Effects of Low Temperature, Nitrogen Starvation and Their Combination on the Photosynthesis and Metabolites of Thermosynechococcus E542: A Comparison Study

Both low temperature and nitrogen starvation caused chlorosis of cyanobacteria. Here, in this study, for the first time, we compared the effects of low temperature, nitrogen starvation, and their combination on the photosynthesis and metabolites of a thermophilic cyanobacterium strain, Thermosynechococcus E542. Under various culture conditions, the growth rates, pigment contents, and chlorophyll fluorescence were monitored, and the composition of alkanes, lipidomes, and carbohydrates were determined. It was found that low temperature (35 °C) significantly suppressed the growth of Thermosynechococcus E542. Nitrogen starvation at 45 °C and 55 °C did not affect the growth; however, combined treatment of low temperature and nitrogen starvation led to the lowest growth rate and biomass productivity. Both low temperature and nitrogen starvation caused significantly declined contents of pigments, but they resulted in a different effect on the OJIP curves, and their combination led to the lowest pigment contents. The composition of fatty acids and alkanes was altered upon low-temperature cultivation, while nitrogen starvation caused reduced contents of all lipids. The low temperature did not affect carbohydrate contents, while nitrogen starvation greatly enhanced carbohydrate content, and their combination did not enhance carbohydrate content, but led to reduced productivity. These results revealed the influence of low temperature, nitrogen starvation, and their combined treatment for the accumulation of phycobiliproteins, lipids, and carbohydrates of a thermophilic cyanobacterium strain, Thermosynechococcus E542.


Introduction
An increase in CO 2 concentration in the atmosphere caused by the exploitation of fossil fuels results in global climate change and threatens the sustainable development of humankind. Carbon sequestration technologies have attracted the wide attention of both academics and industries. Cyanobacteria are prokaryotic photosynthetic micro-organisms able to inhabit various environments and tolerate different stresses, such as high salinity, temperature, pressure, and desiccation [1]. Thanks to their high photosynthetic rates and potential to produce various value-added products and biofuels, cyanobacteria have been promising candidates for biological carbon sequestration [2,3].
Growth temperature and nitrogen supply are two major factors that regulate the photosynthetic rates and biochemical composition of cyanobacteria [4,5]. Mesophilic A two-stage culture strategy was adopted to investigate the effects of growth temperature and nitrogen supply. In the first stage, Thermosynechococcus E542 was cultivated for 24 h in BG-11 medium supplemented with sodium nitrate. At the end of the first stage, the biomass was collected by centrifugation and then resuspended in a fresh BG-11 medium supplemented with or without sodium nitrate. It was then incubated for another 24 h (the second stage), and the results related to the growth rate and biomass yield are shown in Table 1. Growth temperature significantly affected the growth rate and biomass yield of Thermosynechococcus E542. During the first 24 h, when temperature declined from 45 • C to 35 • C, the growth rate was reduced by 42.9% from 2.26 to 1.29 d −1 , and the 48-h average biomass productivity was increased by 123% from 0.132 g L −1 d −1 to 0.296 g L −1 d −1 when growth temperature was elevated from 35 • C to 45 • C. The effect of temperature on growth rate became less pronounced when it was further increased from 45 • C to 55 • C. The growth rate was relatively constant, confirming prior findings [11].
Intriguingly, it was found that nitrogen starvation did not affect the growth rate and biomass productivity at 45 • C and 55 • C. However, combined treatment of nitrogen starvation and low temperature at 35 • C caused the growth rate to decline by 20.7% from 0.82 to 0.65, while biomass productivity also declined from 0.148 g L −1 d −1 to 0.094 g L −1 d −1 .

Variation of Pigment Contents and Chlorophyll Fluorescence
Thermosynechococcus E542 cultivated at 35 • C exhibited lower contents of Chla and PC. The cultures that were grown at 45 • C and 55 • C in nitrogen-rich BG-11 medium contained an abundance of pigments, C-phycocyanin (PC) and allophycocyanin (APC) accounted for 12-13% and 6.8-6.8% of dry biomass, respectively, while Chla reached more than 2% of the biomass (Figure 1). However, at 35 • C, their content was dramatically reduced by 66.1% and 67.4%, respectively, compared with cultures grown at 45 • C. The ratio of PC to Chla did not decline with decreasing growth temperature and was estimated at 5.8, 5.6, and 5.1, respectively, for growth temperatures of 35 • C, 45 • C, and 55 • C. These findings are in contrast to previous research that indicates that PC/Chla is reduced during low-temperature acclimation of mesophilic cyanobacteria strains [13].

55-NS
0.240 ± 0.004 2.19 ± 0.04 0.654 ± 0.018 1.00 ± 0.04 Growth temperature significantly affected the growth rate and biomass yield of Thermosynechococcus E542. During the first 24 hours, when temperature declined from 45 °C to 35 °C, the growth rate was reduced by 42.9% from 2.26 to 1.29 d −1, and the 48-h average biomass productivity was increased by 123% from 0.132 g L −1 d −1 to 0.296 g L −1 d −1 when growth temperature was elevated from 35 °C to 45 °C. The effect of temperature on growth rate became less pronounced when it was further increased from 45 °C to 55 °C. The growth rate was relatively constant, confirming prior findings [11].
Intriguingly, it was found that nitrogen starvation did not affect the growth rate and biomass productivity at 45 °C and 55 °C. However, combined treatment of nitrogen starvation and low temperature at 35 °C caused the growth rate to decline by 20.7% from 0.82 to 0.65, while biomass productivity also declined from 0.148 g L −1 d −1 to 0.094 g L −1 d −1 .

Variation of Pigment Contents and Chlorophyll Fluorescence
Thermosynechococcus E542 cultivated at 35 °C exhibited lower contents of Chla and PC. The cultures that were grown at 45 °C and 55 °C in nitrogen-rich BG-11 medium contained an abundance of pigments, C-phycocyanin (PC) and allophycocyanin (APC) accounted for 12-13% and 6.8-6.8% of dry biomass, respectively, while Chla reached more than 2% of the biomass (Figure 1). However, at 35 °C, their content was dramatically reduced by 66.1% and 67.4%, respectively, compared with cultures grown at 45 °C. The ratio of PC to Chla did not decline with decreasing growth temperature and was estimated at 5.8, 5.6, and 5.1, respectively, for growth temperatures of 35 °C, 45 °C, and 55 °C. These findings are in contrast to previous research that indicates that PC/Chla is reduced during low-temperature acclimation of mesophilic cyanobacteria strains [13]. Nitrogen starvation treatment led to significantly reduced pigment contents in Thermosynechococcus E542 at all tested temperatures. The content of PC was 67-78% lower, while the content of Chla was 51-60% lower than cultures grown in nitrogen-rich BG-11 medium. Combined treatment of low temperature (35 °C) and nitrogen starvation led to the lowest pigment contents among all conditions. Contents of PC, APC, and Chla declined to 1.4%, 0.78%, and 0.34% of dry biomass.
Growth temperature significantly affects the OJIP curves of Thermosynechococcus E542. Figure 2a shows the fluorescence transient curves of Thermosynechococcus E542 cultivated at different temperatures in a nitrogen-rich BG-11 medium. The transient curves showed clear polyphasic rise, with the I and J step appearing at 2 ms and 30 ms, Nitrogen starvation treatment led to significantly reduced pigment contents in Thermosynechococcus E542 at all tested temperatures. The content of PC was 67-78% lower, while the content of Chla was 51-60% lower than cultures grown in nitrogen-rich BG-11 medium. Combined treatment of low temperature (35 • C) and nitrogen starvation led to the lowest pigment contents among all conditions. Contents of PC, APC, and Chla declined to 1.4%, 0.78%, and 0.34% of dry biomass.
Growth temperature significantly affects the OJIP curves of Thermosynechococcus E542. Figure 2a shows the fluorescence transient curves of Thermosynechococcus E542 cultivated at different temperatures in a nitrogen-rich BG-11 medium. The transient curves showed clear polyphasic rise, with the I and J step appearing at 2 ms and 30 ms, respectively, while the P step appeared at 141-281 ms. Significant differences between the OJIP transients for the culture 35-48 h and those of 45-48 h and 55-48 h could be observed. Firstly, the culture 35-48 h has much lower F O fluorescence, 44.6% lower than that of 45-48 h. Similar results were obtained for eukaryotic photosynthetic organisms and mesophilic cyanobacterium Synechococcus sp. PCC 7942 [14]. F O fluorescence in cyanobacteria considerably depends on the cellular phycobilin concentration [15]. In this study, OD 730 of all cultures was adjusted to the same value (0.26 ± 0.01) prior to the measurement of OJIP transients. Hence, the lower F O fluorescence of 35-48 h was due to its lower PC content. Another difference is that the relative variable fluorescence at step J (  Table 2). This indicates that the reoxidation capacity of Q A − is decreased at a growth temperature of 35 • C [16]. Growth temperature also affects the photochemical efficiency of PSII in the dark-adapted state (F v /F m ). F v /F m reaches the maximal value of 0.422 at 45 • C, close to the maximal values of 0.4-0.5 for mesophilic cyanobacteria [6].
(VJ = (FJ -Fo)/ (Fm -Fo)) of the culture 35-48 h is much higher than that of 45-48 h and 55-48 h ( Table 2). This indicates that the reoxidation capacity of QA − is decreased at a growth temperature of 35 °C [16]. Growth temperature also affects the photochemical efficiency of PSII in the dark-adapted state (Fv/Fm). Fv/Fm reaches the maximal value of 0.422 at 45 °C, close to the maximal values of 0.4-0.5 for mesophilic cyanobacteria [6].
Nitrogen starvation also affected the shape of the OJIP transients of Thermosynechococcus E542, as shown in Figure 2b. Due to the degradation of pigments, the FO fluorescence declined to 10,000-15,000 after nitrogen starvation, compared with 25,000-50,000 for cultures replete in nitrogen. Additionally, the OJIP transients almost totally level off after nitrogen starvation treatment. Similar results were observed for the OJIP transients of mesophilic cyanobacteria under other stress factors, such as high salinity [17] and heat stress [18]. Moreover, the photochemical efficiency of PSII also sharply declined to 0.18-0.20 from 0.3-0.4 (Table 2).    Nitrogen starvation also affected the shape of the OJIP transients of Thermosynechococcus E542, as shown in Figure 2b. Due to the degradation of pigments, the F O fluorescence declined to 10,000-15,000 after nitrogen starvation, compared with 25,000-50,000 for cultures replete in nitrogen. Additionally, the OJIP transients almost totally level off after nitrogen starvation treatment. Similar results were observed for the OJIP transients of mesophilic cyanobacteria under other stress factors, such as high salinity [17] and heat stress [18]. Moreover, the photochemical efficiency of PSII also sharply declined to 0.18-0.20 from 0.3-0.4 (Table 2).

Variation of Alkanes and Fatty Acids
Membrane fluidity plays a pivotal role in the temperature acclimation of photosynthetic organisms [8]. Adjustment of the membrane fluidity could be achieved by the production of polyenoic acid or by changing the ratio of saturated fatty acids to unsaturated fatty acids (S/U ratio) in mesophilic cyanobacteria [19]. Recently, it was found that hydrocarbons may also be involved in the adjustment of membrane fluidity during low-temperature acclimation of cyanobacteria [20]. The composition of fatty acids and hydrocarbons of Thermosynechococcus E542 cultivated at different temperatures and nitrogen supply is shown in Figure 3. Under the tested conditions, palmitic acid (C16:0) is the most abundant fatty acid, accounting for 55-60% of total fatty acid methyl esters (FAMEs) and hydrocarbons. Other fatty acids include palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), and myristic acid (C14:0). The hydrocarbons include pentadecane and heptadecane, but the amount of pentadecane is not quantitated due to its limited concentration.
35 °C, respectively. S/U ratio was also altered and was equal to 1.4, 1.7, and 2.5 at the growth temperatures of 35 °C, 45 °C, and 55 °C, respectively. The content of heptadecane also depends on growth temperature. As growth temperature decreased from 55 °C to 45 °C and 35 °C, the amount of heptadecane increased by 25% and 49%, respectively, suggesting an increase in membrane fluidity.
Nitrogen starvation caused a significant decline in the total amount of FAMEs and hydrocarbons from 6-7% to about 4%. However, nitrogen starvation did not affect the correlation between FAMEs and hydrocarbon composition and growth temperature. This is consistent with previous findings that nitrogen starvation caused the degradation of the intracellular membranes of Synechococcus elongates PCC 7942 [9].

Lipidomic Analysis based on LC-MS/MS
In total, 56 polar glycerides in Thermosynechococcus E542 were identified, including 29 monogalactosyldiacylglycerols (MGDG), 20 digalactosyldiacylglycerols (DGDG), 3 sulfoquinovosyl-diacylglycerols (SQDG), and 4 phosphatidylglycerols (PG) ( Table S1). To the best of our knowledge, no prior reports on the lipidome of thermophilic cyanobacteria based on LC-MS/MS have been found in the literature. Besides, our results showed that Thermosynechococcus E542 could synthesise glycerides containing fatty acids with an odd number of carbon atoms, such as C17:0 and C19:1. Glycerides containing odd-number fatty acids were also observed in other cyanobacteria strains. Glycerides containing C17:1, Growth temperature significantly affects the composition of fatty acids and hydrocarbons. The content of C16:1 is inversely proportional to the growth temperature and increased by 61% and 135% as growth temperature decreased from 55 • C to 45 • C and 35 • C, respectively. S/U ratio was also altered and was equal to 1.4, 1.7, and 2.5 at the growth temperatures of 35 • C, 45 • C, and 55 • C, respectively. The content of heptadecane also depends on growth temperature. As growth temperature decreased from 55 • C to 45 • C and 35 • C, the amount of heptadecane increased by 25% and 49%, respectively, suggesting an increase in membrane fluidity.
Nitrogen starvation caused a significant decline in the total amount of FAMEs and hydrocarbons from 6-7% to about 4%. However, nitrogen starvation did not affect the correlation between FAMEs and hydrocarbon composition and growth temperature. This is consistent with previous findings that nitrogen starvation caused the degradation of the intracellular membranes of Synechococcus elongates PCC 7942 [9].

Content and Productivity of Carbohydrate and Elemental Composition of the Biomass
Growth temperature did not affect the content of carbohydrates significantly. As shown in Figure 4, the samples cultivated in nitrogen-rich BG-11 medium contain only 14-17% of carbohydrates, leading to low carbohydrate productivity of 20-40 mg L −1 d −1 . Elemental analysis shows that nitrogen accounts for 8-9% of the dry weight (Table 4), indicating that the biomass comprises more than 50% protein (calculated using a conversion factor of 6.25 from nitrogen to protein). Nitrogen starvation significantly promoted the content and productivity of carbohydrates. Maximal carbohydrate content and carbohydrate productivity were obtained at 45 • C, which was 53.7% and 154 mg L −1 d −1 , respectively. Meanwhile, the nitrogen content in the biomass declined by more than 50% from 8-9% to about 4%. Combined treatment of low temperature and nitrogen starvation did not improve carbohydrate content and carbohydrate productivity.
Nitrogen supply had a significant effect on the contents of various lipids at all tested temperatures. With nitrate depleted, at 35 °C, the contents of MGDG, DGDG, SQDG, and PG were reduced by 31%, 46%, 53%, and 35%, respectively, compared with nitraterepleted culture, while, at 55 °C, they were reduced by 26%, 16%, 39 %, and 41%, respectively. Besides, the structure of fatty acids in specific lipids was also changed. However, these patterns were not as clear as those observed for temperature changes.

Content and Productivity of Carbohydrate and Elemental Composition of the Biomass
Growth temperature did not affect the content of carbohydrates significantly. As shown in Figure 4, the samples cultivated in nitrogen-rich BG-11 medium contain only 14-17% of carbohydrates, leading to low carbohydrate productivity of 20-40 mg L −1 d ±1 . Elemental analysis shows that nitrogen accounts for 8-9% of the dry weight (Table 4), indicating that the biomass comprises more than 50% protein (calculated using a conversion factor of 6.25 from nitrogen to protein). Nitrogen starvation significantly promoted the content and productivity of carbohydrates. Maximal carbohydrate content and carbohydrate productivity were obtained at 45 °C, which was 53.7% and 154 mg L −1 d −1 , respectively. Meanwhile, the nitrogen content in the biomass declined by more than 50% from 8-9% to about 4%. Combined treatment of low temperature and nitrogen starvation did not improve carbohydrate content and carbohydrate productivity.

Discussion
Growth of Thermosynechococcus E542 at 35 • C, 20 • C lower than the optimal growth temperature, led to significantly lower growth rate, biomass productivity, and pigment contents. This is in accordance with the previous study suggesting that photoinhibition of mesophilic cyanobacteria usually occurred at 15 • C lower than their optimal temperature [6]. In addition, the abundance of pigments in mesophilic cyanobacteria could be drastically altered during acclimation to low temperatures. Vonshak et al. found that, when Arthrospira platensis strains M 2 and Kenya were cultivated at 15 • C, the contents of Chla and PC were reduced by 59.6% and 60.9%, 56.5%, and 68.2%, respectively, compared with those at 30 • C [13].
Nitrogen starvation also caused a significant decline in PBSs and Chla in non-diazotrophic mesophilic cyanobacteria [4]. In line with these results, it was found that pigment contents of Thermosynechococcus E542 were also reduced upon nitrogen starvation. Upon nitrogen depletion, not only is synthesis of PBS in cyanobacteria depressed, but, also, rapid degradation of PBS occurs. Nutrient starvation induced the expression of nblA gene, which is essential for PBS degradation in cyanobacteria [24]. Our previous study confirmed the existence of nblA gene in the genome of Thermosynechococcus E542. It was found that nblA is responsible for cell bleaching upon nutrient depletion [11].
Nitrogen starvation led to reduced contents of pigments at all tested growth temperatures. However, at 45 • C and 55 • C, 24-h nitrogen starvation did not affect the growth of Thermosynechococcus E542. This suggests that a considerable proportion of pigments (more than 50%) accumulated during growth in the nitrogen-rich medium can serve as a nitrogen source during growth in the nitrogen-depleted medium. These findings are in accordance with previous studies based on mesophilic cyanobacteria Synechococcus sp. PCC 7942 [25] and Synechococcus sp. PCC 6803 [26]. Although growth at 35 • C also led to reduced pigment contents, they were higher than those observed under nitrogen starvation treatment. However, growth rate and biomass productivity were significantly lower when cultivated at 35 • C than at 45 • C and 55 • C. This indicates that the decline in pigments should not be the cause of reduced growth rate and biomass productivity. Combined treatment of nitrogen starvation and low temperature caused the lowest growth rate, biomass productivity, and pigment contents, suggesting that low temperature acclimation of Thermosynechococcus E542 depends on nitrogen supply. Analysis of Chla fluorescence showed that the impact of nitrogen starvation and low growth temperature on the electron transport chain are different, although both treatments caused reduced pigment contents. The OJIP curves showed typical multiphasic rises at all growth temperatures without nitrogen starvation, but almost totally levelled off after 24-h nitrogen starvation.
The effect of nitrogen starvation and low temperature on the composition of fatty acids and hydrocarbons was also different. During low-temperature acclimation, the ratio of saturated fatty acids to unsaturated fatty acids and the content of heptadecane increased, but the total amount of fatty acids and hydrocarbons was maintained constant. In comparison, after 24-h nitrogen starvation, the total amount of fatty acids and hydrocarbons decreased. Analysis of the lipidome based on LC-MS/MS showed that growth temperature did not affect the absolute amount of various lipids in the biomass. However, the structure of fatty acids in lipids was altered, although in different patterns for different lipid classes. For MGDG and DGDG, species with a fatty acid composition of 16:0/16:1 increased with decreasing temperature. In addition, for PG, the most significant change was that PG (16:0/18:1) sharply increased with reduced temperature. On the other hand, the composition of fatty acids in SQDG was not affected by temperature. In comparison, nitrate depletion caused a significant decline in the absolute contents of various lipids. In contrast, the change in fatty acid structure in specific lipids was not as apparent as observed for temperature.
Low temperature and nitrogen starvation also had different impacts on the content and productivity of carbohydrates. The low temperature did not increase the content of carbohydrates but significantly reduced their productivity. However, the content and productivity of carbohydrates were greatly enhanced upon nitrogen starvation. The content and productivity of carbohydrates were higher than those of mesophilic cyanobacteria strains Synechocystis sp. PCC6803 [26] and Synechococcus elongatus PCC7942 [27].

Cultivation of Cyanobacterium
Unless otherwise stated, cyanobacterium Thermosynechococcus elongatus PKUAC-SCTE542 (hereafter, Thermosynechococcus E542 in abbreviation) deposited in the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-2455) was cultivated in BG-11 medium supplemented with 0.1 M NaHCO 3 , prepared by mixing one portion of twofold BG-11 and one portion of 0.2 M solution of NaHCO 3 in equal volumes. In nitrogen starvation experiments, NaNO 3 free BG-11 (BG11-N hereafter) was used instead of standard BG-11. The strain was cultivated in a shaking incubator (Bluepard, Yiheng Group, Shanghai, China) at 35, 45, or 55 • C, 120 rpm. The shaking incubator was modified with white LED strips to provide two-sided LED lighting. Photosynthetic photon flux density was 109 µmol m −2 s −1 (measured with Lighting Passport, Asense Tek, Tainan, China). The light/dark cycle was 23/1 for all experiments. Samples from precultures were diluted in 150 mL fresh BG-11 medium in 500 mL Erlenmeyer flasks to OD 730 of 0.12 ± 0.1, corresponding to 0.027 gL −1 . After 24 h of incubation, the biomass was collected by centrifugation at 10,000 rpm for 3 min and resuspended in fresh BG-11 or BG-11-N (for nitrogen starvation experiments), supplemented with 0.1 M NaHCO 3 , and incubated for another 24 h. All experiments were conducted in three biological replicas. At specific times, an aliquot of the culture was withdrawn and OD 730 was determined (UV-1800, Shimadzu Corporation, Kyoto, Japan). The culture was diluted by fresh medium if OD 730 exceeded 1.0. At 48 h, the biomass was collected by centrifugation at 10,000 rpm for 3 min, washed with 30 mL deionised water, and freeze-dried for 48 h at −50 • C, 12 Pa (Freezone 2.5, Labcon, USA). Specific growth rates (µ, d −1 ) were determined using biomass concentration (gL −1 ) at the beginning and end of the exponential growth phase using Equation (1).
where X 1 and X 2 are biomass concentration at times t 1 and t 2 , respectively.

Chla Fluorescence Transient Analysis
The fluorescence intensity of photosynthetic organisms shows polyphasic increases upon dark to light transition from a low level of F O via two intermediates F J and F I to a maximum level of F M . Each rise phase can be related to a specific step of the reduction in the ETC, and thus information on the state of the ETC can be obtained via analysis of the OJIP transients. In general, the OJ step is related to the reduction in Q A to Q A − and, hence, the accumulation of Q A − Q B , the JI step is related to the accumulation of Q A − Q B − , and the IP step reflects an accumulation of Q A − Q B 2− [16]. Polyphasic rise of Chla fluorescence transient (OJIP) measurements were conducted using pulse amplitude magnitude (PAM) fluorimeter (AquaPen-C AP-C 100, Photon Systems Instruments, Czech Republic) and FluorPen 1.0 software. All samples were diluted with the same medium of the culture OD 730 of 0.26 ± 0.01 and were incubated in the dark for at least 15 min before measurements. Fluorescence was recorded at the same temperature as the culture by housing the fluorimeter and the sample in the incubator. Red-orange light (620 nm) of 3000 µmol m −2 s −1 was used for saturating light pulses in the fluorimeter. The PAM fluorimeter recorded the Chla fluorescence induced by the saturating pulses between 20 µs and 1 s. The fluorescence intensity at 50 µs, 2 ms, and 30 ms was designated as O, J, and I fluorescence, respectively, whilst P was the maximum fluorescence. For more details about the procedure and parameters of OJIP, please refer to [28].

Measurement of Pigments
To determine Chla and carotenoids, 4 mL aliquot was withdrawn from the culture and transferred to a 15 mL centrifuge tube and centrifuged at 15,000× g for 7 min. The supernatant was discarded and 4 mL methanol precooled to 4 • C was added [29]. The centrifuge tube was covered with aluminium foil and incubated at 4 • C for 20 min. After being centrifuged at 15,000× g for 7 min, Chla and carotenoids dissolved in the supernatant were determined by measuring optical absorbance at 470, 665, and 720 nm using methanol as blank (UV-1800, Shimadzu Corporation, Kyoto, Japan). Concentrations of Chla and carotenoids were calculated according to the following equations [30]: To determine phycobiliprotein, 1.5 mL culture suspension was transferred to centrifuge tubes and was centrifuged at 15,000× g at 4 • C for 5 min. Collected biomass was freeze-dried overnight. Subsequently, it was homogenised with 2 mm glass beads for 15 s on a homogeniser. Then, 1.5 mL PBS buffer (pH 7.4) precooled to 4 ºC was added, and the sample was mixed with PBS for 5 s on the homogeniser. After being kept on ice for 60 min, the sample was centrifuged at 15,000× g and 4 • C for 5 min. Concentrations of phycobiliprotein and APC were quantified by measuring optical absorbance at 615, 652, and 720 nm with a spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) and were calculated as follows [31]:

Determination of FAMEs and Alkane
Analysis of fatty acid methyl esters (FAMEs) by in situ transesterification was conducted according to the procedure published by the National Renewable Energy Laboratory of the USA [32]. In brief, 5 mg biomass was transferred into a 1.5-mL GC vial. Subsequently, 20 µL of internal standard (C19:0), 200 µL of chloroform:methanol (2:1, v/v), and 300 µL of 0.6 M HCl:methanol were added. The tube was then incubated in a water bath at 85 • C for 1 h. After cooling to room temperature, 1 mL n-hexane was added to extract FAMEs and heptadecane. The tube was vortexed and allowed to stand undisturbed for 1 h. FAMEs and alkanes in n-hexane phase were analysed by gas chromatography (7890A, Agilent, Santa Clara, CA, USA) equipped with a flame ionisation detector (FID) and DB-23 column with a film thickness of 0.25 µm (30 m × 0.25 mm, length × internal diameter, Agilent Technologies, Santa Clara, CA, USA). The injection volume was 1 µL with a split ratio of 10:1. The temperature of the inlet and FID was 250 • C and 280 • C, respectively. Helium was used as a carrier gas at a constant flow rate of 1.0 mL·min −1 . Programme of oven temperature: 50 • C for 1 min, 25 • C·min −1 up to 175 • C and hold for 0 min, 4 • C·min −1 up to 230 • C and hold for 5 min. FAMEs and heptadecane were identified by comparing elution time with a calibration standard. The concentration of FAMEs and heptadecane was determined by comparing with internal calibration curves made of 5 points with C19:0 as an internal standard (R 2 > 0.998).

Lipidomic Analysis
For lipid extraction, 1-2 mg freeze-dried biomass was transferred into a 2 mL centrifuge tube. A total of 300 µL methanol was added, and the tube was vortexed for 20 s. Subsequently, 1000 µL MTBE (methyl tertiary butyl ether) was added, and the tube was vortexed for 5 min. Then, 250 µL deionised water was added, and the tube was vortexed for 30 s. The tube was left to stand for 10 min and centrifuged at 14,000 rpm for 2 min. The upper phase was transferred to a 2 mL vial and dried under nitrogen. Dried lipids were solubilised by 1 mL chloroform-methanol (1:1, v/v) and kept at −20 • C. Data Availability Statement: All data generated or analysed during this study are included in this published article.