Introduction

Haematococcus pluvialis is a freshwater, green unicellular microalga, distributed in various areas worldwide. H. pluvialis cells have two growth stages, vegetative (green) and aplanospore (red) stages, which are rich in astaxanthin (3,3′-dihydroxy-diketo-β, β′-carotene-4,4′-dione) at aplanospore stage. H. pluvialis is the main organism for producing astaxanthin with about 0.3–0.5 mg L−1 under normal conditions (Lorenz and Cysewski 2000). Astaxanthin is also founded in other living organisms such as birds, aquatic animals, and yeast, but in a low amount (Ambati et al. 2014). Astaxanthin is one of the most influential and potent antioxidants with several utilizations in the food and pharmaceutical industries due to its strong anti-aging, anti-cancer, anti-bacterial, anti-inflammatory, skin health, and immune-boosting impacts (Boussiba 2000). It can be generated synthetically, but natural astaxanthin has over 20 times more potent antioxidant activity (Capelli et al. 2013). Physical and chemical factors have been previously proved to stimulate astaxanthin production in H. pluvialis. Red-blue LED light increased astaxanthin content (3.04 mg L−1) after 14 days of culture. Salicylic acid (50 mg L−1) and low-intensity light increased astaxanthin content from 0.39 to 2.7 mg L−1 (Gao et al. 2012). On the other hand, biomass production of H. pluvialis has several problems because of genetic differences in strains, contamination, complex cultivation media, and slow growth rate. So, optimization of the cultivation conditions is vital for the promotion of biomass yield and astaxanthin production in H. pluvialis.

Astaxanthin biosynthesis is initiated from isopentenyl pyrophosphate (IPP), as a key mediator for the synthesis of carotenoids, and IPP can be biosynthesized from two separated pathways, mevalonate (MAV) in the cytosol and methylerythritol phosphate (MEP) in the plastid. Pyrophosphates are the common precursors for biosynthesis of monoterpenes and also are required for the production of phytohormone, modification of lipids, proteins, and other biomolecules (Manzano et al. 2004). It has been reported astaxanthin in H. pluvialis microalgae may synthesize through the MEP pathway and be stored in cytoplasmic lipid vesicles (Boussiba 2000; Gwak et al. 2014; Liang et al. 2016). Tetrasodium pyrophosphate (NaPP) is known as an IPP analog and inhibits IPP translocation from cytosol to plastid and visa vera (Fig. 1). It has also been used as a food additive, emulsifier, and buffer (Deshpande 2002). In biology research, it is used to determine biosynthesis pathways and metabolic engineering of secondary metabolites, especially terpenoids (Wang et al. 2003; Ramak et al. 2020). It has been reported that NaPP may change Fe bioavailability from iron compounds in a culture medium resulting in the rise of cellular soluble ionic Fe and its uptake by cells (Cercamondi et al. 2016). Moreover, NaPP stimulated terpenoid production and antioxidant capacity in Satureja khuzistanica (Ramak et al. 2020).

Fig. 1
figure 1

Astaxanthin biosynthesis pathways in plastid and cytosol. GA3P, 3-phosphoglyceraldehyde; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; MVA, mevalonate; MEP, methylerythritol phosphate; NaPP, tetrasodium pyrophosphate

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Static magnetic field (SMF) is a ubiquitous environmental factor that can impact cell growth and development by altering the activity, lifetime, and level of cellular reactive oxygen species (ROS) and influencing the activity of antioxidant enzymes. Magnetic field (MF) can change the membrane permeability and ion exchanges and affect cell division and growth (Latef et al. 2020; Taghizadeh et al. 2019). It has been reported the cellular metabolism of microorganisms, including photosynthetic performance (Hirano et al. 1998), accumulation of pigments, carbohydrates, and amino acids (Small et al. 2012) are changed following SMF. SMF induced the production of Apigenin in the cell suspension culture of Matricaria chamomile (Hassanpour and Niknam 2020). Also, the enhancement of ROS by SMF can stimulate the activity of enzymes and gene expressions related to the biosynthetic pathway of secondary metabolites (Taghizadeh et al. 2019) and increases antioxidant capacity. ROS induced by oxidative stress could induce astaxanthin content in H. pluvialis alga (Kobayashi 2003). There is a lack of information about the impact of SMF and NaPP (an inhibitor of IPP translocation) on the ROS accumulation and antioxidant capacity of H. pluvialis, and this is the first study. In this research, we hypothesized that the alteration in IPP translocation in the MEP/MVP pathways and ROS level can affect astaxanthin biosynthesis and antioxidant capacity of H. pluvialis, So, the impact of SMF and NaPP were investigated on growth, chlorophyll pigments, H2O2 content, the activity of enzymes, and antioxidant compounds. The result of this study may help to improve our knowledge about the response mechanisms of unicellular microalga to SMF and NaPP treatments.

Material and methods

Algae strain and growth condition

H. pluvialis algae were obtained from the Industrial Microbial Biotechnology Department of Khorasan Razavi, Mashad, Iran. Stock cells were initially prepared in a liquid Bold Basal Medium (BBM) and placed in a growth chamber with cool white LED-light intensity of 30 µmol m−2 s−1 with light/dark photoperiod of 14/10 h during the experimental period and 25 ± 2 °C for two weeks. For detection of the growth curve, the cell density of 0.5 × 105 from stock was cultured in BBM medium, and optical density at 750 nm and cell number were investigated every two days for 22 days. Cell numbers were investigated by a hemocytometer under a light microscope (Nikon, E-200).

NaPP and SMF applications

The H. pluvialis cells (optical density of 0. 5 × 105) at the logarithmic phase (day of sixth) were cultured in a liquid BBM medium with different concentrations of NaPP (0, 0.1, 0.2, 0.3, and 0.4 mM) to detect optimum growth and astaxanthin content after day 14 and 21 (Tables 1, 2) and the optimum NaPP concentration was selected to continue studies. Selection of SMF intensity and duration was also conducted according to the procedure described by Hassanpour and Niknam (2020) for 1 h for three days, continuously. Then, the best treatment of NaPP (0.3 mT), SMF (4 mT), and their combinations were applied to the cell suspension culture of H. pluvialis. SMF was applied at the logarithmic phase for three days (from day 5 to day 7), and then NaPP was added to the culture medium. SMF exposure was performed by a locally designed homogenous SMF generator (MFG-13971, OFOGH, Iran) with a 220 V DC power supply (MP-6010, MEGATECH, Iran) for producing various SMF intensities in the range of 0.5 µT-15 millitesla (mT) (Mansourkhaki et al. 2019). This system has consisted of a copper wire (1.1 mm in diameter) wrapped 2300 rounds around a polyethylene tube 12 cm in diameter and 50 cm in length. The container containing algal suspension culture (Erlenmeyer flask) was placed in the middle of the tube to get uniform intensity at all points of the container, and SMF intensities were applied and measured by Tesla meter (MG-3002) with a B-probe type of hall sound. After two weeks of treatment applications, some physiological and biochemical responses were investigated with three or four replications in each treatment.

Table 1 Impact of different NaPP concentrations on optical density and cell count of H. pluvialis microalga
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Table 2 Impact of different NaPP concentrations on chlorophyll a and b, and astaxanthin contents of H. pluvialis microalga
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Chlorophyll and astaxanthin contents

Chlorophyll a and b contents were evaluated spectrophotometrically at 665 and 652 nm using the procedure described by Wellburn (1994). For detection of astaxanthin, the algae cell suspension (20 ml) was centrifuged at 5000 rpm for 5 min, and the cell pellet was added with 5 mL solution of 5% (w/v) KOH in 30% (v/v) methanol in a 75 °C water bath for 10 min to remove the chlorophyll. The colorless pallet was added to 5 ml DMSO in a water bath (70 °C) for 10 min, and the absorbance of the supernatant was read at 429 nm. The astaxanthin content was calculated by the equation described by Li et al. (2012).

Total phenol, flavonoid, and DPPH scavenging activity

Freeze-dried biomass (100 mg) was placed in 80% methanol (v/v) for 48 h, and then was homogenized using an IKA homogenizer, T18 digital ULTRA-TURRAX. The solutions were centrifuged (5000 rpm for 5 min), and the supernatant was used for evaluation of the total flavonoid, phenol, and DPPH scavenging activity.

Total phenolic content was evaluated by the procedure described by Singleton and Rossi (1965). Gallic acid was used to draw the standard curve as mg of equivalent per g fresh weight.

Flavonoid content was determined by the aluminum chloride colorimetric procedure (Hatamnia et al. 2014) and was recorded at 415 nm. Total flavonoid content was shown as mg of rutin equivalent per g fresh weight.

Free radical scavenging potential was assayed based on the procedure described by Patro et al. (2005), and the absorbance was recorded at 517 nm. The percentage of inhibition (reducing activity) was evaluated by the following equation:

  • % Inhibition = Absorbance of DPPH—Absorbance of sample/Absorbance of DPPH × 100.

Hydrogen peroxide (H2O2) content and phenylalanine ammonia-lyase (PAL) activity

The H2O2 evaluation was conducted by the method described by Velikova et al. (2000). The extraction of fresh algae cells (10 mg) was conducted in 2 mL trichloroacetic acid 0.1% (w/v) under cold (4 °C) conditions, and the absorbance of the reaction mixture was recorded at 390 nm.

PAL activity was evaluated with the extraction of fresh alga biomass (50 mg) in 50 mM Tris–HCl buffer (pH 8.8), including 15 mM β-mercaptoethanol (Ochoa-Alejo and Gómez-Peralta 1993). The enzyme activity was recorded at 270 nm in the reaction mixture of 100 µl enzyme extract, 100 mM Tris–HCl buffer (pH 8.8), and 10 mM phenylalanine after incubation at 37 °C for 1 h. Cinnamic acid was used to draw the standard curve, and enzyme activity was expressed as an mg of cinnamic acid per g protein.

Protein and antioxidant enzyme activity

Fresh biomass (100 mg) was ground in 1 M Tris–HCl (pH 6.8) at 4 °C, and the supernatant was separated under 13,000 × g centrifugation for 10 min, and held at − 70 °C for enzyme and protein assays. Protein content was evaluated based on the procedure described by Bradford (1976).

Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined with the reaction mixture of 0.1 mM EDTA (0.1 mM), nitroblue tetrazolium (NBT, 75 μM), potassium phosphate buffer (50 mM), 13 mM methionine, 75 μM riboflavin, and 150 μl of enzyme extract based on the Giannopolitis and Ries (1977) procedure. The absorbance of NBT reduction was recorded at 560 nm.

Catalase (CAT; EC 1.11.1.6) activity was assayed based on Aebi (1984) procedure. The 5 μl enzyme extract was combined with 0.625 ml of 50 mM potassium phosphate buffer (pH 7.0) and 0.075 ml H2O2 (3%). The activity was read at 240 nm.

Statistical analysis

The data were analyzed with a completely randomized design using SPSS (version 18) software. Each data is the mean ± SE (standard error) of three or four replications in each treatment, and the significance of differences among treatments was obtained by Duncan's multiple range test (DMRT) test at P ≤ 0.05. Principal component analysis (PCA) was carried out through the XLSTAT 2021.2.2 software.

Results

Cell growth curve of H. pluvialis

Cell growth of H. pluvialis algae was evaluated by determination of optical density at 750 nm and cell counting every 2 days for about three weeks. The growth rate was slow for the first 4 days (lag phase, 0.21 × 105 cell number), and then the cells entered the logarithmic phase, and noticeable cell growth was observed during this phase. Microalgae vegetative growth reached high cell numbers (3 × 105) on the fourteenth day of cultivation. Then the stationary phase was started on the fifteenth day of cultivation (Fig. 2).

Fig. 2
figure 2

Optical density and cell count of H. pluvialis microalga after 22 days of culture. Each value indicates mean ± SE (n = 3) in each group

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Impact of different NaPP concentrations on growth, chlorophyll, and astaxanthin contents

In the preliminary experiments, different concentrations of NaPP (0, 0.1, 0.2, 0.3, and 0.4 mM) were used to determine the optimum growth rate and astaxanthin content after 14 and 21 days for the next study. The results showed that NaPP changed the optical density and cell count during the growth (Table 1). NaPP at 0.4 and 0.1 mM concentrations showed lower and higher optical density and cell count as compared to the control, respectively. There was no difference between NaPP at 0.3 mM and control for growth rate. Images of the lightning microscope on day 18 of culture are presented in Fig. 3. In the control group, most cells were in the form of green vegetative palmella, and low numbers of them were in transition to aplanospore for astaxanthin accumulation. With increasing NaPP concentrations from 0.1 mM to 0.3 mM, cells went to astaxanthin accumulated aplanospore with red color and larger size than green vegetative cells. Moreover, the cell size and the number of aplanospore cells decreased at 0.4 mM NaPP compared to control and 0.3 mM NaPP exposed cells.

Fig. 3
figure 3

Impact of different NaPP concentrations on cell morphology of of H. pluvialis on day 18 of culture. A, aplanospore; VP, vegetative palmella

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Chlorophyll a and Chlorophyll b contents decreased at all concentrations of NaPP, and their contents markedly reduced with increasing duration time (after 21 days) and concentration of NaPP. The lowest chlorophyll content was observed at 0.4 mM compared to control, and there was no difference between 0.2 and 0.3 mM NaPP (Table 2).

The result of astaxanthin content was in contrast to chlorophyll contents and increased significantly after 21 days (late of stationary phase) compared to 41 days. Moreover, astaxanthin content increased from 0.1 NaPP to 0.3 mM, and then decreased slightly at 0.4 mM. Maximum astaxanthin content (7.19 mg ml−1) was observed at 0.3 mM NaPP after 21 days compared to control (4.37 mg ml−1) (Table 2). According to the studied parameters, 0.3 mM NaPP was selected as the optimum concentration for the next experments.

Impact of NaPP and SMF on chlorophyll and astaxanthin contents

NaPP and its combination with SMF (SMF + NaPP) didn’t change significantly in chlorophyll a content after 21 days of culture, but SMF treatment enhanced chlorophyll a content by 37.74% as compared to control. Similar to chlorophyll a, Chlorophyll b content also increased (30.53%) by SMF treatment as compared to control, and relatively unchanged under NaPP and SMF + NaPP (Fig. 4a).

Fig. 4
figure 4

Impact of NaPP, SMF, and their combinations on chlorophyll a and b (a) and astaxanthin content (b). Each value indicates mean ± SE (n = 4) in each group. Different letters present significant differences at p ≤ 0.05

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Astaxanthin content after 21 days of culture showed a significant increase under NaPP (68.13%) and NaPP + SMF (59.81%) as compared to control. SMF exposure also increased astaxanthin content (28.94%), however, its increased level was lower than NaPP alone (Fig. 4b). The results showed that NaPP had the highest effect on enhancing astaxanthin content in comparison to other treatments.

Impact of NaPP and SMF on the content of total phenol and flavonoid, and activity of DPPH scavenging and PAL

Total phenol content showed a 55.86% and 40.51% increase under NaPP and SMF treatments, respectively as compared to control. NaPP induced the highest total phenol in comparison to other treatments. Moreover, the combination of SMF and NaPP induced total phenol content with an 22.52% enhancement compared to the control (Fig. 5a). Similar to phenol content, a total flavonoid also increased as compared to control, but the enhancement of flavonoid content by SMF and SMF + NaPP treatments was more pronounced as compared to alone NaPP treatment. SMF and SMF + NaPP treatments caused a 1.66 and 1.58-folds increase in flavonoid content compared to control (Fig. 5b). DPPH activity increased significantly by SMF, NaPP, and their combinations, but NaPP treatment markedly enhanced (46.57%) this parameter as compared to control (Fig. 5c). PAL activity increased significantly under NaPP, SMF, and NaPP + SMF treatments, and the maximum activity was observed after SMF application with a 52% increase in comparison to control (Fig. 5d).

Fig. 5
figure 5

Impact of NaPP, SMF, and their combinations on total phenol (a) and flavonoid (b) contents, and activity of DPPH radical scavenging (c) and PAL (d). Each value indicates mean ± SE (n = 3) in each group. Different letters present significant differences at p ≤ 0.05

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Impact of NaPP and SMF on H2O2, protein, and antioxidant enzyme activity

H2O2 content in H. pluvialis under NaPP and SMF + NaPP treatment significantly increased, but unchanged by SMF application compared to the control. H2O2 content in NaPP- treated H. pluvialis was higher than that of SMF + NaPP- treated H. pluvialis. NaPP treatment significantly induced H2O2 content by 47.82% as compared to control (Fig. 6b).

Fig. 6
figure 6

Impact of NaPP, SMF, and their combinations on protein (a) and H2O2 (b) contents, and activity of SOD (c) and CAT (d). Each value indicates mean ± SE (n = 3) in each group. Different letters present significant differences at p ≤ 0.05

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According to our results, all treatments had different effects on protein content. H. pluvialis treated by SMF obtained higher protein content as compared to the control. However, SMF + NaPP treatment did not show a significant effect on protein content as compared to the control (Fig. 6a). NaPP treatment significantly decreased this parameter compared to the control. Application of SMF alone or in combination with NaPP caused a significant increase in SOD activity of H. pluvialis as compared to control and this effect was pronounced (73.85%) by SMF treatment. However, there was no significant difference in the activity of SOD enzyme between the NaPP-treated and control H. pluvialis (Fig. 6c). The SMF and SMF + NaPP treatments significantly augmented the CAT activity by 52.17% and 49.60% in H. pluvialis, respectively, compared to the control. The NaPP treatment had no significant effect on CAT activity compared to untreated H. pluvialis (Fig. 6d).

Discussion

This study was conducted to elucidate the impact of SMF as a physical factor and NaPP as an inhibitor of IPP translocator on the growth response and secondary metabolite production in H. pluvialis algae. In this study, NaPP from 0.1 to 0.3 mM could not significantly influence chlorophyll pigments, cell number, and optical density. But at a concentration of 0.4 mM, NaPP decreased significantly under the mentioned parameters (Tables 1, 2). Photosynthetic pigments respond quickly to stress situations and can be a marker to study the effect of stress on growth parameters. There are various results of NaPP impact on growth and photosynthetic pigments, which can depend on the type of species, growth stage, and NaPP concentrations. Ramak et al. (2020) reported NaPP enhanced the growth parameters, chlorophyll, and carotenoid contents of S. khuzistanica. Similar to our results, Wang et al. (2003) showed dry biomass didn’t change significantly in the NaPP-treated cell culture of Taxus chinensis. The results of this study suggest that carbon assimilation required in the primary metabolism isn’t inhibited by NaPP at optimum concentration, and this concentration can be used to continue studies. SMF treatment enhanced chlorophyll a and chlorophyll b in H. pluvialis (Fig. 4a). Similarly, Hirano et al. (1998) confirmed the positive effect of SMF on chlorophyll content in Spirulina platensis. SMF treatment enhanced chlorophyll a and b contents in Chlorella kessleri (Small et al. 2012). This fact was also confirmed by some research in various plants, including Beta vulgaris L. (Rochalska 2005) and Solanum tuberosum L. (Rakosy-Tican et al. 2005). Increased chlorophyll content can be related to the SMF effect on the stimulation of chloroplast protein synthesis and their protection by improving antioxidant capacity (Hassanpour et al. 2021), which agrees with the stimulation of enzymatic antioxidant activities in H. pluvialis. The PCA test showed the relation between chlorophyll pigments, protein, and antioxidant enzymes under SMF treatment (Fig. 7). However, the combination of NaPP + SMF decreased slightly chlorophyll pigments which may be related to ROS accumulation and its impact on chlorophyll degradation and membrane lipid peroxidation (Azad et al. 2021).

Fig. 7
figure 7

Principal component analysis (PCA) of the evaluated variables under NaPP, SMF, and their combinations in H. pluvialis. Chlorophyll–Chl; catalase–CAT; superoxide dismutase–SOD; phenylalanine ammonia-lyase–PAL

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Astaxanthin content was augmented in H. pluvialis under NaPP, SMF, and their combination, and the highest content was observed by NaPP (Table 2; Fig. 4b). The impact of SMF on the induction of secondary metabolite production has been reported in the cell suspension of M. chamomilla (Hassanpour and Niknam 2020), Physalis alkekengi (Hassanpour and Hassanpour 2021), which is associated with the stimulation of enzymes activities in the biosynthesis pathway of secondary metabolites. Moreover, there are some studies about the impact of NaPP on secondary metabolite production. For example, NaPP application (0.1 and 1 Mm) inhibited Taxol production in the cell suspension culture of Taxus chinensis (Wang et al. 2003). NaPP induced total phenol, flavonoid, and carvacrol productions in S. khuzistanica plants (Ramak et al. 2020). In our research, NaPP induced astaxanthin content markedly after 21 days of culture against 14 days. There is no data about the NaPP impact on secondary metabolites in H. pluvialis, and this is the first study. Application of other elicitors such as selenite and sodium acetate could also induce astaxanthin accumulation in H. pluvialis (Zheng et al. 2017; Zhang et al. 2019). It is widely known that NaPP is a substrate analog of IPP (Soler et al. 1993) and inhibits the translocation of IPP or other substrates of secondary metabolites from the cytoplasm to plastids and vis versa. So, the IPP translocation isn’t required in astaxanthin biosynthesis at the late phase of H. pluvialis growth (after 21 days of culture). Moreover, exogenous application of NaPP can act as a chemical factor for improvement or generating secondary metabolites by impacting MEP pathway (Ramak et al. 2020). Our result suggested that NaPP can act as an elicitor to stimulate astaxanthin biosynthesis in H. pluvialis alga. Moreover, the combination of NaPP and SMF (6.89 mg L−1) stimulated more astaxanthin content compared to SMF (5.54 mg L−1) treatment alone indicating that NaPP could provide a stressful condition to improve secondary metabolite. PCA graph displayed a positive co-relation between H2O2 level, astaxanthin, and antioxidant capacity (Fig. 7).

Algal phenolic compounds were described to be a potential candidate to overcome ROS, which can induce oxidative damage in cells (Adamson et al. 1999; Estrada et al. 2001). Our results revealed that NaPP, SMF, and their combinations could accumulate total phenol and flavonoid in H. pluvialis (Fig. 5a, b). Activation of PAL in response to NaPP and SMF treatments was reflected by the accumulation of total phenol and flavonoid in H. pluvialis. Ramak et al. (2020) reported that NaPP induced total phenol and flavonoid contents in S. khuzistanica plants. Results of this study suggested that NaPP can cause simultaneous induction of both phenol biosynthesis and MEP pathways by stimulating enzyme activities. Also, the impact of SMF on the induction of phenolic compounds has been previously reported by Abdollahi et al. (2019) and Jalilzadeh et al. (2018). PAL is the main enzyme in the metabolism of phenolic compounds and catalyzes the first step of the phenylpropanoids pathway. Moreover, its activity is a crucial regulation point between primary and secondary metabolisms (Heidarabadi et al. 2011). Taghizadeh et al. (2019) showed a positive relation between PAL activity and total phenol under SMF in Dracocephalum polychaetum cell suspension culture, which is in agreement with this study. In the PCA graph, the results of total phenol, flavonoid, and PAL activity were highly closed to the F1 axis and could induce antioxidant capacity (Fig. 7). On the other hand, the DPPH radical scavenging activity increased under both SMF and NaPP treatments and their combinations, which may associate with astaxanthin, phenol, and flavonoid accumulations. These compounds induce antioxidant capacity in plant cells (Ashouri Sheikhi et al. 2016).

Soluble protein has roles such as controlling osmotic pressure and ameliorating stress and thus can show cellular resistance to stress (Hassanpour 2022; Merati et al. 2015). Our research showed that protein content enhanced markedly in SMF-treated H. pluvialis (Fig. 6a). Increased protein content can be associated with the accumulation and synthesis of proteins by affecting SMF on the activity of some enzymes in the protein synthesis pathway (Luo et al. 2021; Shabrangi et al. 2015). On the other hand, NaPP decreased slightly protein content, but in combination with SMF enhanced significantly the parameter. Cells under stress conditions induce H2O2 generation, which can destruct cellular macromolecules like protein, lipids, etc. at high levels (Hassanpour et al. 2016). In this research, the decline in protein content under NaPP may be related to more induction of ROS accumulation and oxidative damage, which was in agreement with the results of NaPP impact on ROS induction.

CAT is a redox enzyme that converts H2O2 into H2O and O2, thus preventing H2O2 accumulation in the cell and SOD acts as a key defensive enzyme against free radicals in catalyzing superoxide dismutation to H2O2 and O2 (Sreenivasulu et al. 2000). In this study, H2O2 content enhanced significantly under NaPP application, but reduced by SMF exposure. In contrast to NaPP, the activity of antioxidant enzymes such as SOD and CAT increased significantly under SMF exposure. The H2O2 level in SMF-treated H. pluvialis was significantly lower than NaPP treatment, which was associated with the high activity of CAT and SOD enzymes (Fig. 6c, d). In wheat, SMF application enhanced significantly antioxidant enzyme activities (SOD and CAT) as compared to the control (Şen and Alikamanoğlu 2016). CAT and SOD enzymes contain paramagnetic elements including Mn, Fe, and Cu in their catalytic domains and can be powerfully activated under the magnetic field (Serrano et al. 2021). On the other hand, NaPP increased markedly H2O2 content and didn’t display a significant impact on the activity of CAT and SOD enzymes. Indeed, NaPP induced a stressful condition for alga cells through cellular ROS accumulation resulting in a decline in chlorophyll pigments and conducting the energy source to the accumulation of secondary metabolites.

Conclusion

The results revealed that NaPP at 0.3 mM act as an elicitor to stimulate astaxanthin production in H. pluvialis after 21 days of culture, and induced more astaxanthin content compared to SMF. Additionally, the inhibitory impact of NaPP on IPP translocation didn’t affect MEP pathway for astaxanthin biosynthesis. Secondary metabolites such as total phenol and flavonoid contents and DPPH scavenging activity enhanced significantly under NaPP, SMF, and their combinations, which were accompanied by the rise of PAL activity. SMF induced chlorophyll pigments with stimulation of SOD and CAT activities to scavenge the cellular H2O2, which indicated the inducive impact of SMF on defense mechanisms in the phenolic pathway of H. pluvialis cells. On the other hand, ROS accumulated by NaPP could markedly induce astaxanthin content in the MEP pathway of H. pluvialis alga. The results of this work can be properly appropriate to manipulate biosynthesis pathways of secondary metabolites in H. pluvialis and other algae. But key enzyme activities in MEP pathway and their gene expressions need to investigate in future works.