Introduction

The natural carotenoid astaxanthin, which is abundant in aquatic organisms, has a powerful antioxidant activity that is ten times more than that of other carotenoid types, including beta-carotene, canthaxanthin, zeaxanthin, and lutein (Zhao et al. 2019; Zhu et al. 2018; Patil et al. 2022). The demand for astaxanthin in medications, cosmetics, and functional foods has been steadily rising since, in addition to its anti-oxidation activity, astaxanthin has been proven to have anti-aging, anti-inflammation, anti-hypertension, anti-cancer, and other bioactivities (Li et al. 2020; Pereira and Otero 2020; Ma et al. 2018). In 2026, it is anticipated that the astaxanthin trade market will be worth $965 million (Henke et al. 2016). Up to now, few natural astaxanthin producers were found, including plant (Adonis), microalgae (Haematococcus pluvialis, Chlorella zofingiensis, Chlorococcum sp.), yeast (Phaffia rhodozyma) and bacteria (Agrobacterium aurantiacum, Paracoccus carotinifaciens). The most promising of them was believed to be the freshwater microalga H. pluvialis (Patil et al. 2022; Ren et al. 2021; Li et al. 2020; Zhu et al. 2018; Nishshanka et al. 2022). The astaxanthin produced on an industrial scale by H. pluvialis is of higher quality and bioactivity than that of other microbial producers (Nishshanka et al. 2022). H. pluvialis is a unicellular autotrophic microalga, fixes carbon through photosynthesis, and intracellularly allocated to synthesize a variety of bio-substances, including astaxanthin, as well as pigments, proteins, lipids and carbohydrates (Pereira and Otero 2020; Ren et al. 2021).

Light condition is one of the most crucial factors for autotrophic microalgae to create biomass and bio-substances since light is their primary source of energy (Maltsev et al. 2021). The value of products made from microalgae can be increased and the cost of culture can be decreased with proper lighting conditions (Yuan et al. 2020; Ren et al. 2021). Microalgae pigments, such as chlorophyll and carotenoid, absorb photons of photosynthetically active radiation (PAR) with peak wavelengths in the red and blue regions (Xue et al. 2020; Blankenship et al. 2011). Our previous studies on various microalgal species had demonstrated the significant potential for manipulating light quality to encourage the growth of microalgae and the synthesis of desired chemicals (Jin et al. 2021; Yu et al. 2022; Dong et al. 2022). On the other hand, it is yet unclear how the manipulation of light quality affects the adaptability and alterations of the microalgal photosynthetic system, improving the production of substances.

Microalgal chlorophyll absorbs light energy similarly to higher plants. Through several intermediate electron transport complexes, a portion of the absorbed energy reduces the principal quinone electron acceptor (QA) to QA−, while the remaining energy dissipates as heat and fluorescence (Guo et al. 2020). Because it is simple, quick, noninvasive, and gives an abundance of information regarding the fundamental performance of the PSII under varying environmental conditions, Chl-a fluorescence is a widely used approach to evaluate photosynthetic activity (mostly in PSII) (Strasser et al. 2004). The fluorescence quenching caused by photosynthesis is called photochemical quenching (qP); the fluorescence quenching caused by heat dissipation is called non-photochemical quenching (NPQ). qP denotes the level of photosynthetic activity, NPQ denotes the ability to dissipate excess light energy into heat, that is, photoprotection ability (Varshney et al. 2020). The multiphase rise of Chl-a fluorescence from minimum to maximum, which is divided into four main steps: O (at 20 or 50 s, depending on the instruments), J (2 ms), I (30 ms), and P (the maximal fluorescence when all reaction centers (RCs) are closed), is caused by the gradual reduction of the completed oxidized electron transfer chain (ETC) (Strasser et al. 2004). Along with the fundamental steps, new interphase steps like L- (~ 150 μs) and K- (~ 300 μs) steps were found under some unique stresses (Strasser et al. 2004). The JIP-test, a quantitative analysis of the O-J-I-P curve of fluorescence rising kinetics that provides information about the structure, conformation, and function of the photosynthetic apparatus, has proven to be an efficient approach to quantify the PSII architecture and photosynthetic activity (Tsimilli-Michael 2019; Guo et al. 2020).

The response of photosynthetic activity in microalgae and cyanobacteria to temperature, salinity, light intensity, or heavy metal toxicity was recorded, however, the response to light quality is rarely addressed (Markou et al. 2016). The limited studies reported the light quality caused changes of general photosynthetic indicators, such as the maximum PSII photochemical efficiency of H. pluvialis (Li et al. 2020). The specific mechanism for the changes in the general indicators, the specific acting sites of light quality on the electron transport chain determined by JIP test, and the link between photosynthetic process and substance productions at various light quality for this economically significant species needs more detailed study.

The aim of this study was to investigate the potential use of the monochromatic red and blue light and their combinations to promote growth and astaxanthin synthesis in H. pluvialis. In addition, the effect on the photosynthetic performance and the status of photosynthetic electron transport chain of H. pluvialis revealed by NPQ analysis and JIP-test will be examined to better understand its response to the variation of light quality.

Materials and methods

Algal species

H. pluvialis FACHB-712 was originally obtained from the Fresh Algal Collection at Institute of Hydrobiology, Chinese Academy of Sciences, and maintained in Marine Science and Technology Institute, Yangzhou University.

Culture condition and experimental design

Nine kinds of LED illuminators were used in the experiment, each was assembled with 7 LED units emitting either white light, monochromic red (peak wavelength 660 nm), or blue light (peak wavelength 455 nm). The total power of each illuminator was 7W, which was composed of 7 LED units with working power of 1W each. The monochromic red and blue light illuminators were described by the formula n Red + m Blue (nRmB, n + m = 7), where n and m stand for the number of red and blue LED units in the illuminator, respectively. White light (W), which had seven white-light-emitting LED units, served as the control. A plant illumination analyzer (PLA-30) was employed to examine the precise spectral quality of each illuminator, and 11.5 ± 0.8 W·m−2 was found to be the actual photosynthetic irradiance used for algal cultivation (Jin et al. 2021). H. pluvialis during logarithmic growth was inoculated in 250 mL conical flask with 150 mL BG11 culture medium. The initial OD690 of H. pluvialis was controlled at about 0.03 (0.118 × 105 cell/mL), and each treatment was set with triplicates. The alga was grown at 24 ± 1 °C without aeration and a 16:8 light:dark cycle. Throughout the 24-day cultivation, the conical flasks were shaken twice each day.

Determination of growth, cell morphology and products of H. pluvialis

Growth, cell morphology and biomass measurement

The cell morphology was observed and photographed with microscope (BX53, Olympus, Tokyo, Japan). The biomass dry weight was measured after 24-day culture. Microalgal culture (120 mL) was centrifuged for 10 min at 2755 × g, washed with 10 mL pure water, transferred to a clean glass bottle for the freeze-drying at -80 °C (FreeZone2.5 plus, Labconco, Missouri, USA) for 24 h, and weighted (Dong et al. 2022).

Determination of Chl-a and astaxanthin content

Microalgal culture (3 mL) was centrifuged at 2755 × g for 10 min, the cells were suspended in 3 mL methanol, and heated in 50 °C water bath for 30 min, the supernatant was then centrifuged at 2755 × g for 10 min. The absorbance of the supernatant was determined at 652 and 665 nm with methanol as blank. The contents of Chl-a were obtained by the following formula (Wellburn 1994).

$${\mathrm{C}}_{\mathrm{Chl}-\mathrm{a}}=16.72\times {\mathrm{A}}_{665}-9.16\times {\mathrm{A}}_{652}$$

where CChl-a is the Chl-a concentration (mg/L), A665 and A652 are the absorbance at 665 and 652 nm, respectively.

Another 3 mL microalgal culture was centrifuged (2755 × g, 10 min), and the cells were suspended in 3 mL solution (methanol: water = 3:7 (v/v), 5% NaOH (w/v)) and kept in dark in a water bath (65℃) for 10 min. The liquid was centrifuged (2755 × g, 10 min), and the precipitate was washed 3 times with pure water and added with 1 mL DMSO. The sample was heated in a water bath (65℃) for 10 min and frozen at -80 °C for 10 min in dark. The supernatant was then collected after centrifugation (2755 × g, 10 min) and the precipitate was added with1 mL DMSO and heated and frozen again for 3 times or more until the supernatant was nearly colorless. The absorbance of the collected and mixed supernatant was determined at 490 nm. Astaxanthin content was calculated according to the following formula (Boussiba and Vonshak 1991).

$${\mathrm{C}}_{\mathrm{Ast}}=(4.5\times {\mathrm{A}}_{490}\times {\mathrm{V}}_{\mathrm{a}})/{\mathrm{V}}_{\mathrm{b}}$$

where CAst is the astaxanthin concentration (mg/L), A490 is the absorbance at 490 nm, Va and Vb are the volumes (mL) of collected DMSO and microalgal culture, respectively.

Determination of protein, carbohydrate and lipid content

Algal culture (3 mL) was centrifuged at 2755 × g for 10 min, washed with 3 mL pure water, disrupted on ice by an ultrasonic cell disruptor (JY92-IID, 200W, ultrasonic 3 s, interval 2 s) for 8 min. The sample was centrifuged and the supernatant was used for determination of protein and carbohydrate by Coomassie brilliant blue method and phenol sulfuric acid method, respectively (Bradford 1976; Dubois et al. 1956).

The lipid was determined by chloroform/methanol extraction method (Liu 2014). The freeze-dried algae powder (about 20 mg) was mixed with 0.8 mL pure water, 1 mL chloroform, and 2 mL methanol, and then ultrasonically disrupted on ice bath for 8 min. After 1 mL chloroform and 1 mL pure water were added, the sample was centrifuged at 2755 × g for 10 min, the chloroform layer was transferred to a dried and weighed glass bottle, and the lipid weight was measured after drying by N2 at room temperature.

Non photochemical quenching (NPQ) analysis

The NPQ protocol was used to quantify photochemical and non-photochemical quenching. Microalgal samples were adapted in dark for 15 min before test. A fluorometer (AquaPen AP110, Photon Systems Instruments, Czech Republic) was used for NPQ analysis. The NPQ protocol started with a measurement of minimal level of fluorescence (Fo) after a dark period (a weak light pulse of 300 μmol·m−2·s−1 applied). A short saturating flash of 600 μmol·m−2·s−1 was then applied to reduce the plastoquinone pool and measure maximum fluorescence (FM) after the dark-adapted state. Next, actinic light with intensity of 70 μmol·m−2·s−1 was on for 200 s to elicit a transient called the Kautsky effect. Meanwhile, a sequence of 10 saturating flashes at 21 s intervals was then applied to probe the non-photochemical quenching (NPQ) and effective quantum yield (QYss) of photosynthesis in light adapted state. After exposure to continuous illumination, the relaxation of NPQ was determined by means of saturating pulses applied within 60 s of the dark phase, with each pulse interval of 21 s. Parameters of qP (coefficient of photochemical quenching), NPQ, QYmax (maximum QY of PSII) and QYss were calculated according to Varshney et al. 2020.

Chl-a fluorescence rise kinetics measurement and JIP-test

Microalgal samples were adapted in dark for 15 min before test. The transient fluorescence rise kinetics were then determined by 2 s pulses of continuous light (600 μmol (photons) m−2 s−1) and were recorded by a fluorometer. The fluorescence intensity was recorded as Ft (t is the time or step) every 10 μs once until 600 μs, then every 1 ms once until 90 ms, and then every 10 ms once until 2 s. The O, J, I, P steps were at 50 μs, 2 ms, 30 ms, and the time with maximum fluorescence intensity. The interphase steps of L and K bands were at 150 and 300 μs, respectively (Strasser et al. 2004; Guo et al. 2020). 1

The calculation of Chl-a fluorescence kinetics in JIP-test

To clarify the behavior of the electron transport chain, the following relative fluorescence parameters were calculated (Guo et al. 2020; Dimitrova et al. 2020).

  • Vt = (Ft – FO)/(FM – FO), is relative variable fluorescence at time t;

  • WOK = (Ft – FO)/(FK – FO), is used to clarify L-band;

  • WOJ = (Ft – FO)/(FJ – FO), is used to show K-band;

  • WOI = (Ft – FO)/(FI–FO), is common to show J-step, as well as WOI ≥ 1 plotted in the linear 30–270 ms time range to show IP phase;

  • Mo = 4(F300μs – FO)/(FM – FO), is approximated initial slope (in ms−1) of the fluorescence transient normalized on the maximal variable fluorescence (FV).

  • Oxygen-evolving-complex (OEC) centers = [1 –(VK/VJ)]treatment/[1 – (VK/VJ)]control, represents the active fraction of OEC centers.

  • δRo = (1 – VI)/(1 – VJ), represents the probability that an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side.

Other JIP test parameters were calculated according to the equation by Strasser et al. 2004.

Data analysis

The data were analyzed using IBM SPSS22 software for One way ANOVA, and multiple comparisons were conducted by using Least Significant Difference (LSD) or Duncan test. In all figures in this article, the different capital letters and lowercase letters indicate extremely significant (p ≤ 0.01) and significant (p ≤ 0.05), respectively. To evaluate the correlation between photosynthetic activity and astaxanthin production when the samples were exposed to the 9 different light quality, the correlation analysis was conducted using the “Correlation plot” application in OriginPro 2021 software (Ikushima et al. 2020).

Results

Effect of light quality on growth and substance productions of H. pluvialis

Within 24 days culture, the cell differentiation of H. pluvialis at nine light quality was shown in Fig. 1. the majority of cells began to become red at the 10th day and deepened by the 15th day. The red color deepened as the culture duration increased. By the 15th day, the disadvantages of W and 7R gradually appeared. At the end of the culture, the cells at 1R6B, 2R5B, 3R4B, and 5R2B had completely turned red. Additionally, there were differences in H. pluvialis biomass dry weight at harvest time at various light conditions (Fig. 2a). The biomass dry weight for control W was 926.36 mg/L, for 7B it was 988.33 mg/L, and for 7R it was only 886.25 mg/L (as the lowest one at 9 light quality). Except for 1R6B, the dry weight at combined monochromatic light 2R5B-6R1B were all above 940 mg/L, with 5R2B having the highest value of 1007.70 mg/L, which was significantly higher than W. Therefore, combined monochromatic light promoted cell maturation and improved biomass dry weight.

Fig. 1
figure 1

Effect of light quality on the cell morphological characteristics of Haematococcus pluvialis FACHB-712

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Fig. 2
figure 2

Effect of light quality on biomass and substance productions of Haematococcus pluvialis FACHB-712. a for biomass and substance production, b for substance content, c for pigment production, and (d) for astaxanthin content. All data are means ± SD, n = 3. Different capital letters indicate a statistical difference (P < 0.01) as determined by LSD test or Dunnett’3 T3 test

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The productions of major substances harvested after 24 days of cultivation and their contents in biomass dry weight of H. pluvialis were showed in Fig. 2a and b, respectively. Protein, carbohydrate, and lipid productions at 7R were 229.60, 188.58, and 99.41 mg/L, respectively, while the corresponding contents reached 25.9, 21.3, and 11.23%. These productions and contents were significantly greater at 7R than at other light quality. Blue light (7B) and combined monochromatic light of 2R5B, 3R4B, 4R3B, and 5R2B led no significant difference in protein and carbohydrates compared to W, but their lipid production and content were significantly lower than W and 7R.

The Chl-a and astaxanthin productions of H. pluvialis were affected by varying the light quality (Fig. 2c). Chl-a production was highest at control W and much lower at other treatments. The production of astaxanthin was lowest (4.91 mg/L) at 7R, followed by 6.99 and 8.20 mg/L at W and 7B, respectively. The astaxanthin production at combined monochromatic light were all higher than 10 mg/L, and significantly higher than W, 7R and 7B. The highest astaxanthin production of 15.28 mg/L was observed at 3R4B, which was 2.2 times of W. The content of astaxanthin was all exceeding 1.2% and significantly higher than those in 7R, 7B and W. The highest astaxanthin content was observed at 3R4B of 1.58%, which was 2.8, 1.9 and 2.1 times of that at 7R, 7B and W, respectively (Fig. 2d). Therefore, the combined monochromatic light, especially 3R4B, was conducive to the accumulation of astaxanthin in H. pluvialis.

Using the production or content of each substance in control W as 100%, the productions and contents at different light quality were converted into control percentages, and the radar chart of the variations in substance production and content was shown in Fig. 3. It was clear that 7R made protein, carbohydrate, and lipid productions and contents higher than other light quality. At the same time, the productions and contents of Chl-a and astaxanthin at 7R were considerably lower than those at W. Other than 7R, Chl-a, lipid production and content were lower than W at all other light quality, while astaxanthin production and content were substantially higher than W. In particular, the production and content of astaxanthin at 3R4B and 6R1B exceeded 200% of W. The production and content of astaxanthin were particularly sensitive to light quality, compared to other substances of H. pluvialis.

Fig. 3
figure 3

Variation of light quality on substance production and content of Haematococcus pluvialis FACHB-712

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Effect of light quality on photosynthetic characteristics of H. pluvialis

In JIP-test, H. pluvialis showed typical multiphasic fluorescence induction, FO, FJ, FI, and FP (the fluorescence values at points of O-J-I-P) was significantly different among 9 treatments, as shown in Fig. 4a. The remarkable feature was that 7R and 7B made OJIP curve moved upward and downward from W, respectively. The OJIP curves of combined monochromatic light were relatively concentrated, but the values of FM were generally reduced. 7R significantly increased FO, while 7B and other light quality decreased FO value compared with control W. The change of FO value was related to the change of structure and order of light collection complex in the process of light energy absorption (Guo et al. 2020).

Fig. 4
figure 4

Effects of light quality on fluorescence rise kinetics of Haematococcus pluvialis FACHB-712 in JIP-test. a Raw Chl-a fluorescence rise kinetics of Haematococcus pluvialis FACHB-712 under different light quality in a logarithmic time scale. b Chl-a fluorescence rise kinetics normalized by FO and FM as Vt = (Ft – FO)/(FM – FO) in a logarithmic time scale. c ΔVt = Vt(treatment) – Vt(control). d The fluorescence rise kinetics normalized by FO and FK as WOK = (Ft-FO)/(FK-FO) (top), and the difference of kinetics ΔWOK = WOK(treatment)—WOK(control) (bottom) in a linear time scale from 0 to 300 μs. e The fluorescence rise kinetics normalized by FO and FJ as WOJ = (Ft-FO)/(FJ -FO) (top), and the difference of kinetics ΔWOJ = WOJ(treatment) -WOJ(control) (bottom) in a linear time scale from 0 to 2 ms. f The fluorescence rise kinetics curves normalized by FO and FI as WOI = (Ft -Fo)/(FI -Fo) (top) and the difference of kinetics ΔWOI = WOI(treatment) – WOI (control) (bottom) in a logarithmic time scale. Each curve is the average of 3 measurements

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The fluorescence curves double normalized between FO (50 μs) and FM were presented as relative variable fluorescence Vt = (Ft-FO)/(FM-FO) at logarithmic time scale (Fig. 4b). In order to clarify the detailed difference among these treatments, ΔVt = Vt(treatment) – Vt(control) at logarithmic time scale was calculated and shown in Fig. 4c. It was clearly observed that ΔVJ at 4R3B, 5R2B, 6R1B, and 7R were higher than that at W, while ΔVJ at 2R5B, 1R6B and 7B were lower than W. This tendency was also found when the parameters from the JIP-test were analyzed and shown in Fig. 5a. VJ increased in the combined monochromatic light with a high proportion of red light, and VJ at 7R was much higher than W. Similarly, the ΔVI of 5R2B, 6R1B, and 7R (with a high proportion of red light) were higher than that at W, while that at 4R3B, 3R4B, 2R5B, 1R6B, and 7B (with a high proportion of blue light) were lower than W in Fig. 4c. And the VI values in Fig. 5a in the treatments with high proportions of red light were higher than those with high proportions of blue light, including 1R6B, 2R5B, and 3R4B, which were significantly lower than W. The rise of the J-step indicated that the electron flow beyond QA in the PSII reaction center (RC) was blocked, and the accumulation of QA− would result in the deactivation of PSII RCs. And MO values represented the net rate of RC closure (Strasser et al. 2004). Therefore, an improvement in the electron flow beyond QA in the PSII RCs was suggested by the drop at J-step and stability of MO in the treatments with high proportions of blue light. The peak of the I step indicated that the electron transfer from plastoquinone (PQ) to the receptor side of PSI was blocked (Strasser et al. 2004). With high proportions of blue light, the VI values at 1R6B, 2R5B, and 3R4B were significantly lower than that at W, indicating a smoother electron flow from PQ to PSI receptor side than that at W.

Fig. 5
figure 5

Effect of light quality on PSII photosynthetic activity of Haematococcus pluvialis FACHB-712. a The parameters of photosynthetic electron transfer and OEC activity, b The quantum yields of PSII, c The performance indices of PSII, and (d) The NPQ relative parameters. All data are means ± SD, n = 3. Different letters indicate a statistical difference as determined by LSD test or Dunnett’3 T3 test, capital letters indicated P < 0.01, and small letters indicated P < 0.05

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L-band was considered as the index of the energy connection between the PSII unit packet or antenna and the PSII RC (Srivastava et al. 1997; Strasser et al. 2004). To clarify the difference at L-band among these treatments, the WOK and ΔWOK were calculated in Fig. 4d. All treatments, with the exception of 7R, were shown to considerably increase WOK and ΔWOK at the L-band as compared to W. This was especially true with 2R5B. The arrangement of PSII units or the energy connection between the antenna and PSII RC were impacted by these treatments (Srivastava et al. 1997; Strasser et al. 2004). The same pattern, with the exception of 7R, was shown in the analysis of the K-band of WOJ and ΔWOJ in Fig. 4e, which shows that the treatments increased the appearance of K-bands in comparison to W. The inactivation of the PSII donor side OEC centers is indicated by an increase in K-step (Srivastava & Strasser 1996; Strasser et al. 2004). The treatments with high proportions of blue light, such as 1R6B, 2R5B, 3R4B, and 4R3B, caused a considerable decline in the values of the OEC centers when compared to the control W, as shown in Fig. 5a. This meant that a portion of the active OEC centers had been damaged by the treatments of 1R6B, 2R5B, 3R4B, and 4R3B.

The maximum amplitude of the WOI curve involves the information about the size of the electron acceptor pool at the end of the PSI receptor side (Strasser et al. 2004). Variation of light quality led to a various increase in the amplitude of WOI curve compared with W, indicating that these treatments increased the size of the electron acceptor pool at the end of the PSI receptor side (Fig. 4f). Generally, the treatments with a higher proportion of red light led to a lower amplitude in WOI curves (WOI ≥ 1). δRo denotes the probability of electron transfer from the electron acceptor among the reduced systems to the final electron acceptor of PSI (Strasser et al. 2004). With the exception of 5R2B, δRo of all treatments were increased compared with W, while 2R5B had a significantly higher δRo than W (Fig. 5a). Therefore, 2R5B not only increased the size of the electron acceptor pool at the receptor side of PSI, but also increased the activity of PSI.

The maximum photochemical quantum yield in the primary photochemical reaction (φPo), quantum yield of trapped excitons transferring electrons to other electron acceptors in the electron transfer chain that exceed QA (Ψo), quantum yield for electron transfer (φEo) and quantum yield for reduction of PSI receptor side terminal electron acceptor (φRo) were calculated in Fig. 5b (Tsimilli-Michael 2019). Treatments of 7B, 1R6B and 2R5B were generally higher than W, meanwhile, their quantum yields of heat dissipation φDo were equal to W but significantly lower than the treatments with higher proportions of red light. The φPo, Ψo, φEo and φRo of 4R3B, 5R2B, 6R1B and 7R were generally equal to or lower than W, and their φDo values were significantly higher than W. For all electron transport quantum yield characteristics, the treatment of 3R4B was modest and relatively close to those of W.

As the PSII performance parameters, PIABS represents the energy diversion from the absorption of light energy to the reduction of electron transport chain between systems, and PITOTAL includes the reduction of PSI terminal electron acceptor (Tsimilli-Michael 2019; Strasser et al. 2004). As shown in Fig. 5c, PIABS and PITOTAL of the treatments with high proportions of blue light such as 7B, 1R6B and 2R5B resulted in the higher values, and were significantly higher than W. The PIABS and PITOTAL in 2R5B were the highest while those in 7R were the lowest among control and all treatments.

The photochemical quenching (qP) in the NPQ analysis (Fig. 5d) represents the share of light energy absorbed by the PSII antenna and utilized for photochemical reaction electron transfer. The share of absorbed light energy that dissipates as heat is known as non-photochemical quenching (NPQ) (Srivastava and Strasser 1996). Light quality had a significant effect on the qP and NPQ of H. pluvialis, indicating that light quality had an impact on the distribution of light energy for photochemical reactions or heat dissipation. The high values of qP were observed at treatments of 2R5B-5R2B. The qP of W was 0.80, and was significantly lower than those at 2R5B-7R, indicating that the light energy used for photochemical reactions increased at 2R5B-7R. The light energy allotted for heat dissipation was increased in the treatments with high proportions of red light and was decreased in the treatments with high proportions of blue light, according to the NPQ of W, which was 0.52 and was significantly lower than that at 7R and higher than those at 1R6B and 2R5B. QYmax is the maximum PSII photochemical efficiency in dark-adapted state, reflecting the photochemical efficiency when the reaction center of PSII is fully open in the presence of heat dissipation. QYss is the actual PSII photochemical efficiency (Srivastava and Strasser 1996). All treatments had similar QYmax to W (0.76), indicating that QYmax was not affected by light quality. In a variety of light quality, the QYss exhibited the same tendency as qP. The treatments of 2R5B-5R2B had the higher QYss values and W had the lowest QYss of 0.55.

Taking photosynthetic characteristics of control W as 100%, the parameters of the treatments were converted into the percentages of control, and a radar chart of the changes in photosynthetic characteristics caused by variation in light quality was shown in Fig. 6. It clearly showed that 7R was the most unique light quality, as seen by its much higher NPQ, φDo, VJ and Mo, and the significantly lower Ψo, φEo, φRo, PIABS and PITOTAL values when compared to W and other light quality. Treatments with high proportions of blue light, including 7B, 1R6B, 2R5B and 3R4B, had lower NPQ, VJ, VI, OEC centers, Mo, and higher φEo, δRo, φRo, PIABS and PITOTAL values when compared to W and other light quality. The NPQ, PIABS and PITOTAL were the most sensitive indicators to light quality.

Fig. 6
figure 6

Variation of light quality on PSII parameters of Haematococcus pluvialis FACHB-712

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The relationship between photosynthetic characteristics and astaxanthin production of H. pluvialis

The correlation analysis between astaxanthin production and the photosynthetic characteristics described by NPQ analysis and JIP-test was conducted, as showed in Fig. 7. For references, the Chl-a and biomass production were also included. Chl-a synthesized by alga was negatively correlated with qP and QYss (correlation coefficient less than -0.66), which indicated that Chl-a synthesis was down-regulated when the alga performed higher photosynthetic activity. Biomass was positively correlated with QYmax (correlation coefficient = 0.70). In the case of astaxanthin production, both the content (% of biomass) and production (mg/L) had a strong and positive correlation with qP and QYss (correlation coefficient higher than 0.76) and the negative correlation with OEC centers. In addition, NPQ had the moderate and negative correlation with astaxanthin production (correlation coefficient less than -0.45). In general, the production of biomass and astaxanthin correlated negatively with NPQ, OEC centers and parameters measuring the degree of reaction center closure and the degree of electron transfer blockage, such as MO, VJ, and VI. In contrast, the production of biomass and astaxanthin correlated positively with photosynthetic activity as measured by quantum yields and performance indices.

Fig. 7
figure 7

The correlation between photosynthetic activity and astaxanthin production of Haematococcus pluvialis FACHB-712 under various light quality

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Discussion

H. pluvialis was previously found to respond to the variations of light quality by regulating the changes of pigment composition, chloroplast membrane lipids and liposomes. The life cycle of H. pluvialis includes typical green planktonic cell stage and red dormant cyst stage. Green planktonic cells have high efficiency and activity of cell division, while during the period of red dormant cyst stage, the cell division rate is decreased and there is a significant astaxanthin accumulation (Fitriana et al. 2021; Aflalo et al. 2007; Li et al. 2020). It had been reported that monochromatic red light prolonged the period of cell proliferation and increased the final biomass yield (Zhu et al. 2018; Pereira & Otero 2020; Suyono et al. 2015). In this study, cells matured at 7R and W relatively later. Monochromatic blue light accelerated cell turning to mature stage. And the advantages of combined monochromatic light were more pronounced in terms of accelerating the cell transformation (Fig. 1). Combined light not only made the cells mature quickly, but also obtained higher biomass than red light (Fig. 2a). This is consistent with research by Li et al. (2020) and Mourya et al. (2023), which both reported that the accumulation of biomass by combined red and blue light was higher than red light only. In particular, the combination of red and blue light at a ratio of 2:1 produced the maximum biomass in an above previous study, which is quite comparable to the condition and result of 5R2B in this study (Li et al. 2020).

It was reported that blue light was more efficiently absorbed and directed to PSII than red light, which resulted in higher photosynthetic activity in the green microalga at monochromic blue light than at monochromic red light (Tamburic et al. 2014). Blue light was also reported responsible for the enhanced production and activity of respiratory enzymes in plants and microalgae (Ruyters 1984). In this study, the composition of the cells at W, 7B, and combined monochromic light did not differ significantly from one another. The notable increase in intracellular carbohydrate, lipid, and protein contents at red light was probably caused by the cells’ maintaining a relatively fast division rate and later transformation as well as the lack of blue light activation of related metabolic enzymes.

The color of the cells depends on the pigment composition, among which Chl-a and other accessory pigments such as carotenoids are responsible for the absorption of blue light (Xue et al. 2020). In this study, W had the highest Chl-a content. At 7R, astaxanthin content was found to be the lowest, while at combined monochromatic light, it was shown to be far higher. This agreed with previous report that the combined monochromatic light had a better effect on astaxanthin accumulation than white light (Mourya et al. 2023). Astaxanthin as a secondary carotenoid, is often induced by stress to protect cells from the damage of reactive oxygen species (ROS) (Aflalo et al. 2007). Blue light may potentially enhance the content of astaxanthin in H. pluvialis (Ma et al. 2018). When compared to red or white light, monochromatic blue light was found to significantly increase the production of astaxanthin, and more astaxanthin accumulated in combinations of blue and other wavelengths (Pereira and Otero 2020). At blue light, the cell division related minD gene of H. pluvialis was significantly suppressed, and astaxanthin biosynthesis genes psy, lcy, crtO, and crtR-B were upregulated (Lee et al. 2018). The results in this study verified that monochromatic blue light allowed H. pluvialis to accumulate more astaxanthin, the content of astaxanthin reached 0.83% (Fig. 2d). This result agreed with the previous study, in which H. pluvialis was exposed to blue light before to ultraviolet light, and the content of carotenoids (including astaxanthin) was 4 times higher than cells subjected to white light before to ultraviolet light on 7th day (Suyono et al. 2015). Moreover, all combined monochromatic light obtained the astaxanthin contents more than 1.2%, especially 3R4B of 1.58% (2.1 times of W). This result is also supported by Mourya et al. (2023) and Li et al. (2020), in which the astaxanthin yield of H. pluvialis under combined blue and red light was 27% higher than that under white light, and the highest content of carotenoids (including astaxanthin) under 2:1 ratio of combined red/blue light was 8.5 mg/L and 44% higher than that of red light, respectively. Therefore, the combination of monochromic blue and red light is a promising approach to increase the astaxanthin accumulation.

The stress conditions make photosynthetic organisms alter their photosynthetic electron transfer rate. When photochemistry proceeds at lower rates, the excess exciton energy may damage PSII by the increased production of ROS (Niyogi 2000). A protection mechanism against such damaging of PSII is the NPQ, which reflects any distributary energy flows other than photochemistry (Einali et al. 2013). In this study, the only difference among the treatments was light quality, which had close relation with algal photosynthesis. And how light quality affected astaxanthin accumulation through photosynthetic process remains unknown. In Fig. 5, light quality affected the Chl-a fluorescence yields obtained during the NPQ protocol, and finally resulted in the decrease of NPQ at light quality of 1R6B, 2R5B and 3R4B as well as the increase of NPQ at 7R. When photochemistry is partially inhibited, the dissipation of energy through NPQ is expected to be enhanced (Juneau et al. 2002). In other words, when the photochemical ability increased, the need of relying on NPQ for energy dissipation decreased. In this study, the qP at 2R5B-7R were significantly higher than W, which reflected that the light quality improved the transfer of the generated electrons beyond QA, and hence increased photochemistry (Markou et al. 2016). Therefore, the increase in qP is one of the reasons for the decrease in NPQ at combined monochromatic light.

Another reason contributing to the decline in NPQ at combined monochromatic light could be astaxanthin's shielding functions. When cells are subjected to combined monochromatic light, the increased astaxanthin's antioxidant effects will efficiently shield them from severe damage (Pereira and Otero 2020). Astaxanthin is believed to function as passive photoprotectors, limiting the quantity of light that may reach the PSII in the light-collecting pigment complex (Solovchenko 2015). Due to the accelerated cell maturation and improved protection provided by the high astaxanthin accumulation under these conditions, the need for NPQ should be decreased. The demand for NPQ increased at red light because astaxanthin accumulation was reduced and cell maturation was delayed.

The primary cause of the increased photochemical activity is the unhindered electron transport. The process of absorbing, transferring, and converting light energy into chemical energy in photosynthesis occurs in the algal pigment protein complex, which has a specific molecular organization, spatial conformation, and associated electron carriers (Wu et al. 2023). Regeneration and reoxidation of intermediate electron carriers can improve the efficiency of electron transfer, thereby further improve the efficiency of light energy conversion into chemical energy (Blankenship et al. 2011). The OJIP transients also reflect the successive reduction of the electron pools (Pheo, QA and QB) of PSII and therefore it is frequently used to characterize the PSII activity. In this study, by focusing on the detailed sites of electron transport chain in Figs. 4 and 5, the combined monochromatic light with high proportions of blue light improved the electron transport from QA to QB (decreased VJ) and from PQ to PSI receptor side (decreased VI) and increased the probability that an electron transferred from the reduced intersystem electron acceptors to final electron acceptors of PSI (increased δRo). It was anticipated that the dissipation and quenching of energy would be reduced given that the pool size of the end electron acceptors on the PSI acceptor side were all increased at non-white light (increased amplitude of the WOI). The combined monochromatic light enhanced both PSII and PSI activity, in contrast to other environmental stresses like high salinity or high temperature, which increased PSI activity while lowering PSII activity (Zhang et al. 2010). In general, treatments with high proportions of monochromatic blue light had the higher quantum yields of photochemistry (φPo, Ψo, φEo, and φRo) and the lower φDo, when compared to W. This is also consistent with that ETR of H. pluvialis under mixed red/blue light was higher than that under monochromatic red light (Li et al. 2020). These results showed that the combined monochromatic light had an advantageous effect on the photosynthetic activity for H. pluvialis. The performance indices, as very sensitive parameters (especially PItotal), have proven to be very useful for evaluating the overall impact of a stress on photosynthetic performance/behavior (Tsimilli-Michael 2019; Gao et al. 2018). The performance indices and quantum yield of photochemistry of PSII in this study mutually confirmed the improvement of combined monochromatic light on photosynthetic activity.

In the case of monochromatic red light, not only the electron transfer from QA to QB but also the electron transfer from PQ to PSI receptor side were blocked (increased VJ and VI). The closure rate of the reaction center increased (increased Mo). The comprehensive performance indices (PIABS and PItotal) decreased as a result of the lowered quantum yields of photochemistry (φPo, Ψo, and φEo), as well as the increased quantum yield of φDo. These facts led to a significant increase in NPQ. In addition, the lack of the shielding function of astaxanthin further increased the demand for NPQ.

It is worth mentioning that no inhibitory effect of light quality on the QYmax (= FV/FM, maximum quantum yield) was monitored. The kinetic data revealed that QYmax kept a constant level at all light quality while the effective quantum yield of PSII (QYss) improved at combined monochromatic light. The results in this study are basically consistent with the report that H. pluvialis grew best under combined light (red/blue = 2:1) with a high FV/FM value (Li et al. 2020). In this study, although 5R2B did not lead to a significantly higher FV/FM than other light quality, its QYss showed the highest value (Fig. 5d). Because lower FV/FM levels caused by general stress indicate a negative effect on the PSII, it is implied that spectrum regulation is distinct from those stresses (Kalaji et al. 2014; Strasser et al. 2004).

The correlation analysis indicated that astaxanthin accumulation had the strong and positive correlation with qP and QYss and the moderate and negative correlation with NPQ and OEC centers (Fig. 7). In H. pluvialis, the K-band increased at the combined monochromatic light with high proportion of blue light, concomitant with a decrease in OEC centers, which reflect the active portion of the oxygen producing complex. The decrease of OEC centers will reduce the proton production, which will change the pH value of the thylakoid lumen and ultimately affect the ΔpH composition. Further, it may lead to a decrease in ATP production and have a negative impact on dark reactions using ATP and NADPH (Markou et al. 2016). Therefore, the decrease in OEC centers partially counteracts the leading positions of the combined monochromatic light with high proportion of blue light in φPo, Ψo, φEo, φRo and φDo, and comprehensively resulted in the outstanding performance of the treatments with moderate proportion of monochromatic blue light in qP and QYss (Fig. 5).

As conclusion, even though a high proportion of monochromatic blue light resulted in a decrease in OEC centers and a slight increase in L-band, the combined monochromatic light with moderate proportion of blue light achieved the increased qP and QYss due to the unimpeded electron transport, decreased NPQ due to high photosynthetic rate, and strong shielding function of astaxanthin, and comprehensively contributed to the high photosynthetic activity, advanced cell maturation and considerable astaxanthin accumulation.