Regulation of different light conditions for efficient biomass production and protein accumulation of Spirulina platensis*

Yufei ZHANG, Xianjun LI, Yuhui LI, Shiqi LIU, Yanrui CHEN, Miao JIA,Xin WANG, Lu ZHANG, Qiping GAO, Liang ZHANG, Daoyong YU,Baosheng GE,**

Abstract Light plays an important role in the photosynthesis and metabolic process of microalgae.However, how different light conditions regulate the biomass production and protein accumulation of microalgae is mostly unknown.In this study, the influence of different light conditions, including light colors, densities, and light：dark cycles on the cell growth and biochemical composition of Spirulina platensis was symmetrically characterized.Under different colored lights, S.platensis all shows an increase trend within the increased light intensity ranges; however, each showing different optimal light intensities.At the same light intensity, different colored lights show different growth rate of S.platensis following the sequence of red＞white＞green＞yellow＞blue.The maximum growth rate and protein accumulation were determined as 21.88 and 5.10 mg/(L·d) when illuminated under red LED.The energy efficiency of different light sources was calculated and ranked as red＞white＞blue≈green＞yellow.Transcriptomic analysis suggests that red light can promote cell growth and protein accumulation by upregulating genes related to photosynthesis, carbon fixation, and C-N metabolism pathways.This study provides a conducive and efficient way to promote biomass production and protein accumulation of S.platensis by regulating light conditions.

Keyword: microalgae; light emitting diode; protein accumulation; biomass production; transcriptomic analysis

1 INTRODUCTION

Spirulinaplatensisis a planktonic, filamentous cyanobacterium found in tropical and subtropic alkaline warm lakes (Castro et al., 2019; Papalia et al., 2019).Due to its fast growth and robust culture conditions, and richness in proteins, polyunsaturated fatty acids, essential amino acids, vitamins, and pigments (Thajuddin and Subramanian, 2005),S.platensishas been widely applied as foods,feeds, nutraceuticals, pharmaceuticals, and cosmeceuticals (Nethravathy et al., 2019; Tang et al., 2020; Pocha et al., 2022), and also been deemed as one of the most promising microalgae strains for wastewater treatment and carbon sequestration from flue gas (Almomani et al., 2019).Therefore, in order to realize the development of a green sustainable circular economy of microalgae (Azmi et al., 2021;Devadas et al., 2021; Lim et al., 2022), efficient cultivation ofS.platensiswith increasing biomass production, lower cost, and higher special nutrients is highly expected.

Light is the most significant factor that controls the photosynthetic efficiency and cell growth of microalgae (Atta et al., 2013).It has been reported that the light quality can finely regulate the synthesized protein content of microalgae, and properly colored LEDs (light emitting diodes) had been found to be crucial for the microalgal growth(Da Fontoura Prates et al., 2018), pigment (Chen et al., 2010), lipid (Teo et al., 2014), and protein accumulation (Da Fontoura Prates et al., 2020).For example, the orange light increased both the growth and phycocyanin content ofS.platensis, while blue light just increased the phycocyanin content, but lower biomass (Chini Zittelli et al., 2022).Similarly,Da Fontoura Prates et al.(2020) reported that the highest protein productivity ofSpirulinasp.was obtained under red and green LED.Different cellular metabolic processes inNannochloropsis oceanicawere also observed under the regulation of different light qualities through transcriptome,proteomics combined with physiology, and biochemistry (Wei et al., 2020).Other than light quality, the intensity and light：dark cycle can also significantly influence cell growth and regulate the cellular metabolism of microalgae (Tayebati et al.,2021).Light intensity can not only affect cell growth of microalgae, but also the accumulation of organic matter, such as carbohydrates, proteins, fatty acids, chlorophyll, etc.(Wang et al., 2014).In addition, microalgae need to synthesize proteins and other bioactive components under dark conditions, a proper light：dark cycle, for example 12-h L：12-h D and 16-h L：8-h D, has found more effective than 24-h L：0-h D (Posten, 2009; Soni et al., 2017).Although several studies have been reported on the application of LEDs as the light source for cultivation of microalgae, but the regulation of light on microalgae is species-specific, which shows different efficacy with different microalgae because of their differences in light-harvesting pigments,metabolic pathways and gene expressions (Schulze et al., 2014; Kim et al., 2019).Therefore, the detailed biomass and protein accumulation mechanism ofS.platensisin response to different light conditions are still needs to be further explored.

In this study, the regulation effect of different light intensities, qualities, and light：dark cycles on the physiology and biochemistry ofS.platensiswas systematically evaluated, and the economic efficiency of different light conditions for cultivation ofS.platensiswas also calculated.The metabolic regulation mechanism ofS.platensisresponding to different light conditions was also analyzed using transcriptome sequencing analysis.Our study provides a convenient and effective strategy for industrial cultivation ofS.platensisusing different LEDs.

2 MATERIAL AND METHOD

2.1 Strain and culture condition

TheS.platensisstrain (SBD1-3-11) was obtained as a donation from Beihai SBD Bioscience Technology Co., Ltd., and cultured in Zarrouk medium (Zarrouk, 1966).A six-channel 2.0-L cylindrical photobioreactor (Guangyu Biological Technology Co., Ltd., China) with different LED lights was used.All cultures were carried out in triplicate for 10 d at 25±1 °C, with bubbling sterile air at a flow rate of 1.5 L/min.Different wavelengths of LEDs were selected as light sources with the spectra range of white LEDs (380-780 nm), red LEDs (600-700 nm), blue LEDs (450-485 nm),green LEDs (500-565 nm), and yellow LEDs(565-590 nm).Different light intensities (18, 36, 54,72, 90, and 108 μmol/(m2·s) for white, red, blue, and green LEDs; 9, 18, 27, 36, 45, and 54 μmol/(m2·s)for yellow LED) and different light：dark (L：D) cycle(8-h L：16-h D, 10-h L：14-h D, 12-h L：12-h D, and 14-h L：10-h D) were optimized for cultivation ofS.platensis.

2.2 Determination of biomass, chlorophyll-a content, and photosystem efficiency

The biomass production ofS.platensiswas monitored at 560 nm using a UV-spectrophotometer(Shimadzu UV-1800, Kyoto, Japan).The dry weight(DW) was then calculated according to the standard curves between OD560and DW ofS.platensis(Supplementary Fig.S1).The chlorophyll-acontent was determined using method according to previous reports (Lichtenthaler and Buschmann, 2001).

The chlorophyll fluorescence parameters of photosystem II (PS II) inS.platensisunder different light conditions were measured using AquaPen FP110 (FluorCam, Czech Republic) with a detection wavelength at 630 nm.The super pulse and actinic pulse were set as 600 and 54 μmol/(m2·s),respectively.Each 3 mL of algal solution was acclimated in the dark for 20 min before being measured for OJIP transients.

2.3 Determination of total protein, phycocyanin,and allophycocyanin contents

The total protein content was determined using the Bradford method (Bradford, 1976).The content of phycocyanin (PC) in mg/L and allophycocyanin(APC) in mg/L was determined according to previous methods and calculated using the following equation(Bennett and Bogorad, 1973):

where OD620and OD652are the optical density of phycocyanin and allophycocyanin at 620 and 652 nm, respectively.

2.4 Economic efficiency of energy to biomass

The economic efficiency of energy to biomass was defined as below (Wang et al., 2007):

whereCnis the biomass concentration (g/L) ofS.platensisharvested on thenthday;krepresents the price of electric power supply (¥/(kW·h));Trepresents the total cultivation times (h) andPis the power of different LEDs (W).The power consumption of various LEDs was measured and listed in Supplementary Table S1.

2.5 RNA extraction and transcriptome analysis

About 200-300 mg of fresh algae cultured under red, white, and blue LED conditions were collected,respectively.After washing twice with sterilized 1×PBS buffer, the samples were immediately transferred to cryogenic vials and cryo-stored in liquid nitrogen until use.Total RNAs were extracted using the mirVana miRNA isolation kit (Ambion)following the manufacturer’s protocol.RNA purification, library construction, and RNA-seq were performed at the Shanghai OE Biotech Co.,Ltd.(Shanghai, China).

Genes withP＜0.05 and |log2(fold change)|＞0.58 were identified as differentially expressed genes(DEGs).The Gene Ontology (GO, http://geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.kegg.jp) (Kanehisa et al., 2008) enrichment analysis of the DEGs were performed by hypergeometric distribution tests to determine the biological functions or pathways that are mainly affected by DEGs.

2.6 Statistical analysis

Statistical analyses were performed using SPSS.22.0.One-way analysis of variance (ANOVA)with the least significant difference (LSD) post hoc test and thet-test were used to analyze the significant differences among different treatments.Origin 2019b, Microsoft PowerPoint 2019, and Adobe Illustrator CS6 were used to draw and modify graphics.Significant differences were declared atP＜0.05.

3 RESULT AND DISCUSSION

3.1 The effect of various light qualities on cell growth and protein accumulation of S.platensis

The cell growth ofS.platensisunder different wavelengths of LEDs (white, red, blue, green, and yellow) was investigated at the constant light intensity of 54 μmol/(m2·s) and 12-h L：12-h D cycle.As shown in Fig.1a, the red LED group showed the highest dry weight ofS.platensis, which was approximately 1.11 times higher than that of blue LED group, and followed by the white, green,yellow, and blue groups.This was coincident with previous reports that red and blue LEDs showed the highest and lowest effect on the growth of different microalgal species, such asS.platensis(Wang et al.,2007; Chen et al., 2010; Tayebati et al., 2021),Haematococcuspluvialis(Pereira and Otero, 2020),andDunaliellasalina(Li et al., 2020).Similarly,the chlorophyll content under red LED was also significantly higher than other light sources(P＜0.05), following the sequence of red＞white＞blue＞yellow＞green (Fig.1b), which is also consistent with previous reports that the chlorophyll-acontent ofS.platensisunder blue light was significantly lower than that of the red light (Tayebati et al.,2021).It is well known that cyanobacteria, such asS.platensis, mainly utilize chlorophylla(the maximum absorption wavelength at 430 & 680 nm),phycocyanin (the maximum absorption wavelength at 620 nm), and allophycocyanin (the maximum absorption wavelength at 650 nm) for light energy harvesting during the cultivation (Su et al., 2010;Schulze et al., 2014), and the spectrum of red light (600-700 nm) is exactly located within the maximum absorption range of chlorophyllaand phycobiliproteins, therefore, the red LEDs showed the best efficacy for promoting the cell growth ofS.platensis.

Fig.1 Effect of light qualities on the content of dry weight (a), chlorophyll a and protein (b), protein concentration (c),phycocyanin and allophycocyanin concentration (d) in S.platensis

For protein accumulation, it can be clearly seen from Fig.1c that the red LEDs group showed the highest protein concentration of 102.73±2.08 mg/L after 10 days’ culture, and then was blue＞green＞white＞yellow LEDs group.Previous report also revealed that the red light had the highest (P＜0.05)protein productivity inSpirulinasp.LEB 18 compared to other lights (blue, green, and white)(Da Fontoura Prates et al., 2020).Although the biomass ofS.platensiswas the lowest under blue LEDs, it achieved the highest protein content of 318.98±3.55 mg/g compared with other LEDs(Fig.1b).Li et al.(2021) had reported that blue light could lead to lower cell division and longer cell doubling time, resulting accumulation of proteins.Kim et al.(2014a) and Tayebati et al.(2021) also demonstrated that under the light of blue and red,the cellular components of microalgae could be changed significantly and the protein contents were more abundant.Taken together, these results suggested that blue light could induceS.platensisto convert more light energy into protein storage instead of promoting cell growth.

To understand the biosynthesis of phycobiliproteins(PBPs) responding to various light illumination, the PC and APC contents under different colored LEDs were measured and shown in Fig.1d.The highest PC and APC contents were both obtained in the red LEDs group (P＜0.05).The contents of PC and APC under different colored LEDs was ranged as red＞white＞blue＞green＞yellow.Chen et al.(2010) had also reported that the PC content ofS.platensiswas the highest under red LEDs compared with other LEDs.The absorption of PC (～620 nm) (Schulze et al., 2014) and APC (～650 nm) (Su et al., 2010),which were well overlapped with the spectrum of red LEDs (600-700 nm), and then could absorb the visible light energy that is poorly utilized by chlorophyllaand transfer the energy to the photosynthetic reaction center for photosynthesis(Hemlata and Fatma, 2009).Therefore,S.platensisis supposed to generate a large amount of PC and APC proteins under red light conditions (Fig.1d),enhancing the absorption of the red light, and maintaining faster cell growth and protein accumulation.

Several studies had shown that different colored lights could also affect the morphology and division of microalgal cells (Izadpanah et al., 2018).Oldenhof et al.(2004) and Li et al.(2021) had reported thatChlamydomonasreinhardtiicells under blue light had longer division cycles and larger size than under red light.Tayebati et al.(2021) also reported that the cell length ofS.platensiswas longer under blue light, medium under white and yellow light, and shortest under red light.However, in this study, no significant difference was found in the morphology ofS.platensisunder different colored LEDs.

3.2 The effect of various light qualities on the photosynthesis of S.platensis

To further study the effect of various light qualities on the photosynthesis ofS.platensis, the chlorophyll fluorescence parameters were measured and summarized in Fig.2.Under the red LEDs, the initial slope of the fluorescence curve (M0) and the fluorescence level of step J (VJ) ofS.platensisdecreased by 7.4% and 6.7% compared with white LEDs, 30.1% and 31.0% decreased compared with blue LEDs, 27.2% and 26.2% decreased compared with green LEDs, and 13.8% and 12.2% decreased compared with yellow LEDs.These results suggested that red LEDs promoted the electron chain between plastoquinone A (Qa) and plastoquinone B(Qb) inS.platensis.Moreover, the maximum electron transfer efficiency (Fv/Fm) of the PS II under red LEDs was slightly increased by 1.3%compared with white LEDs, but no significant change compared to blue light (Fig.2).

PIABS, based on the “vitality” index or survival index of light quantum flux absorption, is the overall expression of photosynthetic activity (Fan et al., 2021).Compared with the white, blue, green,and yellow LEDs, the PIABSvalue of the red LEDs group increased by 18.1%, 69.9%, 63.9%, and 15.2%, respectively (Fig.2), which suggested that red LEDs could significantly increase the activity of PS II inS.platensis.Considering the changes in the specific energy fluxes used for energy absorption(ABS/RC), capture (TRo/RC), transport (ETo/RC),and consumption (DIo/RC), our results showed that the values of absorption, capture, and consumption efficiency were slightly reduced and the electron transfer efficiency was increased by 4.7% under red LEDs compared with white LEDs (Fig.2).

Fig.2 The spider-plot of chlorophyll fluorescence parameters of S.platensis under different light qualities

3.3 The effect of light intensities on the growth and protein accumulation of S.platensis

To explore the effect of light intensity on the growth ofS.platensis, five different colored LEDs with various light intensities were applied forS.platensiscultivation and the growth ofS.platensiswere evaluated both in terms of dry weight and chlorophyll-acontent.From Fig.3a-e it can be seen that within all the tested light intensity ranges of different colored LEDs, theS.platensisall shows an increasing trend with increasing their light intensities.However, the optimal light intensity for different colored LEDs in terms of chlorophyllaand protein content was different (Fig.3f-g).The highest chlorophyllaand protein content were obtained under 18 μmol/(m2·s) of green and white LEDs.

It has been reported that excessive light (over 2 500 μmol/(m2·s)) could induce formation of reactive oxygen species (ROS), destroying PS II proteins and harmful to the growth ofS.platensis(Levin et al., 2021; Tayebati et al., 2021).Within our tested range of light intensities, no significant adverse effect of higher light intensity on the growth and protein accumulation ofS.platensiswas observed.It should be noted that the light intensity used in this study is the averaged light intensity penetrated into culture medium through glass vessel and water solutions, not the original light intensity of LEDs.To summarize, our results indicate that the optimum light intensities of different colored LEDs for cell growth and protein accumulation ofS.platensiswere different, and the variation trend of optical density and chlorophyll-acontent was not completely consistent, which may be caused by the different absorption and utilization efficiency ofS.platensisat different wavelengths.

Fig.3 The growth curve of S.platensis cultivated at different light intensities of white (a), red (b), blue (c), green (d), and yellow (e) LEDs; effect of light intensity on the content of chlorophyll a (f) and protein (g) in S.platensis under different light sources

3.4 The effect of light：dark cycles on the growth and protein accumulation of S.platensis

Microalgae need to synthesize sugar, proteins,and other bioactive components under dark conditions.Therefore, proper light：dark cycle could also significantly influence the growth ofS.platensis(Posten, 2009; Soni et al., 2017; Levin et al., 2021; Tayebati et al., 2021).To illustrate how different light：dark cycles influence the cell growth,chlorophyll, and protein contents ofS.platensis,totally four light：dark cycles (8-h L：16-h D, 10-h L：14-h D, 12-h L：12-h D, and 14-h L：10-h D) were set and compared.

It can be seen from Fig.4a-e that the 12-h L：12-h D group shows the highest cell growth rate under all the white, red, blue, green, and yellow LEDs.Richmond et al.(2003) reported that the increase of light：dark frequency could enhance the microalgal cell densities.Similarly, Ho et al.(2018)found that increase in the light-dark frequency could significantly increase the biomass production ofS.platensis.However, the contents of chlorophyllashowed an increasing trend with the increase of illumination time within different light quality groups (Fig.4f).For protein accumulation, the white,red, blue, green, and yellow LED groups all showed the same expression pattern, and the highest(P＜0.05) protein content was all obtained in photoperiod 14-h L：10-h D (Fig.4g).In general,with the prolongation of illumination time, the algal cells obtain more light energy, which in turn generate more sugar or protein accumulation,possibly reducing the growth rate of the cells.

3.5 The economic efficiency of energy to biomass under various light conditions

To evaluate the economic efficiency of light energy to biomass under various light conditions,S.platensiswas cultured at same intensity of 54 μmol/(m2·s) and light：dark cycle as 12 h：12 h,and the power consumption of different colored LEDs was determined ranging as yellow＞green＞red＞white＞blue (Supplementary Table S1).Then, the economic efficiency (Eeff) of power consumption cost to biomass under various LEDs was calculated.It was found from Fig.5 that the red LEDs group shows the highestEeffcompared to other LEDs, and theEeffof different LEDs was ranked as red＞white＞blue≈green＞yellow.As a summary, the economic benefit of red LED for the cultivation ofS.platensiswas the highest, while the yellow LED displayed the lowest economic efficiency in light energy to biomass conversion.

3.6 The regulation of photosynthetic carbon fixation and light-harvesting antenna proteins pathways responding to red, white, and blue LEDs

To reveal the mechanism of how different LEDs regulated the metabolism ofS.platensis,transcriptional sequencing ofS.platensisunder different LEDs was conducted.As shown in Fig.6,most of the genes in the photosynthetic pathway were significantly up-regulated under red LEDs compared with the white LEDs group, and few DEGs were found in the blue LEDs group compared with the white LEDs group.Notably, the six genes(psaD,psaE,psaI,psaL,psaM, andpsaX) of lightharvesting center proteins: photosystem I subunit II,photosystem I reaction center subunit IV, photosystem I subunit VIII, photosystem I subunit XI, photosystem I subunit XII, and photosystem I 4.8-kDa protein in PS I were drastically up-regulated at their transcriptional level (log2(fold change), loget:0.73-0.92) under the illumination of red LEDs compared with white LEDs group (RL-vs.-WL group, i.e., red LEDs group compared with white LEDs group).But, only thepsaXshowed 0.63 loget up-regulation under blue LED compared with white LED (BL-vs.-WL group, i.e., blue LEDs group compared with white LEDs group).Compared to the blue LEDs group, the transcriptional level ofpsaD,psaE,psaI,psaL,psaM, andpsaFin the red LEDs group were significantly up-regulated (Fig.6;Supplementary Table S2).It has been reported that cyanobacteria normally concentrate most of their chlorophyllain PS I than in PS II to help their better growth under weak light conditions (Fujita,1997).This study of biochemical results shows that red LEDs could induce the enrichment of chlorophylla(Fig.1b) compared with white and blue LEDs group, which is consistent with the transcriptional results that the red LEDs could enhance the transcriptional level of light-harvesting center proteins of PS I and then increase the chlorophyll content ofS.platensis.

Fig.4 The growth curve of S.platensis cultivated at different light cycles of white (a), red (b), blue (c), green (d), and yellow (e) LEDs; effect of light cycles on the content of chlorophyll a (f) and protein (g) under different light sources in S.platensis

Fig.5 Time courses of energy efficiency of S.platensis under different light qualities

Intriguingly, this unbalance of chlorophyll content between PS I and PS II can be compensated by phycobilisomes (PBSs), which is generally associated to PS II (Mullineaux, 2008).The membrane-extrinsic soluble PBSs are responsible for the majority of light capture in cyanobacteria(Adir et al., 2020; Ma et al., 2020), which are composed of PBPs and linker proteins (Adir et al.,2006).As shown in Fig.6, three genes encoding linker proteins, namely phycobilisome core linker protein, phycobilisome rod-core linker protein, and phycoerythrin-associated linker protein, showed significant differences at transcriptional level.The gene (apcC) encoding phycobilisome core linker protein in red LEDs group was significantly upregulated 0.84 loget and the gene (cpeC) encoding phycoerythrin-associated linker protein was significantly down-regulated -0.91 loget compared with white LEDs group (RL-vs.-WL).There were no DEGs found in the blue LEDs group compared to the white LEDs group (BL-vs.-WL).Therefore,apcCandcpcGwere significantly up-regulated in the red LEDs group compared with the blue LEDs group (RL-vs.-BL, i.e., red LEDs group compared with blue LEDs group) (Fig.6; Supplementary Table S2).Although DEGs related to the synthesis of PBPs subunits were not detected, these upregulated linker proteins also suggested that red LEDs could promoted the biosynthesis and assembly of PBPs.These results were well coincident with previous biochemical results in Fig.1d, which showed that the PC and APC contents ofS.platensiswere the highest illuminated under red LEDs compared to the other LEDs.

According to the photosynthetic electron transport pathway, two homologs’ genes (petF) encoding ferredoxin (Fd), as well as one gene (petJ) encoding cytochrome c6 (Cyt c6) were up-regulated under RL-vs.-WL and RL-vs.-BL groups.In addition, the transcriptional level of genes encoding ferredoxin-NADP+reductase (FNR) was increased by 0.87 loget (Fig.6; Supplementary Table S2).FNR in photosynthesis are involved in production of NADPH by pairing electrons provided by photosystem I with FD, and NADPH and ATP generated by the ATP synthase are two main drive forces in the Calvin-Benson cycle for CO2assimilation (Rochaix,2011).The results of this study revealed that 2 genes (prk,xfp) related to carbon fixation were also up-regulated in red LEDs compared with white LEDs group (Fig.6; Supplementary Table S2).That was well consistent with Patelou’s previous report(Patelou et al., 2020) that FNR inNannochloropsis gaditanawas significantly up-regulated under red light compared to full spectrum white light.According to our biophysical and biochemical results, theFv/FmofS.platensisin the red LEDs group was higher than that of the white LEDs group.It is reasonable to be concluded that red LEDs significantly promotes the expression level of key proteins in the photosynthetic electron transport chain and CO2assimilation pathway, enhancing the photosynthetic rate and then maintaining the rapid growth and pigment content ofS.platensis.Similarly, a higher photosynthetic rate of marine cyanobacteriaSynechococcussp.had also been detected under the red light, which was also supposed resulting from the up-regulated expression of the genes that were involved in photosynthetic electron transport (Kim et al., 2014b).

3.7 The regulation of C-N metabolism pathways responding to red, white, and blue LEDs

Fig.6 Transcriptional analysis of different colored LEDs on the cultivation of S.platensis.The transcriptional differences of genes related with photosynthesis (a), calvin cycle (b), glycolysis/gluconeogenesis and tricarboxylic acid (TCA)cycle (c), and nitrogen metabolism (d)

The glycolysis/gluconeogenesis and tricarboxylic acid (TCA) cycle are important metabolic pathways in organisms (Zhang et al., 2021).In particular, the TCA cycle is a central hub for the metabolism of carbohydrates, lipids, and proteins.In RL-vs.-WL and RL-vs.-BL groups, the expression of several key enzyme genes (gap2,pgk,aceE, anddlat,encoding glyceraldehyde-3-phosphate dehydrogenase,phosphoglycerate kinase, pyruvate dehydrogenase E1 component, and pyruvate dehydrogenase E2 component, respectively) involved in glycolysis were significantly up-regulated (Fig.6; Supplementary Table S2).In the RL-vs.-BL group, the transcriptional level of the gene (pepck) encoding phosphoenolpyruvate carboxykinase involved in gluconeogenesis was significantly down-regulated.Therefore, it is supposed that the expression of key genes of glycolysis was up-regulated and genes involved in gluconeogenesis was inhibited, then led to more energy for rapid cell division and growth,which is well coincident with our physiological data, indicating that red LEDs could significantly accelerate the growth of cells (Fig.1a).In BL-vs.-WL group, onlypepckwas significantly upregulated and no DEGs were found in glycolysis.Therefore, it could be speculated that the enhanced gluconeogenesis under blue LEDs reduced the energy required for cell division while making cells grow much slower and cell size became larger.

The TCA cycle begins with the condensation of oxaloacetic acid (OAA) and acetyl-CoA, releases two carbon atoms as CO2through a series of oxidation steps, and ended with the regeneration of OAA (Sweetlove et al., 2010).Acetyl-CoA is an important precursor of the TCA cycle, and the pyruvate dehydrogenase complex catalyzes pyruvate to acetyl-CoA.As shown in Fig.6,aceE(encoding pyruvate dehydrogenase E1 component) anddlat(encoding pyruvate dehydrogenase E2 component)were both up-regulated to enhance the synthesis of acetyl-CoA.In RL-vs.-WL and RL-vs.-BL groups,the transcriptional level of genescs,idh, andsucD,encoding citrate synthase, isocitrate dehydrogenase,and succinyl-CoA synthetase alpha subunit respectively, were significantly up-regulated (Fig.6;Supplementary Table S2).Moreover, the intermediates of glycolysis and the TCA cycle (e.g., pyruvate,oxaloacetic acid, 2-oxoglutarate, and succinyl-CoA)are linked to amino acid synthesis, which was consistently with our previous results that the biomass concentration and total proteins remained the highest under red LEDs compared to the other LEDs (Fig.1a & c).These suggests that red light can enhance the expression of key enzyme genes of glycolysis and TCA cycle, and then accelerate the synthesis and consumption of three intracellular nutrient sources.

Nitrogen metabolism is another essential nutrient for all organisms required for the biosynthesis of macromolecules, such as proteins, nucleic acids, and chlorophyll (Hockin et al., 2012), and it is crucial forS.platensisto maintain the balance of intracellular carbon and nitrogen metabolism.In the pathway of nitrogen metabolism, the expression of related genes, includingnrt,nrtA,nrtC,narB,nirA,andnorB, were significantly up-regulated under red LEDs compared to white LEDs and blue LEDs groups (Fig.6; Supplementary Table S2).It could be deduced that cellsS.platensisunder red LEDs enhance the metabolic regulation of nitrogen to maintain the homeostasis of C and N.In both RL-vs.-WL and RL-vs.-BL groups, the gene (gdhA)encoding glutamate dehydrogenase markedly showed-1.81 and -1.99 loget down-regulation, respectively(Fig.6; Supplementary Table S2).As the synthetic precursor of amino acids, ammonia has two pathways to generate L-glutamate or arginine, respectively.It could be speculated that the down-regulated expression ofgdhAcould suppress the nitrogen flow to L-glutamate and promote the synthesis of arginine.

On the contrary, in the BL-vs.-WL group the expression of genes, includingnrt,nrtA,nrtC,narB,andnirA, were significantly down-regulated.According to our physiological results, it has been found that the protein content under blue LEDs was significantly increased compared to white LEDs(Fig.1b).As we know, nitrogen can flow to proteins,nucleic acids, or chlorophyll.In Fig.1a and 1b, we have shown that the blue LEDs group gives less biomass and chlorophyll content compared to the white LEDs group, and cells grew slowly and became larger, which was conducive to protein accumulation.It could be speculated that the blue light down-regulated the nitrogen assimilation pathway ofS.platensisin order to sustain the homeostasis of C and N.

4 CONCLUSION

The biomass production and protein accumulation can be significantly regulated by different light conditions, including light quality, intensity, and light：dark cycles.TheEeffof different colored LEDs was calculated and ranked as red＞white＞blue≈green＞yellow.Transcriptional analysis suggested that under red LEDs the expressional level of genes involved in photosynthesis, light-harvesting antenna proteins, and C-N metabolism pathways are significantly up-regulated, and therefore increase their cell growth and protein contents.Our study provided an efficient and convenient way to regulate the growth and protein accumulation ofS.platensisusing different light strategies.In the future work,more advanced light strategies, such as mixed colored LEDs, alternative different colored LEDs or photo-carbon synergy strategies, can be developed for efficient regulation and low-cost cultivation ofS.platensis.

5 DATA AVAILABILITY STATEMENT

All data supporting the findings are available in this published article and the supplementary files.