Dierent nitrogen sources and growth responses of Spirulina platensis

in microenvironments
Jorge Alberto Vieira Costa*, Karla Leal Cozza, Lucielen Oliveira and Glenio Magagnin
Laboratory of Biochemical Engineering, Department of Chemistry, Federal University Foundation of Rio Grande,
P.O. Box 474, RS 96201-900, Brazil
*Author for correspondence: Tel.: +55-53-233 8653, Fax: +55-53-232 9716, E-mail: dqmjorge@super.furg.br
Received 15 June 2000; accepted 16 May 2001

Keywords: Cyanobacteria, microenvironments, nitrogen, Spirulina

Summary
Spirulina platensis was cultivated, in comparative studies, using several sources of nitrogen. The standard source
used (sodium nitrate) was the same as that used in the synthetic medium Zarrouk, whereas the alternative nitrogen
sources consisted of ammonium nitrate, urea, ammonium chloride, ammonium sulphate or acid ammonium
phosphate. The initial nitrogen concentrations tested were 0.01, 0.03 and 0.05 M in an aerated photobioreactor at
30 C, with an illuminance of 1900 lux, and 12 h-light/12 h-dark photoperiod over a period of 672 h. Maximum
biomass was produced in medium containing sodium nitrate (0.010.030.05 M), followed by ammonium nitrate
(0.01 M) and urea (0.01 M). The nal biomass concentrations were 1.992 g l)1 (0.03 M sodium nitrate), 1.628 g l)1
(0.05 M sodium nitrate), 1.559 g l)1 (0.01 M sodium nitrate), 0.993 g l)1 (0.01 M ammonium nitrate) and
0.910 g l)1 (0.01 M urea). This suggested that it is possible to utilize nitrogen sources other than sodium nitrate for
growing S. platensis, in order to decrease the production costs of scaled up projects.
Introduction
The rst tribes of hunter-collectors used to pick up
microalgae for food on margins of lakes, many of them
saline, e.g., the collection of Spirulina in Lake Chad,
Africa (Henrikson 1994). Many advantages are recognized in microalgae cultivation, such as: high protein
biomass (above 60%), absence of parts to be discarded
during processing, use of arid or semi-arid areas around
the world, and growth in saline waters without competing with traditional agriculture. However, there are
some disadvantages, mainly due to the costs of the
mineral sources required (Richmond 1990).
The microalga Spirulina platensis is a multicellular
lamentous cyanobacterium, whose cells (diameter from
1 to 12 lm) are organized in helicoidal trichomes
without ramications (Richmond 1990). Several species
of Spirulina are able to inhabit hostile environments,
such as saline lakes (Henrikson 1994).
According to Richmond (1990) the rst known Spirulina pills are going to be substituted slowly by candies,
gums, biscuits and pastas enriched with this microalga.
The presence of free amino acids, vitamin B12, thiamin,
riboavin, niacin, phycocyanin, carotenoids, and essential unsaturated fatty acids make Spirulina a promising
product to the food industry. Many therapeutic applications, such as stimulus to the immune system (Hayashi
1996), regulation of blood pressure and cholesterol

synthesis (Nayaka 1988; Iwata 1990), and zinc deciency

(Huang et al. 1982) could also be quoted.
The aim of this study was, through using dierent
nitrogen sources and varying them quantitatively, to
obtain new alternative substrates for S. platensis cultivation.
Material and methods
Microorganism and media
Spirulina platensis LEB-52 was supplied by the Faculty
of Pharmaceutical Sciences, University of Sao Paulo
(Costa et al. 2000). Medium composition was the one
established by Zarrouk in which the nitrogen source
available to the microorganism (sodium nitrate) was
replaced by ammonium nitrate, urea, ammonium chloride, ammonium sulphate or acid ammonium phosphate. The concentrations of nitrogen tested were 0.01,
0.03 and 0.05 M in all media.
Cultivation
The experiment was carried out at a scale of 2000 ml
and started with a biomass concentration of 0.1 g l)1. It
was run in a sterile aerated photobioreactor at 30 C.
The aeration rate was 20 l h)1, and a 12 h-light/12

Figure 1. Diagram showing S. platensis growth experiment using a

sterile aerated photobioreactor.

h-dark photoperiod was used. Illumination was obtained from uorescent lights at an illuminance of
1900 lux. A schematic diagram of the experimental
device used is shown in Figure 1.
Analytical and statistical procedures
Samples were taken aseptically each 24 h, to monitor
pH, carbonates and bicarbonates according to AOAC
(1995), and the cellular concentration growth was
determined by optical density measurements at
750 nm. All experiments were carried out in duplicate
or triplicate. Statistical treatment was done with NewmanKeuls analysis within the 95% condence level.
Results and discussion
Results obtained with dierent nitrogen sources could
help our understanding of the development of these
microalgae over 672 h. Since nitrogen is an important
nutritional source, S. platensis response varied with
dierent sources and amounts. Spirulina platensis grew
successfully in six out of 18 media, which can be seen in
Table 1. There were no statistical dierences (at the
95% condence level) in the NewmanKeuls test among
media with sodium nitrate in all three concentrations
tested. However, there were statistical dierences between the nitrate and the three other media tested. The
nal S. platensis biomass concentrations in dierent
nitrogen sources were given in Figure 2.
Table 1. Final biomass concentrations of S. platensis when grown for
672 h in modied Zarrouk media with varying concentrations of
dierent nitrogen sources.
Medium

Acid ammonium phosphate

Ammonium chloride
Ammonium nitrate
Ammonium sulphate
Sodium nitrate
Urea

Figure 2. Time course of S. platensis concentrations with dierent

nitrogen sources: (a) sodium nitrate, (b) urea, and (c) ammonium
nitrate. Symbols: (d) 0.01 M, (h) 0.03 M, (s) 0.05 M.

Final biomass concentration (g l)1)

0.01 M

0.03 M

0.05 M

0.924
0.056
0.993
0.081
1.559
0.910

0.046
0.028
0.038
0.029
1.992
0.091

0.043
0.042
0.032
0.017
1.628
0.059

The best environmental phosphate concentrations for

growth of microorganisms follow a wide range of
tolerance according to the specic species used and do
not depend on the concentrations of the other nutrients
(Becker 1994). The average phosphorus tolerance of
many species, according to Becker (1994) is situated
between 0.050 and 20 mg l)1. During this study, the

Table 2. Maximum specic growth rate (lmax ), doubling time (td ) and
correlation coecient (r) for the best growth results as a function of
the nitrogen source studied.
Medium

lmax (day)1)

td (day)

Ammonium nitrate (0.01 M)

Urea (0.01 M)
Sodium nitrate (0.03 M)
Sodium nitrate (0.01 M)
Sodium nitrate (0.05 M)

0.132
0.116
0.084
0.064
0.047

5.2
5.9
8.2
10.8
14.6

0.9959
0.9922
0.9690
0.9679
0.9781

phosphorus concentration utilized was above Becker's

concentrations and varied from 89 mg l)1 (pattern medium) to 243 mg l)1 at 0.01 M acid ammonium phosphate. The nal concentrations were even higher for 0.03
and 0.05 M ammonium acid phosphate media. Nevertheless, microalgal phosphate assimilation depends on
many other factors such as pH, sodium concentration,
potassium concentration, magnesium concentration and
the presence of heavy metals in the medium, that could
have aected the concentration-dependence results development. Previous reports in the literature on growth
rates of Spirulina under dierent nitrogen sources can
be found at Cohen et al. (1987), Kaplan et al. (1990),
Richmond (1990), Manabe et al. (1992), Olgu n et al.
(1994), Gibbs (1995), Chen & Zhang (1997) and Torres
et al. (1998), where the microalgae were capable of
metabolizing nitrate, ammonia, and urea.
The biomass production in medium containing
0.01 M urea was better than those with 0.01 M acid
ammonium phosphate and 0.01 M ammonium nitrate
media at the 95% condence level. But, when the eects
of acid ammonium phosphate and ammonium nitrate
were compared to each other, no statistical dierence
was found.
The medium with 0.01 M urea was signicantly
dierent from others with 0.03 and 0.05 M urea, but
not statistically dierent, at the 95% condence level,
from the medium with ammonium acid phosphate
0.01 M. However, through microscopic observation,
the medium with ammonium acid phosphate 0.01 M
was found to contain many broken trichomes and lysed
cell walls. The data, in this case, were considered
overestimated when compared to other media results.
Media with sodium nitrate seemed more adequate than
the others for S. platensis growth, but ammonium
nitrate and urea could be alternative nitrogen sources,
since this cyanobacterium showed adaptation at low
concentrations (0.01 M) of these nitrogen sources.
Table 2 shows that a smaller doubling time (td) was
found at 0.01 M ammonium nitrate (td 5:2 days) and
0.01 M urea (td 5:9 days). Notwithstanding, the nal
biomass concentrations in these media were, respectively, 0.993 and 0.910 g l)1, due to the dierent media
adaptation by S. platensis (approximately 240 h to urea
and ammonium nitrate, as seen in the respective lag
phases). Nevertheless, the nal biomass concentrations
in sodium nitrate at the various concentrations studied
surpassed 1.5 g l)1, but the td was between 8.2 and

14.6 days. These dierences in doubling time suggest a

preference of Spirulina for nitrogen sources as ammonia
or urea, and nitrate consumption only when ammonium
is not available, as previously quoted by Becker (1994)
for Chlorella, Scenedesmus and Spirulina. Besides it was
noticed that S. platensis growth showed a time lag when
ammonium nitrate was used as the source of nitrogen
(Figure 2c).
Growth in ammonium nitrate had shorter td than in
other media, since S. platensis is capable of assimilating
both nitrate and ammonium ions. According to Becker
(1994), assimilation of ammonia is easier due to the
simplicity of its molecule, and its presence inhibits the
nitrogenase activity. Urea is a good nitrogen source,
according to Meeks et al. (1983). Urea is metabolized by
cyanobacteria through the activity of enzymes such as
urease and urea starch lyase. In addition, Faintuch et al.
(1992) believe that urea yields two atoms of nitrogen,
whereas nitrate yields only one, contributing to more
successful assimilation of the former by microalgae.
Spirulina is still capable of utilizing nitrogen from nitrate
because, according to Odum (1983), this structure is the
most common in nature.
Conclusions
The cultivation of the microalga S. platensis was feasible
using other nitrogen sources such as urea or ammonium
nitrate instead of sodium nitrate. These alternative
sources can reduce the price of the inputs and would
be an economic improvement for large-scale cultivation.
The best results as biomass obtained were: sodium
nitrate (0.03 M) 1.992 g l)1, sodium nitrate (0.05 M)
1.628 g l)1, sodium nitrate (0.01 M) 1.559 g l)1, ammonium nitrate (0.01 M) 0.993 g l)1 and urea (0.01 M)
0.910 g l)1, and as td were: ammonium nitrate (0.01 M)
5.2 day, urea (0.01 M) 5.9 day, and sodium nitrate
(0.03 M) 8.2 day.
The production of Spirulina biomass over 1.00 g l)1
reiterates the value of this cyanobacterium as an
alternative source of protein in regions where protein
scarcity compromises people's quality of life. Moreover,
Spirulina biomass could be used indirectly, as animal
feed, which becomes later available as dierent protein
sources for food.

Acknowledgements
The authors are grateful to Dr Antonio Liborio
Philomena and Jose Carlos Patta Alves for attention
and valuable support.
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