Light-dependent utilization of two radiolabelled carbon sources, sodium bicarbonate and sodium acetate, and relationships with long chain polyunsaturated fatty acid synthesis in the microalga Pavlova lutheri (Haptophyta)

Abstract

Pavlova lutheri is a common member of the Pavlovophyceae (Haptophyta), often used as a food source for aquatic filter-feeders and cultured in laboratories to produce high levels of polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively), which are known to have benefits for human health. Consequently, we investigated the pathways involved in the biosynthesis of long chain polyunsaturated fatty acids (LC-PUFAs) in this alga during photosynthesis in relation to light intensity. Using two radiolabelled carbon sources, [¹⁴C] sodium bicarbonate and [1-¹⁴C] sodium acetate, we obtained data suggesting that P. lutheri is able to synthesize LC-PUFAs by successive elongation and desaturation steps. It converts palmitic acid into palmitoleic acid by Δ7-desaturation. Moreover, significant incorporation of [1-¹⁴C] acetate (organic carbon) and its subsequent use in lipid metabolism suggest that P. lutheri may have a mixotrophic capacity for carbon assimilation. Synthesis of lipids, including galactolipids and phospholipids, increased with light intensity when the cells were incubated with [¹⁴C] bicarbonate (inorganic carbon), but was less sensitive to differences in light intensity when incubated with [1-¹⁴C] acetate, a heterotrophic carbon source that stimulates the synthesis of monounsaturated fatty acids, such as oleic acid. In the case of n-3 fatty acids, EPA and DHA synthesis was lower at high light (340 µmol photons m⁻² s⁻¹) with the [¹⁴C] bicarbonate, but did not vary with [1-¹⁴C] acetate. Finally, P. lutheri seems to have two distinct enzyme pools involved in LC-PUFA synthesis, one is intra-chloroplastidic and dependent on light intensity, and the other is extra-chloroplastidic and independent of light.

Keywords:

• carbon source
• Haptophyta
• LC-PUFA synthesis
• light intensity
• lipid classes
• liquid chromatography
• Pavlova lutheri
• radiolabelling

Introduction

In their natural environments, marine photosynthetic organisms are exposed to changes in light intensity resulting from several factors, such as clouds, daily variations, seasonal fluctuations, as well as vertical migration due to mixing of water masses and flagellate movement. In microalgae, variations in light regime impose numerous adaptation mechanisms involving changes in pigment content, biochemical composition, and the capacities of the photosynthetic apparatus (Klyachko-Gurvich et al., 1999; Mouget et al., 1999; Tremblin et al., 2000). Specifically, significant changes in lipid content and fatty acid profile are observed in response to changes in the light intensity (Thompson et al., 1990; Blanchemain & Grizeau, 1996; Tzovenis et al., 2003; Guihéneuf et al., 2008). In most of the species, high light conditions induce an increase in cellular lipid content, which is correlated with a reduction in the percentage of long chain polyunsaturated fatty acids (LC-PUFAs) (Sukenik et al., 1989, 1993).

The production of lipids by microalgae can reach a yield per unit area that is more than 30 times higher than that of oilseeds, and may amount to more than 30 to 80% of their dry weight under certain conditions (Chisti, 2007). Because of their high levels of n-3 fatty acids, microalgae are at the heart of many industrial projects as a potential source for animal and human nutrition (Apt & Behrens, 1999; Ward & Singh, 2005; Spolaore et al., 2006; Cardozo et al., 2007). LC-PUFAs, and especially n-3 fatty acids such as eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively), are recognized as conferring benefits for human health, e.g. playing a major role in preventing cardiovascular diseases and promoting the development of the nervous system (Dyerberg & Jorgensen, 1982; Lagarde et al., 1986; Rousseau et al., 2003; Doughman et al., 2007). The high lipid content of microalgae means that they also constitute a promising potential biological source for biodiesel production (Chisti, 2007; Cadoret & Bernard, 2008).

Pavlova lutheri is principally used in aquaculture to feed marine organisms (Thompson et al., 1996; Leonardos & Lucas, 2000; Ponis et al., 2006a , 2006b). It is known to produce high levels of LC-PUFAs, specifically of EPA and DHA (Tatsuzawa & Takizawa, 1995; Carvalho & Malcata, 2000). The effects of environmental factors (light intensity, temperature and nutrients) on the fatty acid composition of this species have previously been reported (Tatsuzawa & Takizawa, 1995; Carvalho & Malcata, 2000; Carvalho et al., 2006; Guihéneuf et al., 2009). In most microalgal species, and particularly in P. lutheri, the highest proportions of EPA are obtained at low light intensities, whereas DHA levels are highest in response to high light intensities (Thompson et al., 1990; Brown et al., 1993; Guihéneuf et al., 2009). High EPA levels have been observed at low light intensities, suggesting that the biochemical composition of chloroplast membranes adapts to low light by increasing PUFA synthesis (Sukenik et al., 1989; Khotimchenko & Yakovleva, 2005; Guihéneuf et al., 2009).

In this context, we set out to explore, in vivo, the effects of light intensities on lipid and fatty acid (especially EPA and DHA) synthesis pathways by using two different radiolabelled carbon sources, [¹⁴C] sodium bicarbonate (inorganic carbon) and [1-¹⁴C] sodium acetate (organic carbon). Using in vivo radiolabelling experiments, Schneider & Roessler (1994) had previously shown that [¹⁴C] bicarbonate and [1-¹⁴C] acetate are used in fatty acid and lipid synthesis in Nannochloropsis spp. (Eustigmatophyceae); they suggested that the acetate is incorporated into fatty acids not only by de novo synthesis, but also as a result of elongation at other (extra-chloroplastidic) sites that are easily accessible to exogenous sodium acetate. Consequently, similarities to the trends of carbon source incorporation observed in Nannochloropsis spp. are expected in P. lutheri. In this paper, the radioactivity derived from [¹⁴C] sodium bicarbonate (autotrophic pathway) or from [1-¹⁴C] sodium acetate (heterotrophic pathway) incorporated into whole cells, total lipids, major lipid fractions and fatty acids was determined in P. lutheri growing in low, medium and high light intensities.

Materials and methods

Microalgal cultures

Pavlova lutheri cells were obtained from the Culture Collection of Algae and Protozoa (http://www.ccap.ac.uk/) as clone CCAP 931/6. After three successive cultures with a 1% antibiotic–antimycotic mix (A5955, Sigma–Aldrich, St. Quentin Fallavier, France; formulated to contain 10 000 units ml⁻¹ penicillin G, 10 mg ml⁻¹ streptomycin sulphate, 25 µg ml⁻¹ amphotericin B), an axenic culture was obtained. The axenicity of the culture was confirmed on two culture media: A (bactopectone, 3 g l⁻¹; yeast extract, 1 g l⁻¹; ammonium sulphate, 1 g l⁻¹; sodium glycerophosphate, 25 mg l⁻¹; iron (Fe³⁺)-EDTA, 6 mg l⁻¹; completed to 1000 ml with artificial sea water 50%), 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; completed to 1000 ml with artificial sea water 75%), which are used to grow fungi and bacteria, respectively. The microalgae were then grown axenically under batch conditions in 500 ml Erlenmeyer flasks sealed with cotton plugs that allowed gas exchange with the atmosphere (carbon dioxide and oxygen released during microalgal 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), and modified according to Perkins et al. (2006). The culture medium contained 33.6 µg l⁻¹ sodium bicarbonate or 32.8 µg l⁻¹ sodium acetate as the carbon source. These two concentrations correspond to 0.4 µM, which is the concentration of [¹⁴C] bicarbonate and [1-¹⁴C] acetate used in artificial seawater to radiolabel microalgal cells during incubation. The medium was prepared with deionized water, and autoclaved at 121°C for 20 min. Exponentially growing cells were used as inocula, after two generations without the antibiotic–antimycotic, and with an initial density of 10⁵ cells ml⁻¹. For each carbon source, the cultures were maintained at 16 ± 1°C under the three light intensities: 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 a 14 : 10 h light : dark cycle. Light intensity was measured at the middle of the culture flask 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 light intensity, and with each of the carbon sources.

Algal growth

Growth was monitored daily by taking a 2 ml sample under a laminar flow hood and making spectrophotometric measurements of the optical density of the cell suspensions at 750 nm (y = 10⁻⁷ x, where y is the optical density at 750 nm, and x the cell density), using a 2.5 ml plastic cuvette. The cell number was determined with an improved Malassez bright-line haemacytometer, after immobilizing the cells with 5% Lugol's solution. No special precautions were taken to prevent carbon dioxide re-equilibration during the daily subsampling.

Radiolabelling experiments

[¹⁴C] Sodium bicarbonate (99% radiochemical purity; 1.96 GBq mmol⁻¹ specific activity) and [1-¹⁴C] sodium acetate (98.5% radiochemical purity; 2.11 GBq mmol⁻¹ specific activity) were purchased from Radiochemical Centre (GE Healthcare, Amersham, UK).

In the mid-exponential growth phase, the cells were gently harvested by centrifuging at low-speed (1200× g, 10 min) at 15°C using a Sigma 4K15 centrifuge (Bioblock Scientific, Illkirch, France), and then resuspended in fresh growth medium without any added bicarbonate. As described by Moreno et al. (1979), [¹⁴C] bicarbonate or [1-¹⁴C] acetate was added to produce a final concentration of 0.4 µM for each compound. The incubations were carried out at each light intensity for 10 h during the light phase of the light : dark cycle, and photosynthesis was stopped by placing the Erlenmeyer flasks in ice and darkness. Cells were then harvested as previously described, washed three times with fresh medium, and the pellets obtained were frozen and stored at −20°C prior to analysis.

Extraction of total lipids

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 by volume) after adding 200 µl of 2.8 g l⁻¹ NaCl, a modified version of Bligh & Dyer's (1959) method, using manual crushing (Dounce cell grinders) coupled with ultrasonication (twice, for 15 and 30 min respectively). Chloroform (1 ml) was added between the two ultrasonication steps to produce phase separation. The chloroform layer, which contained the lipids, was collected and a 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 then dissolved in 200 µl of chloroform. The lipid extracts were stored at −20°C under nitrogen gas (N₂) until analysed, in order to limit oxidization.

Separation of the classes of lipids

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 by volume), and the phospholipids were recovered in 30 ml of methanol (Sukenik et al., 1993). The fractions were reduced in volume by evaporating under a stream of N₂. Thin-layer chromatography (TLC) of the total lipid extract was carried out in order to check the purity of each fraction (Henderson & Tocher, 1992), using non-radiolabelled samples.

Preparation and fatty acid analyses

The solvent was then evaporated under N₂, and the total fatty acids were extracted after saponifying with methanolic sodium hydroxide 0.5 M at 80°C for 20 min, as previously described by Slover & Lanza (1979). Total fatty acid methyl esters (FAMEs) were formed directly by treating the total extracts with methanolic boron trifluoride (14%) (Sigma–Aldrich, St. Quentin Fallavier, France) at 80°C for 20 min, and extracted with iso-octane and 35% NaCl. The iso-octane was evaporated from all the samples under a stream of N₂. The samples were then dissolved in 200 µl of acetone. The FAMEs were separated by reverse-phase high performance liquid chromatography (HPLC) following the method of Narce et al. (1998), using a P1500 high pressure pump (Thermo Separation Products, Les Ulis, France), equipped with a 410 differential refractometer (Waters, Milford, USA) and a Lichrocart column (Lichrospher 100 RP-18, 5 µm, 250 mm × 4 mm internal diameter, Merck, Darmstadt, Germany). The samples were eluted using a acetonitrile : water solvent system (95 : 5 by volume), and the FAMEs were collected at the detector outlet. Each individual peak was identified using pure standards (Sigma–Aldrich, St. Quentin Fallavier, France).

Radioactive counting

The radioactivity in whole cells, the total lipid, major lipid fractions, and each individual FAME were counted using ESR (External Standard Ratios) with a Wallac 1410 scintillation counter (ECG Instrument, Perkin–Elmer, USA), using a liquid scintillation cocktail for aqueous samples (ACS, GE Healthcare, Amersham, UK).

Statistical analysis of the data

Two-factor ANOVA was used to investigate the effect of the irradiance level (LL-20, ML-100 and HL-340) and the radiolabelled carbon source (bicarbonate and acetate) on the radioactivity incorporated into whole cells, total lipids, major lipid fractions (NL: neutral lipids, GL: galactolipids, PL: phospholipids) and fatty acids. The irradiance level and carbon source were taken to be fixed factors. P < 0.05 was accepted as the threshold for significant differences. Post hoc analyses were carried out using the Student–Newman–Keuls (SNK) test. All statistics were performed with SigmaStat (version 3.1) software (SPSS Incorporation, Erkrath, Germany).

Results

Incorporation of ¹⁴C into whole cells and total lipids

The results reported in Fig. 1a show that P. lutheri cells had a significant ability to incorporate from both carbon sources, [¹⁴C] bicarbonate (inorganic carbon) and [1-¹⁴C] acetate (organic carbon). However, regardless of the light intensity, twice as much [¹⁴C] bicarbonate was incorporated as [1-¹⁴C] acetate. The highest light intensities promoted the incorporation of both the carbon sources. Under HL-340, the percentages of [¹⁴C] bicarbonate and [1-¹⁴C] acetate incorporated into the whole cells reached 74 and 42%, respectively, of the initial radioactivity (¹⁴C) present in the culture medium. Under LL-20, the percentage of radioactivity incorporated did not exceed 39% with [¹⁴C] bicarbonate, and 21% with [1-¹⁴C] acetate.

Fig. 1. Effect of light intensity on the radioactivity incorporated (pmol ¹⁴C × 10⁻⁶ cells) into whole cells (a) and total lipids (b) after incubating for 10 h with [¹⁴C] bicarbonate (Bc, unshaded columns) and [1-¹⁴C] acetate (Ac, hatched columns). For explanation of statistical assessments of significance, see footnote to Table 1.

To assess the fraction of carbon from [¹⁴C] bicarbonate or [1-¹⁴C] acetate used in lipid metabolism and not in the synthesis of other compounds (such as proteins, amino acids, etc.), we determined the percentage of ¹⁴C incorporated into total lipids (Table 1). The results show that 30 to 40% of the ¹⁴C incorporated into the cells was used in the synthesis of lipids. The highest proportion of ¹⁴C incorporated into lipids was observed at HL-340 with [¹⁴C] bicarbonate, whereas the highest proportion with [1-¹⁴C] acetate was observed at LL-20. When the cells were grown under LL-20 and HL-340, no significant differences were observed between the carbon sources.

Table 1.  Effect of light intensity on radioactivity incorporated into total lipids, expressed as the percentage of ¹⁴C incorporated into lipids out of the total radioactivity incorporated into the cells, after incubating for 10 h with [¹⁴C] bicarbonate and [1-¹⁴C] acetate.¹ Results are expressed as mean ± SD (n = 3). 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.

Light intensity	LL-20		ML-100		HL-340
Carbon source (^14C)	Bicarbonate	Acetate	Bicarbonate	Acetate	Bicarbonate	Acetate
% radioactivity	36.6 ± 0.3^a	37.4 ± 3.7^β	34.4 ± 2.8^a	29.7 ± 1.0^α,*	40.2 ± 3.5^b	34.5 ± 5.2^αβ
^1After two-way ANOVA, Student–Newman–Keuls (SNK) multiple comparison test results are arranged in increasing order: a < b < c (effect of light intensity with bicarbonate) and α < β < γ (effect of light intensity with acetate). For each light intensity, mean acetate values designated by an asterisk (*) are significantly different from those for bicarbonate (SNK test, P < 0.05).

Both carbon sources, [¹⁴C] bicarbonate and [1-¹⁴C] acetate, were used by the algae to synthesize lipids. Lipid synthesis, expressed in pmol ¹⁴C × 10⁻⁶ cells (Fig. 1), increased significantly with light intensity and with both carbon sources. However, lipid synthesis was 55% lower when the cells were incubated with [1-¹⁴C] acetate than when they were incubated with [¹⁴C] bicarbonate.

Incorporation of ¹⁴C into the major lipid fractions

The distribution of ¹⁴C within polar lipids (GL and PL) and NL lipids, expressed as a percentage of total radiolabelled lipids, is shown in Table 2. The percentage of ¹⁴C incorporated into NL was lower under HL-340 in comparison with LL-20 for both carbon sources ([¹⁴C] bicarbonate: 23.6 versus 15.4%, [1-¹⁴C] acetate: 29.8 versus 19.0% of total radiolabelled lipids). Meanwhile, the proportions of radiolabelled GL increased with light intensity, from LL-20 to HL-340 ([¹⁴C] bicarbonate 58.5 versus 64.0%, and [1-¹⁴C] acetate 50.8 versus 61.2% of total radiolabelled lipids). In PLs, the highest proportions were obtained under ML-100 ([¹⁴C] bicarbonate: 22.9%, [1-¹⁴C] acetate: 23.6% of total radiolabelled lipids). Whatever the carbon source, the percentage of polar radiolabelled lipids (GL + PL) increased with light intensity, from LL-20 to HL-340 ([¹⁴C] bicarbonate: 76.4 versus 84.6%, [1-¹⁴C] acetate: 70.2 versus 81.0% of total radiolabelled lipids).

Table 2.  Effect of light intensity on the distribution (%) of the radioactivity incorporated into the different lipid fractions, expressed as percentage of total radiolabelled lipids, after incubating for 10 h with [¹⁴C] bicarbonate and [1-¹⁴C] acetate. For explanation of statistical assessments of significance, see footnote to Table 1.

Light intensity	LL-20		ML-100		HL-340
Carbon source (^14C)	Bicarbonate	Acetate	Bicarbonate	Acetate	Bicarbonate	Acetate
NL	23.6 ± 0.2^c	29.8 ± 0.5^β,*	18.7 ± 0.7^b	23.9 ± 3.5^α	15.4 ± 0.1^a	19.0 ± 6.0^α
GL	58.5 ± 1.0^a	50.8 ± 2.2^α,*	58.4 ± 1.2^a	52.5 ± 3.0^α,*	64.0 ± 0.7^b	61.2 ± 4.2^β
PL	18.0 ± 1.2^a	19.4 ± 2.6^α	22.9 ± 0.5^b	23.6 ± 0.5^β	20.6 ± 0.6^ab	19.8 ± 1.8^α
Sum GL + PL	76.4 ± 0.2^a	70.2 ± 0.5^α,*	81.3 ± 0.7^b	76.1 ± 3.5^β	84.6 ± 0.1^c	81.0 ± 6.0^β
NL: neutral lipids, GL: galactolipids, PL: phospholipids.

The effect of light intensity on the radioactivity incorporated into the different lipid fractions, and expressed as pmol ¹⁴C × 10⁻⁶ cells (Table 3), were used to measure the synthesis activity of each lipid class (NL, GL and PL). The results presented in Table 3 show an increase of both GL and PL synthesis with light intensity. Under HL-340, the quantities of ¹⁴C derived from [¹⁴C] bicarbonate or [1-¹⁴C] acetate that were incorporated into GL were 1.8 times higher than those incorporated under LL-20. Similarly, between HL-340 and LL-20, the quantities of ¹⁴C derived from [¹⁴C] bicarbonate or [1-¹⁴C] acetate incorporated into PL were 1.9- and 1.5-fold higher respectively. We did not see any change in NL synthesis. Similarly to the synthesis of total lipids, the quantities of ¹⁴C incorporated in each lipid class was lower in the algae cultured with [1-¹⁴C] acetate than in those grown with [¹⁴C] bicarbonate.

Table 3.  Effect of light intensity on the radioactivity incorporated into the different lipid fractions, expressed as pmol ¹⁴C × 10⁻⁶ cells, after incubating for 10 h with [¹⁴C] bicarbonate and [1-¹⁴C] acetate. For explanation of statistical assessments of significance, see footnote to Table 1.

Light intensity	LL-20		ML-100		HL-340
Carbon source (^14C)	Bicarbonate	Acetate	Bicarbonate	Acetate	Bicarbonate	Acetate
NL	9.9 ± 0.4	6.0 ± 1.1*	9.6 ± 0.2	4.8 ± 1.3*	10.4 ± 1.0	6.0 ± 3.0*
GL	24.4 ± 0.9 ^a	10.4 ± 2.1^α,*	29.9 ± 1.1^b	10.5 ± 0.6^α,*	43.3 ± 3.4^c	18.5 ± 2.6^β,*
PL	7.5 ± 0.9 ^a	3.9 ± 0.1^α,*	11.8 ± 0.1^b	4.7 ± 0.5^αβ,*	14.0 ± 1.6^c	6.0 ± 0.7^β,*
NL: neutral lipids, GL: galactolipids, PL: phospholipids.

Distribution of ¹⁴C among total fatty acids

Table 4 shows the distribution of ¹⁴C from [¹⁴C] bicarbonate or [1-¹⁴C] acetate incorporated into the fatty acids of P. lutheri as a function of the light intensity. After incubating for 10 h with [¹⁴C] bicarbonate, the major radiolabelled fatty acids were 14:0, 16:0, 16:1 n-7 and 20:5 n-3 respectively (>14% of total radiolabelled fatty acids). When the cells were incubated with [1-¹⁴C] acetate, the ¹⁴C was also mainly incorporated into fatty acids 14:0, 16:0, 16:1 n-7 and 20:5 n-3, but also into 18:1 n-9 (between 31 and 36% of total radiolabelled fatty acids). Consequently, the proportions of ¹⁴C incorporated into fatty acids 14:0, 16:0, 16:1 n-7, 20:5 n-3 and 22:6 n-3 were significantly lower in cells incubated with [1-¹⁴C] acetate than in those incubated with [¹⁴C] bicarbonate. With both radiolabelled carbon sources, high light intensities led to an increase in the percentages of ¹⁴C incorporated into fatty acids 14:0 and 16:1 n-7, and a decrease in those incorporated into 16:0. With regard to the fatty acids 20:5 n-3 and 22:6 n-3, the proportions of ¹⁴C from [¹⁴C] bicarbonate incorporated in these n-3 fatty acids decreased with light intensity, whereas the proportions of ¹⁴C from [1-¹⁴C] acetate remained unchanged.

Table 4.  Effect of light intensity on distribution of the radioactivity incorporated into each fatty acid, expressed as percentage of total radiolabelled fatty acids, after incubating for 10 h with [¹⁴C] bicarbonate and [1-¹⁴C] acetate. For explanation of statistical assessments of significance, see footnote to Table 1.

Light intensity	LL-20		ML-100		HL-340
Carbon source (^14C)	Bicarbonate	Acetate	Bicarbonate	Acetate	Bicarbonate	Acetate
14:0	17.5 ± 1.8^a	14.0 ± 0.5^α,*	20.6 ± 0.9^ab	15.9 ± 1.1^α,*	22.0 ± 0.5^b	19.6 ± 2.1^β,*
16:0	29.8 ± 0.4^c	20.3 ± 0.8^γ,*	26.1 ± 0.8^a	16.3 ± 1.2^α,*	28.3 ± 0.8^b	17.8 ± 0.6^β,*
18:0	0.7 ± 0.3	1.2 ± 0.2	0.4 ± 0.1	1.3 ± 0.6	0.5 ± 0.1	1.4 ± 1.1
16:1 n-7	18.9 ± 0.9^a	11.4 ± 0.6^α,*	21.9 ± 0.9^b	11.9 ± 0.7^α,*	25.1 ± 0.8^c	13.1 ± 0.5^β,*
18:1 n-9	2.9 ± 0.4	33.9 ± 1.3^β,*	2.4 ± 0.1	35.6 ± 1.8^β,*	2.8 ± 0.1	31.0 ± 0.8^α,*
18:3^1	1.9 ± 0.6^b	1.6 ± 0.4^β	1.3 ± 0.1^ab	1.4 ± 0.4^αβ	0.9 ± 0.1^a	0.9 ± 0.2^α
18:4 n-3	1.5 ± 0.3	1.1 ± 0.4	1.8 ± 0.2	1.2 ± 0.2*	1.3 ± 0.1	1.1 ± 0.3
20:4^1	1.3 ± 0.4^b	1.3 ± 0.4^β	0.9 ± 0.4^ab	1.0 ± 0.4^αβ	0.6 ± 0.1^a	0.7 ± 0.1^α
20:5 n-3	19.1 ± 0.6^b	11.3 ± 0.7*	18.3 ± 0.5^b	11.1 ± 0.2*	14.4 ± 0.9^a	10.9 ± 0.4*
22:6 n-3	6.4 ± 0.8^b	4.0 ± 0.4*	6.3 ± 0.3^b	4.3 ± 0.1*	4.2 ± 0.5^a	3.5 ± 0.8
^1The n-3 and n-6 isomers of linolenic (18:3) and eicosatetraenoic (20:4) acid are not separated by the HPLC system used.

Discussion

Incorporation of carbon substrates and total lipid synthesis

During the thermochemical phase of photosynthesis, ¹⁴C was autotrophically incorporated into whole cells and lipids of P. lutheri from [¹⁴C] bicarbonate, and also heterotrophically incorporated from [1-¹⁴C] acetate. Indeed, during photosynthesis, in response to the action of RuBisCO, the carboxylation phase of the Calvin–Benson cycle allows CO₂ from [¹⁴C] bicarbonate to be fixed, and glyceraldehyde-3-phosphate (G3P) to be formed. This is then used to synthesize organic molecules, such as lipids, carbohydrates and proteins (Masojidek et al., 2004). Moreover, sodium acetate is a precursor of actyl-CoA, which is involved in lipid metabolism, especially de novo fatty acid synthesis.

The radioactivity measured in cells and in total lipids show that P. lutheri is able to incorporate [¹⁴C] bicarbonate (inorganic carbon) and [1-¹⁴C] acetate (organic carbon), and to synthesize lipids from both these substrates. Whatever the light intensity, the fraction of carbon derived from [¹⁴C] bicarbonate or [1-¹⁴C] acetate incorporated into lipids was estimated to be 30–40% of the total ¹⁴C fixed by cells. This implies that some of the radioactivity incorporated into the cells must have been used to synthesize other macromolecules, such as proteins, polysaccharides, but also low molecular weight compounds such as amino acids (Rivkin, 1989; Smith & D'Souza, 1993; Beardall et al., 1994). In autotrophic microalgae, as in plants, inorganic carbon can be fixed by RuBisCO via the Calvin–Benson cycle and also by β-carboxylation, using different enzymes such as phosphoenol pyruvate carboxylase (PEPC), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate carboxylase (Tremblin & Robert, 2001; Rech et al., 2008). As sodium acetate is used in the synthesis of the cellular metabolites of P. lutheri, the mixotrophic capacity of P. lutheri suggested by Guihéneuf et al. (2009) was confirmed by the study reported here.

The amount of ¹⁴C from [¹⁴C] bicarbonate incorporated into the cells and total lipids of P. lutheri was always greater than that from [1-¹⁴C] acetate. This could be explained by the fact that cells need twice as much bicarbonate (one carbon) than acetate (two carbons) to synthesize organic molecules, in particular fatty acids. However, in our study, this explanation is unlikely because only one of two molecules of carbon was labelled for acetate. Consequently, the higher amount of ¹⁴C from [¹⁴C] bicarbonate incorporated into the cells and total lipids can be partially explained by active mechanisms of CO₂ accumulation, such as carbonic anhydrase, which some microalgae have developed alongside RuBisCO activity in order to facilitate the conversion of to CO₂ (Raven & Beardall, 2003; Morant-Manceau et al., 2007). Otherwise, the conversion of acetyl-CoA to malonyl-CoA, which is generally regarded as the first step in fatty acid synthesis, is catalysed by acetyl-CoA carboxylase (ACCase) and requires a large amount of CO₂, which could result from carbonic anhydrase activity. Acetate is mainly absorbed by passive diffusion (Lee, 2004), which along with other mechanisms could explain why smaller quantities of ¹⁴C were incorporated by the whole cells cultured with acetate. In plants, and probably in microalgae, the synthesis of [¹⁴C] acetyl-CoA from [1-¹⁴C] acetate (free acetate) is catalysed by acetyl-CoA synthetase (ACS) and carnitine acetyl-transferase (CAT) (Zeiher & Randall, 1991; Ke et al., 2000), and so the synthesis of lipids may depend on the activities of both these enzymes.

In P. lutheri, the incorporation of inorganic (bicarbonate) or organic (acetate) carbon and lipid synthesis have all been shown to be promoted by higher light intensities. Rivkin (1989) and Lafarga-De la Cruz et al. (2006) obtained similar results in Dunaliella tertiolecta, Thalassiosira rotula and Rhodomonas sp., and showed that carbon fixation ([¹⁴C] bicarbonate) is directly correlated to the light intensity during the exponential growth phase. In plants, acetyl-CoA is not imported by chloroplasts, but must generally be synthesized in this organelle (Rawsthorme, 2002). Thus, in P. lutheri, the conversion of photosynthesis products into acetyl-CoA could depend on the light intensity, which would account for the hypothetical intra-chloroplastidic acetyl-CoA synthesis pathway. In the case of [1-¹⁴C] acetate, when cells were grown under high light intensity, the amount of ¹⁴C incorporated into whole cells and then into total lipids could be increased by changes in ACS activity. Moreover, regardless of whether the carbon source involved [¹⁴C] bicarbonate or [1-¹⁴C] acetate, an increase in the ¹⁴C incorporated into lipids could be induced by the influence of light on the activity of ACCase, which catalyses the formation of malonyl-CoA (a substrate for fatty acid elongation). Indeed, several studies have already reported the light : dark cycle dependence of the mechanisms that regulate ACCase activity in plants (Rivkin, 1989; Post-Beittenmiller et al., 1991, 1992; Ohlrogge & Browse, 1995). For example, Hunter & Ohlrogge (1998) showed that the ACCase activity of isolated spinach chloroplasts incubated in light is twice as great as when they are incubated in darkness.

Synthesis of major lipid classes

In this study, we found that ¹⁴C from both carbon sources, [¹⁴C] bicarbonate and [1-¹⁴C] acetate, was incorporated into all fatty acids, and all lipid classes. However, it can also be incorporated into polar constituents (sugars and amino acids), and the glycerol body of lipids (Harwood & Jones, 1989), which can lead to an overestimation of the ¹⁴C incorporated into lipids. In P. lutheri, whichever radiolabelled carbon source was used, increasing the light intensity induced a decrease in the relative percentage of radioactivity (¹⁴C) incorporated into NL, and an increase of that incorporated into GL and PL. Under our conditions, the percentage of radioactivity (¹⁴C) included in polar lipids (GL + PL) was between 76.4 and 84.6% with [¹⁴C] bicarbonate, and between 70.2 and 81.3% with [1-¹⁴C] acetate. These results corroborate those previously obtained (Guihéneuf et al., 2009) for the levels of each lipid classes in P. lutheri (polar lipids, 60–71% of total lipids; neutral lipids, 29–40% of total lipids).

Concerning the amount of ¹⁴C incorporated into the different lipid fractions, the results can also be used to estimate the synthesis activities of lipid classes in P. lutheri. Whichever radiolabelled carbon source was used, higher light intensities induced an increase of ¹⁴C in GL and PL, indicating an increase in the synthesis of polar lipids. However, by radiolabelling of Nannochloropsis sp. with [¹⁴C] bicarbonate, Sukenik et al. (1993) showed that TAG synthesis was greater and monogalactosyl-diacylglycerol (MGDG) synthesis was lower at high light intensities. As previously suggested (Guihéneuf et al., 2009), high light intensities are potentially damaging to algal photosynthetic systems (Brown et al., 1993), and may induce an increase in polar lipid synthesis, especially GL, in response to acclimation to different light intensities.

Distribution of the radioactivity incorporated into fatty acids

In this study, the percentage of labelled PUFA was generally higher after cells had been incubated with [¹⁴C] bicarbonate than with [1-¹⁴C] acetate. The main radiolabelled fatty acids found in the presence of [¹⁴C] bicarbonate were 14:0, 16:0, 16:1 n-7 and 20:5 n-3. When the cells were incubated with [1-¹⁴C] acetate, a high percentage of ¹⁴C in 18:1 n-9 was observed, and the relative proportions of ¹⁴C incorporated into the other fatty acids were lower. These findings could be explained by the fact that the formation of acetyl-CoA from [¹⁴C] bicarbonate is mainly intra-chloroplastidic, whereas it is mainly extra-chloroplastidic when the ¹⁴C is provided by [1-¹⁴C] acetate. Indeed, in some other (non-photosynthetic) microorganisms, such as yeast Saccharomyces cerevisiae Hansen, ACS is located in the mitochondria and microsomes (Klein & Jahnke, 1971) and in the cytosol (Kispal et al., 1991), although it has not so far been identified or located in microalgae.

This implies that fatty acids synthesized from chloroplastidic acetyl-CoA using [¹⁴C] bicarbonate could be metabolized by enzymes embedded in thylakoid membranes, such as desaturases (Browse & Somerville, 1991). This would facilitate the elongation and desaturation steps in chloroplasts, which could explain the higher percentage of ¹⁴C incorporated into EPA and DHA. In addition, our findings show that the proportion of ¹⁴C incorporated into the LC-PUFAs, EPA and DHA, was lower under high light intensity, confirming the hypothesis of a light-dependent intra-chloroplastidic LC-PUFA synthesis. In chloroplasts, the enzymatic activities that result in the elongation and desaturation of fatty acids could be sensitive to differences in light intensity.

When the cells were incubated with [1-¹⁴C] acetate, the radioactivity incorporated into fatty acids may have been derived from the cytosolic acetyl-CoA formed, which allows us to suggest the possibility of extra-chloroplastidic synthesis of fatty acids. The radioactivity incorporated into LC-PUFA, EPA and DHA may result from the elongation and desaturation of fatty acids synthesized in the cytosol. Several studies (Khozin et al., 1997; Khozin-Goldberg et al., 2002) have shown that LC-PUFA biosynthesis products, such as EPA, may be produced in the cytosol and/or chloroplasts of microalgae. Moreover, non-radiolabelled fatty acids may have incorporated ¹⁴C by elongation, as a result of extra-chloroplastidic malonyl-CoA formation. Indeed, Schneider and Roessler (1994) suggest that fatty acids radiolabelled by [1-¹⁴C] acetate may not only result from de novo synthesis, but could also have incorporated ¹⁴C by elongation in extra-chloroplastidic sites accessible to exogenous acetate. However, this implies that there are two isoforms of acetyl-CoA carboxylase, one intra-chloroplastidic and another extra-chloroplastidic; and therefore that two distinct metabolic pools of malonyl-CoA are involved in fatty acid synthesis. With [1-¹⁴C] acetate, no light-intensity related difference in the relative proportions of ¹⁴C incorporated into EPA and DHA was observed. This means that the radioactivity incorporated into fatty acids was independent of light when the cells were incubated with [1-¹⁴C] acetate. These findings support the hypothesis of the extra-chloroplastidic synthesis of intermediates (acetyl-CoA and malonyl-CoA) involved in the elongation reaction and synthesis of fatty acids.

Moreover, the percentage of ¹⁴C incorporated into 16:1 n-7 shows that this fatty acid was formed by Δ7-desaturation of 16:0. Several studies have already shown that P. lutheri contains high levels of fatty acids 16:1 n-7, accounting for between 12 and 25% of the total fatty acids (Eichenberger & Gribi, 1997; Carvalho et al., 2006; Guihéneuf et al., 2009).

Concerning LC-PUFA, the percentages of radiolabelled EPA and DHA allow us to suggest that P. lutheri possesses all the enzymatic equipment required to synthesize n-3 LC-PUFA by successive elongation and desaturation steps (Δ9, Δ12, Δ15, Δ6, Δ5, and Δ4-desaturases). Indeed, several authors have already demonstrated that microalgae can synthesize LC-PUFAs, such as EPA (Moreno et al., 1979; Schneider & Roessler, 1994; Mimouni et al., 2003). However, the low percentage of ¹⁴C incorporated into n-6 fatty acids (<1% of total fatty acids labelled) suggests that the n-3 fatty acid synthesis pathway is predominant in P. lutheri.

Furthermore, the radiolabelled fatty acids 20:3 n-6 [1-¹⁴C] and 20:4 n-6 [1-¹⁴C] are being used to highlight the influence of light intensity on the enzymatic activities involved in the final steps of EPA and DHA synthesis, and to show the possible relationships between n-6 and n-3 fatty acid pathways.

Acknowledgements

This work was funded by the Ministère de l’Enseignement Supérieur et de la Recherche. The authors would like to thank M. Ghosh for reviewing the English text.