Growth, Nutrient Uptake And Chemical Composition Of Sp. And .

Effects of nitrogen starvation on two microalgae2002), as well as to the experimental conditionsapplied, like temperature (Durmaz et al., 2009), lightintensity (Lourenço et al., 2008), and culture medium(Huerlimann et al., 2010), especially in batch cultures.Due to the interaction of the organisms with the culturemedium, a batch culture is under continuous chemicalchange. These variations reflect on the cell metabolismand consequently on their chemical composition(Lourenço et al., 2002). Thus, the chemical composition of a given species may vary widely underdifferent growth conditions, and such changes may berelated to the growth phase of the culture (Costard etal., 2012). However, studies focusing sampling indifferent growth phases are relatively scarce; most ofthe papers report the chemical profile of given speciesin a fixed momentum of the cultivation, ignoring thecontinuing process of interaction between microalgaeand the medium.Nitrogen is added to most culture media in highconcentrations, and changes in N supply are known toinfluence strongly both growth and chemical compositionof microalgae (Valenzuela-Espinoza et al., 1999;Harrison & Berges, 2005). Studies of the effects ofnitrogen sources on the chemical composition indifferent growth phases may give important informationon species metabolism (Fidalgo et al., 1995). On theother hand, this knowledge is also important for bothaquaculture activities and biotechnological applications,in which different culture media (with nutrients invarious chemical forms) may be used (Sepúlveda et al.,2015). Under many culture conditions, microalgae mayexperience nitrogen starvation at least in part of theirgrowth, especially in the stationary growth phase ofbatch cultures (Lourenço et al., 2004). Photosyntheticactivity and nitrogen metabolism are processesintegrally coupled, and a nitrogen limitation affects thephotosynthesis in microalgae by reducing theefficiency of light collection due a decline in cellpigment content (Geider et al., 1993). This physiological stress may promote strong effects on the chemicalcomposition of a given microalga, such as demonstratedby several authors (e.g., Silva et al., 2009; Jiang et al.,2011; Urreta et al., 2014). Production and accumulationof protein, carbohydrate, lipid, and carotenoids are ofparticular importance if the microalgae are cultivatedeither to feed marine animals or to produce specificvaluable substances, for instance (Machado & Lourenço,2008).This paper aimed to assess growth, nutrient uptake,and chemical composition of two marine microalgae,the trebouxiophycean Chlorella sp. and the eustigmatophycean Nannochloropsis oculata under standardized culture conditions and under nitrogen starvation.Growth under nitrogen starvation is thought to induce277protein and chlorophyll decreases, and increments incarbohydrate and lipid productivity of microalgae, butthe intensity of these processes vary widely dependingon the species tested and experimental conditions. Theeffects of the culture conditions on the two microalgaewere discussed in the context of possible use of themicroalgae as food-species in mariculture and asfeedstocks for biofuel production.MATERIALS AND METHODSThe microalgae testedTwo strains were used in this study: Chlorella sp.(division Chlorophyta, class Trebouxiophyceae, strainCMEA BS04), and Nannochloropsis oculata (divisionHeterokontophyta, class Eustigmatophyceae, strainCMEA MO08). Both strains are available at ElizabethAidar Microalgae Culture Collection, FluminenseFederal University, Brazil (Lourenço & Vieira, 2004).Culture conditionsStarter cultures of 150-250 mL in mid-exponentialgrowth phase were inoculated into 5 L of seawater,previously autoclaved at 121 C for 60 min in 6-Lborosilicate flasks. The microalgae were cultured intwo experimental conditions:a) Seawater enriched with Conway nutrient solutions(Walne, 1966) in its original concentrations andcontinuously bubbled with filtered air at a rate of 2 Lmin-1. This experimental condition is designed as“control”.b) The same conditions described in item a, including anew addition of Conway nutrient solutions withoutnitrogen in the 7th day of growth. This procedurepromoted the enrichment of the culture medium withthe theoretical concentrations of all elements of theConway’s receipt (e.g., 128 μM NaH2PO4.H2O, 1.82μM MnCl2.4H2O, 4.81 μM FeCl3.6H2O, etc.), exceptNaNO3. The actual concentrations of the chemicalcomponents available to the microalgae from the 7thday of cultivation corresponded to the sum of theenrichment done plus the residual concentrations of theoriginal substances that had not been taken up yet. Thisexperimental condition is designed as “N–”.Each experiment was carried out in four cultureflasks (n 4), exposed to 350 μmol photons m-2 s-1measured with a Biospherical Instruments QuantaMeter, model QLS100, provided from beneath by 40 Wfluorescent lamps (Sylvania daylight tubes), on a 12:12h light:dark cycle. Mean temperatures in experimentswere 21 1 C and the salinity was 33.0. Growth wasestimated based on daily microscopic cell countingwith Neubauer chambers. Cultures were not buffered

278Latin American Journal of Aquatic Researchand pH was determined daily, at the beginning (15-30min after the start of the photoperiod), in the middle (6h after the start of the photoperiod), and in the end ofthe light period (11 h after the start of the photoperiod).All samplings for cell counts occurred during the first30 min of the light period. The initial cell densities ofcultures were 3.0x104 cell mL-1 for Chlorella sp. and2.5x105 cell mL-1 for N. oculata. The experiments werecarried out for 13 days. Growth rates were calculateddaily using the following equation:r lnNt - lnNt-1 twhere r is the growth rate; ln is the logarithm of thenumber of cells in a given day, in a standardized time(beggining of the photoperiod), calculated using thebasis 2; Nt is the number of cells recorded in a givenday (t); Nt–1 is the number of cells 24 h before thecounting carried out in the time t (in days); t is thedifference, in days, between the two cell countings.The biovolumes of the cells were measured usingthe equation provided by Hillebrand et al. (1999),assuming a spherical shape for both microalgae. Meanvolumes were based on measurements of 30 cells ineach culture flask, giving four mean values used forstatistics (n 4).Sampling procedureEach culture was sampled twice for chemical analysis(on the 7 and 13th days of growth), corresponding tolate-exponential and stationary growth phases. Samplesof 1.6 to 2.3 L were concentrated by centrifugation at8,000 x g for 9.0 min, at least once, using a Sigmacentrifuge, model σ-15, to obtain highly concentratedpellets. Before the last centrifugation, cells werewashed with artificial seawater (Kester et al., 1967)prepared without nitrogen, phosphorus and vitamins,and adjusted to salinity of 10. All supernatants obtainedfor each sample were combined and the number of cellswas determined (using Neubauer chambers) to quantifypossible cell losses. The pellets were frozen at -18 Cand then freeze-dried (using a Terroni-Fauvel, modelLB 1500 device), weighed and stored in desiccatorsunder vacuum and protected from light until thechemical analyses. Samples to be analyzed for chlorophyll and carotenoid were obtained by filtering thecultures under vacuum onto Whatman GF/F glassmicrofibre filters (0.7 μm nominal pore size). Thefiltered samples were kept at -18 C in flasks containingsilica-gel until analysis. All sampling for chemicalanalysis was done during the first 90 min of the lightperiod.Daily samples of the culture medium were taken toevaluate the uptake of dissolved nutrients by themicroalgae. At the first 30 min of the photoperiod,samples of 40 to 60 mL of the cultures from each flaskwere collected. The samples were filtered in the samemanner as described above for photosynthetic pigments.The filtered samples of culture media were kept at-18 C in polyethylene flasks until analysis of dissolvednutrients.Chemical analysisTotal nitrogen and phosphorus were determined byperoxymonosulfuric acid digestion, using a Hach digestor (Digesdhal , Hach Co., model 23.130-20) (Hach etal., 1987). Calibration curves were prepared usingNH4Cl and NaHPO4 as standards of nitrogen andphosphorus, respectively. See Lourenço et al. (2005)for further details.The Lowry et al. (1951) method was used toevaluate protein in the samples, with bovine serumalbumin as a protein standard, following the extractionprocedures proposed by Barbarino & Lourenço (2005).Spectrophotometric determinations were done at 750nm, 35 min after the start of the chemical reaction. Totalcarbohydrate was extracted with 80% H2SO4, according to Myklestad & Haug (1972). The carbohydrateconcentration was determined spectrophotometricallyat 485 nm, 30 min after the start of the chemicalreaction, by the phenol-sulfuric acid method (Dubois etal., 1956), using glucose as a standard. Total lipid wasextracted following Folch et al. (1957), and determinedgravimetrically after solvent evaporation.Chlorophyll-a and carotenoid were extracted in90% acetone at 4 C for 20 h, after grinding the filterswith the samples. Spectrophotometric determination ofchlorophyll-a was carried out as described by Jeffrey &Humphrey (1975), and the determination of totalcarotenoid was carried out as described by Strickland& Parsons (1968).Determinations of ammonia ammonium followedthe procedure proposed by Aminot & Chaussepied(1983), nitrite and nitrate analyses were performedfollowing Strickland & Parsons (1968), and phosphatewas determined according to Grasshoff et al. (1983).All nutrients were analyzed spectrophotometrically.Statistical analysisThe results of growth and chemical composition werecompared using Student's t-test (Zar, 1996), adopting alevel of significance 0.05.All experiments were performed twice in order toconfirm the results. The chemical analyses were alsodone twice, using samples generated by all experimentscarried out. In this paper, we show the results of onlyone set of experiments.

Effects of nitrogen starvation on two microalgae279RESULTSGrowth curves of Chlorella sp. and Nannochloropsisoculata in the two experiments are shown in Figures 1and 2, respectively. For both species, final cell yieldswere similar in the control and in the treatment withnitrogen starvation (P 0.05). Cell densities at the endof the experiments were 6.43x106 cell mL-1 (control)and 5.97x106 cell mL-1 (N-) for Chlorella sp., and6.96x107 cell mL-1 (control) and 6.52x107 cell mL-1 (N-)for N. oculata. For each species, similar growth rateswere found in the experiments throughout theexponential growth phase. For Chlorella sp., growthrates fluctuated around 1.0-1.1 (with basis on log2 ofcell numbers) throughout the exponential growth phase,which generated increases of 2.0 to 2.3-fold of celldensities every 24 h. In the stationary growth phaserates were lower (typically 0.15). For N. oculata,growth rates fluctuated around 1.2-1.3 (with basis onlog2 of cell numbers) throughout the exponentialgrowth phase, which generated increases of 2.3 to 2.7fold of cell densities every 24 h. In the stationarygrowth phase rates were lower (typically 0.10), withsimilar values for the control and the N- experiment.Cell biovolumes of both species did not varythroughout the exponential growth phases of allexperiments (P 0.23, Figures 3-4). However, in thestationary growth phase significant differences werefound, with larger cell biovolumes in N- treatment (P 0.01, Figures 3-4). Chlorella sp. exhibited nosignificant variations in cell biovolumes in the control,with average values around 61.5 µm3 throughout theexperiment. Similar values were found for Chlorella sp.in the exponential growth phase of the N- treatment, butincreases in cell biovolumes were detected throughoutthe stationary growth phase, achieving a peak of 88.7µm3 in the last day of cultivation. Similarly, N. oculatadid not show variations in cell biovolumes in thecontrol, with an average value of 14.1 µm3. In the Ntreatment, cell biovolumes were smaller in theexponential growth phase (average of 14.0 µm3),increasing throughout the stationary growth phase toachieve 18.7 µm3 in the 13th day of cultivation.In both control and N- experiments, pH valuesfluctuated widely throughout the photoperiod (data notshown). Measurements of pH at the start of thephotoperiod gave always lower values, typically ca.8.0. The values of pH increased throughout thephotoperiod and achieved 8.9 after 11 h of light in thesecond half of the exponential growth phase (from day3 to day 7 of growth). In the stationary growth phase(from the 9th day of growth) daily maximum values ofFigure 1. Growth curves of Chlorella sp. cultured in twodifferent experimental treatments: standardized conditions(control) and under nitrogen limitation from the 7th day ofgrowth (N-). Arrows indicate the moments in which cellswere sampled to perform chemical analyses. Each point inthe curves represent the mean of four replicates standarddeviation (n 4).pH were lower (typically 8.8) than in the exponentialphase.There was a remarkable trend of decreasing theconcentrations of dissolved nitrate and phosphatethroughout the experiments (Figs. 5-6). Nitratedepletion occurred in the 10 or 11th day of cultivationin all experiments. Phosphate uptake was almost totalin the control for N. oculata, achieving 1.3 μM at the13th day of cultivation (Fig. 6). For Chlorella sp., in thecontrol the average phosphate concentration was 9.4μM at the 13th day of cultivation, which is equivalent to 92.7% consumption of the inicial concentration ofphosphate added to the culture medium (Fig. 5). In theexperiments N- the enrichment with phosphateoccurred when the cultures still had some 27-34 μM ofthe original dissolved phosphate, generating peaks ofphosphate ( 155 μM) in 7th day of growth, as a resultof the addition of Conway nutrient solutions withoutNaNO3. A high concentration of phosphate was stillpresent in the culture medium at the end of the Nexperiments: 73 μM for Chlorella sp. and 81 μM for N.oculata. Variable concentrations of nitrite (0.0-29.6μM) and ammonia/ammonium (0.0-3.7 μM) were also

280Latin American Journal of Aquatic ResearchFigure 2. Growth curves of Nannochloropsis oculatacultured in two different experimental treatments:standardized conditions (control) and under nitrogenlimitation from the 7th day of growth (N-). Arrows indicatethe moments in which cells were sampled to performchemical analyses. Each point in the curves represent themean of four replicates standard deviation (n 4).detected in all experiments throughout growth (Figs. 56). Nitrite concentrations tended to increase from thebeginning to the end of the exponential growth phase,progressively decreasing until the end of the stationarygrowth phase. Variations in the ammonia/ammoniumconcentrations did not show a clear pattern in theexperiments, with high fluctuations throughout growth,but the values were always negligible in comparison tothe nitrate concentrations.Total concentration of nitrogen and phosphorus incells of Chlorella sp. and N. oculata are showed inTable 1. Results refer to the stationary growth phaseonly. For both species, total nitrogen concentrationswere higher in the control. Conversely, totalphosphorus concentrations were significantly higher atthe end of the N- experiments.Values for protein, carbohydrate, lipid, chlorophylla, and total carotenoid are shown in Tables 2 and 3. Theprotein content did not vary in the control e7xperimentsfor both species (P 0.05). However, the proteincontent decreased significantly from the exponential tothe stationary growth phase for both microalgaecultured in the N- experiments (P 0.01). Thecarbohydrate content increased throughout growth ofboth species in all treatments. In Chlorella sp.,increments of the carbohydrate content from theexponential to the stationary growth phase were moreintense than in N. oculata (Tables 2 and 3), with a peakconcentration of 54.5% of the dry matter (d.m.) in theN- experiment. There was no variation in the lipidcontent of Chlorella sp. in different treatments andgrowth phases, with values fluctuating around 14.5%d.m. (P 0.29). On the other hand, significantvariations occurred in the lipid content of N. oculata inboth treatments and growth phases (P 0.01), withincreases of the lipid content from the exponential tothe stationary growth phase (Table 3). The highestconcentration of total lipid was found in N. oculata inthe N- experiment (33.7% d.m.). Chlorophyll-a concentrations decreased for both species in allexperiments from the exponential to the stationarygrowth phase (P 0.05). Chlorella sp. tended to showhigher values for chlorophyll-a than N. oculata. Novariations in total carotenoid content were found in thecontrol experiments for both species (P 0.19).However, in the N- experiments of both species, totalcarotenoid contend increased from the exponential tothe stationary growth phase (P 0.01). Absoluteconcentrations of total carotenoid tended to be higherin Chlorella sp. than in N. oculata.DISCUSSIONGrowth, nutrient consumption, and cell biovolumesChlorella sp. and Nannochloropsis oculata exhibitedsimilar growth curves in the treatments tested here. Thechemical composition of the culture medium and theinput of carbon in the experimental flasks are two of themain factors that influence the growth of microalgae incultures (Wood et al., 2005). Current experiments wereperformed using the Conway culture medium, whichshows a nutrient-rich composition with 1.18 mM ofnitrogen and 128 µM of phosphorus, besides highconcentrations of trace-metals and other components.In this context, the depletion of nutrients is unlike tooccur quickly in comparison to what typically occurswith other culture media, such as the f/2 culturemedium (Guillard, 1975). By comparison, the f/2culture medium shows only 880 µM of nitrogen and 36µM of phosphorus. The availability of higherconcentrations of nutrients tends to generate a fastergrowth, especially if the cultures are supplied withcarbon sources. Our experiments were run with theaddition of constant aeration, which promotes the inputof CO2 in the culture medium. In aerated cultures aconstant input of carbon is obtained by dissolving moreCO2 from the air into the culture medium. Considering

Effects of nitrogen starvation on two microalgae281Figure 3. Measurements of cell biovolumes of Chlorella sp. cultured in two different experimental treatments: standardizedconditions (control) and under nitrogen limitation from the 7 th day of growth (N-). Values are expressed as μm3, andrepresent the mean of four replicates standard deviation (n 4).Figure 4. Measurements of cell biovolumes of Nannochloropsis oculata cultured in two different experimental treatments:standardized conditions (control) and under nitrogen limitation from the 7th day of growth (N-). Values are expressed asμm3, and represent the mean of four replicates standard deviation (n 4).the coupling of carbon and nitrogen metabolism, asdemonstrated by Turpin (1991), the availability ofcarbon should make the assimilation of nitrogen faster,supplying cells with carbon for amino acid synthesis.As a consequence, aerated cultures tend to run out ofnitrogen faster than non-aerated cultures, especially inthe stationary growth phase of batch cultures (Lavín &Lourenço, 2005). The faster assimilation of nitrogen,coupled to the greater availability of carbon, is probablythe main factor to promote higher final cell yields inaerated cultures. Previous experiments performed byLourenço et al. (2004) with 12 algal species, including

282Latin American Journal of Aquatic ResearchFigure 5. Variations of the a) nitrate, b) nitrite, c) ammonia/ammonium, and d) phosphate concentrationsthroughout the growth of Chlorella sp. in two experimental treatments: standardized conditions (control) andunder nitrogen limi

on growth, nutrient uptake, and gross chemical composition of Chlorella sp. and Nannochloropsis oculata. The control experiments were performed with Conway culture medium in 13-day batch cultures, 12 h photoperiod, and aeration. A second experimental condition was the addition of the nutrients except nitrogen, one week after