Combined effects of irradiance level and carbon source on fatty acid and lipid class composition in the microalga Pavlova lutheri commonly used in mariculture

Journal of Experimental Marine Biology and Ecology 369 (2009) 136–143

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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j e m b e

Combined effects of irradiance level and carbon source on fatty acid and lipid class composition in the microalga Pavlova lutheri commonly used in mariculture Freddy Guihéneuf a,b, Virginie Mimouni a,b,⁎, Lionel Ulmann a,b, Gérard Tremblin a,c a b c

Ecophysiologie et Métabolisme des Microalgues, EA 2160 qMer, Molécules, Santéq, Université du Maine rs Département Génie Biologique, IUT de Laval, 52 rue des D Calmette et Guérin, BP 2045, 53020 Laval Cedex 9, France Faculté des Sciences ex Techniques, Avenue O. Messiaen, 72085, Le Mans Cedex 9, France

a r t i c l e

i n f o

Article history: Received 4 November 2008 Received in revised form 10 November 2008 Accepted 11 November 2008 Keywords: Carbon source Irradiance level Lipid classes n-3 fatty acids Pavlova lutheri

a b s t r a c t Pavlova lutheri, a marine Pavlovophyceae, has been well documented as it is commonly used as a food source in mariculture. In this study, we investigated the combined effects of carbon sources and irradiance levels on the growth, lipid classes and fatty acid profiles of this microalga. The microalga was cultured at 15 °C with a 14 h photoperiod in artificial seawater containing bicarbonate or acetate as carbon source. The growth and lipid composition of P. lutheri were more sensitive to variations in light intensity than in carbon source. However, P. lutheri seems to be able to use acetate to growth cell and lipid metabolism. With the both carbon source, the lowest cellular lipid contents were obtained under low light intensity. The proportions of PUFAs, especially EPA, were significantly higher under low light, and saturating fatty acids and DHA levels were significantly higher under high light. In P. lutheri, galactolipids, a major component of chloroplast lipid membranes, made up approximately 54-66% of total lipids. The highest PUFA levels, such as those of EPA, were predominantly found in the galactolipid fraction when the cells were grown at low light, regardless of the carbon source. The consequent accumulation of n-3 fatty acids in the galactolipids could facilitate thylakoid membrane fluidity, and therefore the velocity of electron flow involved in photosynthesis during light acclimatization. These results could be used to optimize the culture conditions and the nutritional value of this microalga, which is used to feed marine invertebrates and fish larvae in mariculture hatcheries, and to produce n-3 fatty acids for human health care and nutrition. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Microalgae are currently used as food sources for larval and juvenile molluscs, crustaceans and fish species in mariculture hatcheries (Brown et al., 1997). Their nutritional value is often correlated to their biochemical composition; especially their lipid content and fatty acid composition (Thompson et al., 1996). Several microalgal species are known to synthesize and accumulate large amounts of polyunsaturated fatty acids (PUFAs), specifically eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3) (Moreno et al., 1979; Volkman et al., 1989). Long chain n-3 fatty acids are also recognized as having a number of important neutraceutical and pharmaceutical applications (Apt and Behrens, 1999; Ward and Singh, 2005). They are known to have health benefits for the human organism, and to play a major role in preventing medical disorders in three areas: heart and circulation (Dyerberg and Bang, 1979; Din et al., 2004), inflammatory conditions (Babcock et al., 2000), and cancer (Bourre, 2007). As a result, a

⁎ Corresponding author. Département Génie Biologique, IUT de Laval, 52 rue des Drs Calmette et Guérin, BP 2045, 53020 Laval Cedex 9, France. Tel.: +33 243594953; fax: +33 243594958. E-mail address: [email protected] (V. Mimouni). 0022-0981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2008.11.009

number of studies have focused on microalgal culture conditions with the aim of improving the nutritional value of algal foods (aquaculture) and/or to boost PUFA production (pharmaceutical industry). It is well known that environmental factors (temperature, salinity, light, nutrients, etc.) and culture time affect the growth and biochemical composition of microalgae (Reitan et al., 1994; De Castro Araújo and Tavano Garcia, 2005; Liang et al., 2006; Ranga Rao et al., 2007). Specifically, significant variations in lipid contents and fatty acid profiles are observed in response to differing growth conditions (Yongmanitchai and Ward,1991; Carvalho et al., 2006; Petkov and Garcia, 2007). Much of the published data have reported the influence of temperature on the fatty acid composition of microalgae (Nagashima et al., 1995; Rousch et al., 2003). It is generally recognized that an increase in PUFAs might be one of the ways marine algae acclimatize to low-temperature conditions and maintain membrane fluidity (Tatsuzawa and Takizawa, 1995; Jiang and Gao, 2004). The fatty acid composition is also known to be affected by the level of irradiance (Blanchemain and Grizeau, 1996; Guihéneuf et al., 2008). However, the light conditions that produce the highest proportion of the essential fatty acids are species specific (Thompson et al., 1990). In most species, the highest proportions of EPA were obtained at low levels of irradiance, whereas DHA levels were highest under high light intensities (Thompson et al., 1990; Brown et al., 1993). High PUFA

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levels were observed at low light, suggesting that the biochemical composition of chloroplasts was adapting to low irradiance intensities by increasing PUFA synthesis (Sukenik et al., 1989; Khotimchenko and Yakovleva, 2005). Previous studies have also reported that the carbon source affects growth and fatty acid composition in numerous species (Wood et al., 1999; Wen and Chen, 2003). Under natural conditions, the carbon source usually used for photosynthesis by algae is sodium bicarbonate (NaHCO3) and/or carbon dioxide (CO2). Furthermore, many microalgae are able to grow rapidly and heterotrophically in the dark using organic carbon sources such as sugars or organic acids (Wen and Chen, 2003; Cerón García et al., 2005). When the cells are exposed to light, some microalgae can utilize both inorganic and organic carbon simultaneously, and develop a mixotrophic metabolism (Poerschmann et al., 2004). Wood et al. (1999) have previously reported that acetate is a good carbon source for photoheterotrophic growth, yielding reasonably high PUFA levels in freshwater algae. However, acetate has a negative effect on the growth of some marine microalgae (Wood et al., 1999), such as Phaeodactylum tricornutum (Cerón García et al., 2005). Consequently, strategies to improve algal biomass and fatty acid production under indoor conditions whilst keeping production costs low (dependent on the need for light) involve mixotrophic, photoheterotrophic or heterotrophic algal growth systems (De Swaaf et al., 1999; Wood et al., 1999; Cerón García et al., 2005). Pavlova lutheri (Droop) Green, the organism investigated here, is a common Pavlovophyceae used in microalgal laboratories, known to produce high levels of PUFAs, especially EPA and DHA (Tatsuzawa and Takizawa, 1995; Carvalho et al., 2006), and which is commonly used in the aquaculture industry to feed marine organisms (Thompson et al., 1996; Leonardos and Lucas, 2000; Ponis et al., 2006a,b). Some growth conditions, such as temperature and nutrients, have already been described for this microalga (Tatsuzawa and Takizawa, 1995; Carvalho and Malcata, 2000; Carvalho et al., 2006). However, no studies have so far reported the combined effects of light intensity and carbon source on the lipid composition of P. lutheri. We set out to investigate the simultaneous impacts of irradiance level and carbon source on growth, lipid class and fatty acid composition of P. lutheri. To do this, cells were cultured at different irradiance levels after changing the carbon source in the culture medium by replacing sodium bicarbonate with sodium acetate.

(1.11 µM f.c.). The culture medium contains 0.174 g l- 1 sodium bicarbonate or 0.169 g l- 1 sodium acetate as the carbon source. These two concentrations correspond to 2.07 mM which is the usual concentration of bicarbonate in artificial seawater. The medium was prepared with deionized water, and had been autoclaved at 121 °C for 20 min. Inocula were carried out using exponentially growing cells, after two generations without antibiotic/antimycotic, and with an initial density of 105 cell ml- 1. For each carbon source, the cultures were maintained at 15 ± 1 °C under three irradiance levels: low light intensity (LL-20) with 20 µmol photons m- 2 s- 1; medium light intensity (ML-100) with 100 µmol photons m- 2 s- 1; and high light intensity (HL-340) with 340 µmol photons m- 2 s- 1; provided by cool-white fluorescent lamps (PHILIPS 18 W), attenuated by distance and/or neutral density screening, and using 14/10 h light/dark cycle. Light intensity was measured as PAR (Photosynthetic Active Radiation) using a 4π US-SQS/L Quantum Sensor (Walz Instruments, Effeltrich, Germany), coupled to a LI-189 Data Logger (LI-COR Biosciences, ScienceTec, Les Ulis, France). Three replicate cultures were grown under each irradiance level and with each carbon source.

2. Material and methods

2.3. Extraction of total lipids

2.1. Microalgal cultures

All chemicals used in the experiments were of analytical grade, and were purchased from Carlo Erba (Val de Reuil, France). Total lipids were extracted with methanol/chloroform (2/1, v/v) after adding 200 µL of 2.8 g l- 1 NaCl, a modified version of Bligh and Dyer's (1959) method, using manual crushing (Dounce cells grinders) coupled with ultrasonication (twice, for respectively 15 and 30 min). Chloroform (1 ml) was added between the two ultrasonication steps in order to allow phase separation. The chloroform layer, containing the lipids, was collected and second extraction was carried out by adding 2 ml of chloroform to the remaining methanol/water phase. The solvents were removed by evaporating under vacuum, and all samples were dissolved in a known volume of chloroform. The lipid extracts were stored at -20 °C under nitrogen gas (N2) to limit oxidization until analysis.

Pavlova lutheri cells were obtained from the microalgal culture collection of the “Centre de Ressources Biologiques” (Université de Nantes, France). After three successive cultures with 1% antibiotic/ antimycotic (A5955, Sigma-Aldrich, St. Quentin Fallavier, France; formulated to contain 10000 units ml- 1 penicillin G, 10 mg ml- 1 streptomycin sulfate, 25 µg ml- 1 amphotericin B), an axenic culture was obtained. Culture “axenicity” was tested on two medium, A (bactopectone, 3 g l- 1; yeast extract, 1 g l- 1; ammonium sulfate, 1 g l- 1; sodium glycerophosphate, 25 mg l- 1; iron (Fe3+)-EDTA, 6 mg l- 1; ASW 50%, qsp 1000 ml) and B (bacto-pectone, 4 g l- 1; yeast extract, 500 mg l- 1; sodium glycerophosphate, 25 mg l- 1; iron (Fe3+)-EDTA, 6 mg l- 1; ASW 75%, qsp 1000 ml), respectively conductive to the growth of fungi and bacteria. The microalgae were then grown under batch conditions in 500 ml Erlenmeyer flasks sealed with cotton plugs that allow exchanges with atmosphere (carbon dioxide and oxygen released during microalgae respiration and photosynthesis, respectively), using a working volume of 300 ml. The culture medium was artificial seawater (Harrison et al.,1980) complemented following De Brouwer et al. (2002). The medium enrichment was composed as previously described by Perkins et al. (2006): Fe-NH4-citrate (1.37 µM final concentration), CuSO4 5H20 (0.04 µM f.c.), folic acid (0.18 nM f.c.), nicotinic acid (0.0325 µM f.c.), thymine (0.95 µM f.c.), Ca-d-pantothenate (8.39 nM f.c.) and inositol

2.2. Growth, chlorophyll a and lipid contents After taking a sample of 2 ml in a laminar flow hood, growth was monitored daily by spectrophotometric measurements of the optical density of cell suspensions at 750 nm (Y = 10- 7 X, with Y the optical density at 750 nm and X the cell density), by using a 2.5 ml plastic cuve. The cell number was determined with a Malassez improved bright-line hemocytometer, after immobilizing the cells with Lugol 5%. No special precautions have been taken to prevent carbon dioxide re-equilibration during daily sub-sampling. Specific growth rates (µ, d- 1) were calculated from the increase in cell density during the exponential growth phase, between days 12 and 19, according to the equation µ = ln (N2/N1)/(t2-t1), where N is cell density and t is the time. Pigments were extracted in dimethylformamide, and total chlorophyll a (Chl a) was determined by spectrophotometry (Speziale et al., 1984). Cells were gently harvested by centrifuging at low-speed (1200 g, 10 min) using a Sigma 4K15 centrifuge (Bioblock Scientific, Illkirch, France). The pellets obtained were then frozen, and stored at - 70 °C prior to analysis. Chl a and lipid contents were estimated during the mid-exponential growth phase.

2.4. Lipid class separation Total lipid extracts were fractionated on reversed phase silica gel columns (Sep-Pak Plus silica cartridges, Waters, St. Quentin en Yvelines, France) after an activation step with 20 ml of methanol followed by 20 ml of chloroform. Neutral lipids were eluted using 20 ml of chloroform, polar lipids such as galactolipids were eluted with 40 ml of chloroform/methanol (5/1, v/v), and the phospholipids were recovered in 30 ml of methanol (Sukenik et al., 1989). The fractions were reduced in volume by evaporating under a stream of N2.

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Fig. 1. Cell density (CD, day 26) expressed as ×106 cell ml- 1 (□), and mid-exponential growth phase lipid content expressed as µg × 106 cell- 1 ( ) of P. lutheri cultured at 15 ± 1 °C and 100 µmol photons m- 2 s- 1, in artificial seawater without added carbon, and with two different bicarbonate (A) and acetate (B) concentrations (2.07 mM and 5 mM). Results are expressed as the mean ± SD (n = 3). For each parameter, after one-way ANOVA, Student-Newman-Keuls (SNK) test results are arranged in increasing order from left to right: a b b.

Thin-layer chromatography (TLC) of the total lipid extract was carried out in order to check the purity of each fraction (Henderson and Tocher, 1992). 2.5. Quantification of lipids Lipids were quantified using the sulfuric acid charring method (Marsh and Weinstein, 1966), using palmitic acid as a standard. The sulfuric acid charring method gave reproducible results, and made it possible to carry out quantitative determinations of lipid contents in small culture volumes. 2.6. Preparation and fatty acid analyses The solvent was then evaporated under N2, and the total fatty acids were extracted after saponifying with CH3OH-NaOH 0.5 M at 80 °C for 20 min, according to the method of Slover & Lanza (1979). Total fatty acid methyl esters (FAMEs) were formed directly by treating the total extracts with boron trifluoride-methanol (BF3-MeOH, Sigma-Aldrich, St. Quentin Fallavier, France) at 80 °C for 20 min. The FAMEs were then extracted with iso-octane and 35% NaCl. All samples were analyzed with a FOCUS gas chromatography apparatus (Thermo Electron Corporation, Les Ulis, France) equipped with a flame ionization detector, and a fused-silica capillary column (CP Sil-88 25 m × 0.25 mm id capillary column, Varian, Les Ulis, France). Samples were injected using an autoinjector AI 3000 (Thermo Electron

Corporation, Les Ulis, France). The injector and detector temperatures were 250 and 280 °C, respectively, and the oven temperature was increased from 120 to 220 °C at a rate of 6 °C min- 1. N2 was used as the carrier gas. Pure standards (Sigma-Aldrich, St. Quentin Fallavier, France) were used to identify the fatty acids by comparing the peak retention times of the samples and standards. Pentadecanoic acid was used as an internal standard to quantify the fatty acid content. 2.7. Using of acetate A preliminary study has been also realized in order to test the use of acetate by P. lutheri. The microalgae were grown under batch conditions at 15 ± 1 °C and 100 µmol photons m- 2 s- 1, in artificial seawater without added carbon, and with two different bicarbonate and acetate concentrations (2.07 mM and 5 mM). Then the growth was followed daily and lipid content quantified during mid-exponential growth phase. For each carbon condition, cell density (CD) was determined at the end of experiment at the same time (day 26). 2.8. Data statistical analysis The results of carbon concentration on the cell density (CD, day 26) and mid-exponential growth phase lipid content (Fig. 1A and B) were analysed by one-way analysis of variance (ANOVA). Student-NewmanKeuls (SNK) multiple comparison test were used to test the differences

Fig. 2. Growth curves of P. lutheri cultured with bicarbonate (A) or acetate (B) as carbon source. Results are expressed as the mean ± SD (n = 3). Cells were illuminated with three irradiance levels: LL-20 (●), ML-100 (▲), HL-340 (■).

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between treatment groups. Differences were considered significant at a probability level 0.05. Two factor ANOVA was used to examine the effect of irradiance level (LL-20, ML-100 and HL-340) and carbon source (bicarbonate and acetate) on the growth rates (µ), chlorophyll a content (Chl a), fatty acid compositions and relative proportions of the major lipid fractions (neutral lipids, galactolipids and phospholipids). Irradiance level and carbon source were all considered fixed factors. P b 0.05 was accepted for significant differences. Post hoc analyses were made by StudentNewman-Keuls (SNK) test. All statistics were performed with SigmaStat (version 3.1) software (SPSS Incorporation, Erkrath, Germany). 3. Results 3.1. Preliminary study: effect of bicarbonate and acetate concentration on cell growth and lipid content The result reported in Fig. 1A and B show that cell density (day 26) and lipid content (mid-exponential growth phase) were significantly enhanced with increasing acetate and bicarbonate concentrations. Moreover, P. lutheri can sustain cell growth in artificial seawater without added carbon. In this condition, cell densities account for 86 and 83% of cell densities respectively obtained with bicarbonate and acetate concentrations of 5 mM. 3.2. Algal growth and chlorophyll a content The results reported in Fig. 2A and B are the growth curves when P. lutheri was grown with bicarbonate or acetate, at different irradiance levels. These curves show growth significant differences between the three irradiance levels tested (LL-20, ML-100 and HL-340). Indeed, these results are confirmed by Table 1 that shows variations in the average growth rate (µ) and Chl a content. Few significant differences were observed between the two carbon sources with regard to growth rate. However, the growth rates measured at ML-100 (0.173 ± 0.008 d- 1 and 0.181 ± 0.011 d- 1 with bicarbonate and acetate, respectively) were significantly higher than those observed at LL-20 and HL-340. Finally, the results also show that the cellular Chl a content increased with decreasing irradiance levels when the cells were cultured with either carbon source. The highest Chl a content was observed at LL-20 in presence of acetate. 3.3. Lipid content and major lipid fractions The results reported in Fig. 3 show that cellular lipid contents were significantly lower under growth-limiting irradiance than under the

Fig. 3. Lipid content (µg × 106 cell- 1) in P. lutheri cells cultured with different carbon sources (Bc: bicarbonate, and Ac: acetate) and three irradiance levels A. Results are expressed as the mean ± SD (n = 3). A See footnote to Table 1.

other light intensities. The proportions of the major lipid fractions of P. lutheri grown at the three irradiance levels with different carbon sources are shown in Fig. 4. Under all culture conditions, galactolipids represented the major lipid fraction with 54-66% of total lipids. The neutral lipids and phospholipids were estimated as 29-40% and 5-11% of the total lipid, respectively. The most obvious effect of HL-340 was an increase in galactolipids correlated with a decrease in neutral lipids with bicarbonate. However, no significant difference was observed in the different lipid fractions depending on the irradiance with acetate. Consequently, under HL-340, the percentages of neutral lipids were significantly higher, and those of galactolipids were significantly lower with acetate than with bicarbonate. Finally, a small decrease in the proportions of phospholipids was also obtained at LL-20 and HL-340 with bicarbonate. 3.4. Fatty acid composition Table 2 shows the total lipid fatty acid composition as a function of the irradiance level, for P. lutheri cells grown with bicarbonate and acetate. Whatever the culture conditions, PUFAs corresponded to approximately 45-55% of the TFA, while saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) never reached more than 30% and 23%, respectively. Fatty acids were more sensitive to variations in light intensity than in carbon source. The SFAs were significantly increased at HL-340, while only small changes in MUFAs

Table 1 Growth rate and chlorophyll a content of P. lutheri cultured under different irradiance levels and with different carbon sources A Irradiance level

Carbon source

µ (d- 1)

Chl. a

LL-20

Bicarbonate Acetate Bicarbonate Acetate Bicarbonate Acetate

0.118 ± 0.008 a 0.111 ± 0.011 α 0.173 ± 0.008 c 0.181 ± 0.011 γ 0.145 ± 0.009 b 0.136 ± 0.002 β

0.36 ± 0.02 c 0.40 ± 0.02 γ,⁎ 0.26 ± 0.01 b 0.265 ± 0.01 β 0.233 ± 0.01 a 0.233 ± 0.02 α

ML-100 HL-340

1

(µg 106 cell- 1)

Results are expressed as the mean ± SD (n = 3). µ: growth rates; Chl a: chlorophyll a content. LL-20: 20 µmol photons m- 2 s- 1; ML-100: 100 µmol photons m- 2 s- 1; HL-340: 340 µmol photons m- 2 s- 1. 1 Chl a content was estimated during mid-exponential growth phase under ML-100 and HL-340 conditions, and at the end of culture under LL-20. A After two-way ANOVA, Student-Newman-Keuls (SNK) multiple comparison test results are arranged in increasing order from left to right: a b b b c (effect of irradiance level with bicarbonate) and α b β b γ ( effect of irradiance level with acetate). For each irradiance level, acetate mean values assigned with asterisk (⁎) are significantly different from bicarbonate (SNK test, P b 0.05).

Fig. 4. Relative proportions (% of total lipids) of the major lipid fractions in P. lutheri cells cultured with different carbon sources (Bc: bicarbonate, and Ac: acetate) and irradiance levels. Results are expressed as the mean ± SD (n = 3). Total lipids were divided into three fractions: neutral lipids ( ), galactolipids (■) and phospholipids ( ).

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Table 2 Total lipid fatty acid composition (% molar) of P. lutheri cultured under different irradiance levels and with different carbon sources A Irradiance level Carbon source

LL-20

ML-100

HL-340

Bicarbonate

Acetate

Bicarbonate

Acetate

Saturated fatty acids 14:0 16:0 18:0 Sum SFAs

Bicarbonate

Acetate

10.1 ± 2.2 14.1 ± 0.3 a 1.7 ± 0.8 a 25.8 ± 1.2 a

9.8 ± 0.8 12.6 ± 1.3 α,⁎ 1.5 ± 0.5 α 23.9 ± 1.6 α

10.4 ± 0.2 14.4 ± 0.3 a 1.8 ± 0.1 a 26.6 ± 0.4 a

10.5 ± 0.2 14.1 ± 0.3 1.7 ± 0.4 26.3 ± 0.5

9.8 ± 1.5 16.0 ± 0.8 b 3.2 ± 0.2 b 29.0 ± 1.3 b

8.7 ± 0.6 16.4 ± 1.0 γ 4.6 ± 0.1 β,⁎ 29.7 ± 1.4 γ

Monounsaturated fatty acids 16:1 n-7 18:1 n-7 + 18:1 n-9 Sum MUFAs

15.3 ± 1.1 2.1 ± 0.3 17.3 ± 0.8 a

15.6 ± 1.1 α 2.3 ± 0.3 α 17.9 ± 1.3 α

17.0 ± 0.6 3.7 ± 0.3 20.6 ± 0.8 b

17.8 ± 0.5 β 4.7 ± 1.8 β 22.6 ± 1.7 β,⁎

16.4 ± 0.4 2.9 ± 0.2 19.3 ± 0.6 b

15.8 ± 1.1 α 3.8 ± 0.4 β 19.6 ± 0.7 α

Polyunsaturated fatty acids 18:2 n-6 18:3 n-3 18:4 n-3 20:4 n-6 20:5 n-3 22:5 n-6 22:6 n-3 Sum PUFAs Unidentified n-3 n-6

0.8 ± 0.1 a 0.5 ± 0.1 b 9.2 ± 0.2 c Tr 28.9 ± 0.4 b 0.7 ± 0.3 12.6 ± 0.2 a 52.7 ± 0.8 b 4.1 ± 1.2 51.2 ± 0.5 b 1.5 ± 0.4

1.0 ± 0.2 α 0.5 ± 0.1 α 11.2 ± 0.8 γ,⁎ Tr 28.0 ± 1.5 β 1.0 ± 0.9 13.2 ± 0.4 α 54.9 ± 2.0 β 3.3 ± 1.2 52.9 ± 2.6 β 2.0 ± 0.7 α

1.3 ± 0.1 b 0.9 ± 0.1 c 6.7 ± 0.1 b 0.2 ± 0.1 22.7 ± 0.1 a 0.7 ± 0.1 16.6 ± 0.9 b 48.6 ± 0.9 a 4.2 ± 0.2 46.4 ± 1.1 a 2.2 ± 0.2

1.3 ± 0.2 β 0.7 ± 0.1 β 6.9 ± 0.2 β 0.2 ± 0.1 22.1 ± 0.5 α 1.6 ± 0.9 14.6 ± 0.2 α,⁎ 47.4 ± 1.1 α 3.7 ± 0.3 44.3 ± 0.9 α 3.1 ± 1.1 β,⁎

1.0 ± 0.1 a 0.2 ± 0.3 a 5.2 ± 0.5 a Tr 22.5 ± 1.2 a 0.6 ± 0.1 19.2 ± 0.5 c 48.7 ± 2.1 a 3.1 ± 0.8 47.1 ± 2.0 a 1.7 ± 0.1

1.1 ± 0.1 α 0.2 ± 0.2 α 4.6 ± 0.3 α 0.2 ± 0.2 21.7 ± 2.0 α 0.6 ± 0.1 18.0 ± 1.8 β 46.3 ± 0.5 α 4.4 ± 0.3 44.5 ± 0.6 α 1.8 ± 0.2 α

β α β

Results are expressed as the mean ± SD (n = 3). SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; LL-20: 20 µmol photons m- 2 s- 1; ML100: 100 µmol photons m- 2 s- 1; HL-340: 340 µmol photons m- 2 s- 1. A See footnote to Table 1.

were observed. The proportions of PUFAs, specifically 18:4 n-3 and 20:5 n-3 (EPA), were significantly higher under LL-20. However, the DHA level revealed a significant increase at HL-340. Finally, under LL20, the highest n-3 fatty acid levels accounted for more than 50% of TFA whatever the carbon source. The results reported in Table 3 show the fatty acid composition in the neutral lipids of P. lutheri. The main fatty acids in this fraction were SFAs and MUFAs, which corresponded 33-39% and 33-47% of the TFA, respectively. The highest MUFA levels were observed at low irradiance with bicarbonate whereas the lowest MUFA proportions were obtained at maximum irradiance with acetate. When cells are cultivated with bicarbonate, the percentage of DHA was increased with growing irradiance whilst EPA levels kept constant. However, maximum irradiance with acetate rendered low both EPA and DHA levels by comparison with bicarbonate. Nevertheless, EPA and DHA never accounted for more than 8.1% of the TFA in neutral lipids. Table 4 shows the fatty acid composition in galactolipid fraction when P. lutheri cells were cultured in medium with acetate or bicarbonate at all three irradiance levels. The main fatty acids in this fraction were SFAs and PUFAs, which accounted for 25-35% and 3949% of TFA respectively. A significant increase in SFAs was observed as the irradiance level increased with acetate. Whatever the carbone source, the highest percentages of PUFAs in galactolipids, and specifically those of EPA, were obtained at LL-20; whereas the highest

DHA levels were obtained at HL-340. A significant n-3 PUFA level decrease (specifically EPA) was observed at HL-340, in presence of acetate by comparison with bicarbonate. Table 5 shows the impact of the irradiance level and carbon source on the phospholipid fraction fatty acid composition of P. lutheri. The major fatty acids in this fraction were PUFAs and SFAs, which corresponded to 58-70% and 21-28% of TFAs, respectively. The results show that the EPA and DHA levels reached 21-25% and 34-45% respectively of the TFAs in phospholipids of P. lutheri. The percentage of DHA in phospholipids reached approximately 45% when the algae were grown at HL-340 with bicarbonate. In summary, the highest PUFA levels (particularly those of DHA) in phospholipids were obtained with bicarbonate at ML-100 and HL-340. Finally, in Pavlova lutheri, EPA was mainly found in galactolipids, 57-67% of total EPA content; while DHA was mainly present in the phospholipids, 61-82% of total DHA content in total lipids (data not shown). 4. Discussion 4.1. Using acetate by algae Adding acetate as adding bicarbonate in artificial seawater without added carbon could allow the increase in growth and lipid content of

Table 3 Neutral lipid fatty acid composition (% molar) of P. lutheri cultured under different irradiance levels and with different carbon sources A Irradiance level Carbon source

LL-20 Bicarbonate

Acetate

Bicarbonate

Acetate

Bicarbonate

Acetate

SFA MUFA PUFA Unidentified 20:5 n-3 22:6 n-3 n-3 n-6

33.4 ± 4.1 47.4 ± 0.1 b 8.4 ± 5.6 a 10.8 ± 1.5 b 2.2 ± 2.0 a 2.2 ± 1.4 a 5.9 ± 4.8 a 2.5 ± 0.7 a

34.5 ± 0.7 39.7 ± 2.0 β,⁎ 16.9 ± 4.4 α,⁎ 8.9 ± 1.7 4.2 ± 0.9 α 4.2 ± 1.6 11.9 ± 3.1 α 5.1 ± 1.3

35.7 ± 6.9 37.1 ± 5.4 a 22.1 ± 2.5 b 5.2 ± 1.3 a 6.6 ± 1.8 b 5.4 ± 1.8 b 17.3 ± 4.0 b 4.8 ± 2.2 b

33.0 ± 0.9 37.1 ± 3.2 β 23.5 ± 2.3 β 6.5 ± 0.2 6.3 ± 0.4 β 5.1 ± 0.1 18.9 ± 1.7 β 4.6 ± 0.7

33.8 ± 3.2 33.3 ± 1.4 a 27.0 ± 1.7 b 5.9 ± 0.4 a 6.5 ± 0.3 b 8.1 ± 0.2 c 22.2 ± 0.8 b 4.9 ± 0.9 b

38.5 ± 2.3 29.8 ± 1.1 α 25.1 ± 2.2 β 6.6 ± 0.7 3.7 ± 0.8 α,⁎ 5.6 ± 1.3⁎ 18.4 ± 2.5 β 6.8 ± 0.5

A

See footnote to Table 2.

ML-100

HL-340

F. Guihéneuf et al. / Journal of Experimental Marine Biology and Ecology 369 (2009) 136–143

141

Table 4 Galactolipid fatty acid composition (% molar) of P. lutheri cultured under different irradiance levels and with different carbon sources A Irradiance level Carbon source

LL-20 Bicarbonate

Acetate

Bicarbonate

Acetate

Bicarbonate

Acetate

SFA MUFA PUFA 20:5 n-3 22:6 n-3 Unidentified n-3 n-6

27.8 ± 1.3 a 17.8 ± 1.9 a 49.6 ± 2.0 c 31.3 ± 0.6 c 4.4 ± 3.5 a 4.9 ± 0.4 b 47.9 ± 1.7 c 1.7 ± 0.3

25.6 ± 0.1 α 19.1 ± 0.5 α 50.7 ± 1.1 β 29.1 ± 1.6 γ 4.6 ± 0.1 α 4.6 ± 0.5 48.5 ± 1.4 β 2.3 ± 0.4

32.4 ± 0.4 b 23.0 ± 0.4 b 40.3 ± 0.8 a 20.3 ± 0.8 a 4.1 ± 0.7 a 4.3 ± 0.6 b 35.9 ± 3.1 a 4.4 ± 2.3

30.2 ± 2.7 β 24.7 ± 0.8 γ,⁎ 41.2 ± 3.1 α 22.6 ± 1.4 β 3.1 ± 1.6 α 4.0 ± 0.5 38.1 ± 3.9 α 3.1 ± 0.9

30.6 ± 0.3 b 21.7 ± 0.8 b 44.5 ± 0.9 b 23.6 ± 0.8 b 7.8 ± 0.9 b 3.2 ± 0.3 a 42.0 ± 1.5 b 2.5 ± 0.6

34.2 ± 0.5 γ,⁎ 22.9 ± 0.4 β 38.9 ± 0.3 α,⁎ 18.5 ± 0.6 α,⁎ 9.2 ± 0.4 β 4.1 ± 0.1 36.9 ± 0.6 α,⁎ 2.0 ± 0.3

A

ML-100

HL-340

See footnote to Table 2.

P. lutheri (Fig. 1A and B). These findings support the hypothesis that P. lutheri could use acetate to cell growth and lipid metabolism. However, the capacities of P. lutheri to grow in artificial seawater without added carbon could be explain by the experimental method. Indeed, it seems possible that algal respiration is the source of CO2 which can be dissolved in the medium throughout the cell growth. These results support that P. lutheri could function mixotrophically with inorganic carbon (dissolved CO2) and organic carbon (added acetate), simultaneous. However, doubts may therefore exist because dissolved inorganic carbon (CO2) could hide the possible photoheterotrophic mechanisms. Further work is in progress to confirm the use of acetate by P. lutheri, then to investigate the effects of irradiance levels on synthesis of fatty acids by labeling cells in vivo with [14C]bicarbonate or [14C]-acetate. 4.2. Combined effects of irradiance level and carbon source on algal growth Similar growth of P. lutheri cultured with bicarbonate or acetate was observed, whatever the irradiance levels tested (Fig. 2A and B). Moreover, the use of acetate (organic carbon source) did not increase algal growth (Table 1) under LL-20. In fact, it looks as though that the usually autotrophic alga P. lutheri was able to grow in presence of acetate, but was unable to increase its growth under conditions close to heterotrophy (acetate and LL-20). The average growth rates observed at 15±1 °C suggested that the cells were light-limited at LL-20, and lightsaturated at HL-340. Under similar conditions (18 °C, from 60 to 240 µmol photons m- 2 s- 1), Carvalho and Malcata (2003) have also reported a decrease in growth parameters suggesting that the effect of irradiance level was correlated with cell biomass in P. lutheri. 4.3. Combined effects of irradiance level and carbon source on lipid and total fatty acid contents Previous studies of the biochemical composition have already showed that marine microalgae can acclimatize themselves to variations in light by changing the content of structural and storage substances such as lipids (Khotimchenko and Yakovleva, 2005). As described by

Brown et al. (1993) for Isochrysis galbana (Haptophyceae) and Sukenik et al. (1989) for Nannochloropsis sp. (Eustigmatophyceae), the lowest cellular contents of total lipids in P. lutheri occurred at LL-20. The specific function of each lipid class in algae is well known, and depends on their chemical structure (Khotimchenko and Yakovleva, 2005). According to the results observed by Volkman et al. (1989) in nine different algal species, during the logarithmic growth phase, the results show that polar lipids (galactolipids plus phospholipids) constitute the major lipid class in P. lutheri (60-71% of total lipids) while neutral lipids (triacylglycerols) account for only 29-40% of total lipids. This finding is comprehensible because polar lipids, especially galactolipids, play key roles during the exponential growth phase as structural components of cellular chloroplastic and thylakoidal membranes associated with the regulation of photosynthesis mechanisms (Mock and Kroon, 2002; Khotimchenko and Yakovleva, 2005). In our study, the proportions of neutral lipids and polar lipids were only slightly affected by irradiance levels. In general, the neutral lipids tended to increase, whereas the galactolipids decreased under HL-340 (Sukenik et al., 1989; Sukenik and Yamaguchi, 1993; Khotimchenko and Yakovleva, 2005), especially during the stationary phase (Brown et al., 1996; Mansour et al., 2003). In contrast, during the exponential phase, our results showed an increase in galactolipids linked to a decrease in neutral lipids when cells were exposed to high light intensity with bicarbonate as carbon source. High irradiance levels are potentially damaging to algal photosynthetic systems (Brown et al., 1993), and may induce synthesis of the galactolipids involved in thylakoid membranes in response to varying light acclimatization. 4.4. The influence of irradiance level on fatty acid composition is closely linked to photosynthesis mechanisms The influence of irradiance levels on the biochemical composition could be related to the carbon source. When algae are cultured with inorganic carbon as their sole carbon source, growth depends on the availability of light for all their energy and biomass production needs (Wood et al., 1999). Consequently, the lipid and fatty acid compositions are affected by light intensity. Some species, such as Chaetoceros simplex, Tichocarpus crinitus and Nannochloropsis spp., are known to

Table 5 Phospholipid fatty acid composition (% molar) of P. lutheri cultured under different irradiance levels and with different carbon sources Irradiance level Carbon source

LL-20 Bicarbonate

Acetate

Bicarbonate

Acetate

Bicarbonate

Acetate

SFA MUFA PUFA 20:5 n-3 22:6 n-3 Unidentified n-3 n-6

28.5 ± 0.8 b 11.0 ± 3.3 58.4 ± 4.6 a 21.0 ± 2.2 33.6 ± 2.8 a 2.1 ± 2.0 55.9 ± 4.3 a 2.6 ± 0.3

25.2 ± 3.9 7.5 ± 0.6 66.5 ± 5.1 ⁎ 23.0 ± 3.4 38.9 ± 0.7 α,⁎ 0.8 ± 0.7 63.0 ± 4.2 ⁎ 3.6 ± 0.9

21.8 ± 1.4 a 7.5 ± 1.2 70.4 ± 2.3 b 25.2 ± 1.8 41.9 ± 2.5 b 0.2 ± 0.4 67.7 ± 2.1 b 2.7 ± 0.3

24.6 ± 2.4 10.6 ± 2.9 64.1 ± 1.6 ⁎ 23.6 ± 1.7 37.4 ± 0.5 α,⁎ 0.7 ± 1.1 61.8 ± 1.1 ⁎ 2.4 ± 0.6

21.3 ± 1.2 a 7.3 ± 0.8 71.3 ± 1.2 b 23.0 ± 1.2 44.9 ± 2.6 b 0.2 ± 0.3 68.7 ± 1.5 b 2.6 ± 0.5

25.8 ± 1.3 ⁎ 8.7 ± 0.1 65.5 ± 1.2 ⁎ 21.0 ± 1.7 41.2 ± 0.4 β nd 63.2 ± 1.2 ⁎ 2.3 ± 0.1

A

See footnote to Table 2.

ML-100

A

HL-340

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produce higher levels of EPA at low light intensity (Sukenik et al., 1989; Thompson et al., 1990; Khotimchenko and Yakovleva, 2005). In contrast, the diatom Skeletonema costatum has been shown to contain lower EPA levels at low light intensity (Blanchemain and Grizeau, 1996; Guihéneuf et al., 2008). Thompson et al. (1996) reported that P. lutheri contained higher levels of the main fatty acids 16:0 and 22:6 n-3 (DHA) and less 18:4 n-3 and 20:5 n-3 (EPA) when light intensity was high than when it was low, suggesting that the increase in total SFA was correlated to the decrease in total PUFA under high irradiance levels. With regard to the cellular Ch a content, the data reported (Table 1) show higher Chl a at lower irradiance. This phenomenon has frequently been identified in other microalgae (Mouget et al., 1999). Under low light intensity, the cells increase their photosynthetic pigments, such as Chl a, in order to maximize their ability to harvest light (Mock and Kroon, 2002). An association between fatty acids and pigments has been also suggested by the positive correlation between cellular Chl a content and PUFA levels (Sukenik et al., 1989; Thompson et al., 1990; Blanchemain and Grizeau, 1996). When P. lutheri was grown with bicarbonate, our results show a relationship between Chl a and PUFAs (especially EPA), which supports the hypothesis that links may exist between the phytol chain of Chl a and PUFAs in microalgae (Kates and Volcani, 1966; Thompson et al., 1990; Blanchemain and Grizeau, 1996). PUFAs could help to protect algae against the photooxidation reactions associated with photosynthetic activity. According to previous studies, the highest proportions of DHA are obtained under growth saturating irradiance in most species (Thompson et al., 1990). This could be explained by the percentage of DHA found in each lipid class (2-8% in neutral lipids, 3-8% in galactolipids, and 33-45% in phospholipids; Tables 3–5), which were greatest under HL-340. This suggested that high DHA levels in the phospholipids, and its reduction during light limitation, could play a central role in the physiology of the plasmic membrane and energy storage. Changes in fatty acid composition in algae are often related to the proportions of the different lipid classes, which have distinctive fatty acid compositions (Sukenik and Yamaguchi, 1993; Brown et al., 1996). In P. lutheri, our data show that the fatty acid composition of galactolipids, which include the main lipid classes present, is closely correlated to the fatty acid composition of total lipids. Generally, cells grown under high light intensity are characterized by high triacylglycerol synthesis, resulting in a low PUFA content, and high levels of 16:0 and 16:1 n-7 fatty acids (Sukenik et al., 1989). In our study, when the cells were cultured under low light intensity, the highest levels of PUFAs, such as those of EPA (20:5 n-3), are predominantly found in galactolipids, which constitute a major component of the chloroplast lipid membranes (Thompson et al., 1990; Thompson et al., 1996, Mock and Kroon, 2002). The results reported here show that the galactolipids in P. lutheri have been enriched with EPA to adapt the cells to functioning at LL-20. Indeed, an accumulation of n-3 fatty acids has been observed in thylakoid membranes, indicating thylakoid expansion, and thus an adaptation of the cells to environmental conditions (Mock and Kroon, 2002), such as light intensity (Sukenik et al., 1989). Thus, EPA may preserve the fluidity of thylakoid membranes, and therefore the velocity of the electron flow involved in photosynthesis (Mock and Kroon, 2002). 4.5. Carbon source and fatty acid composition With regard to the influence of the carbon source on the total fatty acid composition, no significant variations have been reported. This suggests that the minor variations obtained in the various different lipid fractions seem to be masked by fatty acid compensation between the different lipid classes. At LL-20, the highest PUFA levels were obtained with acetate (specifically in neutral lipids and phospholipids); while at HL-340, the highest PUFA levels were obtained with bicarbonate (specifically in galactolipids and phospholipids). These findings suggest that P. lutheri cells were using acetate as a carbon

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