EPA ratio of Phaeodactylum tricornutum

Aquaculture 452 (2016) 311–317

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Effect of culture conditions on growth, fatty acid composition and DHA/EPA ratio of Phaeodactylum tricornutum Hongjin Qiao a, Chao Cong a,b, Chunxiao Sun a, Baoshan Li a, Jiying Wang a,⁎, Limin Zhang a,⁎ a b

Shandong Provincial Key Laboratory of Restoration for Marine Ecology, Shandong Marine Resource and Environment Research Institute, Yantai 264006, PR China College of Fisheries and Life, Shanghai Ocean University, 201306 Shanghai, PR China

a r t i c l e

i n f o

Article history: Received 16 April 2015 Received in revised form 3 November 2015 Accepted 7 November 2015 Available online 10 November 2015 Keywords: Phaeodactylum tricornutum Growth Fatty acid composition DHA/EPA ratio

a b s t r a c t The effect of growth phase and environmental factors on growth, fatty acid composition and DHA/EPA ratio of Phaeodactylum tricornutum Bohlin was studied. Microalgae were grown in laboratory batch cultures in f/2 medium. Cultures were grown at different salinities (15, 20, 28, and 35 ppt), nitrogen (N) concentrations (1.24, 12.35, 24.70 and 49.40 mg L−1), light intensities (50, 100 and 150 μmol m−2 s−1; 14:10 h light:dark cycle) and temperatures (15, 20 and 25 °C), and sampled at different points of the respective growth phases (inoculums, earlylinear, middle-linear and late-linear phases). Samples were analyzed for biomass weight, fatty acid composition and total fatty acid content (TFAC). The main fatty acids in all culture conditions were C14:0 (5.25%–6.04%), C16:0 (13.96%–14.78%), C16:1n-7 (19.09%–35.73%), C18:1n-9 (5.56%–9.01%) and eicosapentaenoic acid (EPA, 22.81%– 30.72%). The percentage of polyunsaturated fatty acids (PUFAs) was reduced while that of monounsaturated fatty acid acids (MUFAs) and TFACs increased with culture time. Salinity had no serious effect on fatty acid composition, however, a significant decline of TFAC was observed at the lowest salinity (p b 0.05). Significant (p b 0.05) increases of the relative contents of SFAs and MUFAs and a decrease of PUFAs were observed under N-limitation condition. The percentage of docosahexaenoic acid (DHA) was significantly enhanced with increasing light intensity (p b 0.05), while that of DHA, EPA and PUFA decreased significantly with increasing temperature (p b 0.05). DHA/EPA ratio tended to rise initially and fall later with increasing growth time, and reached the highest level with the lowest salinity, and the lowest temperature and initial N concentration, revealing a possible cell response to the stress brought from the unfavorable conditions. In conclusion, this study demonstrates the variation of growth, fatty acid composition and DHA/EPA ratio with growth phase and environmental factors in P. tricornutum, benefiting the production of PUFA-rich microalgae, with a DHA/EPA ratio optimal for aquaculture live food. Statement of relevance We conducted detailed analysis on the effect of the culture conditions on growth, fatty acid composition and DHA/EPA ratio in Phaeodactylum tricornutum, a widely used microalga for aquaculture feedstuff in China. We found that the environmental stress conditions increased DHA/EPA ratio. The research will benefit the production of PUFA-rich and DHA/EPA-ratio-optimal microalgae for aquaculture feedstuff. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Microalgae provide essential polyunsaturated fatty acids (PUFAs), in particular, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for the larvae of aquaculture animals (Borowitzka, 1997; Duerr et al., 1998; Guedes and Malcata, 2012). Recently, it has been also reported that moderate DHA/EPA ratio is vital on the egg and larval quality (Henrotte et al., 2010), nonspecific immunity and disease resistance

⁎ Corresponding authors. E-mail addresses: [email protected] (J. Wang), [email protected] (L. Zhang).

http://dx.doi.org/10.1016/j.aquaculture.2015.11.011 0044-8486/© 2015 Elsevier B.V. All rights reserved.

(Zuo et al., 2012), and n-3 LC-PUFA retention (Codabaccus et al., 2012). Since microalgae are the initial food for larvae, their DHA and EPA content and the DHA/EPA ratio are important for the development of aquaculture animals. However, the fatty acid profile and content of microalgae vary depending on culture conditions (Richmond and Hu, 2013). Therefore, it is important to find out the effect of these environmental factors on DHA and EPA content and DHA/EPA ratio of microalgae for the production of aquaculture feedstuff. The main environmental factors reported to influence the fatty acid composition of microalgae include: 1) growth phase (Brown et al., 1996; Liang and Mai, 2005; Liang et al., 2006); 2) nutrient source (Terry et al., 1985; Flynn et al., 1992; Lourenço et al., 2002); 3) salinity

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H. Qiao et al. / Aquaculture 452 (2016) 311–317

(Renaud and Parry, 1994; Xu and Beardall, 1997); 4) light (Liang et al., 2001; Carvalho and Malcata, 2005; Guedes et al., 2010); and 5) temperature (Henderson and Mackinlay, 1989; Zhu et al., 1997; Renaud et al., 2002). Although these studies demonstrated the relations between fatty acid composition and culture conditions, most of these relations are species-specific. For example, PUFA and EPA have been reported that some diatoms show a rapid decline with culture age (Liang and Mai, 2005), however, the relative proportions of EPA and DHA have been shown to increase slightly with culture age in other microalgae (Hallegraeff et al., 1991; Shamsudin, 1992). Additionally, more information is needed on the relation between culture conditions and the variations of the DHA/EPA ratio. In this work, we conducted a detailed analysis on the effect of culture conditions on growth, fatty acid composition and DHA/EPA ratio in P. tricornutum, a microalga widely used as live food in Chinese aquaculture (Shi et al., 2008), to screen what factors have the maximal effect on growth, fatty acid composition and DHA/EPA ratio for aquaculture purposes.

2. Materials and methods 2.1. Strain and culture P. tricornutum Bohlin was obtained from the Microalgae Culture Center (MACC), Ocean University of China. Cultures were grown in 500-mL flasks with f/2 medium (Guillard and Ryther, 1962), and shaken at 20 ± 1 °C and 100 rpm under illumination from LED lamps with an irradiance of 100 μmol m−2 s−1 on a 14:10 h light:dark cycle. Salinity and pH of the medium was adjusted to 28 and 8.0, respectively before sterilization. Cultures in the late-exponential growth phase were centrifuged at 4000 g for 5 min and resuspended into the fresh medium for inoculums. Bacterial contamination was checked by inoculating f/2 medium plus 1.5% yeast extract with 1 mL of the culture.

2.2. Salinity, nitrogen, light and temperature Single factorial experiments were conducted in 500 mL glass flasks with 300 mL of f/2 medium and mean initial cellular concentration of 105 mL−1. All cultures were sampled on day 7 for biomass and fatty acids analysis. Four salinities (15, 20, 28, and 35 ppt) were used, diluting seawater (28 ppt) with distilled water to 15 and 20 ppt, or adding natural sea salt to 35 ppt. Sodium nitrate was used as nitrogen source in f/2 medium, resulting in different initial N concentrations of 1.24, 12.35, 24.70 and 49.40 mg L−1, respectively corresponding to 10, 100, 200 and 400% the strength of f/2 medium. Light was provided by LEDarrays with irradiances of 50, 100 and 150 μmol m−2 s−1 on a 14:10 h light:dark cycle. Finally, cultures were kept at different temperatures of 15, 20 and 25 °C. All treatments were performed in triplicate. Light and temperature treatments were carried out in three incubators with manual shaking four times a day for 2 min each time. Culture conditions of other treatments were the same as the stock culture. 2.3. Growth phase Cultures were sampled at every day for cell density determination with a hemocytometer (Qiujing, Shanghai, China). Cultures under normal growth conditions (salinity of 28 ppt, N concentration of 12.35 mg L−1, irradiance of 100 μmol m− 2 s− 1 and temperature of 20 °C) were sampled on days 0, 3, 7 and 16 for fatty acid analysis, respectively. Growth rates were calculated according to Kratz and Myers (1955). 2.4. Biomass analysis Cell growth was determined using optical density (OD) readings at 680 nm with a UV-2010 (Hitachi, Japan) spectrophotometer in a 1 cm-

Fig. 1. Growth curves of Phaeodactylum tricornutum grown at different conditions. The units for salinity (S), irradiance (I), initial nitrogen concentration (N) and temperature (T) are ppt, μmol m−2 s−1, mg L−1 and oC, respectively. Cell density is cell numbers per milliliter.

H. Qiao et al. / Aquaculture 452 (2016) 311–317

light-path cuvette. OD680nm values were converted to biomass using the linear regression y = 0.39 OD680nm + 0.01 (R2 = 0.99, biomass (g L−1) = y), which had been determined before the experiments. Biomass weight was analyzed according to Griffiths et al. (2010).

313

Table 2 Fatty acid composition (% of total fatty acids) and total fatty acid content (TFAC, mg g−1 total dry weight) of Phaeodactylum tricornutum at different growth phases. Inoculums (INO), 0 d; early-linear phase (ELP), 3 d; middle-linear phase (MLP), 7 d; and late-linear phase (LLP), 16 d. Growth phase1

2.5. Fatty acid analysis

Fatty acid

Direct transesterification was used for the fatty acid analysis according to Griffiths et al. (2010). Wet pellets of 0.1 g with 0.1 mg glyceryl triheptadecanoate as internal standard were transesterified by a sequential combination of base and acid catalysts. Fatty acid methyl esters were then analyzed on a fused silica capillary column (Supelco SP-2560: 100 m × 0.25 mm, film thickness 0.20 μm) in a gas chromatograph (GC2010, Shimadzu, Tokyo, Japan) equipped with an auto injector (AOC20i, Shimadzu, Tokyo, Japan). Temperatures of injector and flame ionization detector were controlled at 260 °C with nitrogen as the carrier gas. The column temperature was programmed from 140 to 240 °C at a rate of 4 °C/min. Fatty acids were identified by comparing the relative retention time with the reference standards (Supelco, Bellefonte, PA, USA).

C14:0 C16:0 C16:1n-7 C16:2n-7 C16:2n-4 C16:3n-4 C18:0 C18:1n-9 C18:1n-7 C18:2n-6 C18:3n-3 C18:4n-3 ARA C20:4n-3 C24:0 EPA C22:5n-3 DHA others SFA MUFA PUFA DHA/EPA TFAC

2.6. Statistical analysis Unless otherwise specified, all results were reported as mean ± SD of three replicate groups. Comparison between means was by oneway ANOVA followed by multiple comparisons using Duncan's multiple range test (Duncan, 1955) and significance was accepted at p b 0.05. Statistical analysis was carried out using the SPSS program version 11 for Windows (SPSS Inc., Chicago, IL, USA).

INO

ELP

MLP

LLP

5.25 ± 0.23a 14.54 ± 0.13ab 24.99 ± 0.09a 1.24 ± 0.05a 0.55 ± 0.03a 3.58 ± 0.02a 0.59 ± 0.08ab 7.61 ± 0.15a 0.45 ± 0.00 2.11 ± 0.04a 0.38 ± 0.01a 0.50 ± 0.01a 0.31 ± 0.07 0.36 ± 0.06a 1.34 ± 0.08ab 28.27 ± 0.47a 0.21 ± 0.01a 0.52 ± 0.02a 7.26 ± 0.41a 21.72 ± 0.20a 33.06 ± 0.07a 39.29 ± 0.55a 0.018 ± 0.001a 120.6 ± 12.13a

5.81 ± 0.06bc 14.19 ± 0.46ab 19.09 ± 0.77b 1.52 ± 0.21b 0.52 ± 0.09a 3.18 ± 0.17b 0.90 ± 0.25a 9.01 ± 0.67b 0.43 ± 0.04 1.57 ± 0.08b 0.22 ± 0.03b 0.87 ± 0.09b 0.39 ± 0.28 1.40 ± 0.13b 1.35 ± 0.19ab 30.26 ± 1.28b 0.73 ± 0.05b 0.98 ± 0.13b 7.61 ± 0.44a 22.24 ± 0.49ab 28.53 ± 1.26b 45.03 ± 2.06b 0.032 ± 0.003b 48.7 ± 3.56b

5.69 ± 0.08b 13.96 ± 0.16a 21.97 ± 3.13ab 1.66 ± 0.17b 0.51 ± 0.05a 3.36 ± 0.21ab 0.64 ± 0.2ab 7.51 ± 0.74a 0.42 ± 0.04 1.74 ± 0.23b 0.25 ± 0.07b 0.57 ± 0.17a 0.41 ± 0.26 0.86 ± 0.56ab 1.52 ± 0.09a 30.72 ± 1.1b 0.47 ± 0.27b 0.86 ± 0.23b 6.89 ± 0.34a 21.81 ± 0.36a 29.9 ± 2.58b 44.07 ± 3.01b 0.028 ± 0.007b 76.54 ± 18.69c

6.04 ± 0.14c 14.78 ± 0.56b 35.73 ± 1.68c 1.03 ± 0.04a 0.84 ± 0.08b 2.38 ± 0.22c 0.55 ± 0.03b 5.56 ± 0.17c 0.48 ± 0.04 1.13 ± 0.12c 0.18 ± 0.01b 0.22 ± 0.02c 0.54 ± 0.16 0.38 ± 0.03a 1.22 ± 0.04b 22.81 ± 1.16c 0.09 ± 0.02a 0.45 ± 0.01a 5.61 ± 0.26b 22.59 ± 0.42b 41.77 ± 1.54c 31.06 ± 1.46c 0.02 ± 0.001a 145.65 ± 48.34a

1 Values (mean ± SD of three replicates) within the same row with different letters are significantly different (p b 0.05).

3. Results 3.1. Growth phase After a 1-day lag phase, cells under all conditions entered an exponential phase (EP) until day 2 and a long phase of linear growth until day 16 (Fig. 1). The specific growth rates were compared at EP and day 7 (middle-linear phase, MLP). The maximal growth rates of EP were obtained at salinity of 28 ppt, irradiance of 100 μmol m−2 s− 1, N N 1.24 mg L−1 or temperature of 20 °C, respectively (Table 1). However, the specific growth rates of MLP did not change greatly as that of EP, except at N concentration of 1.24 mg L−1. The specific growth rate at N concentration of 1.24 mg L−1 were reduced 31.6% compared with that at N N 12.35 mg L−1 (Table 1). Fatty acid composition of cells under normal growth conditions from day 0 (inoculums, INO), 3 (early-linear phase, ELP), 7 (MLP) and 16 (late-linear phase, LLP) were analyzed. The main fatty acids were C14:0 (5.25%–6.04%), C16:0 (13.96%–14.78%), C16:1n-7 (19.09%– 35.73%), C18:1n-9 (5.56%–9.01%) and EPA (22.81%–30.72%) in all growth phases (Table 2). The percentage content of C16:1n-7 showed the most obvious increase from 19.09% in ELP to 35.73% in LLP, while that of EPA decreased most obviously from 30.72% in MLP to 22.81% in LLP. The saturated fatty acids (SFAs) showed a similar percentage content in different growth phases. DHA/EPA ratio and PUFA percentage increased at first but later decreased, however, monounsaturated fatty

acid (MUFA) percentage showed an opposite tendency. The total fatty acid content (TFAC) declined to the lowest level at ELP then recovered to the original level at LLP. 3.2. Salinity The biomass displayed a trend of rising first and then falling with increasing salinity, and reached the highest when salinity was 28 ppt in MLP (Fig. 2). The fatty acid composition did not show so great change as that at different growth phases. SFA, MUFA and PUFA percentage content slightly fluctuated under different salinities (Table 3). However, DHA/EPA ratio decreased from 0.034 at 15 ppt to 0.025 at 35 ppt. TFAC at 15 ppt was significantly lower (p b 0.05) than those at other salinities. 3.3. Nitrogen Sharp reduction of the initial nitrogen source in medium resulted in significant decrease of biomass, as shown in Fig. 2. However, biomass remained stable when initial N concentration was beyond 12.35 mg L−1. The fatty acid composition also showed significant variation under the lowest initial N concentration (1.24 mg L−1), including the significant increase in SFA and MUFA (p b 0.05), and decrease in

Table 1 The specific growth rates (d−1) of Phaeodactylum tricornutum at exponential phase (EP) and middle-linear phase (MLP) on different culture conditions. Salinity (ppt)

Irrdiance (μmol m−2 s−1)

Nitrogen (mg L−1)

Temperature (°C)

Growth phase EP MLP

15

20

28

35

50

100

150

1.24

12.35

24.7

49.4

15

20

25

1.41 0.39

1.39 0.37

1.61 0.38

1.29 0.35

1.43 0.35

1.50 0.34

1.39 0.34

1.28 0.26

1.56 0.38

1.53 0.37

1.56 0.37

1.26 0.34

1.42 0.33

0.96 0.31

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Fig. 2. Biomass content (dry mass) of cultures on day 7 at different salinities, irradiances, initial nitrogen concentrations and temperature. Column values (mean ± SD of three replicates) with different letters are significantly different (p b 0.05).

PUFA (p b 0.05) compared with the higher initial N concentrations (≥12.35 mg L−1) (Table 4). DHA/EPA ratio significantly increased from 0.025 to 0.042 with the sharp reduction of initial N concentration from 12.35 mg L−1 to 1.24 mg L−1. TFAC increased almost three-fold at initial N concentration of 1.24 mg L−1 than those at higher initial N concentrations.

3.4. Light

Table 3 Fatty acid composition (% of total fatty acids) and total fatty acid content (TFAC, mg g−1) of Phaeodactylum tricornutum at different salinities on day 7.

Table 4 Fatty acid composition (% of total fatty acids) and total fatty acid content (TFAC, mg g−1) of Phaeodactylum tricornutum at different initial nitrogen concentrations on day 7.

Salinity (ppt)1

Fatty acid

Fatty acid 15 C14:0 C16:0 C16:1n-7 C16:2n-7 C16:2n-4 C16:3n-4 C18:0 C18:1n-9 C18:1n-7 C18:2n-6 C18:3n-3 C18:4n-3 ARA C20:4n-3 C24:0 EPA C22:5n-3 DHA others SFA MUFA PUFA DHA/EPA TFAC

20 a

28 a

35 b

Biomass growth showed a decreasing trend with increasing light intensity (Fig. 2). SFA, MUFA and PUFA percentage content and TFAC showed no significant differences among three groups (Table 5). DHA/ EPA ratio also maintained at a relatively constant level. However, DHA

b

6.83 ± 0.16 6.14 ± 0.37 5.89 ± 0.24 6.93 ± 0.25 11.37 ± 0.26a 12.06 ± 0.38ab 13.05 ± 1.17bc 13.46 ± 0.36c a a b 22.69 ± 0.33 22.11 ± 0.09 20.84 ± 0.57 20.52 ± 0.18b 1.60 ± 0.15 1.58 ± 0.06 1.49 ± 0.17 1.61 ± 0.03 0.72 ± 0.02a 0.58 ± 0.08b 0.51 ± 0.01b 0.78 ± 0.04a 4.00 ± 0.20a 3.95 ± 0.08ab 3.76 ± 0.37ab 3.53 ± 0.15b 0.72 ± 0.12 0.96 ± 0.31 0.90 ± 0.32 0.74 ± 0.05 7.12 ± 0.15a 7.52 ± 0.28ab 7.89 ± 0.59b 7.69 ± 0.05ab 0.40 ± 0.03 0.46 ± 0.06 0.45 ± 0.03 0.47 ± 0.05 1.57 ± 0.02a 1.85 ± 0.05b 2.18 ± 0.04c 1.56 ± 0.02a 0.23 ± 0.03a 0.22 ± 0.01a 0.23 ± 0.02a 0.38 ± 0.05b 0.81 ± 0.06a 0.94 ± 0.11ab 1.12 ± 0.14bc 1.14 ± 0.06c – 0.11 ± 0.02a 0.24 ± 0.01b 0.33 ± 0.06c 0.63 ± 0.04a 0.54 ± 0.08ab 0.49 ± 0.04b 0.49 ± 0.04b 1.65 ± 0.11 1.53 ± 0.12 1.47 ± 0.29 1.62 ± 0.08 31.38 ± 0.13 30.83 ± 0.93 32.18 ± 0.88 31.50 ± 0.52 0.38 ± 0.00 0.30 ± 0.07 0.35 ± 0.09 0.28 ± 0.05 a a b 0.98 ± 0.06 0.85 ± 0.04 0.80 ± 0.04b 1.08 ± 0.09 6.67 ± 0.17 6.78 ± 0.09 6.20 ± 1.13 6.88 ± 0.29 20.66 ± 0.46 21.38 ± 0.68 21.55 ± 1.48 21.71 ± 0.14 30.10 ± 0.26a 29.18 ± 1.11ab 28.68 ± 0.12b 30.21 ± 0.23a 45.39 ± 0.52 44.31 ± 0.91 45.72 ± 1.36 44.89 ± 0.18 0.032 ± 0.001a 0.026 ± 0.001b 0.025 ± 0.002b 0.034 ± 0.003a 98.03 ± 4.92a 119.67 ± 8.56b 139.39 ± 18.95b 124.66 ± 5.91b

1 Values (mean ± SD of three replicates) within the same row with different letters are significantly different (p b 0.05).

C14:0 C16:0 C16:1n-7 C16:2n-7 C16:2n-4 C16:3n-4 C18:0 C18:1n-9 C18:1n-7 C18:2n-6 C18:3n-3 C18:4n-3 ARA C20:4n-3 C24:0 EPA C22:5n-3 DHA others SFA MUFA PUFA DHA/EPA TFAC

Initial nitrogen concentration (mg L−1)1 49.40

24.70 a

12.35 a

1.24 b

5.69 ± 0.28 5.17 ± 0.15 5.57 ± 0.18 12.82 ± 0.22a 12.37 ± 0.30b 12.69 ± 0.11ab 20.92 ± 0.72 21.73 ± 0.81 21.35 ± 0.49 1.49 ± 0.14 1.31 ± 0.20 1.39 ± 0.08 0.64 ± 0.02a 0.54 ± 0.01a 0.62 ± 0.03a 3.52 ± 0.11a 3.53 ± 0.21a 3.06 ± 0.02b 0.74 ± 0.13a 0.59 ± 0.05b 0.58 ± 0.01b 7.83 ± 0.60ab 8.42 ± 0.97a 7.08 ± 0.04b 0.40 ± 0.03a 0.41 ± 0.04a 0.36 ± 0.02a 2.44 ± 0.11a 2.39 ± 0.18ab 2.21 ± 0.05b 0.28 ± 0.10a 0.34 ± 0.06a 0.37 ± 0.02a 0.45 ± 0.09 0.54 ± 0.09 0.40 ± 0.07 0.46 ± 0.07a 0.64 ± 0.04a 0.55 ± 0.19a 0.33 ± 0.02a 0.34 ± 0.03a 0.35 ± 0.05a 2.03 ± 0.67a 1.70 ± 0.62a 1.78 ± 0.09a 31.52 ± 0.30a 31.74 ± 0.61a 31.5 ± 0.16a 0.20 ± 0.02a 0.25 ± 0.06a 0.26 ± 0.03a 0.85 ± 0.02a 0.77 ± 0.01b 0.80 ± 0.04b 7.44 ± 0.97a 6.79 ± 1.61a 9.47 ± 0.20b 21.16 ± 0.36a 20.35 ± 0.77ab 20.21 ± 0.21b 29.15 ± 0.11a 30.56 ± 0.32b 28.79 ± 0.49a 44.35 ± 0.65a 44.32 ± 0.81a 43.65 ± 0.03a 0.027 ± 0.001a 0.024 ± 0.001b 0.025 ± 0.001ab 106.2 ± 4.05a 120.76 ± 3.37a 110.76 ± 6.55a

4.51 ± 0.12c 32.38 ± 0.24c 43.55 ± 0.34 0.26 ± 0.00b 0.60 ± 0.02c 1.14 ± 0.02c 4.64 ± 0.06c 0.50 ± 0.01c 0.78 ± 0.00c 0.08 ± 0.00c 0.46 ± 0.03 0.11 ± 0.03b 0.13 ± 0.00b 0.49 ± 0.02b 7.89 ± 0.18b 0.05 ± 0.00b 0.33 ± 0.01c 2.09 ± 0.33c 38.52 ± 0.30c 48.69 ± 0.41c 11.47 ± 0.21b 0.042 ± 0.00c 362.43 ± 46.33b

1 Values (mean ± SD of three replicates) within the same row with different letters are significantly different (p b 0.05).

H. Qiao et al. / Aquaculture 452 (2016) 311–317 Table 5 Fatty acid composition (% of total fatty acids) and total fatty acid content (TFAC, mg g−1) of Phaeodactylum tricornutum at different irradiances on day 7.

Table 6 Fatty acid composition (% of total fatty acids) and total fatty acid content (TFAC, mg g−1) of Phaeodactylum tricornutum at different temperatures on day 7.

Irradiance (μmol m−2 s−1)1

Temperature (°C)1

Fatty acid C14:0 C16:0 C16:1n-7 C16:2n-7 C16:2n-4 C16:3n-4 C18:0 C18:1n-9 C18:1n-7 C18:2n-6 C18:3n-3 C18:4n-3 ARA C20:4n-3 C24:0 EPA C22:5n-3 DHA others SFA MUFA PUFA DHA/EPA TFAC

315

Fatty acid 50

100

150

5.35 ± 0.19 14.41 ± 1.18 25.24 ± 1.01ab 2.22 ± 0.14 0.58 ± 0.08 3.38 ± 0.41 0.75 ± 0.15 5.66 ± 0.43 0.45 ± 0.04 3.20 ± 0.21a 0.26 ± 0.03 0.27 ± 0.01ab 0.40 ± 0.13 0.50 ± 0.23 2.03 ± 0.12a 28.49 ± 1.98 0.35 ± 0.04 1.01 ± 0.05a 5.43 ± 0.1 22.55 ± 1.61 31.35 ± 0.54 43.41 ± 1.94 0.036 ± 0.004 88.75 ± 5.23

5.48 ± 0.29 15.33 ± 1.08 26.31 ± 0.44a 2.22 ± 0.33 0.49 ± 0.11 3.10 ± 0.35 0.81 ± 0.02 4.99 ± 0.20 0.50 ± 0.11 3.43 ± 0.05a 0.31 ± 0.05 0.32 ± 0.06a 0.45 ± 0.08 0.64 ± 0.17 2.32 ± 0.11b 26.39 ± 0.96 0.34 ± 0.01 1.07 ± 0.04ab 7.04 ± 3.11 23.94 ± 1.46 31.79 ± 0.65 41.57 ± 1.54 0.041 ± 0.002 87.64 ± 4.31

5.83 ± 0.41 14.11 ± 0.59 24.44 ± 0.47b 2.46 ± 0.11 0.63 ± 0.17 3.77 ± 0.50 0.92 ± 0.16 6.15 ± 1.02 0.47 ± 0.05 2.76 ± 0.11b 0.30 ± 0.03 0.18 ± 0.05b 0.43 ± 0.07 0.32 ± 0.08 2.27 ± 0.14ab 28.60 ± 0.96 0.38 ± 0.01 1.12 ± 0.06b 4.86 ± 1.43 23.13 ± 0.67 31.06 ± 0.52 43.95 ± 1.18 0.039 ± 0.003 85.25 ± 12.87

C14:0 C16:0 C16:1n-7 C16:2n-7 C16:2n-4 C16:3n-4 C18:0 C18:1n-9 C18:1n-7 C18:2n-6 C18:3n-3 C18:4n-3 ARA C20:4n-3 C24:0 EPA C22:5n-3 DHA others SFA MUFA PUFA DHA/EPA TFAC

15

20

25

5.21 ± 0.17 14.99 ± 0.16a 20.75 ± 0.62a 2.23 ± 0.10a 0.44 ± 0.06a 3.23 ± 0.25 1.84 ± 0.07a 3.20 ± 0.29a 0.28 ± 0.02a 6.15 ± 0.17a 0.32 ± 0.01 0.62 ± 0.26 0.43 ± 0.11 1.20 ± 0.15a 2.75 ± 0.22a 29.52 ± 0.27a 0.84 ± 0.09a 1.70 ± 0.10a 4.40 ± 0.92a 24.79 ± 0.14a 24.22 ± 0.49a 51.78 ± 0.31a 0.058 ± 0.003a 74.43 ± 7.65a

5.48 ± 0.22 13.59 ± 0.54b 23.77 ± 1.78b 2.43 ± 0.08b 0.69 ± 0.02b 3.60 ± 0.07 1.25 ± 0.14b 5.54 ± 0.32b 0.50 ± 0.08b 2.92 ± 0.27b 0.33 ± 0.07 0.48 ± 0.26 0.49 ± 0.12 0.46 ± 0.10b 2.33 ± 0.11b 27.31 ± 0.62b 0.30 ± 0.12b 1.13 ± 0.03b 7.38 ± 1.22b 22.66 ± 0.29b 29.82 ± 1.74b 42.99 ± 0.49b 0.041 ± 0.002b 89.66 ± 8.84a

5.37 ± 0.31 18.57 ± 0.55c 23.28 ± 0.58b 2.51 ± 0.11b 0.58 ± 0.17ab 3.30 ± 0.16 1.37 ± 0.20b 5.55 ± 0.15b 0.53 ± 0.10b 2.46 ± 0.26b 0.40 ± 0.08 0.29 ± 0.12 0.56 ± 0.06 0.34 ± 0.04b 1.85 ± 0.20c 25.05 ± 1.42c 0.27 ± 0.05b 1.07 ± 0.05b 6.65 ± 1.64ab 27.15 ± 1.01c 29.37 ± 0.68b 39.50 ± 1.66c 0.043 ± 0.004b 109.52 ± 7.59b

1 Values (mean ± SD of three replicates) within the same row with different letters are significantly different (p b 0.05).

1 Values (mean ± SD of three replicates) within the same row with different letters are significantly different (p b 0.05).

percentage was significantly enhanced with increasing light intensity (p b 0.05).

et al., 1989; Siron et al., 1989; Chrismadha, 1993; Tonon et al., 2002; Liang et al., 2006). We also found the general tendency for increased percentage of MUFA and reduced PUFA with increasing culture time in P. tricornutum. This tendency is probably due to the fact that the short chain C16:1n-7, which forms a major proportion of triacylglycerols (neutral lipids) in P. tricornutum (Arao et al., 1987; Yongmanitchai and Ward, 1992) is prone to catabolism for energy supply in rapid growth phase and anabolism for energy storage in stationary phase (Alonso et al., 2000; Miller et al., 2014), while EPA mainly correlated to the proportions of polar lipid in P. tricornutum (Arao et al., 1987; Yongmanitchai and Ward, 1992, 1993) plays an important role in the photosynthetic membranes' activity which increases in rapid growth phase and decreases in stationary phase (Harwood and Russell, 1984; Alonso et al., 2000; Liang et al., 2006). However, considering TFAC, both C16:1n-7 and EPA actually increased and reached the maximum content in stationary phase. The marked rise of total fatty acids in stationary phase of microalgae had been reported to result from the exhausted N and/or P (Alonso et al., 2000; Giordano et al., 2001), as indicated by the significantly enhanced TFAC in the N limitation condition (Table 4). There was no general pattern in the variation of fatty acid composition as functions of salinity. The degree of fatty acid saturation was either increased or decreased with increasing salinity (Ben-Amotz et al., 1985; Al-Hasan et al., 1990; Renaud and Parry, 1994; Xu and Beardall, 1997). P. tricornutum basically maintains the same fatty acid composition at different salinities. However, a significant decline of TFAC was observed at the lowest salinity level, which was consistent with some reports of marine microalgae (Ben-Amotz et al., 1985; Richmond and Hu, 2013). The variation of fatty acid in P. tricornutum under nitrogen-limitation has been reported by others (Alonso et al., 2000; Yongmanitchai and Ward, 1991; Terry et al., 1985; Hu et al., 2008), which was typically shown by the increased percentage of SFA and MUFA as well as TFAC, and decreased percentage of PUFA, as indicated in the present study. These variations were probably resulted from the significant accumulation of triacylglycerols under N-deprivation as reported in above-

3.5. Temperature Biomass at 20 °C was significantly higher than that at 25 °C (p b 0.05), but not significantly higher than that at 15 °C (p N 0.05) (Fig. 2). MUFA percentage increased significantly from 24.22% at 15 °C to 29.82% and 29.37% at 20 and 25 °C, respectively (Table 6). However, DHA, EPA and PUFA percentage decreased significantly with increasing temperature (p b 0.05). DHA/EPA ratio (0.058) at 15 °C was significantly higher than those at 20 and 25 °C (p b 0.05). TFAC at 25 °C was significantly higher than those at 15 and 20 °C (p b 0.05). 4. Discussion 4.1. Biomass growth It was interesting that initial N concentrations higher than 12.35 mg L− 1 did not result in more biomass, in agreement with the previous report of Xu et al. (2001) on Ellipsoidion sp. with nitrogen concentration of 17.92 mg L−1, Converti et al. (2009) on Chlorella vulgaris with nitrogen concentration of 12.35 mg L− 1, and Jiménez and Niell (1991) on Dunaliella viridis with nitrogen concentration of 70 mg L−1. However, it was found that the final biomass increased with increasing nitrogen source concentrations in P. viridis (Li et al., 2005), N. oculata (Converti et al., 2009) and Neochloris oleoabundans (Li et al., 2008). These results suggest species-specific responses to N concentration, possibly due to different forms of nitrate reductase in marine microalgae, which is thought to be a rate-limiting step for nitrogen assimilation (Eppley et al., 1970; Berges and Harrison, 1995). 4.2. Fatty acid composition Several studies have reported that the percentage of certain fatty acids varied over the growth phase of P. tricornutum (Fernández-Reiriz

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mentioned literatures. Furthermore, SFA and MUFA were the major proportion of triacylglycerols while PUFA only occupied a very small fraction of triacylglycerols (Yongmanitchai and Ward, 1992; Yongmanitchai and Ward, 1993). Reports in the literature on the effect of light on fatty acid composition of P. tricornutum are variable. For example, Chrismadha and Borowitzka (1994) observed the percentage content of EPA was enhanced with increasing irradiance, but Liang et al. (2001) reported an opposite trend, while Thompson et al. (1990) documented a trend of rise first then fall with increasing light intensity. However, a constant EPA percentage content was observed in the present study. This variability might be caused by algal strains, the range of light intensity, harvest stage and culture mode (batch or photobioreactor) that different researchers used. Compared to the variability of EPA, DHA was observed to increase steadily with increasing light intensity (14–150 μmol m−2 s−1) in the present study and others (Liang et al., 2001; Thompson et al., 1990). The effect of temperature on fatty acids of marine microalgae has been shown by the significant rise of saturation degree with increasing temperature (Mortensen et al., 1988; Thompson et al., 1992; Renaud et al., 1995; Renaud et al., 2002). P. tricornutum in the present study followed this trend. SFA and MUFA were increased and PUFA was decreased with increasing temperature (Table 5). It is becoming increasingly clear that an increase in the degree of fatty acid unsaturation is to acclimatize to low-temperature conditions by increasing the fluidity of cell membrane phospholipid layers (Harwood, 1988; Tatsuzawa and Takizawa, 1995; Jiang and Gao, 2004).

4.3. DHA/EPA ratio DHA/EPA ratio is a major concern in the essential fatty acid research for its regulatory role in the animal metabolism (Bhattacharya et al., 2007; Zuo et al., 2012; Codabaccus et al., 2012; Ma et al., 2014). However, there were few reports on the DHA/EPA ratio of microalgae, which might also played an important role in the metabolism of microalgae. In the present study, we found DHA/EPA ratio showed a trend of rising first then falling with growth time, and reached the highest level under the lowest salinity, nitrogen concentration or temperature condition. Since DHA and EPA are mainly distributed in the polar lipid and acting in the membrane activities, the increase of DHA/EPA ratio might be a cell response to oxidative stress brought from the unfavorable conditions as above mentioned (Sies, 1997; Mohan and Das, 1997; Ďuračková and Gvozdjáková, 2008). In conclusion, this study clearly demonstrates the variation of fatty acid composition with growth phase and environmental factors in P. tricornutum. DHA/EPA ratio was found to increase under the stress conditions, which might play important roles in the stress response. These results may be useful for the production of PUFA-rich and DHA/ EPA-ratio-optimal microalgae for aquaculture feeds or supplements.

Acknowledgments This project was supported by the National Natural Science Foundation of China (Grant no. 31201973); the Research Award Fund for Outstanding Middle-aged and Young Scientists of Shandong Province of China (Grant no. BS2013HZ018); the Science and Technology Development Plan Project of Shandong Province (Grant no. 2014GHY115006); the Taishan Scholars Station of Aquatic Animal Nutrition and Feed (Grant no. HYK201004) and the National Marine Public Welfare Research Project of China (Grant no. 201205025); the Marine Biological Industry of China: Aquatic Animal Nutrition and Feed Research and Innovation Demonstration Platform (201301003); and the Modern Agricultural Industry System of Shandong Province of China: Industrial Innovation Team of Sea Cucumber (2012–2014).

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