The microalga H. pluvialis used in this study was provided by the Laboratory of Phycology, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Science. The strain was cultivated in an MCM medium at 20 °C under 50 µmol photons·m⁻²s⁻¹ with a 12:12-h light/dark photoperiod (Boussiba and Vonshak, 1991). The algae in exponential growth phase were used for the experiments. The algal cells were harvested by centrifugation (8000× g for 3 min), washed twice in double-distilled water, and re-suspended in the corresponding culture conditions: (1) MCM medium, under the same conditions as those indicated earlier (control); (2) MCM medium, under 350 µmol photons·m⁻²s⁻¹ light intensity (HL); (3) nitrogen-free medium, under optimal light intensity (-N); and (4) nitrogen-free medium, under 350 µmol photons·m⁻²s⁻¹ light intensity (HL-N). Each experiment was performed for 14 days and at least three independent repetitions for each treatment were carried out. The H. pluvialis cells were sampled every 2 days for the subsequent analyses.

H. pluvialis cells were harvested and lyophilized using a freezer dryer for analyzing the total lipid content. A total of 50 mg of freeze-dried samples were treated with 1000 µL of methanol at 4 °C for 15 min. The extraction was repeated three times to obtain all the neutral lipids. Subsequently, the methanol extract was treated with peroxide-free diethyl ether (containing 0.01% butylhydroxytoluene (BHT), hexane, and water) up to a final ratio of 1:1:1:1 (v/v/v/v). After centrifugation of the mixture (3000 g for 5 min), the upper phase was collected. The pH of the lower phase was adjusted to 3–4 with acetic acid, and was subjected to re-extraction with a mixture of diethyl ether and hexane (at a ratio of 1:1, v/v). Subsequently, the combined phases were evaporated to dryness in nitrogen and stored at −20 °C. Four different treatments were performed after 14 days and at least three independent repetitions for each extraction were carried out.

The fatty acid analysis was performed as described by An et al. (2013). A total amount of 20 mg of each lyophilized sample was added to a conical flask containing 30 mL of petroleum ether. The solution was placed in an ultrasound bath (40.0 kHz, 600 W) for 30 min at 50 °C, and this operation was repeated twice. Then, the solvent was moved from a rotary vacuum evaporator at 50 °C until the weight was unchanged. The total fatty acids were transmethylated to fatty acid methyl esters (FAMEs) with 5 mL of 0.4 M KOH:Methanol (v/v) at room temperature (25 °C). The analysis of the resulting FAMEs was carried out using a Finnigan Trace GC-MS (Agilent Technologies, USA). The FAMEs were identified by comparison with authentic standards (Sigma Chemicals Co., USA), and peaks were integrated with DPS software Version 7.05 (Zhejiang University, China).

Cell growth was determined by counting the cell numbers using a hemocytometer. Photosynthetic capability was determined by means of the DIVING-PAM (Walz, Effeltrich, Germany) connected to a PC with WinControl software, using the pulse–amplitude modulated method. Before measurement, the samples were kept in the dark for 15 min and the original fluorescence (F₀) was determined under a low measuring light. A saturation light pulse was applied to obtain maximum fluorescence (F_m) in the dark-adapted samples. The F_m yield in the illuminated samples was denoted as F_m’, and the real-time fluorescence yield was indicated as F_t. The maximal PSII quantum yield (F_v/F_m) was calculated according to the equation: F_v/F_m = (F_m − F₀)/F_m. The effective PSII quantum yield (Y(II)) was calculated as follows: Y(II) = (F_m’ − F_t)/F_m’. The non-photochemical quenching of chlorophyll fluorescence (NPQ) was calculated based on the following equation: NPQ = (F_m − F_m’)/F_m’. All the measurements were performed at room temperature.

For pigment analysis, 50 mL of the H. pluvialis culture were harvested at different intervals of stress induction and ultrasonic decomposition. The pigment was extracted from the algal cells by adding dimethyl sulfoxide (DMSO) at 70 °C for 5 min. The total carotenoids and chlorophyll contents were determined by UV–Vis spectrophotometers (Purkinje General, China) using the coefficients mentioned by Solovchenko et al. (2010). The chlorophyll and carotenoid concentrations were expressed in mg of chlorophyll per liter and mg of carotenoid per liter, respectively.

Each experiment was repeated three times. All the observations and calculations were made separately for each set of experiments. The data were expressed as means with standard deviation (SD). Statistical analyses were performed using SPSS 17.0 for Windows (SPSS, Chicago, IL, USA). Variance among treatments was tested using a one-way ANOVA. The significance level was p<0.05 for all tests unless otherwise indicated.

In the control culture, the sample showed a maximum cell concentration of 9.01×10⁵ cells·mL⁻¹ after 14 days and exhibited an exponential phase from the 2^nd to the 10^th day. The cells exposed to the control culture retained their flagella for longer periods compared to that under stress conditions. In the [HL] cultures, after being cultured for 4 days, the algae entered the exponential growth phase with the highest cell concentration of 3.94×10⁵ cells·mL⁻¹ after 14 days. In the [-N] cultures and in the [HL-N] cultures, the maximum cell concentrations were 2.15×10⁵cells·mL⁻¹ and 1.67×10⁵cells·mL⁻¹, respectively. Although as the stress advanced, all the treatments showed an increase in biomass yield; the cultures stressed with treatments showed lower cell concentrations than the control cultures. The cells under nitrogen-deficiency treatment and the high light combined with the nitrogen-deficiency treatment did not exhibit an obvious exponential phase (Figure 1). The culture stressed with high-light produced significantly more biomass than the cultures under other treatments.

Figure 2 shows the contents in total lipid in the cultures. Under control condition, the total lipid content of H. pluvialis gradually increased with increasing cultivation time, the maximum total lipid content was 13.60% (percentage dry weight = % dw) at the 10^th day. The total lipid content in the [-N] cultures increased sharply in the first 6 days and raised slowly in the following 4 days, then declined after the 10^th day. A similar result was observed in the [HL-N] cultures. Thus, the highest lipid productivities were obtained after 10 days, allowing for a production of up to 46.71% and 46.87%, respectively. In the [HL] culture, a sharp increase in total lipid content in the first 8 days was observed and a similar increase, although less pronounced, was observed in the following 2 days. The highest amount of total lipid content was obtained after 10 days’ cultivation, which was significantly higher than that in the longer cultivated culture (Figure 2). The maximum productivity for total lipid accumulation in high-light-exposed cells was 56.92%, which was almost six-fold over the control cells. The cellular contents of total lipid declined after 10 days regardless of any culture condition (Figure 2). The results suggest that the 10^th stress day may be the optimal time for lipid production.

To identify the change in the fatty acid composition of the H. pluvialis cells grown under stress, a typical profile of lipids extracted from the lyophilized cells of H. pluvialis was developed, as shown in Table 1. The major fatty acids in H. pluvialis were palmitic acid (C16:0), linoleic acid (C18:2n6), and linolenic acid (C18:3n3), which altogether were found to represent about 60–70% of the TFA. Under stress conditions, all of them increased, especially the palmitic acid content (C16:0) under HL-N. Additionally, the oleic acid (C18:1) increased by 2-fold, 1.2-fold and 1.4 fold, respectively, under HL, -N, and HL-N culture conditions, accompanied by a decrease in C16:2, C16:4 and EPA fatty acids. The percentage of saturated fatty acids (SFA) was shown to be significantly higher in cultures grown under the [HL] culture (30.80%), the [-N] culture (29.11%) and the [HL-N] culture (30.79%) conditions compared to the control (27.81%). In general, the polyunsaturated fatty acid (PUFA) content presented a downward trend, whereas the monounsaturated fatty acid (MUFA) content increased under stress conditions.

The influence of stress on carotenoid accumulation and chlorophyll was examined in the present study in order to understand whether the accumulation of lipid and carotenoid was synchronized. The dynamic changes in chlorophyll and carotenoids content are presented in Figure 3. The cellular content of total carotenoids in the control culture did not change appreciably. In the [-N] culture, the total carotenoid content increased moderately. In the [HL] culture, the content in total carotenoids increased slightly on the first 6 days, continued to increase sharply between day 6 and day 10, and subsequently remained stable (Figure 3A). The carotenoid content peaked on the 10^th day, to 36.12 mg·L⁻¹ in the [HL] cultures and to 27.21mg·L⁻¹ in the [-N] cultures, respectively. The carotenoid content in the [HL-N] culture was more than two- times higher than that observed in the control culture (Figure 3A).

In the [HL] culture and [-N] culture, the chlorophyll content increased sharply after the first several days of incubation and then moderately increased, reaching a maximum value of 18.95 mg·L⁻¹ on day 6 and 19.94 mg·L⁻¹ on day 8, respectively. After 14 days, the chlorophyll content decreased to a similar level in both the cultures (Figure 3B). On the other hand, the [HL-N] culture exhibited the highest chlorophyll content, which reached a value of approximately 17.31 mg·L⁻¹ on the fourth day of cultivation and declined slightly thereafter (Figure 3B).

Since the relationship between the change in photosynthetic efficiency and the production of lipids has not been widely studied in algae, we've paid special attention to the changes in the PSII photosynthetic characteristics. F _v/F _m, Y(II) and NPQ were measured to evaluate changes in the photosynthesis efficiency. The F _v/F _m, Y(II) and NPQ differed among the four samples during the course of incubation. The control showed no distinct changes in F _v/F _m, Y(II) and NPQ. F _v/F _m of the [HL] cultures; the [-N] cultures and the [HL-N] cultures decreased by 47.6%, 55.9% and 55.67% after a treatment of 10 days (Figure 4A); Y(II) of the stress treatments decreased by 45.2%,30.4% and 63.6% (Figure 4B); and NPQ of the three stress cultures was found 11.09, 14.2 and 6.9 times higher, respectively, than the initial value (Figure 4C).

A good correlation was found between contents of individual carotenoid and total lipid. As shown in Table 2, it provided high correlation factors ranging from R = 0.786 to R = 0.862. In stress conditions, the positive correlation of carotenoid and total lipid presents a more obvious trend compare to the control condition.

The parameters of the PAM Flourometer (F _v/F _m, Y(II), NPQ) recorded significant physiological stress induced by different stress conditions. (Figure 5 A, B, C)

The algal samples in the control exhibited a tight, nonlinear relationship with total lipid content whether F _v/F _m (R²=0.586), Y(II) (R²=0.289), NPQ (R²=0.580) (Figure 5; Table 2). In the [HL] cultures, the [-N] cultures and the [HL-N] cultures, NPQ were well correlated with content of total lipids.