyll a with increasing PFD,especially under N-deficient conditions. The ratio be-

tween phycoerythrin content in plants grown underN-sufficient conditions and that in plants grown un-der N-deficient conditions is plotted as a function ofPFD in Figure 3. The same is done for phycocyanin

under the two treatments. Figure 3 shows that there

was a more dramatic loss in phycoerythrin than inphycocyanin with increasing PFD, as reflected in the

steeper slope of the phycoerythrin curve.

C: N ratio

There were no signihcant diflerences in either carbon

or nitrogen content ol plants grown at different tem-

peratures under N-sufficient conditions. The C :Nratio as a function of PFD for both nitrogen treat-

ments is illustrated in Figure 4. Under N-sufhcient

conditions there was no variation in either C- or N-content at the various PFD, and the C: N ratio re-

mained about 13 (Figure 4). Under N-deficient con-

r-NC:N'iNC:N

200 300

Photon flux density (p mol m-2 s-r )

Fig.4. C: N ratio in Gelidium latifolium grown at nitrogensufficient (+N, n:10) and nitrogen deficient (-N, n:3)conditions. Vertical bars indicate * SD.

12

Fig. 2. Total phycobiliprotein:chlorophyll a ratio in Gelidium

latfolium grown at nitrogen sufficient (+N) and nitrogen de-

ficient (-N) conditions.

o

=10Oc'68oo-^=b-oo34so-

6.oF

60

50

40

30

20

10

0

coE'ioo-xoz

l

cOE

ooXoz+

^+NPE/-NPE. +NPC/-NPC

1 00 200

Photon flux density (p mol m-2 s-l)

Fig. 3. Phycoerythrin and phycocyanin ratios in Gelidium la-

tifulium grown at nitrogen sulficient (+N) and nitrogen defi-cient (-N) conditions.

45

40

2tr

30

)R

o

zd

20

10100

Botanica Marina I YoI.32 I 1989 I Fasc. 6

Fredriksen and Rueness: Culture studies ol Gelidium latifulium from Norway 543

li.?.3

==E-

ooCA

'==

OL

o_

50

40

c en'a

=€20E

z.{10

caor'63!a_ac'oo.9=-o1o

EooE

B 10 12 14 16 18

Pg N/mg drY weight

Fig. 5. Correlation between nitrogen- and total phycobilipro-tein content in Gelidium latifolium grown at nitrogen dehcientconditions.

ditions there was no variation in carbon content withvarying PFD, while the nitrogen content increased

markedly with decreasing PFD. This is reflected as

an increase in the C: N ratio from about 20 at thelowest PFD to 35 - 40 at the three highest PFD tested(Figure 4). When the nitrogen content is plottedagainst the content of phycobiliproteins under N-deficient conditions, a positive correlation (r : 0.98,

p < 0.01) between the two was found (Fig. 5). Nosuch correlation existed when nitrogen was in excess.

Free amino acid content

The amino analyzer used in this experiment did notenable us to separate between threonine, asparagineand glutamine. This means that the integrated valuesfor threonine in Table III include all these three aminoacids. From Table III it is clear that the increase inthe total free amino acid content is mainly caused bythe increase in the threonine-complex. There is a con-

0 100 200 300 400 500

Hours

Fig. 6. Nitrogen-, phycobiliprotein- and amino acid content inGelidium latifolium from nitrogen starved plants and after trans-fer to nitrogen sufficient conditions.

current slight decrease in glutamic acid content, whilethere are only minor changes in other amino acids.

When the total free amino acid content is plottedagainst phycobiliprotein- and nitrogen content, thepigment content shows a marked increase after 96

hours (Fig. 6). At this time the amino acid contenthas reached its maximum . Later, the phycobiliproteincontent increases towards a threshold level while the

amino acid content decreases towards the initialamount. These results indicate that phycobiliproteinsrather than free amino acids serve as internal nitrogenstorage compounds, although there is a slight increase

in the pool of amino acids in the algae. Nitrogencontent reaches a maximum level at ca 45 pg/mg DWafter 8 days. There was a positive correlation between

nitrogen- and phycobiliprotein content (r : 0.99,

p < 0.001) while no such correlation was seen between

nitrogen and amino acid content.

-+0600

Table III. Content of free amino acids in G. latfolium determined before and after transfer from N-starvation to N-sufficientconditions. Values given in pglg fresh weight.

Amino acids Hours alter nitrogen addition

528t929624 360

Aspartic acidThreonine*Glutamic acidSerineGlycineAlanineValineIsoleucineLeucineTyrosinePhenylalanineHistidineLysineArginine

2434

34617

19

9

30t425233941

4928

41

519JJJ

0

2514

18f

11

JJ438824

0

45554160

015

2420

0l116232918

0

56308429

0222024

7

17

19

3826

9

0

371078

137

02015

200

10

3015

4229

7

55t343

1490

2611

31

J

12

3227

1088741

47807178

02413

19

01429JO

972315

35

410282

0241418

02438397635't 8

698 975 915 1440 1915 t302 1152 1013

* including asparagine and glutamine.

Botanica Marina / Yol.32 I 1989 / Fasc. 6

544 Fredriksen and Rueness: Culture studies of Gelidium latfolium lrom Norway

Discussion

Maximum growth rates (8 -9oh day 1) of G. latfol-ium occurred at the highest PFD tested (300 pmol

- ' r t), while an increase in temperature from 14'Cto 28 "C had little influence on growth rates. Thismay seem in contrast to that previously reported forthe same species by Rueness and Tananger (1984),

who recorded an increase in growth rates from about4o/o day t to 7o day 1 between 15'C and 25"C ata PFD of 40 pmol m ' s t. We ascribe these differ-ences to variation in experimental conditions. Rueness

and Tananger (1984) grew the alga in Petri dishes

without agitation and with illumination from aboveand growth rates were determined over a 14-day pe-

riod. It may well be that the differences in growthrates recorded at various temperatures under these

experimental conditions were due to artifacts caused

indirectly by differences in gas exchange and nutrientuptake. In the present experiments, such differencesbetween various temperatures were eliminated by con-stant aeration and agitation ofthe cultures. In a recentreview, Raven and Geider (1988) point out that thereis a considerable variation in the elfect of temperatureon specific growth rate under light limiting conditions.The 'expected' effect is for light limited growth to be

much less temperature sensitive than is resource sat-urated growth. Also Santelices (1978) demonstratedthat the effect of temperature on growth rate was less

important than the effects of light intensity and watermovement in a study of some Hawaiian Gelidiales.Most studies of Gelidium have reported temperatureoptima in the range of 20 to 30 'C (Yokohama 79J2,

Oliger and Santelices 1981, Santelices et al. 7987,

Carler 1985, Macler and West 1987).

The number of lateral branches varied as a functionof PFD. Since G. latifolium,like most red algae has

an apical meristem, a strong relationship exists be-

tween growth rate and the number of lateral branches.

However, self-shading increases with time as the

fronds become bushy. By continued culturing ofplants kept in suspension by aeration, balllike thalliwere produced. Santelices (1986) and Santelices et ql.

(1981) also observed this phenomenon when growing

Gelidium and Pterocladia under free floating condi-tions. DAntonio and Gibor (1985) also observed in-creased branching with increased PFD in their studyof germlings of Gelidium robustum (Gardner) Hollen-berg et Abbott.

Pigment composition in red algae has frequently been

reported (e.g. Brody and Emerson 7959, Calabrese

and Felicini 7973,Waaland et al.7974,Lapointe and

Duke 1984, Macler and West 1987). In the present

experiments we found that both phycobiliprotein and

chlorphyll a content decreased towards the higherphoton flux densities and that plants grown under N-deficient conditions contained lower levels of pig-ments than plants grown under N-sufficient condi-tions (Table II and Fig. 2). This observation suggests

that phycobiliproteins are used as a nitrogen source

when nitrogen becomes limiting. As shown in Figure3, the steeper slope of the phycoerythrin curve sug-

gests that phycoerythrin rather than phycocyaninserves as internal nitrogen source. This could be due

to either that the algae contain more phycoerythrinthan phycocyanin or the location of the two phycob-iliproteins in the phycobilisomes. Due to the molec-lular organization of the phycobilisomes as describedby Glazer (1977), phycoerythrin, which is located inthe periphery of the phycobilisome, could be used as

a nitrogen source without affecting the energy trans-fere to chlorophyll a. A corresponding situation is

described in the cyanobacteria Synechococczrs sp. byYamanaka and Glazer (1980).

The total phycobiliprotein: chlorophyll a ratio also

decreased with increasing irradiance (Fig. 2). A similardecrease in the phycobiliprotein: chlorophyll a ratiowas found in the red alga Grffithsia pacifica Kylinby Waaland et al. (1974). They demonstrated that thenumber of phycobilisomes per unit of photosyntheticthylakoid changes in direct proportion to the pigmentratio and in an inverse proportion to light intensity,thereby suggesting that the size of a phycobilisome is

ideal for energy transfer. However, it remains to be

shown whether the number of phycobilisomes is re-

duced when plants are grown under N-deficient con-

ditions.

The ability to store nitrogen when in excess may varyaccording to species as do the compounds that serve

as a nitrogen pool. According to Laycock and Craigie(1911) high amounts of citrulline and the dipeptidecitrullinylarginine may serve as nitrogen storage com-pounds in Chondrus crispus Stackh. Ji et al. (1981)

demonstrated accumulation of free amino acids withincreasing nitrogen availability in Porphyra yezoensis

Ueda. Lignell and Peders6n (1987) showed that nitro-gen in excess resulted in an increase in lree aminoacids in Gracilaria secundata Harv. Akatsuka (1986)

reported on amino acid content in Gelidium amansii(Lamouroux) Lamouroux from Japan, and Macler(1986) reported that amino acid content of G. coulterichanged with varying nitrogen availability. In plantsgrown with nitrogen in excess the content of the aminoacids serine and glycine was four-fold higher than innitrogen-starved plants. Glutamine and asparagine

were undetectable in nitrogen starved plants (Macler1e86).

!Botanica Marina / Vol.32 / 1989 / Fasc.6

Fredriksen and Rueness: Culture studies ol Gelidium latifolium from Norway 545

We were not able to separate glutamine and aspara-gine from threonine. This threonine-complex was themost important in the increase in free amino acidcontent in our experiment. These results suggest thatG. latfolium has a somewhat different way of storingnitrogen than G. coulteri when nitrogen is available.Our results suggest that phycobiliproteins could serve

as nitrogen storage compounds and that the rapiddecrease in amino acid content could be due to thebiosynthesis of phycobiliproteins (Fig. 6). While thre-onine is important in the biosynthesis ol porphyrins(Tait 1968), another possibility could be that aspar-agine and especially glutamine (Syrett 1962), are di-rectly involved in the assimilation of reduced nitrogen.If so, their levels could be expected to rise with theaddition of nitrogen as precursors to other aminoacids and to proteins, including phycobiliproteins.

The ecological advantage of a high content of phy-cobiliproteins may be the ability to absorb maximumlight in periods with low irradiance (overcast andtwilight periods). At the same time the phycobilipro-teins may protect against photooxidation of chloro-phyll a during periods of high irradiance. Since nitro-gen is commonly limiting in the ocean, the ability tostore nitrogen even if growth is not favourable wouldbe an advantage.

The C :N ratio in macroalgae has been reported tovary from 5 to 40 (Niell 1976, Hanisak 1919). At-tempts have been made to assess the nitrogen statusof macroalgae using this ratio (Niell 1976, DeBoerand Ryther 7911, Jackson 1977, Lapointe and Ryther7919, Hanisak 1919 and 1983). Critical C:N ratiosfor macroalgae are between 10 and 15, while highervalues indicate N-limitation and lower values indicateN-storage. In our experiment we found that whenplants were grown under N-sufficient conditions, theC:N ratio showed no significant variation with vary-

References

Akatsuka, I. 1986. Japanese Gelidiales (Rhodophyta), especiailyGelidium. Oceanogr. Mar. Biol. Ann. Rev. 24:1'11-263.

Beer, S. and A. Eshel. 1985. Determining phycoerythrin andphycocyanin concentrations in aqueous crude extracts ofred algae. Aust. J. Mar. Freshw. Res. 36:785-792.

Bird, K. T. 19'/6. Simultaneous assimilation of ammonium andnitrate by Gelidium nudifrons (Geiidiales: Rhodophyta). "/.Phycol. 12:238-241.

Bird, K. T., C. Habig and T. DeBusk. 1982. Nitrogen allocationand storage patterns in Gracilaria tikvahiae (Rhodophyta).J. Phycol. 1B:344-348.

Bogorad, L. 1975. Phycobiliproteins and complementary chro-matic adaptation. Ann. Rev. Plant Physiol. 26: 369-401.

Boi1lot, A. 1963. Recherches sur le mode de d6veloppement desspores du genre Gelidium (Rhodophyc6es, G6lidiales). Rer,.Gen. Bot.70:130-137.

Botanica Marina I Yol.32 I 1989 I Fasc. 6

ing PFD. Since a decrease in phycobiliprotein contentwas observed in the same light gradient, there mustbe nitrogen containing compounds others than phy-cobiliproteins. The amount of water soluble proteinsin G. amansii account lor ca 8oh of total protein(Ochiai et al. 1987) while Lapointe and Duke (1984)found that phycoerythrin accounted for ca 20o/o oftotal nitrogen in Gracilaria tikvahiae McLachlan.Even though Bogorad (1915) claims that phycobili-proteins may account for up tlll 600 of the totalsolqble cellular protein in red algae, other storageproieins could be avilable. In such a case nitrogencould be routed through amino acids to these storageproteins, thus the concentration of amino acids showsa transient increase, then falls (Fig. 6).

The relatively high C :N ratio (ca 13) is probably dueto Gelidium's natural high carbon content, mainlypresent as cell wall constituents. When G. latifoliumwas grown under N-deficient conditions a C:N ratioas high as 42 was observed. This clearly suggests thatC: N ratio could be used as an indication of nitrogenstatus. But it is important to keep in mind that PFDalso influences the C: N ratio since the content ofphycobiliproteins depends on PFD. This is alsostressed by Lapointe (1981) and Lapointe and Tenore(1981). In addition, other nitrogen constituents (free

amino acids, inorganic nitrogen reserves) may accu-mulate under 1ow photon flux densities, due to re-duced growth demands (Lapointe et al. 1984).

Acknowledgements

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