Hydrobiologia 398/399: 315–320, 1999.

315
J.M. Kain (Jones), M.T. Brown & M. Lahaye (eds), Sixteenth International Seaweed Symposium,
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

Effects of nitrogen source, N:P ratio and N-pulse concentration

and frequency on the growth of Gracilaria cornea
(Gracilariales, Rhodophyta) in culture

Leonardo Navarro-Angulo & Daniel Robledo∗

CINVESTAV-IPN, Unidad Mérida A.P. 73 Cordemex 97310 Mérida, Yucatán México
Fax: [+52] 99 81 29 17. E-mail: robledo@kin.cieamer.conacyt.mx

Key words: cultivation, Gracilaria cornea, nitrogen metabolism

Abstract
The effects of nitrogen source, nitrogen:phosphorus ratio, nitrogen pulse concentrations and pulse frequency on
Gracilaria cornea growth were investigated under laboratory cultures. No significant differences in growth rate
were detected between nitrogen sources, the mean growth rate decreased from ca. 14 to 11% d−1 over 8 weeks.
Our results indicate that G. cornea can efficiently grow either with inorganic (NH4 -N, NO3 -N, NO3 NH4 ) or organic
(urea) nitrogen. The N:P ratio had a significant effect on G. cornea specific growth rate at 10:1 treatment (8.53%
d−1 ) when compared with ambient phosphate concentration (10:0), which produced the lowest growth rate (2.88%
d−1 ). Neither nitrogen pulse concentration nor pulse frequency showed a significant effect on the specific growth
rate, however, pulse frequency significantly affected biomass increase at 50 µM nitrogen (p < 0.05). Nitrogen
sources containing NH4 –N produced the highest phycoerythrin and protein contents being the most important
N storage in G. cornea. The nitrogen storage capacity of G. cornea allows it to grow over a 7 day period with
low nitrogen concentrations (< 50 µM). The understanding of nitrogen enrichment in G. cornea cultivation can
be applied to manipulate pigment content or agar synthesis, and give the basis for its use in on-land biofiltering
systems.

Introduction Niell, 1993). This capacity has been utilized in cultiv-

Seaweeds belonging to the genus Gracilaria are be-
ing increasingly used in the production of food grade
agar (Armisén, 1995). Its availability has greatly in-
creased mainly through the development of cultiva-
tion techniques in several countries (Critchley, 1993).
Successful large scale cultivation followed laborat-
ory studies where the physiological characteristics of
Gracilaria were studied (DeBoer, 1979; Lignell &
Pedersén, 1987; Friedlander et al., 1990). Gracilaria
has the capacity to take up and store nitrogen in ex-
cess of immediate requirements, and use it to sustain
growth during subsequent periods of nutrient defi-
ciency (Lapointe, 1985). Storage can be in the form of
inorganic nitrogen (Chapman & Craigie, 1977) and/or
metabolites such as proteins and pigments (Vergara &
∗ Author for correspondence.
ation of seaweeds to minimize the growth of epiphytes
and provide the physiological basis for pulse feeding
(Lapointe, 1985).
Nutrient supply is an important operating para-
meter in the management of seaweed cultivation sys-
tems (Lignell & Pedersén, 1987). The development of
a separate management strategy is required for each
species under cultivation since their physiological re-
quirements differ. Nutrient limitation in temperate
algae may occur at a different level than in trop-
ical species which experience oligotrophic conditions
(Hanisak, 1990).
Gracilaria cornea J. Agardh has been recently
considered as a maricultural candidate in the Yucatan
Peninsula due to its high agar yield and quality (Freile-
Pelegrín & Robledo, 1997a,b). Since nitrogen limita-
tion affects both phycocolloid content and growth rate,
the nitrogen requirements of cultured seaweeds have
316
to be determined, depending on the end use of the sea-
weed. The present study was designed to address the
dynamics of nitrogen in G. cornea as a function of ni-
trogen source, nitrogen:phosphorus ratio and nitrogen
concentration and frequency during pulse feeding.
Material and methods
Plant material
Unialgal cultures of G. cornea were obtained from
carpospores under laboratory conditions. Tetrasporo-
phytic thalli were grown in 1 l flasks at a density 1 g
l−1 with filtered sterilized seawater (35 ‰) under con-
trolled laboratory conditions of temperature (27 ± 1.3
◦ C), photon irradiance (100 µmol photons m−2 s−1 ),
and photoperiod (12:12, L:D). These conditions were
previously found to optimize growth of G. cornea tet-
rasporophytes (Orduña-Rojas, 1996). Algae from the
culture were preincubated 7 days before each experi-
mental treatment under the same conditions described
above. Continuous aeration was applied to the culture
medium throughout the study. The fresh weight of
algae was determined weekly for each treatment, ad-
justed to initial density and culture medium renewed.
Relative daily growth rate (R = % d−1 ) was calculated
as
ln Wt − ln W0
R= • 100,
t
where W0 is the initial biomass and Wt the biomass
at day t (Evans, 1972). Three replicates were used for
each tested condition.
Nitrogen source
To test the effect of nitrogen enrichment on G. cornea
growth rate, plant material was cultured with the fol-
lowing sources NO3 –N (added as NaNO3 ), NH4 –N
(added as NH4 Cl), a combination of both NO3 and
NH4 (in the form of NH4 NO3 ) and organic nitrogen
source (added as urea - NH2 CONH2 ). Nitrogen was
added to the other Provasoli Enriched Seawater me-
dia (PES) ingredients to give a final concentration of
824 µM (Starr & Zeikus, 1993). Each treatment was
carried out under conditions described above for eight
weeks. Medium was changed every 7 days.
N:P ratio
The effect of N:P ratio on growth was studied in
laboratory cultures under defined conditions. Enriched
seawater (PES) stock solution without phosphate was
used. Phosphate was added separately as Na2 HPO4
to give four different concentrations 824, 412, 82.4
and < 0.01 µM (ambient phosphorus concentration),
corresponding to the following N:P ratios 10:10, 10:5,
10:1 and 10:0, respectively. Growth rate was recorded
each week during a three week period.
Nitrogen pulse concentration and frequency
This experiment determined the growth rate of G.
cornea at four nitrogen fluxes using a factorial design
with two levels of nitrogen concentrations (50 and 150
µM NH4 –N) and two pulse frequencies, one every two
weeks (each 14 day = 1) and one every week (each
7 day = 2). This combination gave a total amount of
N available to the plants equal to 3.57, 7.14, 10.71
and 21.42 µM NH4 –N g fresh wt d−1 . Each exper-
imental treatment was maintained under unenriched
seawater after a 24 h pulse. The experimental period
was extended under conditions described above for ten
weeks.
Analytical methods
Chemical and biochemical analyses on triplicate
samples were performed as follows: chlorophyll a
(Jeffrey & Humphrey, 1975); phycobiliproteins (Beer
& Eshel, 1985) were determined in all experimental
procedures. Protein content (Lowry et al., 1951) and
carbohydrate content (Dubois et al., 1956) were de-
termined for the nitrogen source trial.
Statistical analysis
Significant differences for R between treatments were
determined with an ANOVA. Homogeneity of vari-
ances (Bartlett’s test) was tested and transformation
applied when necessary (log x). A Tukey HSD test at
95% of significance was used to assess the effect of
nitrogen source on growth rate. Significant differences
between each combination of nitrogen pulse concen-
tration and frequency were determined with a Duncan
test (Zar, 1984).
Results
The effect of nitrogen source on growth is shown in
Figure 1. No significant difference was found for relat-
ive growth rate and biomass increase; the growth rates
ranged from 11.13 to 12.77% d−1 . In general, growth
 317

Figure 1. Relative growth rate (% d−1 ) of Gracilaria cornea as a function of nitrogen source during eight weeks of cultivation. Bars show
mean values ± SD.

rates decreased from 13.64 ± 1.59 to 10.34 ± 1.11%

d−1 between treatments at the end of the experiment.
Phycobiliproteins and chlorophyll a contents were
not affected (p > 0.05); both maximum phycoerythrin
and phycocyanin contents were detected for NH4 Cl
and NH4 NO3 , respectively (Table 1). In general, cul-
tures supplemented with urea had lower pigment con-
tent. The concentration of pigments did not seem to
be influenced by nitrogen source since no significant
difference was found among treatments.
The only significant effect of nitrogen source on
chemical composition was that protein content was
highest in the presence of the NH4 ion (Table 1).
Figure 2. Relative growth rate (% d−1 ) of Gracilaria cornea as a
Maximum protein was found in NH4 Cl and NH4 NO3 , function of PO4 −3 level in N:P experiment. Each symbol corres-
while it was significantly lower for NaNO3 and urea. ponds to a significant different mean in accordance with Tukey HSD
Carbohydrate did not differ significantly between any test. Bars show mean values ± SD.
of the nitrogen source treatments (p > 0.05).
The nitrogen to phosphorus ratio (N:P) had a sig-
red fronds were observed, with 10:5 and 10:10 they
nificant effect on the relative growth rate (Figure 2)
showed a brownish colour, and a yellowish coloration
which ranged from 2.88 ± 0.70 to 8.53 ± 2.95%
was evident in treatment 10:0.
d−1 . The best growth rates were obtained for N:P
Neither nitrogen pulse concentration nor pulse fre-
ratios of 10:1 and 10:5 (p < 0.05). The treatment
quency had a significant effect on the relative growth
with the N:P ratio of 10:0 had the lowest growth, and
rate of Gracilaria cornea (Table 2); however pulse
did not differ significantly from the 10:10 ratio. Two
frequency significantly affected (p < 0.05) weight in-
weeks after starting the experiment plant coloration
crease at 50 µM nitrogen concentration (Figure 3)
changed for each treatment. For the 10:1 ratio, deep
with a mean value of 3.52 ± 1.01 g at 2 pulses per
318
Table 1. Chemical composition of G. cornea cultured with different nitrogen sources. Means
with different superscript are significantly different at p < 0.05 (ANOVA, Tukey HSD).
Standard deviation indicated, n = 8.

Treatments
Variables NH4 Cl NaNO3 NH4 NO3 Urea

Phycoerythrin 9.70 ± 3.95a 7.97 ± 4.29a 8.81 ± 3.25a 6.52 ± 2.00a

(mg g dry wt−1 )
Phycocyanin 1.44 ± 0.71a 1.28 ± 0.90a 1.45 ± 0.75a 1.12 ± 0.45a
(mg g dry wt−1 )
Chlorophyll a 300 ± 58.4a 304 ± 56.1a 291 ± 61.1a 267 ± 22.1a
(µg g dry wt−1 )
Protein 16.01 ± 1.86a 13.47 ± 1.50b 15.58 ± 2.23a 13.7 ± 1.51b
(% dry wt−1 )
Carbohydrate 32.77 ± 4.84a 35.57 ± 4.02a 33.39 ± 4.56 a 37.02 ± 3.44 a
(% dry wt−1 )

Efficient use of different nitrogen sources at differ-

Figure 3. Fresh weight increment (g) of Gracilaria cornea as a
function of nitrogen pulse concentration (NH4 -N) and pulse fre-
quency. One pulse per 2 weeks at 50 µM (N) and 150 µM ();
and 2 pulses per 2 weeks pulse at 50 µM ()and 150 µM (•). Bars
show mean values ± SD.
2 weeks. Phycobiliprotein content was not affected,
whereas the chlorophyll a content was only affected
by pulse frequency (Table 2). A highly significant
correlation was found between the nitrogen flux and
phycoerythrin content (r = 0.995, p< 0.01).
Discussion
Gracilaria cornea can grow with inorganic (NH4 –
N, NO3 –N) or organic (urea) nitrogen sources. The
preference for NH4 -N has been described for other
Gracilaria species and red algae (DeBoer et al., 1978).
ent absorption rates with preference for NH4 -N rather
than NO3 -N have been shown for Gracilaria foliifera
(Førsskäl) Børgesen (D’Elia & DeBoer, 1978), Gra-
cilaria tikvahiae McLachlan (Hanisak, 1990) and
Gracilaria tenuistipitata Chang et Xia (Haglund &
Pedersén, 1993); however relative growth rates in G.
cornea did not change significantly between different
nitrogen sources in the present study. Similarly, G. tik-
vahiae showed similar growth rates when cultured in
NH4 –N or NO3 –N enriched seawater (Hanisak, 1990).
Haglund & Pedersén (1993) did not find any signi-
ficant difference when using KNO3 or NH4 NO3 in
the culture media, with growth rates between 4 and
9% d−1 . DeBoer et al. (1978) reported similar growth
rates for Chondrus crispus Stackh. and G. tikvahiae,
with either NH4 –N or NO3 –N, being higher for the
former.
The preference for nitrogen source depends on ab-
sorption rate and is influenced by the nitrogen status
of the algae (Hanisak, 1990). In Gracilaria sp. ab-
sorption rate for NH4 is higher than that for NO3
since NH4 is directly incorporated into the amino
acid pool (Haglund & Pedersén, 1993; Lobban &
Harrison, 1994). Inhibition of NO3 or NO2 absorp-
tion have been observed, depending on ammonium
concentration (Hanisak, 1990).
Phycobiliproteins, together with free amino acids,
are important as nitrogen storage in red algae
(Lapointe, 1981; Ryther et al., 1981), phycoerythrin
being the pigment which responds faster to nitrogen
availability in the medium (Vergara & Niell, 1993). In

Table 2. Growth and chemical composition of G. cornea cultured under different treat-
ments of nitrogen pulse concentration (50, 150 µM) and pulse frequency (1, 2). Each
combination gave the following NH4 -N flux (µM N g fresh wt d−1 ): 1 × 50 = 3.57; 2 ×
50 = 7.14; 1 × 150 = 10.71 and 2 × 150 = 21.34. Means with different superscript are
significantly different at p < 0.05 (ANOVA, Tukey HSD). Standard deviation indicated, n
= 10.
Variables 1 × 50
R 3.33 ± 2.07a
(% d−1 )
W increase 2.99 ± 0.73a
(g)
Phycoerythrin 0.90 ± 0.29a
(mg g dry wt−1 )
Phycocyanin 38.7 ± 11.3a
(µg g dry wt−1 )
Chlorophyll a 78.2 ± 4.2a
(µg g dry wt−1 )
Gracilaria cornea phycoerythrin constituted approx-
imately 85–87% of total phycobiliproteins. On the
other hand, chlorophyll a is not considered a N pool,
representing approximately 2% of total nitrogen in tis-
sue (McGlathery et al., 1996). Increases in chlorophyll
a content, with increases in cellular nitrogen, are also
well known (Bird et al., 1982).
The best growing conditions for G. cornea were
found for the N:P ratio of 10:1. Lower inorganic nu-
trient concentration including phosphorus reduces the
algal photosynthetic capacity in tropical waters hence
limiting their growth and productivity as has been
shown for G. tikvahiae (Lapointe, 1987). In this re-
gard, ambient phosphate concentrations supplemented
with nitrogen (10:0) limited G. cornea growth rate.
The reduction of growth observed in G. cornea with
10:10 ratio could be related to a detrimental effect
of high phosphate concentrations. Phosphate levels of
about 1 mM were found to inhibit growth of Gracil-
aria conferta (Schousboe ex Montagne) Feldmann et
Feldmann (Friedlander & Ben Amotz, 1991) while an
increase of N:P ratio (2.5–20) in G. conferta caused
a significant growth rate enhancement (Friedlander &
Levy, 1995).
The nitrogen storage capacity of G. cornea allows
it to grow over 7 day period with low nitrogen con-
centration. Similar to our results, the internal nitrogen
pool is used for growth in G. chilensis Bird, McLach-
lan et Oliveira (Pickering et al., 1993) and G. gracilis
(Stackhouse) Steentoft, Irvine et Farnham (Smit et
319
Treatments
1 × 150 2 × 50 2 × 150
3.17 ± 1.79a 4.34 ± 1.69a 3.66 ± 1.43a
2.79 ± 0.72a 3.52 ± 1.0b 3.01 ± 0.85a
0.97 ± 0.16a 1.10 ± 0.26a 1.52 ± 0.67a
64.8 ± 51a 41.9 ± 31a 73.5 ± 39a
77.1 ± 9.6a 122.4 ± 21.7b 125.4 ± 53.2ab
al., 1997). Slow growth rate in G. cornea at one N
pulse may suggests that nitrogen reserves were used
for metabolic maintenance with lower chlorophyll a
content, while with two pulse treatments a higher
chlorophyll a content was found suggesting that the
nitrogen pool was maintained with respect to the same
growth level. Smit et al. (1997) showed similar results
for G. gracilis using two pulses per week. Phycoeryth-
rin constituted the major nitrogen reserve in G. cornea
representing up to 60% of total soluble protein. In G.
gracilis nitrogen reserves in order of importance are
protein, phycoerythrin, chlorophyll a and carotenoids
(Smit et al., 1997). Increasing the nitrogen flux has
been shown to increase protein content and the inverse
relationship has been described for carbohydrates and
agar content in G. tikvahiae (Bird et al., 1982).
Though the present results were obtained under
laboratory conditions the knowledge on the regulation
of the N concentration and the utilization of indicators
to monitor the nutrient status of G. cornea should in-
crease the efficiency and success of its mariculture and
the capability of increasing phycocolloid production.
Acknowledgements
This work has been supported by CONACYT (N◦
2198P-B). L. Navarro-Angulo acknowledges CON-
ACYT (Scholarship N◦ 90037) and a fellowship from
Fondo Yucatan. Both authors are grateful to Ma. Zal-
320
divar Romero and Eloy Gil Trava for their assistance
in laboratory analysis.
References
Armisén, R., 1995. World-wide use and importance of Gracilaria.
J. appl. Phycol. 7: 231–243
Beer, S. & A. Eshel, 1985. Determining phycoerythrin and phycocy-
anin concentrations in aqueous crude extracts of red algae. Aust.
J. mar. Freshwat. Res. 36: 785–792.
Bird, K. T., C. Habig & T. DeBusk, 1982. Nitrogen allocation and
storage patterns in Gracilaria tikvahiae (Rhodophyta). J. Phycol.
18: 344–348.
Chapman, A. R. O. & J. S. Craigie, 1977. Seasonal growth in Lam-
inaria longicruris: relations with dissolved inorganic nutrients
and internal reserves of nitrogen. Mar. Biol. 40: 197–205.
Critchley, A. T., 1993. Gracilaria (Rhodophyta, Gracilariales):
an economically important agarophyte. In Ohno, M. & A. T.
Critchley (eds), Seaweed Cultivation and Marine Ranching.
JICA. Kanagawa International Fisheries Training Center. Japan:
89–111.
D’Elia, C. F. & J. A DeBoer, 1978. Nutritional studies of two red
algae. II. Kinetics of ammonium and nitrate uptake. J. Phycol.
14: 266–272.
DeBoer, J. A., 1979. Effects of nitrogen enrichment on growth rate
and phycocolloid content in Gracilaria foliifera and Neoagrd-
hiella baileyi (Florideophyceae). Proc. int. Seaweed Symp. 9:
263–271.
DeBoer, J. A., H. J. Guigli, T. L. Israel & C. F. D’Elia, 1978. Nu-
tritional studies of two red algae. I. Growth rate as a function of
nitrogen source and concentration. J. Phycol. 14: 261–266.
Dubois, M., F. A. Gilles, J. K. Hamilton, D. A. Rebers & F. Smith,
1956. Colorimetric method for the determination of sugar and
related substances. Anal. Chem. 28: 350–356.
Evans, G. C., 1972. The quantitative analysis of plant growth.
Blackwell, Oxford, 734 pp.
Freile-Pelegrín, Y. & D. Robledo, 1997a. Effects of season on the
agar content and chemical characteristics of Gracilaria cornea
from Yucatán, México. Bot. mar. 40: 285–290.
Freile-Pelegrín, Y. & D. Robledo, 1997b. Influence of alkali treat-
ment on agar from Gracilaria cornea from Yucatán, México. J.
appl. Phycol. 9: 533–539.
Friedlander, M. & A. Ben-Amotz, 1991. The effects of outdoor cul-
ture on growth and epiphytes of Gracilaria conferta. Aquat. Bot.
39: 315–333.
Friedlander, M. & I. Levy, 1995. Cultivation of Gracilaria in
outdoor tanks and ponds. J. appl. Phycol. 7: 315–324.
Friedlander, M., N. Galai & H. Farbstein, 1990. A model of seaweed
growth in an outdoor culture in Israel. Hydrobiologia 204/205:
367–373.
Haglund, K. & M. Pedersén, 1993. Outdoor pond cultivation of the
subtropical marine red alga Gracilaria tenuistipitata in brackish
water in Sweden. Growth, nutrient uptake, co-cultivation with
rainbow trout and epiphyte control. J. appl. Phycol. 5: 271–284.
Hanisak, M. D., 1990. The use of Gracilaria tikvahiae (Gra-
cilariales, Rhodophyta) as a model system to understand the
nitrogen nutrition of culture seaweeds. Hydrobiologia 204/205:
79–87.
Jeffrey, S. W. & G. F. Humphrey, 1975. New spectrophotomet-
ric equations for determining chlorophylls a, b, c1 and c2
in higher plants, algae and natural phytoplankton. Biochem.
Physiol. Pflanzen 167: 191–194.
Lapointe, B. E., 1981. The effects of light and nitrogen on growth,
pigment content, and biochemical composition of Gracilaria fo-
liifera var. angustissima (Gigartinales, Rhodophyta). J. Phycol.
17: 90–95.
Lapointe, B. E., 1985. Strategies for pulse nutrient supply to
Gracilaria cultures in the Florida Keys: interactions between
concentration and frequency of nutrient pulses. J. exp. mar. Biol.
Ecol. 93: 211–222.
Lapointe, B. E., 1987. Phosphorus- and nitrogen-limited photosyn-
thesis and growth of Gracilaria tikvahiae (Rhodophyceae) in
the Florida Keys: an experimental field study. Mar. Biol. 93:
561–568.
Lignell, A. & M. Pedersén, 1987. Nitrogen metabolism in Gracil-
aria secundata Harv. Hydrobiologia 151/152: 431–441.
Lobban, C. S. & P. J. Harrison, 1994. Seaweed ecology and
physiology. Cambridge University Press, 366 pp.
Lowry, O. H., N. J. Rosebrough, A. L. Farr & R. J. Randall,
1951. Protein measurement with the Folin phenol reagent. J. biol.
Chem. 193: 265–275.
McGlathery, K. J., M. F. Pedersen & J. Borum, 1996. Changes in
intracellular nitrogen pools and feedback controls on nitrogen
uptake in Chaetomorpha linum (Chlorophyta). J. Phycol. 32:
393–401.
Orduña-Rojas, J., 1996. Efecto de la radiación y la temperatura en
la liberación y desarrollo de las carposporas del alga roja Gra-
cilaria cornea J. Agardh (Gracilariales, Rodofita). MSc.Thesis
CINVESTAV-Unidad Mérida, Yucatán, México, 43 pp.
Pickering, T. D., M. E. Gordon & L. J. Tong, 1993. Effect of nutrient
concentration and frequency on growth of Gracilaria chilensis
plants and level of epiphytic algae. J. appl. Phycol. 5: 525–533.
Ryther, J. H., T. Corwin, A. DeBusk & L. D. Williams, 1981. Ni-
trogen uptake and storage by the red alga Gracilaria tikvahiae
(McLachlan, 1979). Aquaculture 26: 107–115.
Smit, A. J., B. L. Robertson & D. R. du Preez, 1997. Influence
of ammonium-N pulse concentrations and frequency, tank con-
dition and nitrogen starvation on growth rate and biochemical
composition of Gracilaria gracilis. J. appl. Phycol. 8: 473–481.
Starr, R.C. & J.A. Zeikus, 1993. UTEX. The culture collection of
algae at the University of Texas at Austin. J. Phycol. Suppl. 29:
1–106.
Vergara, J. J. & F. X. Niell, 1993. Effects of nitrate availability and
irradiance on internal nitrogen constituents in Corallina elongata
(Rhodophyta). J. Phycol. 29: 285–293.
Zar, H., 1984. Biostatistical Analysis. Prentice-Hall, Inc., Engle-
wood Cliffs, N.Y., 675 pp.