Effects of the Diurnal Light and Temperature Fluctuations on the Growth, Photosynthesis and Biochemical Composition of Terrestrial Oleaginous Microalga Vischeria sp. WL1 (Eustigmatophyceae)

Abstract

Dynamic changes in diurnal light and temperature are a natural phenomenon, particularly pronounced in arid environments. However, it remains unclear whether the coupling of diurnal light and temperature fluctuations contributes to the capability and evolution of lipid accumulation in arid terrestrial microalgae. Here, we characterized the effects of diurnal light and temperature fluctuations on the growth, photosynthesis, lipids, carbohydrates, proteins and fatty acids in Vischeria sp. WL1, an oil-producing Eustigmatophyceae microalga sourced from an arid steppe. The photosynthetic activity parameters (F_v/F_m, PI_abs, ψ₀ and φ_E0) were the highest, while parameters of damage and energy dissipation (W_k and DI_O/RC) were the lowest, under the diurnal light cycle (DLC) among all conditions and then followed by the diurnal light–temperature cycle (DLTC). DLTC led to reduced growth and biomass but enhanced lipid accumulation, with lipid content reaching a maximum of 54.7% on day 4. This observation may suggest a carbon reallocation from carbohydrates toward lipid synthesis. DLTC resulted in an increase in the saturated fatty acids (SFA) content on day 12 and only in C18:2 among polyunsaturated fatty acids (PUFA) on day 4 while lowering eicosapentaenoic acid (EPA) concentration and content on day 12. Together, our findings will contribute to the understanding of the adaptive strategies of terrestrial microalgae to the arid environment, as well as the beneficial clues for exploring terrestrial microalgae for oil production.

1. Introduction

Microalgae represent a ubiquitous group of organisms across Earth’s ecosystems, endowed with extraordinary resilience that enables them to flourish in a wide array of habitats, including extremes such as arid steppes or deserts, frigid soils and thermally active waters [1]. They are well known for their high photosynthetic efficiency, rapid growth kinetics, high yields of lipids, proteins, polysaccharides and additional high-value metabolites, including carotenoids, phycobiliproteins, polyunsaturated fatty acids and vitamins [2,3,4,5]. It is well documented that abiotic stresses, such as temperature fluctuations, alterations in light intensity [6,7], salt stress and nutrient limitations, are potent triggers for lipid accumulation in microalgae [8,9,10]. Consequently, microalgae have emerged as a pivotal resource across multiple sectors, emerging as a viable substitute to traditional oil crops, and are widely used in agriculture, food processing, pharmaceuticals and bioenergy production [11,12,13]. They offer a sustainable solution to counteract the continuous expansion of conventional agricultural activities [14] as well as offer the nutritional needs of livestock feed and cooking oil [15,16,17].

Among microalgae, the species/strains from arid environments are particularly notable for their robust capability to accumulate lipids [6,18,19]. These organisms thrive in harsh environments characterized by extreme daily/yearly temperature and irradiance fluctuations [20]. To cope with temperature variations, microalgae have evolved mechanisms to regulate the unsaturation of fatty acids, which is crucial for maintaining membrane fluidity and permeability [21,22] and protecting the photosynthetic systems within thylakoid membrane glycolipids [23]. When metabolic processes are limited at low temperatures, the carbon for protein biosynthesis would be restricted and redirected towards the synthesis of triacylglycerols (TAGs) for storage [23,24]. It has been reported that for Scenedesmus sp. Z-4, temperature variations combined with appropriate light/dark cycles are beneficial for lipid accumulation [25]. Studies have also found that a photoperiod 18:6 h (light/dark) resulted in higher lipid content (31.3%) compared to continuous light conditions in Nannochloropsis species [26]. However, it remains unclear whether the natural coupling of diurnal light and temperature fluctuations contributes to the capability and evolution of lipid accumulation in terrestrial microalgae.

We previously isolated an oil-producing microalga Vischeria sp. WL1 from a large biological soil crust (BSC) in an arid environment [19,27]. It was characterized by ~50%, ~60% and ~70% oil yields under nitrogen starvation, NaCl treatment and a low temperature of 9 °C, respectively [27]. In the present study, we grew this strain under three different conditions: control, diurnal light cycles, and diurnal light and temperature fluctuation. Then, we analyzed the growth, photosynthesis, lipid and fatty acids among these conditions. This study provides us with a comprehensive understanding of the effects of diurnal light and temperature fluctuations on lipid accumulation, fatty acids and carbon redirection in Vischeria sp. WL1, thereby providing a theoretical basis for enhancing oil and target fatty acid production in this microalga.

2. Materials and Methods

2.1. Strain and Culture Conditions

The Vischeria sp. WL1 strain (Eustigmatophyceae; Eustigmatales; Chlorobotryaceae; Vischeria) was isolated from a BSC in the eastern side (an arid steppe) of Helan Mountain in Yinchuan, Ningxia Hui Autonomous Region, China in 2019 [19]. The 18S rDNA sequence of this strain was deposited into GenBank (Accession no. MZ373178). The cultures were statically maintained in 250 mL glass flasks containing 150 mL BG-11 medium. Continuous illumination of 100 µmol photons m⁻² s⁻¹ was provided by a light incubator (GDN-600D, Ningbo Yanghui instrument Co., Ltd., Ningbo, China) equipped with LED tri-color light. The temperature was set at 30 °C. The flasks were manually shaken three times a day, and each replicate was randomly placed in the incubator to reduce cell sedimentation and ensure uniform light exposure.

2.2. Experimental Setup

To explore the effects of diurnal light and temperature fluctuation on Vischeria sp. WL1, we designed an experiment by growing it under three specific conditions: 24 h continuous light irradiance at 30 °C (Control); a diurnal light cycle (DLC): 16 h irradiance at 30 °C and 8 h darkness at 30 °C; and a diurnal light–temperature cycle (DLTC): 16 h irradiance at 30 °C and 8 h darkness at 13 °C (the temperature limiting half of the growth rate). Note that 30 °C is the optimal growth temperature for Vischeria sp. WL1 during its short-term growth period [27]. All treatments were maintained at the optimal growth light intensity of 100 µmol photons m⁻² s⁻¹ [27] for 12 days, with each condition replicated three times to ensure statistical reliability. The experimental scheme is shown in Figure 1A. Cultures under different experimental conditions were sampled every 2 days for growth, photosynthetic pigments and photosynthetic parameters, and they were sampled every 4 days for dry weight, oil content, sugar, protein and fatty acids measurement. All samplings, including those for the control group, were conducted 4 h after the onset of illumination in the light/dark cycle of the DLC and DLTC group. These samples were then frozen at −20 °C for further use. Cell density was quantified using a hemocytometer (JSY-SC-031N Boda Biopolymer Company, Guangzhou, China) to assess growth characteristics.

2.3. Measurement of Photosynthetic Pigments and Photosynthetic Parameters

Samples (2 mL) were centrifuged (5000× g, 5 min), and 1 mL 95% ethanol (V:V) was added to the cell pellet along with five 6 mm grinding beads. The photosynthetic pigments were then extracted by breaking the cells with a Tissue Lyser (Wonbio-L, Shanghai, China) for 10 min at 60 Hz by ice cooling in the dark. Following this, the supernatant was obtained through centrifugation (5000× g, 5 min, 4 °C), and their absorbance was measured at 664.1 nm, 648.6 nm and 470 nm by a UV spectrophotometer. The concentration of chlorophyll a (Chl a) and carotenoids (Caro) was calculated according to the equation given by Lichtenthaler [28] as follows:

(1)$\mathrm{Total} \mathrm{Chl} a (\mathsf{\mu }\mathrm{g}/\mathrm{mL}) = 13.36{\mathrm{A}}_{664.1}$

(2)$\mathrm{Total} \mathrm{Caro} (\mathsf{\mu }\mathrm{g}/\mathrm{mL}) = (1000{\mathrm{A}}_{470} - 2.13\mathrm{Chl} a)/209$

where A_664.1 and A₄₇₀ are the absorbance values of the supernatant at 664.1 nm and 470 nm, respectively.

Photosynthetic parameters were measured using a portable PEA fluorometer (FluorPen FP 110, Czech Republic). To assess the photosynthetic parameters of the Vischeria sp. WL1 under different treatments, samples of 2 mL were pre-incubated in the dark for 20 min [29]. The maximum quantum yield of PSII photochemistry (F_v/F_m), performance index on absorption basis (PI_abs), maximum values of quantum yield for electron transport (φ_E0), maximum values of efficiency that a trapped exciton can move an electron into the electron transport chain beyond Q_A⁻ (ψ₀), the change in chlorophyll fluorescence value of the OJIP curve at about 300 μs (W_k), the effective antenna size per reaction center (ABS/RC), the dissipated energy flux per reaction center (DI_O/RC), unit of energy captured by the reaction center for the reduction in Q_a (TR_O/RC), and the light quantum flux transmitted by the initial electron per active reaction center (ET_O/RC) were then measured according to the manufacturer’s instructions.

2.4. Determination of Soluble Sugar and Soluble Protein

Cultures (1 mL) were collected by centrifugation (8000× g for 10 min) on day 4, and the cell pellet was broken by the Tissue Lyser (Wonbio-L, Shanghai, China) for 10 min at 60 Hz. The supernatant was obtained through centrifugation (5000× g, 5 min), and the soluble sugar and soluble protein were determined by the phenol sulfate method [30] and BCA Protein Assay kit (TIANGEN), respectively.

2.5. Determination of Biomass and Total Oil Content

Cultures (50 mL) were harvested from each flask every 4 days by centrifugation (8000× g, 3 min), and the cell pellets were then frozen overnight at −20 °C, followed by a freeze-dried procedure until a constant weight was achieved. The dried pellet was then weighed. The total lipid was extracted as described by Xu [31] with slight modification, where 2 mL of a methanol–chloroform mixture (2:1 v/v) was added to the dried pellets and disrupted using a Tissue Lyser (Wonbio-L, Shanghai, China) at 50 Hz for 10 min. Samples were then centrifuged (5 min, 8000× g), and the supernatant was separated into two phases. The aqueous phase was aspirated off, and the chloroform phase was washed three times with two parts of 1 M KCl each. Finally, the resulting lipid was dried using a nitrogen blower, carefully weighed, and then frozen until analysis. The oil content (%) was calculated using the following formula:

(3)$\mathrm{Oil} \mathrm{content} (\%)={\displaystyle \frac{{\mathrm{w}}_1}{{\mathrm{w}}_2}} \times 100\%$

where w₁ is the total oil weight, and w₂ is the dry weight.

2.6. Analysis of Fatty Acid Composition

The fatty acid composition analysis was conducted as previously described [31]. Briefly, nitrogen blow-dried oil (10 mg) was mixed with 4 mL of iso-octane, and then 200 μL 2 M KOH-CH₃OH was added for methyl esterification, followed by adding about 1 g NaHSO₄ for neutralization after the above reaction for 30 min. The neutralized solution was mixed with 1 mL of distilled water, and the organic phase was collected and filtered using a 0.45 μm filter. The composition of fatty acid was then determined using a GC2010-Pro gas chromatograph (Shimadzu, Japan) equipped with an FBX-10 column (100 m × 0.25 mm × 0.20 μm) (ANPEL Laboratory Technologies (Shanghai) Inc., Shanghai, China). The fatty acids were qualitatively identified according to the chromatographic peaks of a 37-component Fatty Acid Methyl Ester (FAME) mixture (ANPEL Laboratory Technologies (Shang-hai) Inc., Shanghai, China). The relative contents (%) of the individual fatty acids were calculated using the area normalization method [32]. The calculation of the fatty acid unsaturation index was performed using the formula provided earlier [33]. The absolute content of eicosapentaenoic acid (EPA) was determined by an internal standard and calculated by the following standard curve:

(4)$y =6.73x +0.08$

where y is the peak area and x is the EPA concentration (mg mL⁻¹).

2.7. Statistical Analysis

Data were expressed as means ± standard deviations of three replicates and tested for statistical significance using analysis of variance (one-way analysis of variance, ANOVA). Values were considered significantly different when the probability (p) was < 0.05. Statistical analysis was conducted using the SPSS software package (IBM SPSS Statistics 28.0.1.0).

3. Results

3.1. Effects of Different Diurnal Light and Temperature Conditions on Growth, Dry Weight and Oil Content

The effect of diurnal light and temperature fluctuations on growth, dry weight and oil content of Vischeria sp. WL1 are shown in Figure 1. We found that under the diurnal light–temperature cycle (DLTC), Vischeria sp. WL1 grows the slowest, lagging behind those observed under the diurnal light cycle (DLC), with growth rates of 0.13 d⁻¹ and 0.14 d⁻¹ (relative to the control of 0.15 d⁻¹), respectively (Figure 1B). The dry weight per milliliter of Vischeria sp. WL1 under DLTC was significantly lower than that under the other two conditions, especially when grown on day 8 and day 12 (Figure 1C). Although there are no significant differences in the dry weight per cell between the three conditions, and the dry weight per cell shows similar patterns with the dry weight per mL at day 8 and day 12 (Figure 1D). In contrast, the lipid content exhibited a reversed pattern of growth and dry weight, peaking under DLTC, followed by DLC. Notably, while no significant differences were discernible amongst different conditions on day 4, a clear divergence became apparent on day 8, where the maximum lipid content reached an impressive 64.6% under DLTC (Figure 1E).

3.2. Effects of Different Diurnal Light and Temperature Conditions on Photosynthetic Pigments

The effects of different diurnal light and temperature conditions on photosynthesis pigments are shown in Figure 2. We found that chlorophyll a concentration among the three conditions increased similarly, while at different magnitudes (Figure 2A). Specifically, the lowest chlorophyll a concentration was observed under DLTC and comparatively higher under control condition than under DLC. However, the cellular content of Chl a maintained consistent throughout the experimental period except for a decline under DLTC on day 8 (Figure 2B). Generally, the concentration of carotenoids adheres to a similar profile as chlorophyll a concentration (Figure 2C). The cellular content of carotenoid shows similar patterns to the cellular content of Chl a (Figure 2D).

3.3. Effects of Different Diurnal Light and Temperature Conditions on Photosynthetic Parameters

We explored the impact of different diurnal light and temperature conditions on the photosynthetic parameters (Figure 3). We found that values of F_v/F_m, PI_abs, ψ₀ and φ_E0 were the highest under DLC at most the time points, followed by DLTC, and the lowest observed under the control, with the most notable increase of 68.6% observed for PI_abs on days 8 and 10 (Figure 3A–D). In contrast to above the parameters, the change in chlorophyll fluorescence value of the OJIP curve at about 300 μs (W_k), effective antenna size per reaction center (ABS/RC), the trapped energy flux per reaction center (TR_O/RC), the electron transport per active reaction centers (ET_O/RC) and dissipated energy flux per reaction center (DI_O/RC) were the lowest observed under DLC at most the time points, followed by the control, and the highest observed under DLTC (Figure 3E–I).

3.4. Effects of Different Diurnal Light and Temperature Conditions on Fatty Acid Composition

The effects of different diurnal light and temperature conditions on fatty acids of Vischeria sp. WL1 are shown in Figure 4 and Figure 5. We found that C16:1 and C16:0 comprised most of the total lipids, accounting for approximately 52–60% and 7–14% of the total fatty acids, respectively (Figure 4 and Figure 5). Saturated fatty acids (SFA) showed a general declining trajectory across the time under all three conditions, while the decrement was less under DLTC (5.78%) than under DLC (10.74%) and under the control (12.45%). It is worth noting that there are no significant differences in C14:0 between different conditions. A peculiar oscillatory pattern emerged for C16:0 and C18:0, whereby C16:0 underwent a diminution by day 8, contrasted with an augmentation in C18:0 levels during the same period. Subsequently, on day 12, the trend reversed, with C16:0 experiencing a resurgence while C18:0 returned to a steady state (Figure 5A–C). This resulted in a significantly higher SFA under DLTC on day 12,but not on day 4 and day 8 (Figure 4B).

Moreover, we found that monounsaturated fatty acids (MUFAs) maintained relatively constant levels throughout the duration of the experiment under different conditions. Conversely, polyunsaturated fatty acids (PUFAs) tended to increase progressively over time. However, no significant variations were observed for MUFA and PUFA across different conditions over time (Figure 4C,D). These changes together resulted in no difference in MUFAs between different conditions on day 4. When compared to the control, our findings revealed that amongst the PUFAs, only C18:2 displayed a notable variation, showing an increase specifically under DLTC on days 4, respectively (Figure 5E,F). Interestingly, no significant differences in EPA were observed between the three conditions on days 4 and 8. However, on day 12, the EPA levels under DLTC were significantly lower compared to the control, although no significant differences were observed from those under DLC (Figure 5I). Furthermore, compared to the control, DLC induced a decline in the unsaturation index only on day 12, with no significant changes on days 4 and 8 (Figure 5J). In contrast, DLTC led to a decrease in the unsaturation index on days 4 and 12, but interestingly, an increase was observed on day 8.

3.5. Effects of Different Diurnal Light and Temperature Conditions on the Carbon Redistribution

To understand the mechanisms underlying lipid accumulation in Vischeria sp. WL1, we further analyzed the changes in soluble sugar and soluble protein content per unit cell and their ratios (Figure 6). Compared to the control, the soluble sugar content per cell decreased more under DLTC than under DLC on days 4 and 8, except for a parallel decrease on day 12 (Figure 6A). Although there was no significant difference in the soluble protein content per cell across different conditions on days 4 and 8, a notable reduction in the soluble protein per cell under DLTC became evident on day 12 (Figure 6B). Furthermore, as the cultivation time progressed, the proportion of lipids under all conditions gradually increased, paralleled by a diminishing ratio of soluble sugar, while the soluble protein maintained at a constant level over time (Figure 6C). Meanwhile, the proportion of lipids is higher under DLTC than under other conditions, especially on day 12.

4. Discussion

Dynamic changes in diurnal light and temperature are typical and drastic in the arid environment, leading to adaptive evolution in terrestrial organisms. A number of studies have focused on the effects of light and temperature on algal growth, photosynthesis or oil production, both individually and in combination [1,21,27,34]. However, the specific impacts of the combined variation in light and temperature resulting from diurnal rhythms on these parameters remain largely unreported. This study provides evidence for elucidating its effects in Vischeria sp. WL1. We found the following: 1. Diurnal light and temperature fluctuations are important for the repairment of photosynthetic capacity and efficiency. 2. Diurnal light and temperature fluctuations lead to reduced growth and biomass, potentially reallocating resources from proteins to lipids and thus increasing lipid accumulation. 3. Diurnal light and temperature fluctuations affected the composition and saturation of fatty acids, potentially facilitating the maintenance of membrane fluidity and rigidity under fluctuating conditions.

Light and temperature are both important for photosynthesis efficiency and repairment in microalgae. Here, we found that photosynthetic activity parameters (F_v/F_m, PI_abs, ψ₀ and φ_E0) were the highest, while parameters of damage and energy dissipation (W_k and DI_O/RC) were the lowest under the light cycle condition among all conditions. For example, the W_k value serves as an indicator of the degradation extent of the PSII donor-side oxygen-evolving complex (OEC) in microalgae [35]. These results suggest that photosynthetic capacity and efficiency require darkness for repairment incurred during the process, such as the restoration of photosynthetic pigments and complexes [36]. Under the diurnal light and temperature conditions, the above parameters were moderately changed. As low temperatures can slow down the rate of metabolic processes [37] and therefore reduce the efficiency of the repairment, we suggest that the negative impact of low temperature on photosynthesis offsets some of the positive effects of darkness. Additionally, low temperatures can increase the susceptibility of the photosynthetic apparatus to oxidative stress, which can further impair photosynthetic performance [38]. Therefore, the balance between the positive effects of darkness and the negative impact of low temperatures determines the overall photosynthetic activity under diurnal light and temperature cycles.

Studies have demonstrated that low temperatures facilitate the enhancement of lipid accumulation in microalgae [39,40], with some findings indicating optimal light/dark cycles for maximizing these outcomes [41,42], whereas another research suggests that continuous light may be more beneficial [43]. For microalgae isolated from the arid environment, our findings support that light/dark cycles are more conducive to lipid accumulation but not for the biomass increase. Compared to the continuous light, diurnal light and temperature variations resulted in lower biomass but higher lipid content. Considering the positive effect of low temperature on lipid accumulation [27], we suggest that the diurnal light rhythm and nighttime low temperatures synergistically lead to increased lipid accumulation within the cells. Given the relative increase in lipids while the decrease in carbohydrates under both the diurnal light and temperature conditions in Vischeria sp. WL1 (Figure 6), we suggest a reallocation of carbon resources from carbohydrates toward lipid synthesis, and biomass production is limited due to the shorter duration of light exposure for carbon fixation under the diurnal light rhythm. Another explanation is the consumption of the daytime accumulated carbohydrates at night in the dark by respiration and growth [44]. Since Vischeria sp. WL1 accumulates ~40% lipid content under normal conditions without any stress treatment (Figure 1E) [27,45], which is higher than some other Vischeria strains, such as those isolated from field soil [46] under normal conditions and other microalgal species subjected to stressors such as nutrient limitation and salt treatment [47,48]. The high levels of lipids in Vischeria sp. WL1 might be an evolutionary adaptation to the significant diurnal temperature fluctuations in their arid habitat, potentially providing a reserve of energy or membrane lipid that enables rapid metabolic adjustments under unfavorable conditions. Modulation of fatty acid saturation is commonly observed under variations in light intensity, duration and temperature [27,49]. These modulations are believed to have important roles in the adaptation of microalgae to light and temperature variations, such as PUFAs participating in the recovery of photosystem II and regulating membrane fluidity [50,51]. Here, we did not observe changes in SFAs in the Vischeria sp. WL1 under the diurnal light condition. However, we found the increase in SFAs under diurnal light and temperature fluctuations relative to the control and continuous light on day 12 (Figure 4B), and the dynamic change between C16:0 and C18:0, especially like the decrease in C16:0 and the increase in C18:0 on day 8. We suggest that diurnal light and temperature fluctuations induce a potential conversion between C16:0 and C18:0. Intriguingly, fluctuating light and temperature conditions resulted in the decline of EPA on day 12, which is parallel with our previous result that low temperature reduces EPA in Vischeria sp. WL1 on day 8 [27], suggesting that prolonged exposure to low temperature might not be beneficial for the EPA accumulation in Vischeria sp. WL1. Additionally, it is noteworthy that fluctuating light and temperature conditions did not have a significant effect on MUFAs and PUFAs, except for inducing an increase in C18:1 and C18:2 on day 4. This is different than our previous findings [27], suggesting that the response of Vischeria sp. WL1 to low temperature at night on MUFAs and PUFAs might differ from its effect from the constant effect of low temperature. The fluctuating light and temperature conditions elicited a notable increase in the unsaturation index, specifically on day 8, whereas decreases were observed on days 4 and 12. This dynamic pattern may indicate a temporary adjustment in their fatty acid composition and adaptative response to thermal variability [52].

In conclusion, we investigated the effects of diurnal temperature and light fluctuations on the growth, photosynthesis, lipid accumulation and fatty acid composition of Vischeria sp. WL1. The results demonstrate that the balance between the positive effects of darkness and the negative impact of low temperatures determines overall photosynthetic activity under diurnal light and temperature cycles. Specifically, we found that the diurnal variation in light and temperature significantly influenced the saturation levels of certain fatty acids, highlighting their unique roles compared to conditions of constant lower temperatures. This suggests that Vischeria sp. WL1 has developed distinct metabolic strategies to adapt to fluctuating environmental conditions. Overall, these findings contribute to the broader understanding of microalgal physiology and adaptation mechanisms in arid environments. Notably, the high lipid accumulation observed in Vischeria sp. WL1 is likely the outcome of long-term evolutionary adaptation to the remarkable diurnal temperature fluctuations characteristic of arid regions. This adaptive strategy not only enhances the microalga’s survival but also positions it as a promising candidate for biofuel production. From a practical perspective, this research provides a theoretical basis for optimizing oil and target fatty acid production in this strain. By elucidating the metabolic responses to diurnal environmental changes, our study offers valuable insights for developing cultivation strategies that maximize lipid yield under fluctuating conditions. Furthermore, these findings underscore the importance of considering natural environmental variability when designing biotechnological applications for microalgae. In summary, this study advances our understanding of the adaptive mechanisms of Vischeria sp. WL1 and highlights its potential for industrial applications. Future research should explore the genetic and molecular basis of these adaptive traits, as well as their applicability to other microalgal species, so as to further unlock the biotechnological potential of microalgae in the realm of sustainable energy production.

Author Contributions

Conceptualization, Z.L. and X.G. (Xiang Gao); methodology, Z.L., X.G. (Xinhong Guo), D.W. and X.Y.; investigation, X.G. (Xinhong Guo), D.W. and X.W.; writing—original draft preparation, Z.L. and X.G. (Xinhong Guo); writing—review and editing, Z.L., H.X., Q.Z. and X.G. (Xiang Gao); funding acquisition, Z.L. and X.G. (Xiang Gao). All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data will be made available upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures

Figure 1. The growth, dry weight and oil content under different diurnal light and temperature conditions: (A) The experimental scheme. (B) Cell number. (C) Dry weight per milliliter. (D) Dry weight per cell. (E) Oil content. Different letters on the top of each bar in (C–E) represent significant differences (p [less than] 0.05) among treatments for each day. Data are shown as the mean ± SD (n = 3). Control: [L (30 °C, 24 h) + D (30 °C, 0 h)]; a diurnal light cycle (DLC): [L (30 °C, 16 h) + D (30 °C, 8 h)]; a diurnal light–temperature cycle (DLTC): [L (30 °C, 16 h) + D (13 °C, 8 h)]. L, light; D, dark. The statistical analysis of cell numbers across different treatments is presented in Table S1.

Figure 2. The concentration and content of photosynthetic pigments under different diurnal light and temperature conditions: (A) Chlorophyll a concentration. (B) Chlorophyll a content per cell. (C) Carotenoid concentration. (D) Carotenoid content per cell. Data are shown as the mean ± SD (n = 3). Control: [L (30 °C, 24 h) + D (30 °C, 0 h)]; a diurnal light cycle (DLC): [L (30 °C, 16 h) + D (30 °C, 8 h)]; a diurnal light–temperature cycle (DLTC): [L (30 °C, 16 h) + D (13 °C, 8 h)]. L, light; D, dark. The statistical analysis of the concentration and content of photosynthetic pigments across different treatments is presented in Table S2.

Figure 3. The photosynthetic parameter under different diurnal light and temperature conditions: (A) Fv/Fm. (B) PIabs. (C) φE0. (D) ψ0. (E) Wk. (F) ABS/RC. (G) TRO/RC. (H) ETO/RC. (I) DIO/RC. Fv/Fm, the maximum quantum yield of PSII photochemistry; PIabs, performance index on absorption basis; φE0, maximum values of quantum yield for electron transport; ψ0, maximum values of efficiency that a trapped exciton can move an electron into the electron transport chain beyond QA−; Wk, relative variable fluorescence after K point standardization; ABS/RC, the effective antenna size per reaction center; DIO/RC, the dissipated energy flux per reaction center; TRO/RC, unit of energy captured by the reaction center for the reduction in Qa; ETO/RC, the light quantum flux transmitted by the initial electron per active reaction center. Data are shown as the mean ± SD (n = 3). Control: [L (30 °C, 24 h) + D (30 °C, 0 h)]; a diurnal light cycle (DLC): [L (30 °C, 16 h) + D (30 °C, 8 h)]; a diurnal light–temperature cycle (DLTC): [L (30 °C, 16 h) + D (13 °C, 8 h)]. L, light; D, dark. The statistical analysis of the photosynthetic parameter across different treatments is presented in Table S3.

Figure 4. The fatty acid composition of Vischeria sp. WL1 under different diurnal light and temperature conditions: (A) Fatty acid composition. (B) Saturated fatty acids (SFA). (C) Monounsaturated fatty acids (MUFA). (D) Polyunsaturated fatty acid (PUFA). Different letters on the top of each bar in (B–D) represent significant differences (p [less than] 0.05) among treatments for each day. Data are shown as the mean ± SD (n = 3). Control: [L (30 °C, 24 h) + D (30 °C, 0 h)]; a diurnal light cycle (DLC): [L (30 °C, 16 h) + D (30 °C, 8 h)]; a diurnal light–temperature cycle (DLTC): [L (30 °C, 16 h) + D (13 °C, 8 h)]. L, light; D, dark.

Figure 5. Individual fatty acids patterns (A–I) and unsaturation index (J) of Vischeria sp. WL1 under different diurnal light and temperature conditions: Numbers in the upper middle indicate the fatty acid classes shown in each panel. Different letters on the top of each bar represent significant differences (p [less than] 0.05) among treatments for each day. Data are shown as the mean ± SD (n = 3). Control: [L (30 °C, 24 h) + D (30 °C, 0 h)]; a diurnal light cycle (DLC): [L (30 °C, 16 h) + D (30 °C, 8 h)]; a diurnal light–temperature cycle (DLTC): [L (30 °C, 16 h) + D (13 °C, 8 h)]. L, light; D, dark.

Figure 6. The soluble sugar; the soluble protein; and the composition between sugar, protein and lipid: (A) The soluble sugar content per cell. (B) Soluble protein content per cell. (C) The ratios of sugar, protein and lipid content on a per-cell weight. Different letters on the top of each bar in (A,B) represent significant differences (p [less than] 0.05) among treatments for each day. Data are shown as the mean ± SD (n = 3). Control: [L (30 °C, 24 h) + D (30 °C, 0 h)]; a diurnal light cycle (DLC): [L (30 °C, 16 h) + D (30 °C, 8 h)]; a diurnal light–temperature cycle (DLTC): [L (30 °C, 16 h) + D (13 °C, 8 h)]. L, light; D, dark.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d17020135/s1, Table S1: Changes in cell number of Vischeria sp. WL1 over time under three culture conditions (Control, DTC, and DLTC); Table S2: Changes in photosynthetic pigment content of Vischeria sp. WL1 over time under three culture conditions (Control, DTC, and DLTC).; Table S3: Changes in photosynthetic parameters of Vischeria sp. WL1 over time under three culture conditions (Control, DTC, and DLTC).

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