Distribution of intracellular nitrogen in marine microalgae: Calculation of new nitrogen-to-protein conversion factors

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Distribution of intracellular nitrogen in marinemicroalgae: Calculation of new nitrogen-to-proteinconversion factorsSergio O Loureno a , Elisabete Barbarino a , Paris L Lavn a , Ursula M Lanfer Marquez b &Elizabeth Aidar ca Departamento de Biologia Marinha, Universidade Federal Fluminense, Caixa Postal100644, Niteri, RJ, Brazil, CEP 24001-970b Departamento de Alimentos e Nutrio Experimental, Faculdade de CinciasFarmacuticas, Universidade de So Paulo, Caixa Postal 66083, So Paulo, SP, Brazil, CEP05315-970c Departamento de Oceanografia Biolgica, Instituto Oceanogrfico, Universidade de SoPaulo, Caixa Postal 66149, So Paulo, SP, Brazil, CEP 05315-970d Fax: E-mail: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 conversionfactors, European Journal of Phycology, 39:1, 17-32

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Distribution of intracellular nitrogen in marine microalgae:

Calculation of new nitrogen-to-protein conversion factors

SERGIO O. LOURENCO1, ELISABETE BARBARINO1, PARIS L . LAV IN 1* ,

URSULA M. LANFER MARQUEZ2 AND ELIZABETH AIDAR3{

1Departamento de Biologia Marinha, Universidade Federal Fluminense, Caixa Postal 100644, CEP 24001-970, Niteroi, RJ, Brazil2Departamento de Alimentos e Nutricao Experimental, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Caixa

Postal 66083, CEP 05315-970, Sao Paulo, SP, Brazil3Departamento de Oceanograa Biologica, Instituto Oceanograco, Universidade de Sao Paulo, Caixa Postal 66149,CEP 05315-970, Sao Paulo, SP, Brazil

(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 constructingbudgets 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 (NO37 , 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 isunsuitable 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 micro-algae are needed for a wide range of applica-tions, such as for biochemical and physiologicalresearch on cultured species and for animalnutrition in aquaculture (Lourenco et al.,2002b). Despite the importance of protein datain phycology, there are still signicant weak-

nesses in the basic knowledge of proteinanalysis in marine microalgae.Extraction is one of the main problems of

protein analysis in microalgae, which is per-formed with variable eciency by dierentmethods. Dierences in cell wall compositionof microalgae and in procedures for proteinextraction have a remarkable inuence on thenal results (Fleurence, 1999). Moreover, themethods most commonly used for proteindetermination in microalgae (methods of Lowryet al., 1951 and Bradford, 1976) are subject tointerference from many factors (Peterson, 1983;Stoscheck, 1990), which are independent of theproblems related to the protein extraction. In

Correspondence to: S. O. Lourenco. Fax: +55 21 2717 2041.

e-mail: [email protected]

*Present address: Departamento de Botanica, Facultad de

Ciencias Naturales y Oceanografa, Universidad de Concepcion,

Casilla 160-C, Concepcion, Chile.

{The authors regret to report that Dr Elizabeth Aidar died on11 September 2000.

Eur. J. Phycol. (2004), 39(1): 17 32.

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

DOI: 10.1080/0967026032000157156

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addition, the amino acid composition of eachspecies is a key factor in interpreting the resultsobtained with dierent methodologies, becauseof the distinct reactivity obtained with dierentamino acids (Lourenco et al., 2002a). Forinstance, in Bradfords method, the Coomassiebrilliant blue dye G-250 binds disproportionatelywith basic and aromatic amino acids, such asarginine and phenylalanine (Compton & Jones,1985). As a consequence, samples of arginine-and/or phenylalanine-rich microalgae could havehigh and incorrect values for protein if quanti-ed by Bradfords method. The same type oflimitation occurs for Lowrys method; the Cu2+

ion present in the reagent is overly sensitive tosome amino acids such as tryptophan andtyrosine (Legler et al., 1985).By contrast, total nitrogen (TN) is relatively

simple to measure, and nitrogen-to-protein con-version factors (N-Prot factors) can then beused to determine crude protein content. TNanalysis, carried out by Kjeldahls method(AOAC, 1990) or Hach techniques (Hach etal., 1987), is fast and inexpensive (Watkins etal., 1987). Data for TN from CHN elementalanalysis can also be directly converted to crudeprotein by the use of conversion factors. Theuse of N-Prot factors allows better comparisonsof protein data among species, as well as morepractical comparisons of results obtained bydierent authors. By the use of conversionfactors, protein is estimated without a demand-ing previous extraction (Fleurence et al., 1995),and possible losses of protein are avoidedduring the preparation of the samples.The major problem involved with this metho-

dology is the establishment of specic N-Protfactors for each species, since the conventionalfactor used to calculate crude protein (6.25) isunsuitable for several materials (Sosulski &Imadon, 1990). The use of the factor 6.25(Jones, 1931) is based on the assumption thatthe samples contain protein with 16% nitrogenand a negligible concentration of non-proteinac-eous nitrogen (NPN). Nevertheless, plant materi-als and seaweeds normally have large contents ofNPN (Conklin-Brittain et al., 1999; Levey et al.,2000; Lourenco et al., 2002a), and commonlydeviate from a N-content of 16% in total protein(Mosse, 1990; Yeoh & Truong, 1996b). The sameconcerns apply to nitrogen distribution andamino acid composition of marine microalgae(Lourenco et al., 1998).Because of the wide applications of data on

nitrogen and protein composition, the calculationof specic nitrogen-to-protein conversion factorsis important to many elds of science. Plantmaterials and fungi have been especially studied

in the last few years. Average N-Prot factors of5.51, 3.59, 5.64 and 3.24 were proposed for appleower buds (Khanizadeh et al., 1992), sweetpotato (Yeoh & Truong, 1996a), wild fruits fromsoutheastern USA (Levey et al., 2000) andcassava roots (Yeoh & Truong, 1996b), respec-tively. N-Prot factors calculated by Wu et al.(1995) for processed kidney beans ranged from5.63 to 5.67. Danell & Eaker (1992) proposed aN-Prot factor of 4.38 for the mushroom Canthar-ellus cibarius, while Mattila et al. (2002) estab-lished an average N-Prot factor of 4.70 for fourmushrooms cultivated in Finland. Aitken et al.(1991) determined a factor of 5.00 for two speciesof the edible red alga Porphyra from NewZealand, in a study focussing on the seasonalvariations in amino acid composition. Lourencoet al. (2002a) calculated an average factor of 4.92for nineteen species of tropical seaweeds (ninered, six green and four brown algae) from Brazil.All these studies derived N-Prot factors lowerthan 6.25, and some of them detected thepresence of high concentrations of NPN in thespecies studied by nitrogen budgets.Marine microalgae may also accumulate high

concentrations of NPN, such as inorganicnitrogen (Dortch et al., 1984) and nucleic acids(Machado et al., 1999). Lourenco et al. (1998)quantied the nitrogen distribution in aminoacids, chlorophylls (a, b and c), RNA, DNA,and inorganic intracellular ions (nitrate, nitriteand ammonia), and published the rst paper inwhich specic N-Prot factors were proposed forten marine microalgae. In that study an overallmean N-Prot factor of 4.58 was established. Allspecies were cultured using Conway culturemedium (Walne, 1966), a nitrogen-rich mediumcommonly used in aquaculture, without aeration.The authors suggested that the experimentalconditions adopted could have stimulated theaccumulation of large amounts of non-proteinac-eous nitrogen, and recommended further studiesto establish N-Prot factors, which could beapplied to species cultured in other growthconditions.In the present study we have quantied the

budgets for protein and the main NPN com-pounds, showing the nitrogen distribution in cellsof twelve species of marine microalgae. Ten specieswere cultured under two growth conditions, andtwo species were cultured under three dierentconditions. The results are discussed in relation tothe availability of carbon and nitrogen, majorpoints in the experimental design. We propose newconversion factors from total nitrogen to crudeprotein for each species in each treatment andgrowth phase, as well as average conversionfactors.

18S. O. Lourenco et al.

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Materials and methods

The microalgae tested

All strains were obtained from the Microalgae CultureCollection of the Department of Marine Biology,Federal Fluminense University, Brazil. The followingspecies were studied: Amphidinium carterae (Dinophy-ceae, Gymnodiniales; strain NO1), Chlorella minutissima(Chlorophyceae, Chlorococcales; strain CN1), Dunaliellatertiolecta (Chlorophyceae, Volvocales; strain FR1),Hillea sp. (Cryptophyceae, Cryptomonadales; strainPB1), Isochrysis galbana (Prymnesiophyceae, Isochrysi-dales; strain TH1), Nannochloropsis oculata (Eustigma-tophyceae, Eustigmatales; strain KGH1),Phaeodactylum tricornutum (Bacillariophyceae, Bacillar-iales; strain UB7), Prorocentrum minimum (Dinophy-ceae, Prorocentrales; strain CN3), Skeletonema costatum(Bacillariophyceae, Biddulphiales; strain CF1), Synecho-coccus subsalsus (Cyanophyceae, Chroococcales; strainUB2), Tetraselmis gracilis (Prasinophyceae, Tetraselmi-dales; strain CN1), and Thalassiosira oceanica (Bacillar-iophyceae, Biddulphiales; strain ST1). All strains wereisolated from Brazilian coastal waters except for fourobtained from foreign institutions: Amphidinium carterae(University of Oslo, Norway), Dunaliella tertiolecta(Universite DAix-Marseille II, France), Isochrysis gal-bana, and Nannochloropsis oculata (both from Kagoshi-ma Oika Fisheries Research Center, Japan).

Culture conditions

Starter cultures of 10 50 ml in mid-exponential growthphase were inoculated into 2 l of seawater, previouslyautoclaved at 1218C for 30 min in 3 l borosilicate asks.Each species was cultured in two experimental condi-tions:(a) seawater enriched with Conway nutrient solution

(Walne, 1966) in its original concentrations

(N=1178 mM) and bubbled with ltered air at2.0 l min7 1;

(b) seawater enriched with 20% of the originalconcentration of nitrogen of Walnes medium(235 mM) and no aeration.

In addition, Amphidinium carterae and Thalassiosiraoceanica were cultured with the original Walnes mediumwithout aeration, as in our previous study (Lourenco etal., 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 m7 2 s7 1 (mea-sured with a Biospherical Instruments quantum meterQLS100), provided from beneath by uorescent lamps(Sylvania daylight tubes), on a 12 : 12 h light : dark cycle.Mean temperatures during experiments were 23+ 28C inthe light and 20+ 18C in the dark. Salinity in theexperiments was 32.0%. Growth rates were calculateddaily by direct microscopic cell counting with Agasse-Lafont, Fuchs-Rosenthal, Malassez or Thoma cham-bers. Cultures were not buered and pH was determineddaily. All sampling for cell counts and pH measurementsoccurred during the rst 10 min of the light period.

Sampling procedure

Each culture was sampled four times for both chemicaland biochemical analysis, in dierent growth phases:mid-exponential, late-exponential, early stationary andlate-stationary growth phases (Fig. 1), except forAmphidinium carterae and Thalassiosira oceanica, whichwere sampled only once in the stationary growth phase.Samples of 300 to 400 ml were concentrated bycentrifugation at 7000 g and 158C for 10 min, at leastonce, to obtain highly concentrated pellets. Before thelast centrifugation, cells were washed in articial sea-water (Kester et al., 1967) prepared without nitrogen,phosphorus and vitamins, and adjusted to 15% salinity.All supernatants obtained for each sample were com-bined and the number of cells was determined to

Fig. 1. Growth curves for Amphidinium carterae, Nannochloropsis oculata, Phaeodactylum tricornutum, and Tetraselmis gracilisin dierent treatments, based on cell counts. Each point represents the mean of four replicates+SD. Arrows indicate thetimes 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 min7 1); N/5indicates cultures grown with 20% of the original concentration of nitrogen of Walnes medium and no aeration.

19Intracellular nitrogen in marine microalgae

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quantify possible cell losses. The pellets were frozen at7 208C and then freeze dried, weighed and stored indesiccators under vacuum and protected from light untilanalysed for CHN elemental composition and totalamino acids. Samples to be analysed for DNA, RNA,intracellular inorganic nitrogen and chlorophylls wereobtained by ltering the cultures under vacuum ontoWhatman GF/F1 glass microbre lters (0.7 mm nom-inal pore size), previously exposed to a temperature of4008C for 4 h in a mue furnace. Samples for bothchlorophyll and intracellular inorganic nitrogen assayswere kept at 7 208C in asks containing silica-gel untilanalysis, whereas those for nucleic acid determinationwere stored in liquid nitrogen. All sampling for chemicaland biochemical analysis was done during the rst90 min of the light period. For both chemical andbiochemical analysis, samples from three out of the fourexperimental asks were analysed (n=3); the fourthsample was kept as a reserve.

Biochemical analysis

Chlorophyll was extracted in 90% acetone at 48C for20 h, after grinding the lters with the samples. Spectro-photometric determination of pigments was carried outas described by Jerey & Humphrey (1975). Nitrogenconcentrations were obtained by multiplying chlorophylla, b and c content by 0.0628, 0.0618 and 0.0916,respectively, corresponding to the nitrogen content ofthese chlorophylls.Nucleic acids were analysed by reaction with

Thiazole Orange (Aldrich Co.) and Hoechst 332581

(Sigma Co.) stains and measurement in a spectro-uorometer (PTI model QM-1; Machado et al., 1999).Nitrogen concentration in the nucleic acids wascalculated according to Dortch et al. (1983). Assumingequal proportions of the four major nucleotides, thenitrogen content of DNA is 16.84% and that of RNAis 16.12%.Amino acid analysis was carried out by ion-exchange

chromatography in a Beckman, model 7300, equippedwith an automatic integrator. Samples containing 5.0 mgof protein were acid hydrolysed with 1.0 ml 6 N HCl invacuum-sealed hydrolysis vials at 1108C for 22 h.Norleucine was added as an internal standard. Trypto-phan and cystine+cysteine are completely lost with acidhydrolysis, while methionine could be destroyed tovarying degrees by this procedure. The following valuesfor the N content of each amino acid were used tocalculate 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-Ndetermination, the content of ammonia was included inthe calculation of protein-nitrogen retrieval, as it comesmainly from glutamine and asparagine degradationduring acid hydrolysis (Mosse, 1990; Yeoh & Wee,1994). The NH3 values presented in Table 1 are alreadycorrected for the free intracellular ammonia concentra-tions (part of the inorganic N concentration, see Table2). The ammonia-N content was calculated by multi-

plication of the concentrations determined for ammoniaby 0.824 (NH3=82.4% of N).

Chemical analysis

Intracellular inorganic nitrogen (IIN) was measuredspectrophotometrically and represents the sum of theconcentrations of ammonia+ammonium (accordingto Aminot & Chaussepied, 1983), nitrate, and nitrite(according to Parsons et al., 1984) within cells.Samples were kept frozen at 7 208C until chemicalanalysis. The IIN extraction procedure was as de-scribed by Lourenco et al. (1998), except that the rststep of the process was carried out at 48C instead of158C, in order to minimise bacterial contamination ofthe samples.Total nitrogen was quantied in a CHN elemental

analyser Perkin-Elmer, model 2400. Helium was used asa 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 indierent growth phases by the ratio of amino acidresidues to TN of the sample: N-Prot factor=Aa-Res/TN. Thus, a sample with 16.21 g of amino acid residuesand 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 eachspecies and treatment) were analysed by one-wayanalysis of variance (ANOVA; Zar, 1996) followed,where applicable, by a Tukeys multiple comparison test.ANOVA (for Amphidinium carterae and Thalassiosiraoceanica) or Students t-test were used for comparingeach variable in two or more treatments at a xed time ofobservation.

Results

The results for four representative species (Am-phidinium carterae, Nannochloropsis oculata,Phaeodactylum tricornutum, and Tetraselmis gra-cilis) are shown in Figs. 1 6, and for three otherspecies (Chlorella minutissima, Prorocentrum mini-mum, and Thalassiosira oceanica) in Tables 2 and3.

Growth, C :N ratio and TN

All species showed signicantly higher nal yieldswhen cultured with aeration (Fig. 1). Growth ofAmphidinium carterae and Thalassiosira oceanicawith original Walnes medium and with reducedinitial nitrogen (N/5) were similar, and generatedsimilar nal yields.


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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 aminoacids 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 acidsdestroyed during acid hydrolysis. Values are the mean of three replicates+ SD

Amino

acids

Amphidinium

carterae

Prorocentrum

minimum

Synechococcus

subsalsus

Chlorella

minutissima

Dunaliella

tertiolecta

Tetraselmis

gracilis

Isochrysis

galbana Hillea sp.

Nannochloropsis

oculata

Phaeodactylum

tricornutum

Skeletonema

costatum

Thalassiosira

oceanica

Mean of

species

Asp 8.9+ 0.2 9.8+ 0.8 10.0+ 0.2 10.6+ 0.1 10.1+ 0.2 10.4+ 0.2 10.2+ 0.2 11.1+ 0.4 9.8+ 0.5 11.9+ 0.2 11.2+ 0.6 11.0+ 0.0 10.4Thr 5.2+ 0.2 5.7+ 0.3 5.7+ 0.1 5.6+ 0.1 5.7+ 0.0 5.8+ 0.0 5.6+ 0.1 6.5+ 0.0 6.2+ 0.2 6.4+ 0.0 5.8+ 0.4 5.2+ 0.1 5.8Ser 6.2+ 0.5 4.6+ 0.2 4.1+ 0.1 5.2+ 0.0 4.7+ 0.1 4.9+ 0.1 5.2+ 0.1 4.8+ 0.2 5.2+ 0.4 5.8+ 0.0 5.8+ 0.5 7.3+ 0.1 5.3Glu 13.0+ 0.5 15.6+ 0.8 14.2+ 0.4 13.3+ 0.2 13.4+ 0.7 14.2+ 0.1 13.3+ 0.3 14.5+ 0.4 13.0+ 0.7 15.5+ 0.4 13.1+ 0.3 11.4+ 0.0 13.7Pro 5.2+ 0.6 5.7+ 0.2 3.8+ 0.1 4.6+ 0.2 4.5+ 0.5 5.2+ 0.2 4.7+ 0.1 4.2+ 0.0 4.8+ 0.2 4.6+ 0.2 4.4+ 0.1 4.6+ 0.1 4.7Gly 4.8+ 0.1 6.2+ 0.4 5.5+ 0.1 6.0+ 0.1 6.1+ 0.2 6.9+ 0.1 6.0+ 0.1 6.0+ 0.3 6.3+ 0.2 6.5+ 0.1 6.0+ 0.4 6.2+ 0.0 6.0Ala 7.4+ 0.2 7.8+ 0.1 8.4+ 0.2 7.9+ 0.3 8.2+ 0.2 7.8+ 0.2 8.2+ 0.2 7.9+ 0.2 7.7+ 0.4 8.2+ 0.1 6.9+ 0.6 7.6+ 0.2 7.8Val 6.6+ 0.5 5.8+ 0.0 5.7+ 0.1 6.2+ 0.1 6.3+ 0.2 6.1+ 0.2 6.2+ 0.2 6.0+ 0.6 5.9+ 0.2 7.0+ 0.2 6.0+ 0.5 6.1+ 0.0 6.1Met 1.7+ 0.4 0.6+ 0.0 1.3+ 0.0 0.4+ 0.1 1.6+ 0.2 0.5+ 0.1 1.0+ 0.0 1.6+ 0.3 0.3+ 0.1 1.0+ 0.1 1.3+ 0.3 1.6+ 0.2 1.1Ile 4.0+ 0.5 4.2+ 0.3 5.0+ 0.1 4.4+ 0.2 4.5+ 0.0 4.1+ 0.2 4.6+ 0.1 4.2+ 0.1 4.0+ 0.2 5.0+ 0.0 4.6+ 0.3 4.7+ 0.1 4.4Leu 8.6+ 0.4 8.4+ 0.5 9.2+ 0.2 9.2+ 0.3 9.6+ 0.4 9.6+ 0.2 9.4+ 0.2 8.0+ 0.1 9.2+ 0.2 9.0+ 0.1 7.9+ 0.6 8.9+ 0.1 8.9Tyr 3.4+ 0.2 2.3+ 0.2 2.3+ 0.1 2.7+ 0.1 2.6+ 0.4 2.4+ 0.1 2.0+ 0.1 2.7+ 0.0 2.4+ 0.2 2.2+ 0.2 2.3+ 0.5 4.0+ 0.2 2.9Phe 5.4+ 0.3 4.8+ 0.1 5.4+ 0.1 5.7+ 0.2 5.9+ 0.0 6.4+ 0.0 5.3+ 0.1 5.0+ 0.1 5.2+ 0.1 6.1+ 0.1 6.2+ 0.5 6.5+ 0.0 5.6His 1.9+ 0.2 1.8+ 0.2 1.9+ 0.0 1.8+ 0.0 2.2+ 0.4 3.6+ 0.6 4.5+ 0.1 3.8+ 0.5 1.9+ 0.1 1.5+ 0.2 2.1+ 0.5 2.4+ 0.1 2.6Lys 7.0+ 0.0 6.1+ 0.5 5.0+ 0.1 6.3+ 0.0 5.5+ 0.5 7.2+ 0.2 6.5+ 0.2 5.3+ 0.1 6.2+ 0.3 4.7+ 0.2 7.2+ 0.5 6.6+ 0.1 6.1Arg 5.1+ 0.4 6.6+ 0.3 5.2+ 0.1 5.0+ 0.7 4.8+ 0.1 5.4+ 0.4 5.5+ 0.1 5.5+ 0.2 5.8+ 0.2 4.9+ 0.2 5.8+ 0.4 5.0+ 0.3 5.4Ammonia 1.0+ 0.3 2.0+ 0.4 2.0+ 0.0 1.5+ 0.1 1.6+ 0.1 1.4+ 0.2 1.9+ 0.0 1.7+ 0.1 1.5+ 0.0 2.2+ 0.1 2.3+ 0.3 1.2+ 0.2 1.7Total 94.3+ 5.3 96.0+ 4.8 92.7+ 2.4 95.0+ 2.9 95.7+ 4.4 100.6+ 3.2 98.2+ 2.2 97.1+ 3.6 93.9+ 4.2 100.4+ 2.4 96.4+ 6.3 99.0+ 1.8 96.8

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Variations in C :N ratio over time indicatesome common trends for most of the species(Fig. 2). C :N values tended to be low in theexponential growth phase, and increased withtime, reaching highest values in the late station-ary growth phase, especially in aerated condi-tions. The lowest C :N ratio was found inChlorella minutissima (2.8, N/5), and the highestin Nannochloropsis oculata (18.3, air). Chlorellaminutissima, Isochrysis galbana, Skeletonema cost-atum, and Thalassiosira oceanica cultured with N/5 resembled Nannochloropsis oculata (Fig. 2) inshowing little change in C :N ratio over time (asdid T. oceanica cultured with original medium),but increasing C :N values during growth inaerated treatments. Amphidinium carterae showedsimilar values for C :N ratio in the dierenttreatments, except in the stationary growth phase,in which mean values with N/5 were signicantlyhigher than in the other treatments (p5 0.05).For Phaeodactylum tricornutum (Fig. 2) andSynechococcus subsalsus, signicant dierencesbetween the treatments were found only in late-exponential phase (p5 0.01). Only Tetraselmisgracilis (Fig. 2) and Dunaliella tertiolecta showed

higher values for C :N ratio in N/5 for at leasttwo sample times.All microalgae showed large changes in total

nitrogen during growth (p4 0.02; Fig. 3). Whencultured with aeration, all species except Thalassio-sira oceanica had higher values of N per cell in theexponential growth phase, which decreased in laterphases of the growth cycle. Some species (Amphi-dinium carterae, Phaeodactylum tricornutum Fig.3, and also Chlorella minutissima, Hillea sp., andThalassiosira oceanica) showed the same trendwhen cultured in N/5, but the N-content of othermicroalgae (Nannochloropsis oculata Fig. 3, andalso Dunaliella tertiolecta, Isochrysis galbana, Pro-rocentrum minimum, and Skeletonema costatum)increased during growth in N/5 (except Tetraselmisgracilis Fig. 3, and Synechococcus subsalsus).When cultured with the original medium, total Nper cell increased over time in Amphidiniumcarterae (p5 0.01; Fig. 3) and decreased inThalassiosira oceanica (p5 0.01). Based on thedata for total N cell7 1 and the initial Nconcentration in the medium, we have calculatedthe consumption of N from the medium at the endof the experiments (data not shown). The uptake of

Table 2. Distribution of nitrogenous components in Chlorella minutissima, Prorocentrum minimum, and Thalassiosira oceanicain dierent growth phases and treatments. Results represent mg N in the substances per 100 mg of total N, and are meansof three replicates+SD. Similar analyses were made for the other species

Species/growth

phase Experiment Amino acid-N Chlorophyll-N Nucleic acid-N Inorganic-N Total NPN

C. minutissima

Mid exponential Air 63.2+ 1.4 0.5+ 0.1 9.5+ 3.0 25.3+ 10.9 35.3+ 14.0N/5 80.7+ 1.5 0.2+ 0.0 2.7+ 0.5 16.7+ 3.7 19.6+ 4.3

Late exponential Air 66.2+ 3.7 1.8+ 0.1 12.2+ 4.6 21.2+ 3.9 35.3+ 8.6N/5 86.3+ 3.2 0.2+ 0.3 1.5+ 0.5 6.9+ 1.1 8.6+ 1.9

Early stationary Air 67.5+ 1.8 1.7+ 0.3 9.5+ 4.2 20.3+ 6.1 31.5+ 10.6N/5 86.3+ 5.8 0.2+ 0.1 1.0+ 0.2 14.7+ 5.5 16.0+ 5.7

Late stationary Air 72.5+ 4.2 0.9+ 0.2 10.6+ 2.9 18.4+ 2.1 29.9+ 4.3N/5 87.3+ 3.9 0.3+ 0.0 1.2+ 0.2 12.9+ 4.5 14.5+ 4.7

P. minimum





T. oceanica

Mid exponential Original 92.2+ 9.4 0.1+ 0.0 0.7+ 0.0 6.5+ 1.3 7.3+ 1.4Air 88.3+ 2.9 0.1+ 0.0 0.6+ 0.1 6.6+ 2.2 7.3+ 2.3N/5 89.4+ 3.3 0.1+ 0.0 0.6+ 0.3 6.1+ 2.3 6.9+ 2.6

Late exponential Original 94.0+ 3.5 0.1+ 0.0 0.7+ 0.2 5.7+ 1.2 6.5+ 1.4Air 93.7+ 7.3 0.2+ 0.0 0.3+ 0.1 2.2+ 0.5 2.7+ 0.6N/5 92.3+ 2.4 0.1+ 0.0 0.9+ 0.1 5.7+ 2.0 6.9+ 2.2

Stationary Original 94.4+ 2.1 0.1+ 0.0 0.8+ 0.1 5.9+ 0.2 6.8+ 0.3Air 93.8+ 7.8 0.1+ 0.0 0.2+ 0.1 1.7+ 0.3 2.0+ 0.4N/5 91.5+ 1.7 0.1+ 0.0 0.9+ 0.1 5.0+ 0.9 6.0+ 1.1


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nitrogen was equivalent to 100% in all aeratedexperiments, but reached only 21.3% and 35.5% ofthe initial N in cultures of Amphidinium carterae

and Thalassiosira oceanica, respectively, with theoriginal medium. The consumption of N from themedium varied from 88.1% (Skeletonema costa-tum) to 99.5% (Amphidinium carterae) in N/5experiments.

Fig. 2. Changes in carbon:nitrogen ratio (by atoms) ofAmphidinium carterae, Nannochloropsis oculata,Phaeodactylum tricornutum, and Tetraselmis gracilis in

dierent treatments. Each point represents the mean of threereplicates+SD. Other details as in Fig. 1.

Fig. 3. Changes in total nitrogen per cell during growth ofAmphidinium carterae, Nannochloropsis oculata,Phaeodactylum tricornutum, and Tetraselmis gracilis in

dierent treatments. Other details as Fig. 2.


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Changes in the concentration of non-proteinaceousnitrogen

Most of the microalgae studied had higherconcentrations of chlorophyll under aeration than

in N/5 experiments (Fig. 4), except Amphidiniumcarterae, Hillea sp., Isochrysis galbana, Prorocen-trum minimum, and Thalassiosira oceanica, forwhich there was no signicant dierence betweenthe treatments. Hillea sp. was the only species thatshowed higher values for chlorophyll cell7 1 whencultured with N/5. Most of the species showed

Fig. 4. Changes in nitrogen contained in chlorophylls during

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

Fig. 5. Changes in nitrogen contained in total nucleic acids(DNA+RNA) during growth of Amphidinium carterae,

Nannochloropsis oculata, Phaeodactylum tricornutum, andTetraselmis gracilis in dierent treatments. Other details asFig. 2.


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higher concentrations of chlorophyll in the expo-nential phase, which decreased with time(p4 0.04), but Chlorella minutissima, Dunaliellatertiolecta, Prorocentrum minimum, and Skeletone-ma costatum, showed no variations in chlorophyll

per cell with time. Phaeodactylum tricornutumshowed a dierent pattern of variation with air,with a peak in chlorophyll concentration in earlystationary phase, decreasing in late stationaryphase to concentrations similar to those recordedin late exponential phase (Fig. 4). Variations inchlorophyll concentration during growth with N/5were small for Nannochloropsis oculata, Phaeodac-tylum tricornutum, and Tetraselmis gracilis. (Fig.4), and also for Dunaliella tertiolecta, Prorocentrumminimum and Synechococcus subsalsus.As with chlorophyll, microalgae tended to show

higher concentrations of nucleic acids (DNA+R-NA) in the exponential phase, which decreasedwith time (Fig. 5), except for Phaeodactylumtricornutum with air, Isochrysis galbana, Skeletone-ma costatum, and Synechococcus subsalsus with N/5. In these species the concentrations of nucleicacids either increased in the stationary growthphase or did not change with time. RNA contentwas higher than DNA in almost all samples (datanot shown). In the exponential growth phase,RNA :DNA ratios varied from 1.5 : 1 (Nannochlor-opsis oculata, N/5) to 8.0 : 1 (Dunaliella tertiolecta,air cultures), with an overall mean of 3.7 : 1. In thestationary phase, however, the RNA :DNA ratiovaried from 0.6 : 1 (Nannochloropsis oculata, air) to5.4 : 1 (Prorocentrum minimum, air), with an overallmean of 1.9 : 1.All species contained intracellular inorganic

nitrogen as nitrate, nitrite and ammonium (datanot shown). However, the ratio among these ionsas well as their respective concentrations variedconsiderably during growth. Ammonium+ammo-nia values were higher than those of nitrate incultures of Chlorella minutissima, Phaeodactylumtricornutum, Synechococcus subsalsus, and Tetra-selmis gracilis. All other species had higherconcentrations of nitrate than of ammoniu-m+ammonia. Nitrite concentrations were alwaysmuch lower than those of the other inorganic ions.Total IIN decreased during growth (Fig. 6), withfew exceptions (Tetraselmis gracilis Fig. 6, andalso Dunaliella tertiolecta and Prorocentrum mini-mum, in air; Dunaliella tertiolecta and Skeletonemacostatum in N/5 cultures). Chlorella minutissimaand Skeletonema costatum showed higher IINvalues in N/5 than in experiments with aeration,as well as Amphidinium carterae and Thalassiosiraoceanica in both late-exponential and stationarygrowth phases. Phaeodactylum tricornutum (Fig. 6)and Isochrysis galbana showed similar concentra-tions of IIN in the two treatments, except in themid-exponential growth phase, in which the valuesfor N/5 were higher. Values of IIN were predomi-nantly higher in aerated cultures of Nannochlor-opsis oculata (Fig. 6), Hillea sp., Prorocentrumminimum and Synechococcus subsalsus.

Fig. 6. Changes in inorganic intracellular nitrogen(nitrate+nitrite+ammonia/ammonium) during growth of

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


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Total amino acid, nitrogen budgets and thecalculation of N-Prot factors

Data on total amino acids are presented for the latestationary growth phase of aerated cultures only(Table 1). Glutamic acid was the most abundantamino acid in all species, followed by aspartic acid.Histidine and methionine were identied as minoramino acids in these species (concentrations oftryptophan and cysteine + cystine not taken intoaccount), and only Isochrysis galbana showed highconcentrations of histidine (4.5% of the totalamino acids) compared to the other species.Percentages of alanine, leucine, isoleucine andthreonine were similar in all species. The concen-tration of arginine was higher in Prorocentrumminimum and that of serine was higher in Thalas-siosira oceanica. Amphidinium carterae, Skeletone-ma costatum, and Tetraselmis gracilis showed highconcentrations of lysine, while the highest percen-tages of tyrosine, proline and valine were found inIsochrysis galbana, Prorocentrum minimum andPhaeodactylum tricornutum, respectively. Higherconcentrations of phenylalanine were found in thethree diatoms and Tetraselmis gracilis. Mean valuesof each amino acid over all the microalgae testedare also shown in Table 1.

The amino acid-N as a percentage of TNincreased from the exponential to the stationarygrowth phase (Table 2). Values ranged from 59.3(Prorocentrum minimum, N/5) to 96.8% (Nanno-chloropsis oculata, N/5) of the TN. Total NPNvaried from 0.8 (Synechococcus subsalsus, N/5)up to 39.0% (Nannochloropsis oculata, N/5) ofthe TN. IIN was the main component of NPNin almost all species in all treatments. Nucleicacid-N represented the second most importantcomponent of NPN, while the contribution ofchlorophyll to TN was always low (less than 1%of TN, in most samples).The actual protein concentration of the sam-

ples was calculated by summing the masses ofthe amino acid retrieved after acid hydrolysis(total amino acid), minus the mass of water (18 gH2O per mol of amino acid). The water isincorporated into each amino acid after thedisruption of the peptide bonds (Mosse, 1990;Lourenco et al., 1998), and it is not present inpeptides and proteins. Consequently, the proteincontent of the samples is indicated as mg of totalamino acid residues (Table 3). The microalgalspecies showed a wide range of protein concen-tration, varying from 3.5 (Chlorella minutissima,

Table 3. Calculation of N-Prot factors for Chlorella minutissima, Prorocentrum minimum, and Thalassiosira oceanica, basedon the amino acid residues to total nitrogen ratio. Data are expressed as percentage of dry matter, and represent the means ofthree replicates+ SD. Similar calculations were made for the other species

Species/growth

phase Experiment Total N Total amino acid

Amino acid

residues Amino acid-N N-Prot factors

C. minutissima





P. minimum





T. oceanica

Mid exponential Original 1.65+ 0.14 9.91+ 1.01 8.56+ 0.88 1.52+ 0.16 5.19+ 0.38Air 2.44+ 0.06 14.45+ 0.47 12.44+ 0.40 2.15+ 0.07 5.10+ 0.28N/5 1.96+ 0.06 11.77+ 0.44 10.13+ 0.38 1.75+ 0.06 5.17+ 0.34

Late exponential Original 1.80+ 0.07 11.40+ 0.43 9.72+ 0.36 1.69+ 0.06 5.40+ 0.41Air 4.42+ 0.07 29.10+ 2.86 24.86+ 1.93 4.14+ 0.32 5.62+ 0.53N/5 2.10+ 0.07 12.71+ 0.33 10.81+ 0.28 1.94+ 0.05 5.15+ 0.31

Stationary Original 1.99+ 0.07 13.00+ 0.28 11.09+ 0.24 1.88+ 0.04 5.58+ 0.32Air 3.57+ 0.06 23.22+ 1.92 19.45+ 1.65 3.35+ 0.28 5.59+ 0.45N/5 2.85+ 0.03 19.27+ 0.35 16.43+ 0.30 2.61+ 0.05 5.77+ 0.16


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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 ninereplicates. Results obtained in our previous study (Lourenco et al., 1998; col. 7) are included in the nal calculations (col. 8)

Taxonomic

Growth phases*

Means across all Overall means from Overall mean values for

groups/species Mid-exponential Late-exponential Early stationary Late stationary growth phases Lourenco et al. (1998) each species**

Cryptophyceae

Hillea sp. 4.28a 4.57ab 4.93b 5.01b 4.74 (n=24) 4.61 (n=6) 4.72 (n=30)

Cyanobacteria

Synechococcus subsalsus 4.96a 5.16a 5.30a 5.57b 5.43 (n=24) 4.66 (n=9) 5.22 (n=33)

Diatoms

Phaeodactylum tricornutum 4.46a 4.86b 4.89b 5.48c 4.93 (n=24) 4.72 (n=9) 4.87 (n=33)

Skeletonema costatum 4.40a 4.55a 4.53a 5.10b 4.73 (n=24) 3.82 (n=3) 4.63 (n=27)

Thalassiosira oceanica 5.19a 5.39b 5.65c 5.40 (n=27) 5.40 (n=27)

Dinoagellates

Amphidinium carterae 4.80a 5.21b 5.39c 5.13 (n=27) 5.13 (n=27)

Prorocentrum minimum 3.76a 4.34ab 4.79b 4.73b 4.60 (n=24) 3.77 (n=6) 4.43 (n=30)

Eustigmatophyceae

Nannochloropsis oculata 4.00ab 4.95a 5.17b 5.59c 4.98 (n=24) 4.87 (n=6) 4.95 (n=30)

Green algae

Chlorella minutissima 3.09a 4.45b 4.57b 4.67b 4.22 (n=24) 4.50 (n=3) 4.25 (n=27)

Dunaliella tertiolecta 3.64a 4.39b 4.39b 4.91b 4.38 (n=24) 3.99 (n=3) 4.34 (n=27)

Tetraselmis gracilis 4.37a 4.75ab 5.10b 5.08b 4.96 (n=24) 4.37 (n=9) 4.80 (n=33)

Prymnesiophyceae

Isochrysis galbana 3.99a 4.31a 5.07b 4.95b 4.74 (n=24) 3.99 (n=6) 4.59 (n=30)

Mean values for each growth phase 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)

*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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N/5) to 34.0% of the dry matter (Phaeodactylumtricornutum, air). Percentages of protein wereusually higher in aerated cultures than in theother treatments. The mass of N within aminoacids ranged from 0.87 (Prorocentrum minimum,mid-exponential phase, N/5) to 5.68% (Phaeo-dactylum tricornutum, late-exponential, air) of thedry matter. The N-Prot factors for the micro-algae were calculated from the ratio of the massof amino acid residues to total nitrogen, bothexpressed as mg contained in 100 mg of samples(Table 3). N-Prot factors ranged 2.53 (Chlorellaminutissima, mid-exponential, N/5) to 5.77 (Tha-lassiosira oceanica, stationary, N/5). For allspecies, N-Prot factors were lower in mid-exponential growth (average=4.33+ 0.72,n=78) and higher in late stationary phase(average=5.23+ 0.40, n=60). An overall aver-age N-Prot factor of 4.86+ 0.63 (n=294) wascalculated from the data for all species, indierent growth phases and treatments. Com-pared with our previous results (Lourenco et al.,1998), the new mean N-Prot factors for thespecies and growth phases under dierent experi-ments were signicantly dierent, with meanvalues for N/5 (4.92) higher than those forOriginal (4.59) and Air (4.75) (F2,350=7.38,p5 0.01). Dierences between the mean valuesfor Original and for Air experiments were notsignicant. An overall average N-Prot factor of4.78 was calculated combining data from bothstudies, and mean values of 4.33, 4.77, 4.99, and5.06 were calculated for mid-exponential, lateexponential, early stationary, and late stationarygrowth phases, respectively (Table 4). Signicantdierences were found among the mean N-Protfactors calculated for the dierent growth phases(F3,350=30.0, p5 0.001). Mean values calculatedfor the mid-exponential growth phase were lowerthan those for the other growth phases(p5 0.01). No dierence was found betweenmean values calculated for early and latestationary growth phases (p=0.61). The meanN-Prot factors calculated for taxonomicallyrelated species (e.g. diatoms) in all treatmentsand growth phases were also compared. Themean N-Prot factors calculated for the threediatoms were signicantly dierent (ANOVA+Tukey, p5 0.05), with higher N-Prot factors forT. oceanica; values for P. tricornutm and S.costatum were not signicantly dierent. The N-Prot factor calculated for the dinoagellate A.carterae was higher than that for P. minimum (t-test, p5 0.001). For the green algae, the meanN-Prot factors of C. minutissima and D. tertio-lecta were not signicantly dierent (p=0.65),but both were signicantly lower than that of T.gracilis (p5 0.01).

Discussion

Eects of the treatments on growth, C :N ratio andTN in the microalgae

Growth responses of all species showed largedierences between nal cell yields obtained in N/5 and aerated experiments. All species showedhigher cell densities in the stationary phase whencultured with aeration. Two factors are inuencingthe trends observed: an eect of nitrogen as alimiting factor in N/5 experiments, and theinuence of the input of carbon in experimentswith aeration. CO2 occurs in low concentrations inthe air (close to 0.04% v/v), but the use ofcontinuous aeration at 2.0 l air min7 1 provides asignicant input of carbon into the cultures, asdemonstrated by Fabregas et al. (1995). N uptakeand assimilation is faster when carbon is notlimiting, and growth is stimulated (Huertas et al.,2000). On the other hand, dierences in nal cellyield were small between experiments with theoriginal culture medium and N/5 for Amphidiniumcarterae and Thalassiosira oceanica. Based on ourprevious results (Lourenco et al., 1998) and on thecurrent data we can assume that N was in excess inthe original culture medium. Nevertheless, our datafrom N/5 experiments show that the uptake of Nfrom the medium was almost complete (data notpresented), with possible N-limitation in thestationary growth phase. We assume that the lackof a C source may have limited growth for themicroalgae in a similar way in both original and N/5 experiments, generating a lower nal yieldcompared to aerated experiments.According to Burkhardt et al. (1999), minor

variations in C :N ratio occur when growth ratesare constant. Our results indicate that all aeratedcultures were limited by nitrogen in stationaryphase and showed wide C :N variations duringgrowth. In these cultures, a constant input ofcarbon was obtained by bubbling with air. Con-sidering the coupling of C and N metabolism, theavailability of carbon should make the assimilationof nitrogen faster, supplying cells with carbon foramino acid synthesis (Turpin, 1991). The fasterassimilation of N, associated with the greateravailability of C, is probably the main factordetermining the higher nal cell yield in aeratedcultures (Fig. 1). Cultures under N/5 had lowergrowth rates and probably were carbon-limited inthe stationary growth phase, since the diusion ofCO2 from the air to the cultures occurs at very lowrates, which were not enough to sustain a rapidgrowth. With low growth rates, the consumption ofnutrients tends to be slow, and the chemicalcomposition of the microalgae under those treat-ments may have changed slightly.


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Total N in cells during growth showed adecreasing trend with few exceptions. This generaltrend seems to be related to the progressively loweravailability of N in the culture medium. Somespecies may display a decrease in cell volume overgrowth, as an eect of either unsuitable carbon ornitrogen supply (Burkhardt et al., 1999). Thishypothesis has support in our data from aeratedexperiments, in which no carbon limitation wasrecorded, and all species except Thalassiosiraoceanica showed a remarkable trend of decreasingtotal N.

Variations in the concentrations of non-proteinaceous substances over growth

The decrease of chlorophyll-N cell7 1 duringgrowth was shown by almost all species andtreatments, and may be a consequence of thedecrease in nutrient availability in the culturemedium, and possibly the decrease in cell volumes.Lourenco et al. (1997) reported a signicantdecrease in chlorophyll concentration in Tetrasel-mis gracilis cells during growth in aerated cultures,and Huertas et al. (2000) did the same forNannochloropsis gaditana and Nannochloris macu-lata. In our experimental design, cultures were keptunder saturating irradiance, and self-shading isunlikely 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 establishprogressively 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 :DNAratio in the exponential phase and lower ratios inthe stationary phase of all cultures.The variations in intracellular inorganic nitrogen

concentrations during growth were similar to thosereported in our previous study (Lourenco et al.,1998). Almost all species showed higher values forIIN in the exponential growth phase, whichdecreased during growth in all treatments. Thehigh concentrations of IIN in the exponential phasereect the rapid N uptake in the rst days ofgrowth, when no factor is limiting (Lavn &Lourenco, unpublished data). However, the assim-ilatory process may be limited by the activity ofenzymes, resulting in an accumulation of highconcentrations of inorganic N. High concentra-tions of nitrite may be built up, and excretion ofthis ion may occur, preventing its toxic eects(Lourenco et al., 1997). The decrease of IINconcentrations seems to be the result of theassimilatory process, since the ratio of PN/TNincreased from exponential to stationary phases.

The IIN pool was consumed when N availabilitybecame smaller, conrming its physiological role asa nitrogen reserve (Dortch et al., 1984).Our data show that the relative importance of

sources of non-proteinaceous nitrogen tends tobecome progressively smaller during growth. Anincrease in the ratio of protein-N to TN occursduring growth, as demonstrated in our N budgetsfor all species in almost all cultures. IIN was themost important NPN component, followed bynucleic acids and chlorophyll. Chlorophyll has aminor importance in the N budgets, representingless than 1% of TN in most cases.

Nitrogen budgets and the calculation of N-Protfactors

The percentages of nitrogen and protein wererelatively low in some samples, mainly those fromexponential growth phases of N/5 experiments(Table 3). In most cases this resulted from theinuence of residual salts from articial seawater(without N) used for washing the samples at theend of the centrifugation. In mid-exponentialphase, cultures normally showed low cell densities,and the pellets generated by centrifugation tendednot to aggregate well. Conversely, the centrifuga-tion of high-density cultures, such as thoseobtained with aeration and in stationary phase ofN/5 experiments, was easier and the inuence ofresidual salts from the medium was negligible.Without the inuence of residual salts, highervalues for both N and protein content could havebeen obtained. The use of freeze-dried samples isessential for the method of amino acid analysisadopted in this study, since 5 mg of protein arerequired to perform the acid hydrolysis. Thisamount of protein would not be easily obtainedon lters, and we had to centrifuge samples foramino acid analysis. Nevertheless, residual saltshave no inuence on the calculation of N-Protfactors, which is based on the Aa-Res to TN ratio,independently of the mass of the other componentsin the samples. For both determinations, sampleswere taken from freeze-dried materials. Non-proteinaceous substances analysed in this study(chlorophyll, IIN, and nucleic acids) were sampledon glass bre lters, and were not inuenced byresidual salts from the culture medium.As with the variations of PN to TN ratio, the N-

Prot factors were lower in exponential growthphase, attaining maximum values in the stationaryphase. Since the relative importance of protein-Nincreases during growth, N-Prot factors tended tobe higher from mid-exponential to late stationaryphase. The concentration of protein per celldecreases during growth, while percentages ofprotein in dry matter show dierent trends. In


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aerated experiments, and in most of N/5 cultures,the percentages of protein were highest in late-exponential or early stationary phase and de-creased in the late stationary phase. In some N/5experiments (Nannochloropsis oculata and Thalas-siosira oceanica) and in cultures with the originalmedium (Amphidinium carterae and Thalassiosiraoceanica), the percentages of protein increasedduring growth, and maximum values were obtainedin the late exponential phase. This trend seems tobe due to the lack of carbon, since these samecultures showed no variations in C :N ratio overgrowth (see discussion in the rst subsection).Nevertheless, the contribution of PN to totalnitrogen increases during growth (Table 2).Quantitative analysis of total amino acids is of

fundamental importance for the determination ofthe actual protein content (Salo-Vaananen &Koivistoinen, 1996). Thus, the calculation ofspecic N-Prot factors is dependent on aminoacid analysis, since its determination depends onthe amount of nitrogen recovered from the aminoacids after acid hydrolysis (Mattila et al., 2002).Excluding the inuence of NPN, samples in whichtotal amino acid-N represent 16% of protein (inmass) 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 asN-Prot factors, respectively. N-Prot conversionfactors calculated for materials and species thatpossess proteins rich in highly nitrogenous aminoacids (e.g. histidine, arginine) tend to be low.Conversely, samples with a large content ofnitrogen-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 (Lourencoet al., 1998, 2002a) tend to show high concentra-tions of non-proteinaceous nitrogen. As thedetermination of TN does not enable one toidentify and distinguish protein-nitrogen fromNPN fractions, uctuations of NPN content havea remarkable inuence on the calculation of N-Prot factors. In NPN-rich species the inuence ofNPN on the calculation of N-Prot factors may beeven more important than that of the total aminoacid prole (Fujihara et al., 1995). Since the1990s, several studies have adopted the construc-tion of nitrogen budgets to validate N-Protfactors. The quantication of the main nitrogen-ous substances within cells by dierent andindependent methods allows the relationshipsamong values to be established. Lourenco et al.(1998) determined the concentrations of NPN in10 microalgae, which ranged from 9.2 to 47.0% ofthe TN, and an overall mean N-Prot factor forthe microalgae tested was 4.58 (n=60). Fujiharaet al. (2001) analysed 20 Japanese vegetables and

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 beinuenced by the presence of free amino acids inthe samples, because total amino acid analysis doesnot distinguish between free amino acids and thosecombined into protein. The extent to which freeamino acids will inuence the overall estimation ofprotein content is likely to vary from species tospecies (Ezeagu et al., 2002), but the inuence offree amino acids is normally low. Free amino acidstypically represent less than 10% of the total aminoacids in microalgae (Dortch et al., 1984) and up to5% of the total amino acids in grasses (Yeoh &Watson, 1982). Moreover, it is necessary toconsider that some amino acids (e.g. tryptophan,cystine, glutamine, methionine, serine) are partiallyor totally destroyed in an acid hydrolysis. Studyingthe edible mushroom Cantharellus cibarius, Danelland Eaker (1992) found that ca. 9% of theninhydrin-detectable N in amino acid analysis byion-exchange chromatography is ammonia, andmost of the ammonia results from amino aciddestruction in acid hydrolysis. Not all amino acidlosses during the hydrolysis are transformed intoammonia and detected in the amino acid analysis.Ezeagu et al. (2002) suggest that side reactionsduring hydrolysis may chelate some amino acid-N,but that some N compounds may be tightly boundwithin the cell-wall matrix and might not bereleased under HCl hydrolysis conditions. Thismeans that the typical losses during acid hydrolysismight compensate for the presence of free aminoacids, at least to some extent. As a consequence, theinuence of free amino acids on the calculation ofN-Prot factors would be minor. Finally, thenitrogen budgets from this study (Table 2) clearlyindicate that the lack of data for free amino acidsdid not signicantly aect the calculation of N-Protfactors. Due to all these arguments, the use of totalamino acid data to estimate protein content with-out determination of free amino acids is a widelyaccepted procedure (Mosse, 1990; Yeoh & Truong,1996b).Despite common trends in the variation of

proteinaceous-N and non-proteinaceous-N for themicroalgae in aerated and N/5 experiments, the N-Prot factors calculated for some species showedlarge dierences (Chlorella minutissima, Nanno-chloropsis oculata, Prorocentrum minimum), mainlyin the exponential phase. These may reectrelatively rapid changes in the biochemical compo-sition of microalgae in the exponential growthphase of batch cultures. Other species (Hillea sp.,Isochrysis galbana, Tetraselmis gracilis) showedsmall to medium dierences in N-Prot values inthe treatments in dierent growth phases. The


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other species (e.g. Synechococcus subsalsus andThalassiosira oceanica) showed similar N-Protfactors for each growth phase in the dierentexperimental conditions, with a small dispersion ofvalues.Establishing mean N-Prot factors for each

species by combining the results obtained fromdierent growth phases and culture conditionsresults in an increased variability of the values.We recognise that culture conditions aect thebiochemical composition of the microalgae, but itmakes no sense to recommend specic N-Protfactors for each species in each dierent growthphase and culture condition. Each species wouldreceive dozens of N-Prot factors, depending on theexperimental conditions used. For practical rea-sons, it is necessary to average N-Prot factorsacross dierent growth conditions to make the N-Prot approach more useful. This has been donesuccessfully for other groups, and the N-Protcalculated for cereals (Mosse, 1990), leaves (Yeoh& Wee, 1994), mushrooms (Mattila et al., 2002),for instance, come from samples obtained indierent places, experiments and/or environmentalconditions.

Conclusions

Our results provide information for a large numberof marine microalgae, in dierent growth condi-tions and growth phases, and permit an assessmentof the variability of the nitrogen distribution incells. Some general trends were found to beindependent of the treatments. The relative pro-portion of PN to TN increases during growth, andthe percentages of NPN are lower in the stationarygrowth phase.The present study establishes that N-Prot factors

are lower than the traditionally used factor of 6.25for all species, regardless of growth phase andculture conditions. The contribution of nitrogenousnon-protein substances was accurately identiedand quantied, as shown in the intracellularnitrogen budgets (Table 2). Possible variations inthe N-Prot factors proposed could be attributed tominor non-protein substances, which were notidentied and quantied. However, concentrationsof these substances seem to be low, since the sum ofthe various forms of nitrogen quantied was alwaysclose to TN.We suggest the use of the mean N-Prot conver-

sion factors calculated for each species in this study(Table 4). Some species (e.g. Synechococcus sub-salsus, Thalassiosira oceanica) show small dier-ences in specic values of N-Prot factors calculatedfor dierent growth phases and treatments. Otherspecies (e.g. Chlorella minutissima, Prorocentrum

minimum) show greater variation among theproposed conversion factors at dierent growthphases and experiments, owing to a markeductuation of NPN in dierent growth conditions.In spite of this variability, the N-Prot factorscalculated here will be more accurate than thetraditional factor 6.25, which overestimates theactual protein content. Researchers can chooseeither the N-Prot factor for each growth phase orthe average N-Prot factor of each species.It is virtually impossible to establish specic N-

Prot factors for all species of marine microalgaecurrently used in research, biotechnology andaquacultural purposes. For microalgal species notyet studied, we recommend the use of the overallmean N-Prot factor of 4.78 for calculating totalprotein concentration from nitrogen content.

Acknowledgements

This work was supported by the Foundation forResearch Support of Rio de Janeiro State (FA-PERJ, grant E-26.171.043/99) and State of SaoPaulo Research Foundation (FAPESP, grant 95/9022-7) and research fellowships to S.O.L. fromFAPERJ and CNPq (National Council for theDevelopment of Science and Technology). Wethank Luzia E. Narimatsu, Adriana Nascimento,and Rosa M.C. Barros (Universidade de SaoPaulo) for their help with CHN and amino acidanalysis, and Dr. Yocie Yoneshigue-Valentin andProf. Ricardo M. Chaloub (Universidade Federaldo Rio de Janeiro) for laboratory facilities. Thanksare due to Dr. John A. Berges (Queens Universityof Belfast, U.K.) and two anonymous referees fortheir critical comments on the manuscript.

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Distribution of intracellular nitrogen in marine microalgae: Calculation of new nitrogen-to-protein conversion factors