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European Journal of Phycology

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Distribution of intracellular nitrogen in marine

microalgae: Calculation of new nitrogen-to-protein
conversion factors
a

Sergio O Loureno , Elisabete Barbarino , Paris L Lavn , Ursula M Lanfer Marquez &
Elizabeth Aidar

Departamento de Biologia Marinha, Universidade Federal Fluminense, Caixa Postal

100644, Niteri, RJ, Brazil, CEP 24001-970
b

Departamento de Alimentos e Nutrio Experimental, Faculdade de Cincias

Farmacuticas, Universidade de So Paulo, Caixa Postal 66083, So Paulo, SP, Brazil, CEP
05315-970
c

Departamento de Oceanografia Biolgica, Instituto Oceanogrfico, Universidade de So

Paulo, Caixa Postal 66149, So Paulo, SP, Brazil, CEP 05315-970
d

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Version of record first published: 20 Feb 2007.

To cite this article: Sergio O Loureno , Elisabete Barbarino , Paris L Lavn , Ursula M Lanfer Marquez & Elizabeth Aidar
(2004): Distribution of intracellular nitrogen in marine microalgae: Calculation of new nitrogen-to-protein conversion
factors, European Journal of Phycology, 39:1, 17-32
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Eur. J. Phycol. (2004), 39(1): 17 32.

Distribution of intracellular nitrogen in marine microalgae:

Calculation of new nitrogen-to-protein conversion factors

S E R G I O O . L O U R E N C O 1 , E L I S A B E T E B A R B A R I N O 1 , P A R I S L . L A V I N 1 * ,
U R S U L A M . L A N F E R M A R Q U E Z 2 A N D E L I Z A B E T H A I D A R 3{
1

Departamento de Biologia Marinha, Universidade Federal Fluminense, Caixa Postal 100644, CEP 24001-970, Niteroi, RJ, Brazil
Departamento de Alimentos e Nutricao Experimental, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Caixa
Postal 66083, CEP 05315-970, Sao Paulo, SP, Brazil
3
Departamento de Oceanograa Biologica, Instituto Oceanograco, Universidade de Sao Paulo, Caixa Postal 66149,
CEP 05315-970, Sao Paulo, SP, Brazil
2

Downloaded by [77.162.204.181] at 01:56 03 January 2013

(Received 14 June 2001; revised 18 July 2003; accepted 30 September 2003)

Nitrogen budgets in microalgae are strongly aected by growth conditions and physiological state of the cultures. As a
consequence, protein N (PN) to total N (TN) ratio may be variable in microalgae grown in batch cultures, and this may
limit the usefulness of the nitrogen-to-protein conversion factors (N-Prot factors), the most practical way of determining
protein content. The accuracy of protein determination by this method depends on the establishment of specic N-Prot
factors, and experimental data are needed to ll this gap. Complementing a previous study, the present work was designed
to quantify the uctuations of the main nitrogenous compounds during the growth of 12 species of marine microalgae, as
well as to determine N-Prot factors for them. The microalgae were cultured in two experimental conditions: (a) using a
N-replete culture medium (initial N concentration, 1.18 mM) and aeration, and (b) with a N-depleted culture medium
(initial N concentration, 235 mM) and no aeration. The distribution of intracellular nitrogen was studied by constructing
budgets of dierent nitrogen pools in dierent growth phases of the cultures. In all species, large variations occurred in the
distribution of PN and non-protein N (NPN) in the treatments tested and in dierent growth phases. Intracellular inorganic
nitrogen (NO3 7 , NO2 7 and NH3 + NH4 + ) was the most important NPN component (0.4 30.4% of TN) in all species,
followed by nucleic acids (0.3 12.2% of TN), and chlorophylls (0.1 1.8% of TN). The relative importance of NPN was
greater in the exponential phase, decreasing during growth. PN ranged from 59.3 to 96.8% of TN. N-Prot factors are
proposed for each of the species studied, based on the ratio of amino acid residues to TN, with values ranging from 2.53 to
5.77. Based on current results and on the previous study, we establish an overall average N-Prot factor for all species,
treatments and growth phases of 4.78 + 0.62 (n = 354). This study conrms that the use of the traditional factor 6.25 is
unsuitable for marine microalgae, and the use of the N-Prot factors proposed here is recommended.
Key words: amino acids, carbon, chlorophyll, intracellular inorganic nitrogen, marine microalgae, nitrogen-to-protein
conversion factors, nucleic acids, nitrogen, protein

Introduction
Data on the protein contents of marine microalgae are needed for a wide range of applications, such as for biochemical and physiological
research on cultured species and for animal
nutrition in aquaculture (Lourenco et al.,
2002b). Despite the importance of protein data
in phycology, there are still signicant weakCorrespondence to: S. O. Lourenco. Fax: +55 21 2717 2041.
e-mail: solourenco@yahoo.com
*Present address: Departamento de Botanica, Facultad de
Ciencias Naturales y Oceanograf a, Universidad de Concepcion,
Casilla 160-C, Concepcion, Chile.
{The authors regret to report that Dr Elizabeth Aidar died on
11 September 2000.

nesses in the basic knowledge of protein

analysis in marine microalgae.
Extraction is one of the main problems of
protein analysis in microalgae, which is performed with variable eciency by dierent
methods. Dierences in cell wall composition
of microalgae and in procedures for protein
extraction have a remarkable inuence on the
nal results (Fleurence, 1999). Moreover, the
methods most commonly used for protein
determination in microalgae (methods of Lowry
et al., 1951 and Bradford, 1976) are subject to
interference from many factors (Peterson, 1983;
Stoscheck, 1990), which are independent of the
problems related to the protein extraction. In

ISSN 0967-0262 print/ISSN 1469-4433 online # 2004 British Phycological Society

DOI: 10.1080/0967026032000157156

Downloaded by [77.162.204.181] at 01:56 03 January 2013

S. O. Lourenco et al.
addition, the amino acid composition of each
species is a key factor in interpreting the results
obtained with dierent methodologies, because
of the distinct reactivity obtained with dierent
amino acids (Lourenco et al., 2002a). For
instance, in Bradfords method, the Coomassie
brilliant blue dye G-250 binds disproportionately
with basic and aromatic amino acids, such as
arginine and phenylalanine (Compton & Jones,
1985). As a consequence, samples of arginineand/or phenylalanine-rich microalgae could have
high and incorrect values for protein if quantied by Bradfords method. The same type of
limitation occurs for Lowrys method; the Cu2 +
ion present in the reagent is overly sensitive to
some amino acids such as tryptophan and
tyrosine (Legler et al., 1985).
By contrast, total nitrogen (TN) is relatively
simple to measure, and nitrogen-to-protein conversion factors (N-Prot factors) can then be
used to determine crude protein content. TN
analysis, carried out by Kjeldahls method
(AOAC, 1990) or Hach techniques (Hach et
al., 1987), is fast and inexpensive (Watkins et
al., 1987). Data for TN from CHN elemental
analysis can also be directly converted to crude
protein by the use of conversion factors. The
use of N-Prot factors allows better comparisons
of protein data among species, as well as more
practical comparisons of results obtained by
dierent authors. By the use of conversion
factors, protein is estimated without a demanding previous extraction (Fleurence et al., 1995),
and possible losses of protein are avoided
during the preparation of the samples.
The major problem involved with this methodology is the establishment of specic N-Prot
factors for each species, since the conventional
factor used to calculate crude protein (6.25) is
unsuitable for several materials (Sosulski &
Imadon, 1990). The use of the factor 6.25
(Jones, 1931) is based on the assumption that
the samples contain protein with 16% nitrogen
and a negligible concentration of non-proteinaceous nitrogen (NPN). Nevertheless, plant materials and seaweeds normally have large contents of
NPN (Conklin-Brittain et al., 1999; Levey et al.,
2000; Lourenco et al., 2002a), and commonly
deviate from a N-content of 16% in total protein
(Mosse, 1990; Yeoh & Truong, 1996b). The same
concerns apply to nitrogen distribution and
amino acid composition of marine microalgae
(Lourenco et al., 1998).
Because of the wide applications of data on
nitrogen and protein composition, the calculation
of specic nitrogen-to-protein conversion factors
is important to many elds of science. Plant
materials and fungi have been especially studied

18
in the last few years. Average N-Prot factors of
5.51, 3.59, 5.64 and 3.24 were proposed for apple
ower buds (Khanizadeh et al., 1992), sweet
potato (Yeoh & Truong, 1996a), wild fruits from
southeastern USA (Levey et al., 2000) and
cassava roots (Yeoh & Truong, 1996b), respectively. N-Prot factors calculated by Wu et al.
(1995) for processed kidney beans ranged from
5.63 to 5.67. Danell & Eaker (1992) proposed a
N-Prot factor of 4.38 for the mushroom Cantharellus cibarius, while Mattila et al. (2002) established an average N-Prot factor of 4.70 for four
mushrooms cultivated in Finland. Aitken et al.
(1991) determined a factor of 5.00 for two species
of the edible red alga Porphyra from New
Zealand, in a study focussing on the seasonal
variations in amino acid composition. Lourenco
et al. (2002a) calculated an average factor of 4.92
for nineteen species of tropical seaweeds (nine
red, six green and four brown algae) from Brazil.
All these studies derived N-Prot factors lower
than 6.25, and some of them detected the
presence of high concentrations of NPN in the
species studied by nitrogen budgets.
Marine microalgae may also accumulate high
concentrations of NPN, such as inorganic
nitrogen (Dortch et al., 1984) and nucleic acids
(Machado et al., 1999). Lourenco et al. (1998)
quantied the nitrogen distribution in amino
acids, chlorophylls (a, b and c), RNA, DNA,
and inorganic intracellular ions (nitrate, nitrite
and ammonia), and published the rst paper in
which specic N-Prot factors were proposed for
ten marine microalgae. In that study an overall
mean N-Prot factor of 4.58 was established. All
species were cultured using Conway culture
medium (Walne, 1966), a nitrogen-rich medium
commonly used in aquaculture, without aeration.
The authors suggested that the experimental
conditions adopted could have stimulated the
accumulation of large amounts of non-proteinaceous nitrogen, and recommended further studies
to establish N-Prot factors, which could be
applied to species cultured in other growth
conditions.
In the present study we have quantied the
budgets for protein and the main NPN compounds, showing the nitrogen distribution in cells
of twelve species of marine microalgae. Ten species
were cultured under two growth conditions, and
two species were cultured under three dierent
conditions. The results are discussed in relation to
the availability of carbon and nitrogen, major
points in the experimental design. We propose new
conversion factors from total nitrogen to crude
protein for each species in each treatment and
growth phase, as well as average conversion
factors.

19

Intracellular nitrogen in marine microalgae

Materials and methods

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The microalgae tested

All strains were obtained from the Microalgae Culture
Collection of the Department of Marine Biology,
Federal Fluminense University, Brazil. The following
species were studied: Amphidinium carterae (Dinophyceae, Gymnodiniales; strain NO1), Chlorella minutissima
(Chlorophyceae, Chlorococcales; strain CN1), Dunaliella
tertiolecta (Chlorophyceae, Volvocales; strain FR1),
Hillea sp. (Cryptophyceae, Cryptomonadales; strain
PB1), Isochrysis galbana (Prymnesiophyceae, Isochrysidales; strain TH1), Nannochloropsis oculata (Eustigmatophyceae,
Eustigmatales;
strain
KGH1),
Phaeodactylum tricornutum (Bacillariophyceae, Bacillariales; strain UB7), Prorocentrum minimum (Dinophyceae, Prorocentrales; strain CN3), Skeletonema costatum
(Bacillariophyceae, Biddulphiales; strain CF1), Synechococcus subsalsus (Cyanophyceae, Chroococcales; strain
UB2), Tetraselmis gracilis (Prasinophyceae, Tetraselmidales; strain CN1), and Thalassiosira oceanica (Bacillariophyceae, Biddulphiales; strain ST1). All strains were
isolated from Brazilian coastal waters except for four
obtained from foreign institutions: Amphidinium carterae
(University of Oslo, Norway), Dunaliella tertiolecta
(Universite DAix-Marseille II, France), Isochrysis galbana, and Nannochloropsis oculata (both from Kagoshima Oika Fisheries Research Center, Japan).
Culture conditions
Starter cultures of 10 50 ml in mid-exponential growth
phase were inoculated into 2 l of seawater, previously
autoclaved at 1218C for 30 min in 3 l borosilicate asks.
Each species was cultured in two experimental conditions:
(a) seawater enriched with Conway nutrient solution
(Walne, 1966) in its original concentrations

(N = 1178 mM) and bubbled with ltered air at

2.0 l min 7 1;
(b) seawater enriched with 20% of the original
concentration of nitrogen of Walnes medium
(235 mM) and no aeration.
In addition, Amphidinium carterae and Thalassiosira
oceanica were cultured with the original Walnes medium
without aeration, as in our previous study (Lourenco et
al., 1998), but in which these species had not been tested.
Each experiment was carried out in four culture asks
(n = 4), exposed to 300 mmol photons m 7 2 s 7 1 (measured with a Biospherical Instruments quantum meter
QLS100), provided from beneath by uorescent lamps
(Sylvania daylight tubes), on a 12 : 12 h light : dark cycle.
Mean temperatures during experiments were 23 + 28C in
the light and 20 + 18C in the dark. Salinity in the
experiments was 32.0%. Growth rates were calculated
daily by direct microscopic cell counting with AgasseLafont, Fuchs-Rosenthal, Malassez or Thoma chambers. Cultures were not buered and pH was determined
daily. All sampling for cell counts and pH measurements
occurred during the rst 10 min of the light period.
Sampling procedure
Each culture was sampled four times for both chemical
and biochemical analysis, in dierent growth phases:
mid-exponential, late-exponential, early stationary and
late-stationary growth phases (Fig. 1), except for
Amphidinium carterae and Thalassiosira oceanica, which
were sampled only once in the stationary growth phase.
Samples of 300 to 400 ml were concentrated by
centrifugation at 7000 g and 158C for 10 min, at least
once, to obtain highly concentrated pellets. Before the
last centrifugation, cells were washed in articial seawater (Kester et al., 1967) prepared without nitrogen,
phosphorus and vitamins, and adjusted to 15% salinity.
All supernatants obtained for each sample were combined and the number of cells was determined to

Fig. 1. Growth curves for Amphidinium carterae, Nannochloropsis oculata, Phaeodactylum tricornutum, and Tetraselmis gracilis
in dierent treatments, based on cell counts. Each point represents the mean of four replicates + SD. Arrows indicate the
times of sampling for chemical and biochemical analysis. Treatments: Original represents cultures grown in Walnes
medium without aeration; Air represents cultures grown in Walnes medium with aeration (2.0 l air min 7 1); N/5
indicates cultures grown with 20% of the original concentration of nitrogen of Walnes medium and no aeration.

S. O. Lourenco et al.
quantify possible cell losses. The pellets were frozen at
7 208C and then freeze dried, weighed and stored in
desiccators under vacuum and protected from light until
analysed for CHN elemental composition and total
amino acids. Samples to be analysed for DNA, RNA,
intracellular inorganic nitrogen and chlorophylls were
obtained by ltering the cultures under vacuum onto
Whatman GF/F1 glass microbre lters (0.7 mm nominal pore size), previously exposed to a temperature of
4008C for 4 h in a mue furnace. Samples for both
chlorophyll and intracellular inorganic nitrogen assays
were kept at 7 208C in asks containing silica-gel until
analysis, whereas those for nucleic acid determination
were stored in liquid nitrogen. All sampling for chemical
and biochemical analysis was done during the rst
90 min of the light period. For both chemical and
biochemical analysis, samples from three out of the four
experimental asks were analysed (n = 3); the fourth
sample was kept as a reserve.

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Biochemical analysis
Chlorophyll was extracted in 90% acetone at 48C for
20 h, after grinding the lters with the samples. Spectrophotometric determination of pigments was carried out
as described by Jerey & Humphrey (1975). Nitrogen
concentrations were obtained by multiplying chlorophyll
a, b and c content by 0.0628, 0.0618 and 0.0916,
respectively, corresponding to the nitrogen content of
these chlorophylls.
Nucleic acids were analysed by reaction with
Thiazole Orange (Aldrich Co.) and Hoechst 332581
(Sigma Co.) stains and measurement in a spectrouorometer (PTI model QM-1; Machado et al., 1999).
Nitrogen concentration in the nucleic acids was
calculated according to Dortch et al. (1983). Assuming
equal proportions of the four major nucleotides, the
nitrogen content of DNA is 16.84% and that of RNA
is 16.12%.
Amino acid analysis was carried out by ion-exchange
chromatography in a Beckman, model 7300, equipped
with an automatic integrator. Samples containing 5.0 mg
of protein were acid hydrolysed with 1.0 ml 6 N HCl in
vacuum-sealed hydrolysis vials at 1108C for 22 h.
Norleucine was added as an internal standard. Tryptophan and cystine + cysteine are completely lost with acid
hydrolysis, while methionine could be destroyed to
varying degrees by this procedure. The following values
for the N content of each amino acid were used to
calculate N from total amino acid analysis: aspartic acid,
0.106; threonine, 0.118; serine, 0.134; glutamic acid,
0.096; proline, 0.123; glycine, 0.188; alanine, 0.158;
valine, 0.120; methionine, 0.095; isoleucine, 0.108;
leucine, 0.108; tyrosine, 0.078; phenylalanine, 0.085;
histidine, 0.271; lysine, 0.193; arginine, 0.322 (Sosulski
& Imadon, 1990). Due to the lack of specic amide-N
determination, the content of ammonia was included in
the calculation of protein-nitrogen retrieval, as it comes
mainly from glutamine and asparagine degradation
during acid hydrolysis (Mosse, 1990; Yeoh & Wee,
1994). The NH3 values presented in Table 1 are already
corrected for the free intracellular ammonia concentrations (part of the inorganic N concentration, see Table
2). The ammonia-N content was calculated by multi-

20
plication of the concentrations determined for ammonia
by 0.824 (NH3 = 82.4% of N).
Chemical analysis
Intracellular inorganic nitrogen (IIN) was measured
spectrophotometrically and represents the sum of the
concentrations of ammonia + ammonium (according
to Aminot & Chaussepied, 1983), nitrate, and nitrite
(according to Parsons et al., 1984) within cells.
Samples were kept frozen at 7 208C until chemical
analysis. The IIN extraction procedure was as described by Lourenco et al. (1998), except that the rst
step of the process was carried out at 48C instead of
158C, in order to minimise bacterial contamination of
the samples.
Total nitrogen was quantied in a CHN elemental
analyser Perkin-Elmer, model 2400. Helium was used as
a carrier gas. Acetanilide (C = 71.09%; N = 10.36%;
H = 6.71%) and/or benzoic acid (C = 68.84%;
H = 4.95%) were used for calibrating the instrument.
Calculation of N-Prot factors
N-Prot factors were determined for each species in
dierent growth phases by the ratio of amino acid
residues to TN of the sample: N-Prot factor = Aa-Res/
TN. Thus, a sample with 16.21 g of amino acid residues
and 3.48 g of total nitrogen for every 100 g (dry weight)
yielded a N-Prot factor of 4.66.
Statistical analysis
The variations of each substance over time (for each
species and treatment) were analysed by one-way
analysis of variance (ANOVA; Zar, 1996) followed,
where applicable, by a Tukeys multiple comparison test.
ANOVA (for Amphidinium carterae and Thalassiosira
oceanica) or Students t-test were used for comparing
each variable in two or more treatments at a xed time of
observation.

Results
The results for four representative species (Amphidinium carterae, Nannochloropsis oculata,
Phaeodactylum tricornutum, and Tetraselmis gracilis) are shown in Figs. 1 6, and for three other
species (Chlorella minutissima, Prorocentrum minimum, and Thalassiosira oceanica) in Tables 2 and
3.
Growth, C : N ratio and TN
All species showed signicantly higher nal yields
when cultured with aeration (Fig. 1). Growth of
Amphidinium carterae and Thalassiosira oceanica
with original Walnes medium and with reduced
initial nitrogen (N/5) were similar, and generated
similar nal yields.

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Amino
acids
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Met
Ile
Leu
Tyr
Phe
His
Lys
Arg
Ammonia
Total

Amphidinium Prorocentrum Synechococcus Chlorella

carterae
minimum
subsalsus
minutissima
8.9 + 0.2
5.2 + 0.2
6.2 + 0.5
13.0 + 0.5
5.2 + 0.6
4.8 + 0.1
7.4 + 0.2
6.6 + 0.5
1.7 + 0.4
4.0 + 0.5
8.6 + 0.4
3.4 + 0.2
5.4 + 0.3
1.9 + 0.2
7.0 + 0.0
5.1 + 0.4
1.0 + 0.3
94.3 + 5.3

9.8 + 0.8
5.7 + 0.3
4.6 + 0.2
15.6 + 0.8
5.7 + 0.2
6.2 + 0.4
7.8 + 0.1
5.8 + 0.0
0.6 + 0.0
4.2 + 0.3
8.4 + 0.5
2.3 + 0.2
4.8 + 0.1
1.8 + 0.2
6.1 + 0.5
6.6 + 0.3
2.0 + 0.4
96.0 + 4.8

10.0 + 0.2
5.7 + 0.1
4.1 + 0.1
14.2 + 0.4
3.8 + 0.1
5.5 + 0.1
8.4 + 0.2
5.7 + 0.1
1.3 + 0.0
5.0 + 0.1
9.2 + 0.2
2.3 + 0.1
5.4 + 0.1
1.9 + 0.0
5.0 + 0.1
5.2 + 0.1
2.0 + 0.0
92.7 + 2.4

10.6 + 0.1
5.6 + 0.1
5.2 + 0.0
13.3 + 0.2
4.6 + 0.2
6.0 + 0.1
7.9 + 0.3
6.2 + 0.1
0.4 + 0.1
4.4 + 0.2
9.2 + 0.3
2.7 + 0.1
5.7 + 0.2
1.8 + 0.0
6.3 + 0.0
5.0 + 0.7
1.5 + 0.1
95.0 + 2.9

Dunaliella
tertiolecta

Tetraselmis
gracilis

Isochrysis
galbana

Hillea sp.

10.1 + 0.2
5.7 + 0.0
4.7 + 0.1
13.4 + 0.7
4.5 + 0.5
6.1 + 0.2
8.2 + 0.2
6.3 + 0.2
1.6 + 0.2
4.5 + 0.0
9.6 + 0.4
2.6 + 0.4
5.9 + 0.0
2.2 + 0.4
5.5 + 0.5
4.8 + 0.1
1.6 + 0.1
95.7 + 4.4

10.4 + 0.2
5.8 + 0.0
4.9 + 0.1
14.2 + 0.1
5.2 + 0.2
6.9 + 0.1
7.8 + 0.2
6.1 + 0.2
0.5 + 0.1
4.1 + 0.2
9.6 + 0.2
2.4 + 0.1
6.4 + 0.0
3.6 + 0.6
7.2 + 0.2
5.4 + 0.4
1.4 + 0.2
100.6 + 3.2

10.2 + 0.2
5.6 + 0.1
5.2 + 0.1
13.3 + 0.3
4.7 + 0.1
6.0 + 0.1
8.2 + 0.2
6.2 + 0.2
1.0 + 0.0
4.6 + 0.1
9.4 + 0.2
2.0 + 0.1
5.3 + 0.1
4.5 + 0.1
6.5 + 0.2
5.5 + 0.1
1.9 + 0.0
98.2 + 2.2

11.1 + 0.4
6.5 + 0.0
4.8 + 0.2
14.5 + 0.4
4.2 + 0.0
6.0 + 0.3
7.9 + 0.2
6.0 + 0.6
1.6 + 0.3
4.2 + 0.1
8.0 + 0.1
2.7 + 0.0
5.0 + 0.1
3.8 + 0.5
5.3 + 0.1
5.5 + 0.2
1.7 + 0.1
97.1 + 3.6

Nannochloropsis Phaeodactylum Skeletonema

oculata
tricornutum
costatum
9.8 + 0.5
6.2 + 0.2
5.2 + 0.4
13.0 + 0.7
4.8 + 0.2
6.3 + 0.2
7.7 + 0.4
5.9 + 0.2
0.3 + 0.1
4.0 + 0.2
9.2 + 0.2
2.4 + 0.2
5.2 + 0.1
1.9 + 0.1
6.2 + 0.3
5.8 + 0.2
1.5 + 0.0
93.9 + 4.2

11.9 + 0.2
6.4 + 0.0
5.8 + 0.0
15.5 + 0.4
4.6 + 0.2
6.5 + 0.1
8.2 + 0.1
7.0 + 0.2
1.0 + 0.1
5.0 + 0.0
9.0 + 0.1
2.2 + 0.2
6.1 + 0.1
1.5 + 0.2
4.7 + 0.2
4.9 + 0.2
2.2 + 0.1
100.4 + 2.4

11.2 + 0.6
5.8 + 0.4
5.8 + 0.5
13.1 + 0.3
4.4 + 0.1
6.0 + 0.4
6.9 + 0.6
6.0 + 0.5
1.3 + 0.3
4.6 + 0.3
7.9 + 0.6
2.3 + 0.5
6.2 + 0.5
2.1 + 0.5
7.2 + 0.5
5.8 + 0.4
2.3 + 0.3
96.4 + 6.3

Thalassiosira
oceanica

Mean of
species

11.0 + 0.0
5.2 + 0.1
7.3 + 0.1
11.4 + 0.0
4.6 + 0.1
6.2 + 0.0
7.6 + 0.2
6.1 + 0.0
1.6 + 0.2
4.7 + 0.1
8.9 + 0.1
4.0 + 0.2
6.5 + 0.0
2.4 + 0.1
6.6 + 0.1
5.0 + 0.3
1.2 + 0.2
99.0 + 1.8

10.4
5.8
5.3
13.7
4.7
6.0
7.8
6.1
1.1
4.4
8.9
2.9
5.6
2.6
6.1
5.4
1.7
96.8

Intracellular nitrogen in marine microalgae

Table 1. Total amino acid content of 12 marine microalgae in the stationary growth phase of experiments carried out with aeration. Results are expressed as percentage of total amino
acids measured in 100 g of algal protein and represent the real recovery of amino acids after analysis. Concentrations of ammonia correspond to nitrogen recovery from some amino acids
destroyed during acid hydrolysis. Values are the mean of three replicates + SD

21

S. O. Lourenco et al.

22

Table 2. Distribution of nitrogenous components in Chlorella minutissima, Prorocentrum minimum, and Thalassiosira oceanica
in dierent growth phases and treatments. Results represent mg N in the substances per 100 mg of total N, and are means
of three replicates + SD. Similar analyses were made for the other species
Species/growth
phase
C. minutissima
Mid exponential
Late exponential
Early stationary
Late stationary
P. minimum
Mid exponential
Late exponential
Early stationary

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Late stationary
T. oceanica
Mid exponential

Late exponential

Stationary

Experiment

Amino acid-N

Chlorophyll-N

Nucleic acid-N

Inorganic-N

Total NPN

Air
N/5
Air
N/5
Air
N/5
Air
N/5

63.2 + 1.4
80.7 + 1.5
66.2 + 3.7
86.3 + 3.2
67.5 + 1.8
86.3 + 5.8
72.5 + 4.2
87.3 + 3.9

0.5 + 0.1
0.2 + 0.0
1.8 + 0.1
0.2 + 0.3
1.7 + 0.3
0.2 + 0.1
0.9 + 0.2
0.3 + 0.0

9.5 + 3.0
2.7 + 0.5
12.2 + 4.6
1.5 + 0.5
9.5 + 4.2
1.0 + 0.2
10.6 + 2.9
1.2 + 0.2

25.3 + 10.9
16.7 + 3.7
21.2 + 3.9
6.9 + 1.1
20.3 + 6.1
14.7 + 5.5
18.4 + 2.1
12.9 + 4.5

35.3 + 14.0
19.6 + 4.3
35.3 + 8.6
8.6 + 1.9
31.5 + 10.6
16.0 + 5.7
29.9 + 4.3
14.5 + 4.7

Air
N/5
Air
N/5
Air
N/5
Air
N/5

77.5 + 7.6
59.3 + 3.5
85.2 + 7.9
86.0 + 7.8
88.6 + 2.7
87.1 + 2.0
89.8 + 11.3
90.8 + 1.7

0.1 + 0.0
0.4 + 0.1
0.1 + 0.0
0.2 + 0.0
0.1 + 0.0
0.1 + 0.0
0.1 + 0.0
0.1 + 0.0

7.7 + 2.5
10.6 + 0.6
5.1 + 0.7
5.0 + 1.3
4.4 + 1.5
3.6 + 0.1
4.1 + 0.4
3.1 + 0.7

8.1 + 1.0
20.3 + 1.7
7.2 + 3.0
9.2 + 2.3
6.5 + 1.5
5.2 + 0.5
8.6 + 2.6
4.5 + 0.3

16.0 + 3.6
31.4 + 2.4
12.4 + 3.7
14.4 + 2.6
11.0 + 3.0
8.9 + 0.6
12.8 + 3.0
7.8 + 1.0

Original
Air
N/5
Original
Air
N/5
Original
Air
N/5

92.2 + 9.4
88.3 + 2.9
89.4 + 3.3
94.0 + 3.5
93.7 + 7.3
92.3 + 2.4
94.4 + 2.1
93.8 + 7.8
91.5 + 1.7

0.1 + 0.0
0.1 + 0.0
0.1 + 0.0
0.1 + 0.0
0.2 + 0.0
0.1 + 0.0
0.1 + 0.0
0.1 + 0.0
0.1 + 0.0

0.7 + 0.0
0.6 + 0.1
0.6 + 0.3
0.7 + 0.2
0.3 + 0.1
0.9 + 0.1
0.8 + 0.1
0.2 + 0.1
0.9 + 0.1

6.5 + 1.3
6.6 + 2.2
6.1 + 2.3
5.7 + 1.2
2.2 + 0.5
5.7 + 2.0
5.9 + 0.2
1.7 + 0.3
5.0 + 0.9

7.3 + 1.4
7.3 + 2.3
6.9 + 2.6
6.5 + 1.4
2.7 + 0.6
6.9 + 2.2
6.8 + 0.3
2.0 + 0.4
6.0 + 1.1

Variations in C : N ratio over time indicate

some common trends for most of the species
(Fig. 2). C : N values tended to be low in the
exponential growth phase, and increased with
time, reaching highest values in the late stationary growth phase, especially in aerated conditions. The lowest C : N ratio was found in
Chlorella minutissima (2.8, N/5), and the highest
in Nannochloropsis oculata (18.3, air). Chlorella
minutissima, Isochrysis galbana, Skeletonema costatum, and Thalassiosira oceanica cultured with N/
5 resembled Nannochloropsis oculata (Fig. 2) in
showing little change in C : N ratio over time (as
did T. oceanica cultured with original medium),
but increasing C : N values during growth in
aerated treatments. Amphidinium carterae showed
similar values for C : N ratio in the dierent
treatments, except in the stationary growth phase,
in which mean values with N/5 were signicantly
higher than in the other treatments (p 5 0.05).
For Phaeodactylum tricornutum (Fig. 2) and
Synechococcus subsalsus, signicant dierences
between the treatments were found only in lateexponential phase (p 5 0.01). Only Tetraselmis
gracilis (Fig. 2) and Dunaliella tertiolecta showed

higher values for C : N ratio in N/5 for at least

two sample times.
All microalgae showed large changes in total
nitrogen during growth (p 4 0.02; Fig. 3). When
cultured with aeration, all species except Thalassiosira oceanica had higher values of N per cell in the
exponential growth phase, which decreased in later
phases of the growth cycle. Some species (Amphidinium carterae, Phaeodactylum tricornutum Fig.
3, and also Chlorella minutissima, Hillea sp., and
Thalassiosira oceanica) showed the same trend
when cultured in N/5, but the N-content of other
microalgae (Nannochloropsis oculata Fig. 3, and
also Dunaliella tertiolecta, Isochrysis galbana, Prorocentrum minimum, and Skeletonema costatum)
increased during growth in N/5 (except Tetraselmis
gracilis Fig. 3, and Synechococcus subsalsus).
When cultured with the original medium, total N
per cell increased over time in Amphidinium
carterae (p 5 0.01; Fig. 3) and decreased in
Thalassiosira oceanica (p 5 0.01). Based on the
data for total N cell 7 1 and the initial N
concentration in the medium, we have calculated
the consumption of N from the medium at the end
of the experiments (data not shown). The uptake of

23

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Intracellular nitrogen in marine microalgae

Fig. 2. Changes in carbon:nitrogen ratio (by atoms) of

Amphidinium carterae, Nannochloropsis oculata,
Phaeodactylum tricornutum, and Tetraselmis gracilis in
dierent treatments. Each point represents the mean of three
replicates + SD. Other details as in Fig. 1.

nitrogen was equivalent to 100% in all aerated

experiments, but reached only 21.3% and 35.5% of
the initial N in cultures of Amphidinium carterae

Fig. 3. Changes in total nitrogen per cell during growth of

Amphidinium carterae, Nannochloropsis oculata,
Phaeodactylum tricornutum, and Tetraselmis gracilis in
dierent treatments. Other details as Fig. 2.

and Thalassiosira oceanica, respectively, with the

original medium. The consumption of N from the
medium varied from 88.1% (Skeletonema costatum) to 99.5% (Amphidinium carterae) in N/5
experiments.

24

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S. O. Lourenco et al.

Fig. 4. Changes in nitrogen contained in chlorophylls during

growth of Amphidinium carterae, Nannochloropsis oculata,
Phaeodactylum tricornutum, and Tetraselmis gracilis in
dierent treatments. Other details as Fig. 2.

Changes in the concentration of non-proteinaceous

nitrogen
Most of the microalgae studied had higher
concentrations of chlorophyll under aeration than

Fig. 5. Changes in nitrogen contained in total nucleic acids

(DNA + RNA) during growth of Amphidinium carterae,
Nannochloropsis oculata, Phaeodactylum tricornutum, and
Tetraselmis gracilis in dierent treatments. Other details as
Fig. 2.

in N/5 experiments (Fig. 4), except Amphidinium

carterae, Hillea sp., Isochrysis galbana, Prorocentrum minimum, and Thalassiosira oceanica, for
which there was no signicant dierence between
the treatments. Hillea sp. was the only species that
showed higher values for chlorophyll cell 7 1 when
cultured with N/5. Most of the species showed

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Intracellular nitrogen in marine microalgae

Fig. 6. Changes in inorganic intracellular nitrogen

(nitrate + nitrite + ammonia/ammonium) during growth of
Amphidinium carterae, Nannochloropsis oculata,
Phaeodactylum tricornutum, and Tetraselmis gracilis in
dierent treatments. Other details as Fig. 2.

higher concentrations of chlorophyll in the exponential phase, which decreased with time
(p 4 0.04), but Chlorella minutissima, Dunaliella
tertiolecta, Prorocentrum minimum, and Skeletonema costatum, showed no variations in chlorophyll

25
per cell with time. Phaeodactylum tricornutum
showed a dierent pattern of variation with air,
with a peak in chlorophyll concentration in early
stationary phase, decreasing in late stationary
phase to concentrations similar to those recorded
in late exponential phase (Fig. 4). Variations in
chlorophyll concentration during growth with N/5
were small for Nannochloropsis oculata, Phaeodactylum tricornutum, and Tetraselmis gracilis. (Fig.
4), and also for Dunaliella tertiolecta, Prorocentrum
minimum and Synechococcus subsalsus.
As with chlorophyll, microalgae tended to show
higher concentrations of nucleic acids (DNA + RNA) in the exponential phase, which decreased
with time (Fig. 5), except for Phaeodactylum
tricornutum with air, Isochrysis galbana, Skeletonema costatum, and Synechococcus subsalsus with N/
5. In these species the concentrations of nucleic
acids either increased in the stationary growth
phase or did not change with time. RNA content
was higher than DNA in almost all samples (data
not shown). In the exponential growth phase,
RNA : DNA ratios varied from 1.5 : 1 (Nannochloropsis oculata, N/5) to 8.0 : 1 (Dunaliella tertiolecta,
air cultures), with an overall mean of 3.7 : 1. In the
stationary phase, however, the RNA : DNA ratio
varied from 0.6 : 1 (Nannochloropsis oculata, air) to
5.4 : 1 (Prorocentrum minimum, air), with an overall
mean of 1.9 : 1.
All species contained intracellular inorganic
nitrogen as nitrate, nitrite and ammonium (data
not shown). However, the ratio among these ions
as well as their respective concentrations varied
considerably during growth. Ammonium + ammonia values were higher than those of nitrate in
cultures of Chlorella minutissima, Phaeodactylum
tricornutum, Synechococcus subsalsus, and Tetraselmis gracilis. All other species had higher
concentrations of nitrate than of ammonium + ammonia. Nitrite concentrations were always
much lower than those of the other inorganic ions.
Total IIN decreased during growth (Fig. 6), with
few exceptions (Tetraselmis gracilis Fig. 6, and
also Dunaliella tertiolecta and Prorocentrum minimum, in air; Dunaliella tertiolecta and Skeletonema
costatum in N/5 cultures). Chlorella minutissima
and Skeletonema costatum showed higher IIN
values in N/5 than in experiments with aeration,
as well as Amphidinium carterae and Thalassiosira
oceanica in both late-exponential and stationary
growth phases. Phaeodactylum tricornutum (Fig. 6)
and Isochrysis galbana showed similar concentrations of IIN in the two treatments, except in the
mid-exponential growth phase, in which the values
for N/5 were higher. Values of IIN were predominantly higher in aerated cultures of Nannochloropsis oculata (Fig. 6), Hillea sp., Prorocentrum
minimum and Synechococcus subsalsus.

S. O. Lourenco et al.

26

Table 3. Calculation of N-Prot factors for Chlorella minutissima, Prorocentrum minimum, and Thalassiosira oceanica, based
on the amino acid residues to total nitrogen ratio. Data are expressed as percentage of dry matter, and represent the means of
three replicates + SD. Similar calculations were made for the other species
Species/growth
phase
C. minutissima
Mid exponential
Late exponential
Early stationary
Late stationary
P. minimum
Mid exponential
Late exponential
Early stationary

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Late stationary
T. oceanica
Mid exponential

Late exponential

Stationary

Experiment

Total N

Total amino acid

Amino acid
residues

Amino acid-N

N-Prot factors

Air
N/5
Air
N/5
Air
N/5
Air
N/5

5.80 + 0.43
1.37 + 0.07
8.17 + 0.27
2.08 + 0.11
7.70 + 0.16
1.38 + 0.11
4.53 + 0.19
2.11 + 0.07

24.79 + 0.57
3.93 + 0.08
36.96 + 2.08
12.21 + 0.45
36.12 + 0.96
8.24 + 0.55
22.46 + 1.29
12.93 + 0.57

21.13 + 0.48
3.47 + 0.07
31.49 + 1.77
10.50 + 0.39
30.80 + 0.82
7.09 + 0.47
19.25 + 1.11
11.12 + 0.49

3.66 + 0.08
1.11 + 0.02
5.41 + 0.30
1.79 + 0.07
5.20 + 0.14
1.19 + 0.08
3.28 + 0.19
1.84 + 0.08

3.64 + 0.36
2.53 + 0.18
3.85 + 0.35
5.05 + 0.46
4.00 + 0.19
5.13 + 0.57
4.25 + 0.42
5.27 + 0.41

Air
N/5
Air
N/5
Air
N/5
Air
N/5

4.67 + 0.29
1.48 + 0.07
5.55 + 0.26
2.18 + 0.11
4.83 + 0.14
2.34 + 0.07
4.44 + 0.10
1.86 + 0.01

24.18 + 2.38
5.36 + 0.32
30.40 + 2.82
10.19 + 0.93
27.44 + 0.85
12.87 + 0.29
26.25 + 3.30
12.90 + 0.33

20.57 + 2.03
4.60 + 0.27
25.97 + 2.41
18.71 + 0.79
23.45 + 0.73
11.04 + 0.25
22.45 + 2.81
9.77 + 0.89

3.62 + 0.36
0.88 + 0.05
4.73 + 0.44
1.60 + 0.15
4.28 + 0.13
2.04 + 0.05
3.99 + 0.50
1.79 + 0.16

4.41 + 0.50
3.11 + 0.33
4.68 + 0.47
4.68 + 0.46
4.86 + 0.29
4.72 + 0.25
5.05 + 0.55
5.25 + 0.52

Original
Air
N/5
Original
Air
N/5
Original
Air
N/5

1.65 + 0.14
2.44 + 0.06
1.96 + 0.06
1.80 + 0.07
4.42 + 0.07
2.10 + 0.07
1.99 + 0.07
3.57 + 0.06
2.85 + 0.03

9.91 + 1.01
14.45 + 0.47
11.77 + 0.44
11.40 + 0.43
29.10 + 2.86
12.71 + 0.33
13.00 + 0.28
23.22 + 1.92
19.27 + 0.35

8.56 + 0.88
12.44 + 0.40
10.13 + 0.38
9.72 + 0.36
24.86 + 1.93
10.81 + 0.28
11.09 + 0.24
19.45 + 1.65
16.43 + 0.30

1.52 + 0.16
2.15 + 0.07
1.75 + 0.06
1.69 + 0.06
4.14 + 0.32
1.94 + 0.05
1.88 + 0.04
3.35 + 0.28
2.61 + 0.05

5.19 + 0.38
5.10 + 0.28
5.17 + 0.34
5.40 + 0.41
5.62 + 0.53
5.15 + 0.31
5.58 + 0.32
5.59 + 0.45
5.77 + 0.16

Total amino acid, nitrogen budgets and the

calculation of N-Prot factors
Data on total amino acids are presented for the late
stationary growth phase of aerated cultures only
(Table 1). Glutamic acid was the most abundant
amino acid in all species, followed by aspartic acid.
Histidine and methionine were identied as minor
amino acids in these species (concentrations of
tryptophan and cysteine + cystine not taken into
account), and only Isochrysis galbana showed high
concentrations of histidine (4.5% of the total
amino acids) compared to the other species.
Percentages of alanine, leucine, isoleucine and
threonine were similar in all species. The concentration of arginine was higher in Prorocentrum
minimum and that of serine was higher in Thalassiosira oceanica. Amphidinium carterae, Skeletonema costatum, and Tetraselmis gracilis showed high
concentrations of lysine, while the highest percentages of tyrosine, proline and valine were found in
Isochrysis galbana, Prorocentrum minimum and
Phaeodactylum tricornutum, respectively. Higher
concentrations of phenylalanine were found in the
three diatoms and Tetraselmis gracilis. Mean values
of each amino acid over all the microalgae tested
are also shown in Table 1.

The amino acid-N as a percentage of TN

increased from the exponential to the stationary
growth phase (Table 2). Values ranged from 59.3
(Prorocentrum minimum, N/5) to 96.8% (Nannochloropsis oculata, N/5) of the TN. Total NPN
varied from 0.8 (Synechococcus subsalsus, N/5)
up to 39.0% (Nannochloropsis oculata, N/5) of
the TN. IIN was the main component of NPN
in almost all species in all treatments. Nucleic
acid-N represented the second most important
component of NPN, while the contribution of
chlorophyll to TN was always low (less than 1%
of TN, in most samples).
The actual protein concentration of the samples was calculated by summing the masses of
the amino acid retrieved after acid hydrolysis
(total amino acid), minus the mass of water (18 g
H2O per mol of amino acid). The water is
incorporated into each amino acid after the
disruption of the peptide bonds (Mosse, 1990;
Lourenco et al., 1998), and it is not present in
peptides and proteins. Consequently, the protein
content of the samples is indicated as mg of total
amino acid residues (Table 3). The microalgal
species showed a wide range of protein concentration, varying from 3.5 (Chlorella minutissima,

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Growth phases*
Taxonomic
groups/species
Cryptophyceae
Hillea sp.
Cyanobacteria
Synechococcus subsalsus
Diatoms
Phaeodactylum tricornutum
Skeletonema costatum
Thalassiosira oceanica
Dinoagellates
Amphidinium carterae
Prorocentrum minimum
Eustigmatophyceae
Nannochloropsis oculata
Green algae
Chlorella minutissima
Dunaliella tertiolecta
Tetraselmis gracilis
Prymnesiophyceae
Isochrysis galbana
Mean values for each growth phase

Mid-exponential

Late-exponential

Early stationary

Late stationary

Means across all

growth phases

Overall means from

Lourenco et al. (1998)

Overall mean values for

each species**

4.28a

4.57ab

4.93b

5.01b

4.74 (n = 24)

4.61 (n = 6)

4.72 (n = 30)

4.96a

5.16a

5.30a

5.57b

5.43 (n = 24)

4.66 (n = 9)

5.22 (n = 33)

4.46a
4.40a
5.19a

4.86b
4.55a
5.39b

4.89b
4.53a
5.65c

5.48c
5.10b

4.93 (n = 24)
4.73 (n = 24)
5.40 (n = 27)

4.72 (n = 9)
3.82 (n = 3)

4.87 (n = 33)
4.63 (n = 27)
5.40 (n = 27)

4.80a
3.76a

5.21b
4.34ab

5.39c
4.79b

4.73b

5.13 (n = 27)
4.60 (n = 24)

3.77 (n = 6)

5.13 (n = 27)
4.43 (n = 30)

4.00ab

4.95a

5.17b

5.59c

4.98 (n = 24)

4.87 (n = 6)

4.95 (n = 30)

3.09a
3.64a
4.37a

4.45b
4.39b
4.75ab

4.57b
4.39b
5.10b

4.67b
4.91b
5.08b

4.22 (n = 24)
4.38 (n = 24)
4.96 (n = 24)

4.50 (n = 3)
3.99 (n = 3)
4.37 (n = 9)

4.25 (n = 27)
4.34 (n = 27)
4.80 (n = 33)

3.99a

4.31a

5.07b

4.95b

4.74 (n = 24)

3.99 (n = 6)

4.59 (n = 30)

4.33a (n = 87)

4.77b (n = 99)

4.99bc (n = 93)

5.06c (n = 75)

4.86 (n = 294)

4.58 (n = 60)

4.78 (n = 354)

Intracellular nitrogen in marine microalgae

Table 4. Summary of mean N-Prot factors for 12 marine microalgae, based on the amino acid residues to total nitrogen ratio. N-Prot factors in columns 2 5 are means of three to nine
replicates. Results obtained in our previous study (Lourenco et al., 1998; col. 7) are included in the nal calculations (col. 8)

*Within each row for the growth phases (columns 2 5), values with the same superscript letters are not significantly different at p = 0.05 (Tukeys test).
**The last column combines the new data and those calculated by Lourenco et al. (1998) for each species.

27

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S. O. Lourenco et al.
N/5) to 34.0% of the dry matter (Phaeodactylum
tricornutum, air). Percentages of protein were
usually higher in aerated cultures than in the
other treatments. The mass of N within amino
acids ranged from 0.87 (Prorocentrum minimum,
mid-exponential phase, N/5) to 5.68% (Phaeodactylum tricornutum, late-exponential, air) of the
dry matter. The N-Prot factors for the microalgae were calculated from the ratio of the mass
of amino acid residues to total nitrogen, both
expressed as mg contained in 100 mg of samples
(Table 3). N-Prot factors ranged 2.53 (Chlorella
minutissima, mid-exponential, N/5) to 5.77 (Thalassiosira oceanica, stationary, N/5). For all
species, N-Prot factors were lower in midexponential
growth
(average = 4.33 + 0.72,
n = 78) and higher in late stationary phase
(average = 5.23 + 0.40, n = 60). An overall average N-Prot factor of 4.86 + 0.63 (n = 294) was
calculated from the data for all species, in
dierent growth phases and treatments. Compared with our previous results (Lourenco et al.,
1998), the new mean N-Prot factors for the
species and growth phases under dierent experiments were signicantly dierent, with mean
values for N/5 (4.92) higher than those for
Original (4.59) and Air (4.75) (F2,350 = 7.38,
p 5 0.01). Dierences between the mean values
for Original and for Air experiments were not
signicant. An overall average N-Prot factor of
4.78 was calculated combining data from both
studies, and mean values of 4.33, 4.77, 4.99, and
5.06 were calculated for mid-exponential, late
exponential, early stationary, and late stationary
growth phases, respectively (Table 4). Signicant
dierences were found among the mean N-Prot
factors calculated for the dierent growth phases
(F3,350 = 30.0, p 5 0.001). Mean values calculated
for the mid-exponential growth phase were lower
than those for the other growth phases
(p 5 0.01). No dierence was found between
mean values calculated for early and late
stationary growth phases (p = 0.61). The mean
N-Prot factors calculated for taxonomically
related species (e.g. diatoms) in all treatments
and growth phases were also compared. The
mean N-Prot factors calculated for the three
diatoms were signicantly dierent (ANOVA +
Tukey, p 5 0.05), with higher N-Prot factors for
T. oceanica; values for P. tricornutm and S.
costatum were not signicantly dierent. The NProt factor calculated for the dinoagellate A.
carterae was higher than that for P. minimum (ttest, p 5 0.001). For the green algae, the mean
N-Prot factors of C. minutissima and D. tertiolecta were not signicantly dierent (p = 0.65),
but both were signicantly lower than that of T.
gracilis (p 5 0.01).

28
Discussion
Eects of the treatments on growth, C : N ratio and
TN in the microalgae
Growth responses of all species showed large
dierences between nal cell yields obtained in N/
5 and aerated experiments. All species showed
higher cell densities in the stationary phase when
cultured with aeration. Two factors are inuencing
the trends observed: an eect of nitrogen as a
limiting factor in N/5 experiments, and the
inuence of the input of carbon in experiments
with aeration. CO2 occurs in low concentrations in
the air (close to 0.04% v/v), but the use of
continuous aeration at 2.0 l air min 7 1 provides a
signicant input of carbon into the cultures, as
demonstrated by Fabregas et al. (1995). N uptake
and assimilation is faster when carbon is not
limiting, and growth is stimulated (Huertas et al.,
2000). On the other hand, dierences in nal cell
yield were small between experiments with the
original culture medium and N/5 for Amphidinium
carterae and Thalassiosira oceanica. Based on our
previous results (Lourenco et al., 1998) and on the
current data we can assume that N was in excess in
the original culture medium. Nevertheless, our data
from N/5 experiments show that the uptake of N
from the medium was almost complete (data not
presented), with possible N-limitation in the
stationary growth phase. We assume that the lack
of a C source may have limited growth for the
microalgae in a similar way in both original and N/
5 experiments, generating a lower nal yield
compared to aerated experiments.
According to Burkhardt et al. (1999), minor
variations in C : N ratio occur when growth rates
are constant. Our results indicate that all aerated
cultures were limited by nitrogen in stationary
phase and showed wide C : N variations during
growth. In these cultures, a constant input of
carbon was obtained by bubbling with air. Considering the coupling of C and N metabolism, the
availability of carbon should make the assimilation
of nitrogen faster, supplying cells with carbon for
amino acid synthesis (Turpin, 1991). The faster
assimilation of N, associated with the greater
availability of C, is probably the main factor
determining the higher nal cell yield in aerated
cultures (Fig. 1). Cultures under N/5 had lower
growth rates and probably were carbon-limited in
the stationary growth phase, since the diusion of
CO2 from the air to the cultures occurs at very low
rates, which were not enough to sustain a rapid
growth. With low growth rates, the consumption of
nutrients tends to be slow, and the chemical
composition of the microalgae under those treatments may have changed slightly.

29

Intracellular nitrogen in marine microalgae

Total N in cells during growth showed a
decreasing trend with few exceptions. This general
trend seems to be related to the progressively lower
availability of N in the culture medium. Some
species may display a decrease in cell volume over
growth, as an eect of either unsuitable carbon or
nitrogen supply (Burkhardt et al., 1999). This
hypothesis has support in our data from aerated
experiments, in which no carbon limitation was
recorded, and all species except Thalassiosira
oceanica showed a remarkable trend of decreasing
total N.
Variations in the concentrations of nonproteinaceous substances over growth

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71

The decrease of chlorophyll-N cell

during
growth was shown by almost all species and
treatments, and may be a consequence of the
decrease in nutrient availability in the culture
medium, and possibly the decrease in cell volumes.
Lourenco et al. (1997) reported a signicant
decrease in chlorophyll concentration in Tetraselmis gracilis cells during growth in aerated cultures,
and Huertas et al. (2000) did the same for
Nannochloropsis gaditana and Nannochloris maculata. In our experimental design, cultures were kept
under saturating irradiance, and self-shading is
unlikely to have occurred.
The very substantial decrease in nucleic acids per
cell is in accordance with ndings of other authors
(Berdalet et al., 1992; Machado et al., 1999).
Chemical changes in batch cultures establish
progressively less favourable conditions for growth;
consequently, levels of nucleic acids, mainly RNA,
tend to decrease in cells. Our results t this trend,
since we recorded higher values for RNA : DNA
ratio in the exponential phase and lower ratios in
the stationary phase of all cultures.
The variations in intracellular inorganic nitrogen
concentrations during growth were similar to those
reported in our previous study (Lourenco et al.,
1998). Almost all species showed higher values for
IIN in the exponential growth phase, which
decreased during growth in all treatments. The
high concentrations of IIN in the exponential phase
reect the rapid N uptake in the rst days of
growth, when no factor is limiting (Lav n &
Lourenco, unpublished data). However, the assimilatory process may be limited by the activity of
enzymes, resulting in an accumulation of high
concentrations of inorganic N. High concentrations of nitrite may be built up, and excretion of
this ion may occur, preventing its toxic eects
(Lourenco et al., 1997). The decrease of IIN
concentrations seems to be the result of the
assimilatory process, since the ratio of PN/TN
increased from exponential to stationary phases.

The IIN pool was consumed when N availability

became smaller, conrming its physiological role as
a nitrogen reserve (Dortch et al., 1984).
Our data show that the relative importance of
sources of non-proteinaceous nitrogen tends to
become progressively smaller during growth. An
increase in the ratio of protein-N to TN occurs
during growth, as demonstrated in our N budgets
for all species in almost all cultures. IIN was the
most important NPN component, followed by
nucleic acids and chlorophyll. Chlorophyll has a
minor importance in the N budgets, representing
less than 1% of TN in most cases.
Nitrogen budgets and the calculation of N-Prot
factors
The percentages of nitrogen and protein were
relatively low in some samples, mainly those from
exponential growth phases of N/5 experiments
(Table 3). In most cases this resulted from the
inuence of residual salts from articial seawater
(without N) used for washing the samples at the
end of the centrifugation. In mid-exponential
phase, cultures normally showed low cell densities,
and the pellets generated by centrifugation tended
not to aggregate well. Conversely, the centrifugation of high-density cultures, such as those
obtained with aeration and in stationary phase of
N/5 experiments, was easier and the inuence of
residual salts from the medium was negligible.
Without the inuence of residual salts, higher
values for both N and protein content could have
been obtained. The use of freeze-dried samples is
essential for the method of amino acid analysis
adopted in this study, since 5 mg of protein are
required to perform the acid hydrolysis. This
amount of protein would not be easily obtained
on lters, and we had to centrifuge samples for
amino acid analysis. Nevertheless, residual salts
have no inuence on the calculation of N-Prot
factors, which is based on the Aa-Res to TN ratio,
independently of the mass of the other components
in the samples. For both determinations, samples
were taken from freeze-dried materials. Nonproteinaceous substances analysed in this study
(chlorophyll, IIN, and nucleic acids) were sampled
on glass bre lters, and were not inuenced by
residual salts from the culture medium.
As with the variations of PN to TN ratio, the NProt factors were lower in exponential growth
phase, attaining maximum values in the stationary
phase. Since the relative importance of protein-N
increases during growth, N-Prot factors tended to
be higher from mid-exponential to late stationary
phase. The concentration of protein per cell
decreases during growth, while percentages of
protein in dry matter show dierent trends. In

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S. O. Lourenco et al.
aerated experiments, and in most of N/5 cultures,
the percentages of protein were highest in lateexponential or early stationary phase and decreased in the late stationary phase. In some N/5
experiments (Nannochloropsis oculata and Thalassiosira oceanica) and in cultures with the original
medium (Amphidinium carterae and Thalassiosira
oceanica), the percentages of protein increased
during growth, and maximum values were obtained
in the late exponential phase. This trend seems to
be due to the lack of carbon, since these same
cultures showed no variations in C : N ratio over
growth (see discussion in the rst subsection).
Nevertheless, the contribution of PN to total
nitrogen increases during growth (Table 2).
Quantitative analysis of total amino acids is of
fundamental importance for the determination of
the actual protein content (Salo-Vaananen &
Koivistoinen, 1996). Thus, the calculation of
specic N-Prot factors is dependent on amino
acid analysis, since its determination depends on
the amount of nitrogen recovered from the amino
acids after acid hydrolysis (Mattila et al., 2002).
Excluding the inuence of NPN, samples in which
total amino acid-N represent 16% of protein (in
mass) would have a N-Prot factor = 6.25 ( = 100/
16); for instance, samples with 15.5% and 19%
total amino acid-N would receive 6.45 and 5.26 as
N-Prot factors, respectively. N-Prot conversion
factors calculated for materials and species that
possess proteins rich in highly nitrogenous amino
acids (e.g. histidine, arginine) tend to be low.
Conversely, samples with a large content of
nitrogen-poor amino acids (e.g. phenylalanine,
tyrosine) tend to yield higher N-Prot factors.
However, plants (Conklin-Brittain et al., 1999),
fungi (Fujihara et al., 1995) and algae (Lourenco
et al., 1998, 2002a) tend to show high concentrations of non-proteinaceous nitrogen. As the
determination of TN does not enable one to
identify and distinguish protein-nitrogen from
NPN fractions, uctuations of NPN content have
a remarkable inuence on the calculation of NProt factors. In NPN-rich species the inuence of
NPN on the calculation of N-Prot factors may be
even more important than that of the total amino
acid prole (Fujihara et al., 1995). Since the
1990s, several studies have adopted the construction of nitrogen budgets to validate N-Prot
factors. The quantication of the main nitrogenous substances within cells by dierent and
independent methods allows the relationships
among values to be established. Lourenco et al.
(1998) determined the concentrations of NPN in
10 microalgae, which ranged from 9.2 to 47.0% of
the TN, and an overall mean N-Prot factor for
the microalgae tested was 4.58 (n = 60). Fujihara
et al. (2001) analysed 20 Japanese vegetables and

30
found an average of 27% of NPN in the samples,
and an average N-Prot factor of 4.39.
The calculation of N-Prot factors based on the
ratio of amino acid residues to TN may be
inuenced by the presence of free amino acids in
the samples, because total amino acid analysis does
not distinguish between free amino acids and those
combined into protein. The extent to which free
amino acids will inuence the overall estimation of
protein content is likely to vary from species to
species (Ezeagu et al., 2002), but the inuence of
free amino acids is normally low. Free amino acids
typically represent less than 10% of the total amino
acids in microalgae (Dortch et al., 1984) and up to
5% of the total amino acids in grasses (Yeoh &
Watson, 1982). Moreover, it is necessary to
consider that some amino acids (e.g. tryptophan,
cystine, glutamine, methionine, serine) are partially
or totally destroyed in an acid hydrolysis. Studying
the edible mushroom Cantharellus cibarius, Danell
and Eaker (1992) found that ca. 9% of the
ninhydrin-detectable N in amino acid analysis by
ion-exchange chromatography is ammonia, and
most of the ammonia results from amino acid
destruction in acid hydrolysis. Not all amino acid
losses during the hydrolysis are transformed into
ammonia and detected in the amino acid analysis.
Ezeagu et al. (2002) suggest that side reactions
during hydrolysis may chelate some amino acid-N,
but that some N compounds may be tightly bound
within the cell-wall matrix and might not be
released under HCl hydrolysis conditions. This
means that the typical losses during acid hydrolysis
might compensate for the presence of free amino
acids, at least to some extent. As a consequence, the
inuence of free amino acids on the calculation of
N-Prot factors would be minor. Finally, the
nitrogen budgets from this study (Table 2) clearly
indicate that the lack of data for free amino acids
did not signicantly aect the calculation of N-Prot
factors. Due to all these arguments, the use of total
amino acid data to estimate protein content without determination of free amino acids is a widely
accepted procedure (Mosse, 1990; Yeoh & Truong,
1996b).
Despite common trends in the variation of
proteinaceous-N and non-proteinaceous-N for the
microalgae in aerated and N/5 experiments, the NProt factors calculated for some species showed
large dierences (Chlorella minutissima, Nannochloropsis oculata, Prorocentrum minimum), mainly
in the exponential phase. These may reect
relatively rapid changes in the biochemical composition of microalgae in the exponential growth
phase of batch cultures. Other species (Hillea sp.,
Isochrysis galbana, Tetraselmis gracilis) showed
small to medium dierences in N-Prot values in
the treatments in dierent growth phases. The

31

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Intracellular nitrogen in marine microalgae

other species (e.g. Synechococcus subsalsus and
Thalassiosira oceanica) showed similar N-Prot
factors for each growth phase in the dierent
experimental conditions, with a small dispersion of
values.
Establishing mean N-Prot factors for each
species by combining the results obtained from
dierent growth phases and culture conditions
results in an increased variability of the values.
We recognise that culture conditions aect the
biochemical composition of the microalgae, but it
makes no sense to recommend specic N-Prot
factors for each species in each dierent growth
phase and culture condition. Each species would
receive dozens of N-Prot factors, depending on the
experimental conditions used. For practical reasons, it is necessary to average N-Prot factors
across dierent growth conditions to make the NProt approach more useful. This has been done
successfully for other groups, and the N-Prot
calculated for cereals (Mosse, 1990), leaves (Yeoh
& Wee, 1994), mushrooms (Mattila et al., 2002),
for instance, come from samples obtained in
dierent places, experiments and/or environmental
conditions.

Conclusions
Our results provide information for a large number
of marine microalgae, in dierent growth conditions and growth phases, and permit an assessment
of the variability of the nitrogen distribution in
cells. Some general trends were found to be
independent of the treatments. The relative proportion of PN to TN increases during growth, and
the percentages of NPN are lower in the stationary
growth phase.
The present study establishes that N-Prot factors
are lower than the traditionally used factor of 6.25
for all species, regardless of growth phase and
culture conditions. The contribution of nitrogenous
non-protein substances was accurately identied
and quantied, as shown in the intracellular
nitrogen budgets (Table 2). Possible variations in
the N-Prot factors proposed could be attributed to
minor non-protein substances, which were not
identied and quantied. However, concentrations
of these substances seem to be low, since the sum of
the various forms of nitrogen quantied was always
close to TN.
We suggest the use of the mean N-Prot conversion factors calculated for each species in this study
(Table 4). Some species (e.g. Synechococcus subsalsus, Thalassiosira oceanica) show small dierences in specic values of N-Prot factors calculated
for dierent growth phases and treatments. Other
species (e.g. Chlorella minutissima, Prorocentrum

minimum) show greater variation among the

proposed conversion factors at dierent growth
phases and experiments, owing to a marked
uctuation of NPN in dierent growth conditions.
In spite of this variability, the N-Prot factors
calculated here will be more accurate than the
traditional factor 6.25, which overestimates the
actual protein content. Researchers can choose
either the N-Prot factor for each growth phase or
the average N-Prot factor of each species.
It is virtually impossible to establish specic NProt factors for all species of marine microalgae
currently used in research, biotechnology and
aquacultural purposes. For microalgal species not
yet studied, we recommend the use of the overall
mean N-Prot factor of 4.78 for calculating total
protein concentration from nitrogen content.
Acknowledgements
This work was supported by the Foundation for
Research Support of Rio de Janeiro State (FAPERJ, grant E-26.171.043/99) and State of Sao
Paulo Research Foundation (FAPESP, grant 95/
9022-7) and research fellowships to S.O.L. from
FAPERJ and CNPq (National Council for the
Development of Science and Technology). We
thank Luzia E. Narimatsu, Adriana Nascimento,
and Rosa M.C. Barros (Universidade de Sao
Paulo) for their help with CHN and amino acid
analysis, and Dr. Yocie Yoneshigue-Valentin and
Prof. Ricardo M. Chaloub (Universidade Federal
do Rio de Janeiro) for laboratory facilities. Thanks
are due to Dr. John A. Berges (Queens University
of Belfast, U.K.) and two anonymous referees for
their critical comments on the manuscript.

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