YU Binqi ,QIAN Shouyuan ,LIU Qing ,JIN Cuili, ,and ZHOU Xiaojian,

1) College of Environmental Science &Engineering, Yangzhou University,Yangzhou 225127,China

2) Jiangsu Key Laboratory of Marine Bioresources and Environment,Jiangsu Ocean University, Lianyungang 222005,China

Abstract Illuminating conditions are crucial factors affecting the microalgal growth and biosynthesis.The effects of illuminating spectral quality on the growth and bio-component production of Nannochloris oculata were investigated.The results indicated that a high proportion of Red-light enhanced the pigments and carbohydrate production but reduced those of the biomass and lipid.Monochromatic Blue-light has advantageous effects on lipid production compared with Red-and White-light.The optimal light spectrum for the protein production was the combination of Red-and Blue-light at a ratio of 4:3 or 5:2.Among the seven fatty acids identified from N.oculata,the contents of C16:0,C18:0,and C18:3(n-3) in the lipid were inhibited by the increased proportion of Red-light while those of C18:2(n-9),C16:2(n-6),and C20:0 were inhibited by Blue-light.Monochromatic Red-and Blue-light and their combinations were proposed as a promising illuminating strategy for the large-scale cultivation aiming for various bio-components.

Key words biomass;bio-component productivity;monochromatic light;Nannochloris oculata

1 Introduction

Microalgae were taken as an important feed in aquaculture,a potential source for food,and a promising source for biofuel production,due to their valuable intracellular biochemical compositions (Garaliet al.,2016;Lopez-Rosaleset al.,2019;Zhanget al.,2019).Proteins,carbohydrates,pigments,and lipids are the major valuable bio-components synthesized in cells,which determine the application values of algae (Maet al.,2016;Zhanget al.,2019).These valuable bio-components are originally produced through photosynthesis,in which microalgae convert CO2into organic carbon (Almutairi,2020).The photosynthetic organic carbon allocates to proteins,lipids,and carbohydrates,in response to variations in environmental conditions (Garaliet al.,2016;Zhanget al.,2019).

Marine microalgaeNannochloris oculatais a species with wide distribution and rapid growth (Parket al.,2012).It has been commonly used as a feed for the cultivation of zooplanktons and a bioremediation species for the rapid removal of ammonium and phosphate from wastewater due to its rapid growth (Parket al.,2012;Wanget al.,2019).It was proposed to be utilized as a protein ingredient in animal or aquatic feed because of its high protein solubility and digestibility (Parket al.,2012;Subhashet al.,2020).Moreover,the high biomass productivity and lipid content ofN.oculatahave also suggested its potential for biodiesel production (Parket al.,2012;Zhanget al.,2019;Bounnitet al.,2020;Subhashet al.,2020;Yuanet al.,2020).

Since light is the unique energy source for the photoautotrophicmicroalgae,illumination conditions are the crucial factors to affect the algal cultivation.The photon density as well as the spectra of light was reported affecting the algal growth as well as metabolite biosynthesis inNannochloris atomus,Nannochloropsis oculate,Chlorellasp.,andNannochloris oculata(Wanget al.,2019;Zhanget al.,2019;Bounnitet al.,2020;Yuanet al.,2020).Photons in the green and orange spectrum range(540–620 nm) were found to be underutilized in many studies.Therefore,optimization of the spectra absorbed by the microalgae in cultivation systems seems to be a notable way of increasing the bio-components productivity of microalgae likeN.oculata(Vadivelooet al.,2015;Zhanget al.,2019).An appropriate illumination condition could not only increase the value of the algal products but also reduce the costs of cultivation (Lamerset al.,2012;Yuanet al.,2020).

Because of its accurate and stable light emission at the specific wavelength as well as its low energy consumption,the light-emitting diode (LED) was proposed as the preferred illumination source for microalgae cultivation(Fuet al.,2013;Hanet al.,2019;Zhanget al.,2019).In response to different monochromatic light of blue,green,or red,emitted by LEDs,the unicellular green algaeChlamydomonas reinhardtiiandChlorella variabiliscells modified the associations between light-harvesting chlorophyll protein complexes and photosystems (Yoshifumiet al.,2019).A red and blue LED illuminated photo-bioreactor was proposed to increase beta-carotene production fromDunaliella salina(Fuet al.,2013).A wavelengthshifting system (blue and red-LED using interchangeably,5 days a cycle) was reported enhancing the cell density and beta-carotene productivity ofD.salina(Hanet al.,2019).Although LED illumination was verified with a strong potential in microalgal cultivation,rare reports have examined the effects of combined monochromatic light on the bio-components production of microalgae(Zhanget al.,2019;Bounnitet al.,2020;Breddaet al.,2020;).

Our previous work onD.salinaindicates that the combined monochromatic light has the strong potential on lipid productivity (Jinet al.,2021).Further understanding how the wavelength and intensity of different illuminators affect the bio-component synthesized byN.oculatamay aid the development of potential scale-up application,cost reduction,and yield boosting (Fuet al.,2013;Hanet al.,2019).The present study investigated the effects of different combinations of monochromatic light on the growth and bio-component production ofN.oculata.Changes in the composition of intercellular lipid were also examined.

2 Materials and Methods

2.1 Illumination System

Fig.1 The spectral characters of used illuminators.

Different LEDs illuminators that emit monochromatic Red light (peak wavelength,660 nm),monochromatic Blue light (peak wavelength,455 nm),and their various combinations were used as light sources to evaluate the effects of spectral qualities onN.oculatagrowth and cellular content,with White light as the control.To avoid the possible light saturation,the illuminators were designed with a relatively low photon density in this study (Liet al.,2020).Each illuminator (customized and obtained from a local LED producer) is composed of seven light-emitting units with working power of 1 watt each,hence each illuminator has a total power of 7 watts (1 watt × 7).The LED illuminators were installed with different combinations of red and blue light-emitting units.The combinations were designed as n Red+m Blue (nRmB),in which n indicates the number of Red light-emitting units and m indicates the number of Blue light-emitting units in an illuminator,while the sum of n and m was 7.The examined combinations included 0R7B,1R6B,2R5B,3R4B,4R3B,5R2B,6R1B,and 7R0B.The exact spectrum charts and CIE 1976 chromaticity charts of illuminators for the algal culture were examined by a plant lighting analyzer (PLA-30,EVERFINE Corporation,China) and shown in Figs.1a and 1b,respectively.Cardboard containers (L × W × H=25 cm × 25 cm × 35 cm) inside-covered by silver paper with a hole (φ=8 cm) on the top of the illuminator installation were prepared.N.oculataculture flasks were placed in the containers under the LED illuminator.The containers were covered with silver paper to eliminate light leakage and protect the samples from any external light.Photon densities of all illuminators for the algal culture were also measured by PLA-30 as 77.26 ±4.32 μmol m-2s-1.

2.2 Algal Strain and Growth Conditions

N.oculataused in this studywas obtained from the Key Laboratory of Mariculture,Ministry of Education,Ocean University of China.Guillard’s f/2 seawater culture medium at 30‰ salinity was used for the incubation of microalgae (Guillard and Ryther,1962).N.oculatacells were repeatedly inoculated into fresh medium at 10-day intervals,and cultured in 250-mL Erlenmeyer flasks containing 150 mL f/2 liquid medium at 25℃ under the illumination with light/dark (L/D)=16 h/8 h and photon density=80μmol m-2s-1by a white fluorescent lamp.N.oculatacells inoculated into fresh medium were then transferred and cultivated under different illumination treatments with 16 h/8 h (L/D) cycles at 25℃ for an incubation period of 16 days.The flasks were shaken twice a day.All the treatments were performed with three repetitions.

2.3 Determination of Cell Growth

The OD values at 690 nm against f/2 medium as well as the cell densities counted under a microscope with a hemocytometer were employed to reflect the growth ofN.oculata(Konget al.,2010).At 2-or 3-day intervals,200 μL cultures from each treatment was transferred to a well on 96 wells plate and OD690values were detected by a plate reader;another 100 μL culture was used for the cell counting under a microscope.The specific growth rate (μ)of the algae under each treatment was calculated as follows:

whereμis the specific growth rate (d-1),1tCandCt2are the cell densities at the incubation timet1andt2,respectively (Konget al.,2010).The doubling rate [K(d-1)=μ/ln2] and the generation time [T(d)=1/K] were subsequently calculated based on the growth rate (Garaliet al.,2016).

2.4 Chlorophyll and Carotenoid Measurement

The content of the pigments in the sample was determined according to the method by Sujithaet al.(2016).In detail,5 mL algal cultures were collected and centrifuged(4000 rmin-1,10 min,4℃) to obtain the cell pellets.After the addition of 5 mL 90% acetone,the algal pellets were re-suspended and kept overnight at 4℃ in dark.Cell contents were lysed on the ice by an ultrasonic cell disruptor for 10 min (each 5 s working with a 3 s interval,700 W).Cell debris was removed by centrifugation (4000 rmin-1,10 min,4℃),and the supernatant was retained to measure the optical density at 470,644,661 nm wavelength.The pigment concentration (mg L-1) was calculated according to the formula by Sujithaet al.(2016).

2.5 Cellular Weight and Cellular Lipid,Carbohydrate,and Protein Contents Determinations

Algal culture with a volume of 100 mL was centrifuged(4000 rmin-1,10 min,4℃),washed with distilled water,freeze-dried in a freeze-drying machine,dried at 50℃ in a vacuum drying oven overnight,and then the dried biomass was weighed.

The extraction of total lipids was performed according to the method of Jinet al.(2021).The dry algae powder(10–50 mg) was accurately weighed before mixing with 0.8 mL distilled water,then 3 mL of a methanol:chloroform mixture (2:1,v/v) was used to extract the lipids overnight.After the same ultrasonic cell disruption treatment as above,the cell contents solution was then centrifuged (4000 r min-1,10 min,4℃) to discard the above water and cell debris.The chloroform layer was once again washed with the equal volume of water.The chloroform phases containing lipids were then collected and transferred to an evaporator at 60℃.After evaporation until stable weight,the dried lipid was weighed to calculate.

The protein and carbohydrate contents were measured using Kaumas blue method and the phenol sulphuric acid method,taking bovine serum albumin (BSA) and D-glucose as standards,respectively (Duboiset al.,1956;Bradfordet al.,1976).In detail,10 mL algal cultures were collected and centrifuged (4000 r min-1,10 min,4℃).The pellets were re-suspended in 10 mL distilled water and lysed on ice by an ultrasonic cell disruptor for 10 min (each 5 s working with a 3 s interval,700 W).After centrifugation at 4000 r min-1,4℃ for 10 min,the supernatant was used for protein or carbohydrate content measurement.

2.6 Lipid Composition Analysis by GC-MS

Lipid samples obtained from various illumination treatments were methylated according to the method of Sukhija and Palmquist (1988) and dissolved in N-hexane before being subjected to GC-MS analysis.The GC-MS analysis was modified and performed according to Qari and Oves (2020).A Thermo Scientific ITQ900 GC-MS system with the setup of a Trace GC Ultra,a ITQ900 MS,and an Al/AS 3000 autosampler (Thermo Scientific,USA)was used.The GC is equipped with a TR-5MS (30 m ×0.25 mm × 0.25 μm) column (Thermo Scientific,USA).The injection volume was 1 μL.The carrier gas was helium (99.999%),and the constant flow rate was 0.8 mL min-1.All samples were injected in splitless mode to the inlet (230℃).The internal oven temperature was maintained increasing order by program at the initial temperature start from 120℃ for 1 min and increased 3℃ min-1up to 240℃ and held for 10 min.The ion source temperature was 220℃.The solvent delay time was 4 min.Mass spectra were acquired in full-scan mode with a mass range from 50 to 800 in 0.68 s.During the analysis,the sample injection sequence was randomly arranged,and one quality control (QC) sample was added every 9 samples.The total ion chromatogram of the sample was obtained.The collected spectra was subjected to peak alignment,peak matching correction,and peak identification.Base on the standards and NIST Mass Spectral Library(2008),each Fatty Acid Methyl Ester (FAME) was identified,and those in a sample with percentages of peak areas higher than 1.5% were recorded.Finally,the results were converted into FAMEs and peak area information.

2.7 Average Productivity

Productivity (mg L-1d-1) for each bio-component was calculated as below (Hanet al.,2016).

whereBis the final biomass (106mL-1);Ciis the concentration (pg cell-1) of componenti,in whichirepresents either the lipids,proteins,or carbohydrates concentration;andTis the total cultivation time (d).

2.8 Statistical Analysis

All experiments were repeated four times and the relevant data were indicated as means ± standard deviation(SD).SPSS 22.0 software was used for statistical analysis.One-way analysis of variance (ANOVA) followed by LSD or Dunnett’s T3 multiple comparisons was used to detect the significant differences among the treatments,whereP<0.05 orP<0.01 was considered significant differences.

3 Results and Discussion

3.1 Effect of Illumination Spectrum on the Growth of N.oculata

AllN.oculatacultured under nine different LED illuminators (White,0R7B,1R6B,2R5B,3R4B,4R3B,5R2B,6R1B,7R0B) showed continuous growths during the incubation time (Fig.2a).Except that the monochromatic 7R0B light showed obvious adverse effects on the growth ofN.oculatain comparison with the control(White),the growth curves for the other eight treatments(including White) were similar to each other.At the end of the incubation,cell densities showed that monochromatic 7R0B light led to a significantly low cell growth (P=0.01),with the lowest final cell density of 10.97×106cell mL-1(Fig.2b).There is no significant difference in the cell densities of the other 8 treatments (P=0.01),while the highest one as 13.30×106cell mL-1was obtained at 2R5B.The specific growth rate (μ),doubling rate (K),and the generation time (T) calculated based on the cell density growth also showed that all treatments achieved similar growths except for the significantly poor one in monochromatic light of 7R0B (Fig.2c).Many publications have also shown that growth of green algae under Red light is weaker than that under Blue light,includingNannochloropsissp.,Chlorella variabilis,andD.salina,which indicates that the algae is sensitive to the spectral quality of cultivation light (Konget al.,2010;Vadivelooet al.,2015;Jinet al.,2021).The present study verifies the above conclusion by gradually changing from Red light to Blue light under the same illumination intensity,and supports that this strain ofN.oculatais sensitive to the spectrum quality of the light source (Severeset al.,2017;Liet al.,2020).

Fig.2 Effect of illumination spectra on growth kinetics of N.oculata (means ± SD,n=3).(a),Growth curves under different treatments;(b),Cell density at harvest under different treatments;(c),Growth rate,doubling rate,and generation time under different treatments.Different letters indicate a statistical difference as determined by LSD test or Dunnett T3 test,capital letters indicate P <0.01,and small letters indicate P <0.05.

3.2 Effect of Illumination Spectrum on Production of Biomass,Protein,Carbohydrate,Pigments,and Lipid Contents of N.oculata

Production of the biomass and specific bio-component is the most directive and obvious indicators concerned by the industrial algal cultivation,and bio-component production is decided by both biomass and cellular content(Zhanget al.,2019;Bounnitet al.,2020).Red light(7R0B) achieved the lowest dried biomass productivity of 16.96 mg L-1d-1,which is significantly lower than those in other treatments (P=0.01,Fig.3a).Blue light of 0R7B achieved the highest dried biomass productivity of 24.34 mg L-1d-1,which has no significant difference with most of the combined monochromatic light treatments (P=0.01).Dried biomass productivity in White light was moderate and was significantly lower than that in 0R7B but higher than that in 7R0B.The biomass productivity suggested the changes in the alga led by the various light spectra were more drastic than that observed from cell growth,the spectral profiles of light might have brought the biochemical and physiological changes in microalgae(Lin and Tseng,2018;Zhanget al.,2020).

The contents (bio-component/dried biomass,%) and productivity (mg L-1d-1) of each bio-component were examined and showed in Table 1 and Fig.3,respectively.White light achieved the lowest protein content of 13.89%(Table 1).Protein contents ranged from 29.27% to 34.59%among the treatments of 2R5B to 6R1B without significant difference,and they achieved significantly higher cellular protein contents than the monochromatic Red(7R0B) or Blue (0R7B) light (P=0.01).The top protein content was observed in the treatment of 3R4B as 34.59%,which was about 3 folds of that in White light.In the protein productivity in Fig.3b,the White light achieved the lowest one as 3.04 mg L-1d-1,while the monochromatic treatments obtained significantly higher protein productivity (P=0.01).Among the monochromatic treatments,there was the top protein productivity observed at 4R3B and 5R2B with values of 8.06 and 7.91 mg L-1d-1.The highest protein productivity in 4R3B treatment was higher than Blue,Red,and White light with the percentages of 57.72%,82.35%,and 165.13%,respectively.In previous studies onNannochloropsissp.andNannochloris oculata,no difference in protein production was detected between the treatments of Red and Blue light(Vadivelooet al.,2015;Yuanet al.,2020).In this study,no significant difference between the protein production for 0R7B and 7R0B,no matter in terms of contents (Table 1) or productivity (Fig.3b),agreed above studies.By stepwise changing in the ratios of Red:Blue in this study,the top protein production ofN.oculatawas observed at the combined monochromatic light of 4R3B and 5R2B(Fig.3b and Table 1),and were significantly higher than those at White,Red,or Blue light (P=0.01).This result indicates that the optimal light spectra for protein production requires a suitable combination of monochromatic Red and Blue light rather than individual ones.To our knowledge,this might be the only report about the optimal combined monochromatic spectra for microalgal protein production examined by a stepwise change in the ratio of monochromatic light.

Table 1 Effect of illumination spectrums on the ratio of bio-component/biomass of N.oculata (%,means ± SD, n=3)

Fig.3 Effects of illumination spectrums on biocomponent productivity of N.oculata (mg L-1 d-1,means ± SD,n=3).Different letters indicate a statistical difference as determined by LSD test or Dunnett T3 test,capital letters indicate P <0.01,and small letters indicate P <0.05.

In the case of carbohydrate contents,the highest value of 35.93% was obtained at 7R0B (Table 1).The carbohydrate contents were increased as the proportion of Red light in the combined monochromatic light increased.The low levels of carbohydrate contents ranged from 22.13%to 22.53% without significant difference were observed in the treatments of White light and those from 0R7B to 4R3B.With the further increased proportion of Red light in the combined monochromatic light,the significant increase in carbohydrate contents was found in 5R2B,6R1B,and 7R0B.In Fig.3c,carbohydrate productivity in 6R1B and 7R0B was 6.01 and 6.09 mg L-1d-1,respectively,and were higher than those in other treatments.Significantly lower carbohydrate production ofN.oculataoccurred under the light with lower proportions of Red light (Fig.3c and Table 1).Blue light is believed to be responsible for the enhanced production and activity of respiratory enzymes in microalgae,which is proportional to the rate of carbohydrate catabolism,hence leading to carbohydrate reduction (Vadivelooet al.,2015).The results in this study agreed with the previous report that the carbohydrate produced by a green alga ofNannochloropsissp.under Red light was significantly enhanced compared to that under Blue (Vadivelooet al.,2015).

In algaeN.oculata,chlorophyll-aserved as the photosynthetic reaction center and shows absorption at 425–475 nm (Blue) and 630–675 nm (Red) of the visible range of the spectra while carotenoids served as the accessory pigments absorb the photons at 400–550 nm and transfer the charges to chlorophyll molecules (Kandilianet al.,2013;Severeset al.,2017;Liet al.,2020).The production of chlorophyll-a and carotenoids was therefore also investigated.For the pigment contents,the lowest values of 0.22% and 0.15% for chlorophyll-aand carotenoid,respectively,were both observed in the treatment of 0R7B(Table 1).The increase of the proportion of Red light in the combined monochromatic light resulted in the increase of pigment contents.Respectively,the highest chlorophyll-aand carotenoid contents were observed as 0.58% and 0.38% in the treatment of 7R0B.In Figs.3d and 3e,the highest productivity of chlorophyll-aand carotenoids was both noticed at 7R0B as 0.098 and 0.064 mg L-1d-1and followed by that in White light treatment.The productivity of both chlorophyll-aand carotenoid behaved with similar patterns that the increase in the ratio of Red:Blue led to the increase of the pigment productivity.Enhanced production in cellular pigments was also observed inNannochloropsis oculatawhen it responded to the light-limited conditions (Kandilianet al.,2013).In this study,the photon densities in all treatments were designed at the same level.The cells under the treatments with a high proportion of red light were at relative ‘lightlimited’ conditions due to their lower utilization efficiency for Red photons than that for Blue ones,which should further led to the higher pigment production(Kandilianet al.,2013).

Lipid production is deeply concerned by the biofuel industry (Bounnitet al.,2020;Liet al.,2020;Jinet al.,2021).In this study,the highest and lowest cellular lipid content of 24.21% and 15.47% was noticed in the treatments of 0R7B and 7R0B,respectively (Table 1).White light and 1R6B achieved the moderate lipid contents of 23.09% and 22.88%,respectively.In treatments with 2R5B to 6R1B,similar cellular lipid contents without significant differences were obtained.The general tendency was the higher proportion of Red light leading to the lower lipid content.In the lipid productivity,the highest and lowest values were also observed in 0R7B and 7R0B as 5.89 and 2.63 mg L-1d-1,respectively (Fig.3f).White light achieved 5.08 mg L-1d-1lipid productivity as a moderate one.In general,the contents (Table 1) and productivity (Fig.3f) of the combined monochromatic Red and Blue light were situated between the two uncombined monochromatic treatments of 0R7B and 7R0B.The general tendency of lipid productivity variation among ng 4R3B,5R2B,6R1B,and 7R0B was the increase in the ratio of Red:Blue led to the decrease of the lipid productivity,although it was not strictly followed by the lipid productivity for 2R5B and 3R4B.These results indicate that the Blue light may be beneficial forN.oculatato acuminate lipid in cells.The higher lipid production in this study occurred at Blue light (0R7B) compared to those at Red light (7R0B) and White light,the results of which also agreed with many published literature working onNannochloropsissp.,Nannochloropsis oculate,Chlorella variabilis,andD.salina,(Vadivelooet al.,2015;Lin and Tseng,2018;Jinet al.,2021).

3.3 Effect of Illumination Spectrum on the Cellular Lipid Composition of N. oculata

The fatty acid composition is a key indicator of suitability for biodiesel production since C16–C18 fatty acids can improve the combustion performance and unsaturated fatty acids proportion can improve biodiesel’s low-temperature flow properties (Anet al.,2020).The lipid compositions of cellular lipid sample obtained from each treatment were listed in Table 2.Totally seven kinds of major Fatty Acid Methyl Ester (FAME) were identified fromN.oculata.The major fatty acids ranked with the descending contents (%) as C18:3(n-3),C16:0,C18:2(n-9),16:3(n-3),C18:0,C16:2(n-6),C20:0.

Generally,the sum of saturated fatty acids (C16:0,C18:0,and C20:0) was less than that of unsaturated fatty acids (C16:2(n-6),16:3(n-3),C18:2(n-9),and C18:3(n-3)).Fatty acids including palmitic acid (C16:0),stearic acid(C18:0),Octadecenoic acid (C18:1),and 9,12-Octadecadienoic acid (C18:2(n=9)) were previously identified inNannochlorissp.by Lopez-Rosaleset al.(2019) and inNannochloropsis oculataby Parket al.(2012) and were also detected in this study (Table 2).The fatty acid of C16:0 was dominant in the saturated fatty acids,which agreed with the above aforementioned study (Parket al.,2012;Lopez-Rosaleset al.,2019).Our analysis found that the dominant unsaturated fatty acid was C18:3(n-3)and C18:2(n-9),which was also noted in anotherNannochlorissp.(Bounnitet al.,2020).For all the treatments in this study,the proportion of C16–C18 fatty acids exceeded 98% of the total fatty acids,and was much higher than the values reported in the literature (Anet al.,2020).The fatty acid profile of the testedN.oculatastrain indicates it has the potential for biofuel production.

Table 2 Effect of illumination spectrums on the lipid composition of N.oculata (Percentage of peak area from GC-MS,%,means ± SD,n=3)

Treatments by various illumination spectrums led to significant changes in the contents of all identified 7 fatty acids (P=0.01,Table 2).To estimate how the illumination spectrums affecting the lipid compositions,Fig.4 was made based on the data obtained from triplicated samples for each treatment.Among the identified 7 fatty acids,16:3(n-3),C18:0,C16:2(n-6),and C20:0 ranked at low contents (less than 8%),while C18:3(n-3),C16:0,and C18:2(n-9) were higher than 20%.Along with the increased proportion of Red light in the combined monochromatic light,the contents of fatty acids changed with different pattern,which was the decrease in C18:3(n-3),C16:0,and C18:0,the increase in C18:2(n-9),C16:2(n-6),and C20:0,and the slight variation in 16:3(n-3).White light achieved moderate percentages for all the fatty acids except for C20:0.The levels of saturated fatty acids of palmitic acid (C16:0) and stearic acid (C18:0) and fatty acid of C18:3(n-3) in thisNannochloris oculatastrain were inhibited by Red light (Fig.4),which is the same as that concluded by Lin and Tseng (2018) inNannochlo-ropsis oculata.The application values of the lipid are contributed by fatty acids composition (Lopez-Rosaleset al.,2019;Zhanget al.,2019;Anet al.,2020;Bounnitet al.,2020).The predictable changing pattern of lipid composition inN.oculataaffected by the step-wise variation Red:Blue provided a potential tool to manipulate the lipids produced byN.oculata(Vadivelooet al.,2015).

Fig.4 The relationship between lipid compositions of N.oculata and the illumination spectrums.Each spot on the plot was a peak area percentage of an identified fatty acid by GC-MS analysis for an independent lipid sample.The identification of the peaks was based on the standards and NIST Mass Spectral Library (2008).The peaks in a sample with percentages of peak areas higher than 1.5% were recorded.Every treatment had triplicated independent samples and thus triplicated spots on the plot.

Although almost all photosynthetic organisms contain chlorophyll-a,different microalgae contain various accessory pigments,which participate in absorbing light of various wavelengths to help increase the total absorption(Vadivelooet al.,2015).Photo and chromatic preference depends on the pigments of microalgae species,moreover,a large difference in pigment expression exists even among the same species (Kandilianet al.,2013).Photons at Blue and Red wavelength potentially are more efficiently utilized by algae ofN.oculatasince its major pigments are chlorophyll-aand carotenoids in Table 1(Kandilianet al.,2013;Yoshifumiet al.,2019;Liet al.,2020).Beside the photosynthesis pathway,stimulation in photoreceptors and consequent cellular processes are also the cause of altered light spectra affecting on the algal bio-component production (Vadivelooet al.,2015).Due to the low cost and energy consumption as well as the inexpensive and convenient utilization of monochromatic LED illuminators,it is an exciting strategy to manipulate the biochemical composition of microalgae aiming for various utilization by using light spectra as a tool (Lamerset al.,2012;Vadivelooet al.,2015).Combined monochromatic Red and Blue light should be the promising illumination strategy for the cultivation of microalgae containing chlorophyll-aand carotenoid,and the employment of various ratios of Red:Blue might manipulate different production of the bio-components.Therefore,the results of this study could provide significant contributions toward the utilization of this microalga,in addition to providing solid evidence on the superiority of combined monochromatic Red and Blue light in algal cultivation.

4 Conclusions

Monochromatic Red light has advantageous effects on pigments and carbohydrate production but disadvantageous effects on cell growth,biomass production,and lipid production forN.oculata.Monochromatic Blue light leads to higher lipid production than Red and White light.The production of fatty acids of C16:0,C18:0,and C18:3(n-3) inN.oculatawas inhibited by Red light while those of C18:2(n-9),C16:2(n-6),and C20:0 were enhanced by Red light.The optimal light spectra for the protein production ofN.oculatarequires a combination of monochromatic Red and Blue light like 4R3B or 5R2B.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Nos.41776156 and 42177459),the Open-End Funds of Jiangsu Key Laboratory of Marine Bioresources and Environment (No.SH20201206).