Bioprocess and Biosystems Engineering

https://doi.org/10.1007/s00449-018-02064-8

RESEARCH PAPER

Role of media composition in biomass and astaxanthin production

of Haematococcus pluvialis under two-stage cultivation
Yongteng Zhao1 · Chenchen Yue1 · Shuxiang Geng2 · Delu Ning2 · Ting Ma2 · Xuya Yu1

Received: 14 September 2018 / Accepted: 15 December 2018

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
In the present study, the effects of four different culture media on the growth, astaxanthin production and morphology of
Haematococcus pluvialis LUGU were studied under two-step cultivation. The interactions between astaxanthin synthesis
and secondary messengers, reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) were also inves-
tigated. In the first green vegetative cell stage, maximal biomass productivity (86.54 mg L−1 day−1) was obtained in BBM
medium. In the induction stage, the highest astaxanthin content (21.5 mg g−1) occurred in BG-11 medium, which was higher
than in any other media. The expressions of MAPK and astaxanthin biosynthetic genes in BG-11 were higher than in any
other media, whereas the ROS content was lower. Biochemical and physiological analyses suggested that the ROS, MAPK
and astaxanthin biosynthetic gene expression was involved in astaxanthin biosynthesis in H. pluvialis under different culture
media conditions. This study proposes a two-step cultivation strategy to efficiently produce astaxanthin using microalgae.

Keywords  Haematococcus pluvialis · Astaxanthin · Two-step cultivation · Reactive oxygen species · Mitogen-activated
protein kinases

Introduction traditional conditions, it has also been cultivated under

Astaxanthin has important applications in the food and
health supplements, and feed additives and colourants [1,
2]. Many species of microalgae have been used as sources
of natural astaxanthin [3]. According to the astaxanthin con-
tent, Haematococcus pluvialis is the richest natural asta-
xanthin source and the main organism that produces this
commercial product [4, 5].
Increasing astaxanthin production yield remains an active
research target. Although it is normally cultured through
Yongteng Zhao and Chenchen Yue have contributed equally to this
work.
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s0044​9-018-02064​-8) contains
supplementary material, which is available to authorized users.
* Xuya Yu
xuya_yu@163.com
1
Faculty of Life Science and Technology, Kunming
University of Science and Technology, Kunming 650500,
China
2 (Table 1). Different media have varying nutrient quantities
Yunnan Academy of Forestry, Kunming 650051, China
adverse environment conditions, such as nutrient starva-
tion, high light, high temperature and UV-C, to store astax-
anthin [6–8]. However, astaxanthin production can still be
enhanced by adapting the conditions favourable for astax-
anthin production.
A two-stage cultivation strategy is one of the crucial areas
relating to improving accumulation of second metabolites
from microalgae [9–12]. Especially related to H. pluvialis,
in this strategy algal cells are cultured in a complete medium
to achieve high cell density under low light at the green
growth stage. After this, seed cells are suspended in a nitro-
gen-depleted medium through a combination of high light
illumination and elicitor addition for the second astaxanthin
accumulation period [12–14]. Aflalo et al. [15] concluded
that the two-stage system yields a richer production of asta-
xanthin than the one-stage system of culturing H. pluvialis
and is more readily upscalable and increasingly amenable to
outdoors production.
Furthermore, variation in culture medium composition
has been found to modify the biochemical composition
of microalgae that can be utilized for producing valuable
products such as pigments, lipids, protein and carbohydrates

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 Bioprocess and Biosystems Engineering

Table 1  The compositions of various freshwater algal culture media

Chemical component CZ-M1 SE BG-11 BBM

NaNO3 750 mg 250 mg 1.5 g 250 mg

MgSO4·7H2O 75 mg 75 mg 75 mg 75 mg
CaCl2·H2O 25 mg 25 mg 36 mg 25 mg
NaCl 25 mg 25 mg – 25 mg
K2HPO4·3H2O 75 mg 75 mg 40 mg –
KH2PO4 175 mg 175 mg – 175 mg
Na2EDTA – 0.2 mg 1 mg 0.75 mg
FeCl3·6H2O 5 mg 0.5 mg – 9.7 µg Fig. 1  The pathway of astaxanthin biosynthetic in Haematococcus
MnSO4·H2O 0.17 mg – – – pluvialis. GA3P 3-phosphoglyceraldehyde, DXS 1-deoxy-d-xylulose
5-phosphate synthase, DXP 1-deoxy-d-xylulose 5-phosphate, IPP
FeSO4·7H2O 69.5 µg – – –
isopentenyl pyrophosphate, GGPP geranylgeranyl pyrophosphate, psy
(NH4)6Mo7O4 12.35 µg – – – phytoene synthase, bkt β-carotene ketolase, chy β-carotene hydroxy-
H3BO3 61 µg 2.86 mg 2.86 mg – lase)
CuSO4·5H2O 2.5 µg 80 µg 79 µg –
ZnSO4·7H2O 287 µg 0.22 mg 222 µg 5 µg
MnCl2·4H2O – 1.86 mg 1.81 mg 41 µg
Co ­(NO3)2·6H2O – 50 µg 49 µg 2 µg
Na2MoO4 – 0. 39 mg 0.39 mg 4 µg
Na2CO3 – – 20 mg –
Citric acid – – 6 mg –
Ferric ammonium citrate – – 6 mg –
Vitamin ­B1 – – – 1 mg
Biotin – – – 0.25 µg 18, 21]. In H. pluvialis, astaxanthin is derived from isopen-
Vitamin ­B12 – – – 0.15 µg tenyl diphosphate (IPP), which is synthesized via the non-
Total volume 1 L 1 L 1 L 1 L mevalonate methyl-d-erythritol 4-phosphate (MEP) pathway.
Phytoene synthase (psy), which catalyses the first committed
step for astaxanthin biosynthesis through condensation of two
that can significantly alter the quantity of cell growth and 20-carbon geranylgeranyl pyrophosphate (GGPP) molecules
secondary metabolites accumulated during cultivation [16, to form a 40-carbon phytoene, the precursor for all other
17]. Thus, it is essential to optimize the appropriate medium carotenoids [22]. β-Carotene ketolase (bkt) in H. pluvialis
composition to obtain a higher yield of astaxanthin from H. that catalyses the introduction of keto functions at position
pluvialis. C-4 of the β-ionone ring of β-carotene and zeaxanthin [23].
Reactive oxygen species (ROS) are generated as a meta- Astaxanthin in H. pluvialis is proposed to be synthesized from
bolic by-product of aerobic growth. In contrast, ROS also the hydroxylation of canthaxanthin catalysed by β-carotene
act as signals to activate defence responses fight multiple hydroxylase (chy) [22]. A linear relationship between the
abiotic stresses and to enhance the metabolites production increase of bkt and chy transcripts and astaxanthin concentra-
in microalgae [7, 18, 19]. In addition, a parallel signalling tion was observed in the H. pluvialis wild type [21, 24]. Hence,
cascade, the mitogen-activated protein kinases (MAPK) sig- bkt and chy genes have been often proposed as rate-limiting
nalling pathway, has also been shown to play critical roles in steps in astaxanthin biosynthesis of Haematococcus pluvialis.
various eukaryotic organisms [20]. Although several studies In the current study, the alga H. pluvialis LUGU was used
have revealed the crucial functions of the ROS and MAPK- to investigate the effects of different media compositions on
signalling pathways in mediating a broad variety of cellular its biomass and biochemical nature under two-step cultivation.
functions, there is no information regarding the functions A high astaxanthin production was obtained, and association
of these signalling cascades in microalgae under different analysis was performed to determine the relationships among
culture media conditions. ROS, MAPK, astaxanthin biosynthetic-related gene expres-
Previous studies elucidated the pathway of astaxanthin bio- sion and astaxanthin accumulation in LUGU under different
synthesis in H. pluvialis with specific inhibitors, and a number culture medium conditions. Our work provides a potential
of involved genes have been identified (Fig. 1). Particularly strategy that can be used in further manipulations of H. plu-
in response to abiotic stress conditions, the transcript expres- vialis for improved biomass and astaxanthin production and
sion of related genes has presented a parallel enhancement offers valuable insights into understanding the regulation of
with increased astaxanthin accumulation in H. pluvialis [14, astaxanthin biosynthesis.

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Bioprocess and Biosystems Engineering
Materials and methods
Microalgae strain and their culture conditions
The Haematococcus pluvialis LUGU for this paper was
obtained from Lugu Lake (27°42′N and 100°47′E) in Liji-
ang, Yunnan, China [25]. The seed cells were cultivated in
5-L flat-bottom flasks with 3 L of medium at 25 ± 1 °C under
continuous light intensity of 30 µmol m−2 s−1.
Experimental conditions
Different media have different effects on the biomass and
metabolites of microalgae, and media optimization is a vital
parameter in assessing the production of a selected strain. To
determine which growth medium is best for accumulating
biomass and astaxanthin content, four different freshwater
media, viz. CZ-M1, SE, BG-11 and BBM, with different
elemental compositions were used. For autotrophic culture,
seed cells in the logarithmic phase were collected through
centrifugation (Eppendorf 5804R, Germany) at 5000×g for
5 min and washed with deionized water to remove nutrients.
The collected cells were diluted to 0.15 g L−1 and incubated
aseptically in a 500-mL Erlenmeyer flask containing 300 mL
of four different types of fresh growth media at 25 ± 1 °C
under continuous light intensity of 30 µmol m−2 s−1.
In the photoinduction step, the seed cells were har-
vested and re-suspended in four different types of fresh
media without nitrogen under high illumination with a
100 µmol m−2 s−1 intensity, with the initial cell concentra-
tion adjusted to 0.2 g L−1. The experiments were performed
in 500-mL flasks.
Analysis of H. pluvialis biomass and astaxanthin
content
The cell dry weight was weighed after freeze-drying
the algae for 12 h. Optical observation of the morphol-
ogy, colour, and astaxanthin accumulation in H. pluvialis
was conducted using an eclipse fluorescence microscope
(DMI6000B, Leica, Germany). Total astaxanthin extraction
and measurement method was developed by Boussiba and
Vonshak [26]. 10 mL of fresh algal culture was harvested
by centrifugation during the induction phase. The cell pel-
let was washed twice with distilled water and then treated
with a solution of 5% (w/v) KOH in 30% (v/v) methanol at
65 °C for 15 min to destroy the chlorophyll. The pellet was
centrifuged for 5 min at 5000×g and washed three times with
distilled water. The treated pellet was ultrasonicated with
5 mL dimethylsulfoxide (DMSO) at 200 W for 10 min and
extracted in a 45 °C water bath for 20 min. The extraction
procedure was repeated as necessary until the pellet became
colourless. Total astaxanthin was spectrophotometrically
determined by measuring absorbance at 490 nm. All experi-
ments were conducted in triplicate. The astaxanthin content
(mg g−1) was calculated according to the following equation:
C mg L−1 = 4.5 × A490 × Va ∕Vb ,
( )
where Va (mL) and Vb (mL) are the volume of DMSO and
the algal samples, respectively, and A490 is the absorbance
of the extracts at 490 nm.
Astaxanthin content mg g−1 = Ct∕DW,
( )
where t (day) is the induction time, Ct (mg L−1) represents
the astaxanthin concentration, and DW (g ­L− 1) is the dry
cell weight.
ROS analysis and western blotting
Endogenous ROS levels were analysed by 2′,7′-dichlorodi-
hydrofluorescein diacetate (DCFH-DA; Beyotime, China)
as previously described [27].
The total protein extraction and western blotting methods
were described by Ding et al. [28].
RNA isolation and quantitative real‑time PCR
The total amount of RNA was extracted with TRIzol (Inv-
itrogen, USA) and was quantified as previously described
[25]. The total RNA quantity was determined by an Ultro-
spec 2100pro spectrophotometer (GE, USA). The resulting
cDNA sequence from mRNA transcripts was prepared using
an RT-PCR kit (TaKaRa, Japan) following the manufactur-
er’s instructions.
The above cDNA was used for quantitative real-time
PCR (qRT-PCR) using a CFX96 Real-Time PCR System
(Bio-Rad, USA). The specific primers of four astaxanthin
biosynthetic genes for qRT-PCR are listed in Table S1. The
housekeeping gene 18S rRNA was used as internal standard
for the normalization of RNA.
Results and discussion
Selection of growth medium for enhanced biomass
concentration
The H. pluvialis LUGU for this study has great potential to
adapt to the local environmental conditions [25]. The micro-
algae have a high astaxanthin yield and could be explored for
natural astaxanthin production. Cell density is also an impor-
tant parameter that regulates the efficiency of microalgae for
13

its use as astaxanthin feedstock [29]; hence, the species was
cultivated in four different culture media.
In order to optimize the alga biomass, H. pluvialis LUGU
was cultivated in CZ-M1, SE, BG-11 and BBM at its growth
stage (Fig. 2a). The biomass concentration was found to be
highest in BBM media (1.38 g L−1), whereas the biomass
production was lowest in CZ-M1 (0.94 g L−1). The biomass
production was at approximately the same level in SE and
BG-11 media. The absence of ­Na2EDTA might be a cause
for the low biomass concentration in CZ-M1. Previous stud-
ies showed that adding EDTA can improve the solubility
of metal ions and enhance their availability to microalgal,
which evidently promotes biomass and metabolite accumu-
lation [30, 31]. Additionally, the highest biomass productiv-
ity was observed at 86.54 mg L−1 day−1 in BBM medium
(Fig. 2b). This may be due to the presence of vitamins in
BBM medium. Ruangsomboon et al. [32] indicated that
the addition of vitamins, particularly vitamin B1, increased
the microalgae biomass. Therefore, BBM medium was
selected as a suitable growth medium for increased biomass
production.
Effects of different induction media on growth
and astaxanthin accumulation
The availability of nutrients is one of the main factors
mediating the cell growth and metabolites accumulation in
microalgae. The maximal astaxanthin content may not initi-
ate from the microalgae biomass with the maximal astaxan-
thin concentration. Biomass production plays a key role in
estimating microalgae astaxanthin production. Accordingly,
it is vital to optimize medium composition appropriately to
obtain the best astaxanthin content in microalgae species.
Fig. 2  Effect of different growth media on the cell growth (a), maximum biomass concentration (BC) and biomass productivity (BP) (b) of H.
pluvialis LUGU under autotrophic culture conditions. The error bars indicate the standard deviations from three independent samples. d days
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Bioprocess and Biosystems Engineering
To study the influence of different media on microalgae
during the induction phase, cultures were exposed to high
light intensities (100 µmol m−2 s−1) and incubated in four
media without nitrogen for 15 days. The growth curves of
all cultures were determined (Fig. 3a). All cultures showed
some growth under induction conditions, where the highest
biomass concentration (0.84 g L−1) was found in CZ-M1
medium on day 13, and it was opposite to the first green
stage. Lowest biomass production (0.47 g L−1) was observed
in cells induced using SE. Under CZ-M1 medium, ­Fe3+ may
play a vital role in the cell growth. The growth rate of micro-
algae was stimulated by an increased bio-available iron con-
centration [33]. Wan et al. [34] also recognized iron as a key
factor in regulating microalgae biomass. Additionally, there
are few nitrogens in CZ-M1 relating to ammonia, and few
carbon sources and vitamins in BG-11 and BBM, respec-
tively. However, SE medium has fewer nutrients than do the
other three media. Therefore, a greater decrease in cell dry
weight was observed after induction day 11. Similar results
were obtained in a previous study, which demonstrated that
nitrogen deficiency inhibits chlorophyll biosynthesis and
favours chlorophyll b degradation, chlororespiration and
cyclic electron transport, while cells cultured in high light
are characterized by a higher destabilization of PSII [6].
Moreover, the astaxanthin accumulation is often accompa-
nied by a reduction in cell density due to the influence of
photooxidative stress [14, 35].
Astaxanthin accumulation was associated with algal cell
morphology, and the colour in all cultures gradually changed
(Fig. 4). Morphological observation showed that the green
cell colour in all cultures began to diminish after inocu-
lation. After 3 days of culturing, most cells became non-
motile. On day 7, the cellular colour in the BG-11 medium
became more visible and redder than in the others. On day
Bioprocess and Biosystems Engineering
Fig. 3  Effect of different induction media on the cell growth (a), astaxanthin production (b) and astaxanthin content (c) of H. pluvialis LUGU
under induction period. The error bars indicate the standard deviations from three independent samples. d days
13, the percentage of red cells was approximately 70%,
100%, almost 90%, and 80% in CZ-M1, BG-11, BBM and
SE cultures, respectively. Furthermore, some cultures, par-
ticularly for BG-11, exhibited a larger cell size (10–25 µm)
and contained more large-size red aplanospores than others,
of which the cell size was ca. 10–20 µm. Meanwhile, all the
cultures began to die and disintegrate. Zhang et al. [13] also
showed such algal cell formation in Haematococcus pluvia-
lis under stress conditions, which demonstrated that the cells
size acclimated under moderate light and nitrogen repletion
conditions is larger than that under nitrogen depletion and
high light. In addition, during the induction phase, massive
astaxanthin accumulation is largely accompanied by a mor-
phological change in motile cells to a cyst cell formation in
H. pluvialis, thereby increasing the mass per unit cell [36].
In the present study, the highest astaxanthin production
and total astaxanthin content were reported in cells grown in
BG-11 (12.8 mg L−1 and 21.5 mg g−1, respectively), which was
also evident relating to morphological observations (Figs. 3b,
c, 4). The lowest content of astaxanthin was occurred in cells
grown in CZ-M1 (8.17 mg g−1), and there was no significant
difference in the content of astaxanthin in alga cells cultured
in BBM and SE (17.4 mg g−1 and 16.86 mg g−1, respectively).
Moreover, the media composition of BG-11 was practical,
simple and economic. Thus, the BG-11 medium was chosen
as an appropriate induction medium for promoting astaxanthin
accumulation. In particular, multiple light-mediated stresses
can trigger excess ROS accumulation, such as the heat stress-
driven Haber–Weiss reaction that can simultaneously increase
the production of biomass and astaxanthin in H. pluvialis [7].
ROS association with the astaxanthin synthesis
process
The endogenous ROS (­ O2− and H
­ 2O2) overgenerated during
photoinduction were measured under growth with four ND
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 Bioprocess and Biosystems Engineering

Fig. 4  Effect of different induction media on the cell morphology of H. pluvialis LUGU under induction period. a–d Correspond to the algal
cells in CZ-M1, BG-11, BBM and SE. Bars 20 µm. d days

media with the cell-permeable fluorescent dye ­H2DCFDA,
which is mainly oxidized by H
­ 2O2 among other ROS to form
DCF [37]. Figure 5a depicts the ROS levels during photoin-
duction in the four culture media. The results showed that
the ROS content increased in cells exposed to high light
and nitrogen deficiency stresses. DCF content increased
rapidly in cells exposed to high light and decreased with
astaxanthin accumulation. The highest DCF content
occurred in CZ-M1 medium, and the lowest was observed
in cells grown in BG-11 medium. This phenomenon may
be relevant to higher astaxanthin production; most antioxi-
dants are the metabolites and can considerably decrease
excess ROS. Such antioxidants are existing in H. pluvialis,
which can produce triacylglycerol and astaxanthin under

Fig. 5  Effect of different induc-

tion media on the reactive oxy-
gen species (ROS) content (a)
and MAPK expression (b) of H.
pluvialis LUGU under induction
period. d days

13
Bioprocess and Biosystems Engineering
photooxidative stress conditions [38]. These results were
similar to those observed in algal cells under heat stress
[7]. Additionally, ­O2− and ­H2O2 may affect the expression
of carotenogenic genes via negative association with oxi-
dative stress protection and carotenoids accumulation [21,
35]. ­H2O2 may directly inactivate sensitive enzymes at a low
concentration, thereby depressing the expression of astaxan-
thin biosynthesis-related genes [39]. However, the specific
molecular mechanism of ROS and astaxanthin biosynthesis
in H. pluvialis LUGU requires further investigate.
MAPK levels in different induction media
The MAPK cascade is a key conserved signalling pathway
used to transduce extracellular induces into intracellular
responses in eukaryotes [40] and plays crucial roles in plant
developmental and physiological control under biotic and
abiotic stresses [20]. The MAPK content under the four
media during the induction period is shown in Fig. 5b. In
contrast to ROS, the highest level of MAPK was obtained
in BG-11 medium, followed by BBM and SE. The CZ-M1
medium showed the lowest MAPK level. This result was
associated with astaxanthin accumulation and ROS levels
(Figs. 3b, 5a). Asai et al. [40] indicated that MAPK can
regulate the radical burst in Nicotiana benthamiana. MAPK
can promote glycerol production in Dunaliella tertiolecta
under osmotic stress [41]. Previous study revealed that
enhancement of the MAPK level increased the production
of astaxanthin in H. pluvialis, and vice versa [28]. On the
other hand, MAPK play an important role in abiotic stress
responses in microalgae [40, 41]. Signal transmission com-
plexity via MAP kinases is further layered by the fact that
MAP kinases are multitiered gene families recruited for mul-
tiple functions such as development, immune defense sys-
tem, hormones signalling and responses to biotic and abiotic
environmental stresses [20]. Additionally, Ding et al. (2018)
indicated that MAPK is a target of NO action in physiologi-
cal processes in H. pluvialis. MAPK also can modulate ROS
and ABA signallings in plant responses to various stresses
[42]. Thus, we deduced that MAPK regulate astaxanthin
production and the possible involvement of the four media
in mediating different MAPK-signalling pathways of H. plu-
vialis in response to the high light and nitrogen starvation
stresses. However, the MAPK-signalling pathway and the
correction between MAPK and ROS should be investigated
in further studies.
The expression levels of astaxanthin biosynthetic
genes
To gain insight into the molecular mechanisms underlying
astaxanthin accumulation under four different media, the
transcript levels of four key astaxanthin biosynthetic genes
(ptox2, psy, bkt and chy) were examined by qRT-PCR using
CZ-M1 as the standard. The results showed that the tran-
script patterns and induction ratios of these four genes were
different between CZ-M1 and algae grown in the other three
media (Fig. 6). Plastid terminal oxidase (PTOX) functions
as a stress protein by inhibiting the over-reduction of the
plastoquinone (PQ) pool and provides a safety regulator for
excess electrons under abiotic stress conditions [35, 43, 44].
The expression level of ptox2 in BG-11 medium was shown
to exhibit transient upregulation, where the highest transcript
level of ptox2 occurred in BG-11 and was 3.16-fold higher
than its levels in CZ-M1 on day 13 (Fig. 6a). Furthermore,
the highest levels of ptox2 in BBM and SE media were 1.98-
and 1.39-fold higher than those in CZ-M1. Ding et al. [44]
indicated that ptox2 upregulation ensures a safe level of elec-
trons involved in the photosynthetic electron transport chain
to reduce the redox-potential-dependent generation of ROS
under stress. Li et al. [43] showed that astaxanthin overpro-
duction in H. pluvialis can occur by overexpression of ptox2.
The results agreed with the results relating to ROS levels and
astaxanthin in our study (Fig. 5a).
Compared to CZ-M1, psy and bkt gene expression showed
transient increases and then decreases (Fig. 6b, c). The maxi-
mal expression level of chy occurred on day 13 (Fig. 6d).
psy and bkt expression also increased in the first 24 h and
then decreased under high light stress [25]. Ding et al. [44]
showed that bkt has a sharp increase at induction day 1,
and the chy gene was not upregulated in the early induc-
tion phase. Those results demonstrated that psy and bkt may
increase astaxanthin accumulation at post-transcriptional
levels and that chy may upregulate it at the transcript level.
The maximal transcript levels of psy, bkt and chy under
BG-11 were 7.41-, 3.18- and 4.4-fold higher than those
under CZ-M1 on days 13, 1 and 13, respectively (Fig. 6).
The expressions levels of these genes under BG-11 were also
higher than those under BBM and SE, which were closely
linked to astaxanthin accumulation (Figs. 3b, 6). Under high
light and nitrogen deficiency conditions, different genes have
variable transcription levels under different induction media.
This result may be connected to different binding sites in the
promoters of genes related to astaxanthin biosynthesis in
response to exposure to different media [25].
Conclusion
In H. pluvialis LUGU, high and efficient astaxanthin content
was significantly achieved with the two-stage induction strat-
egy. The maximum biomass productivity and astaxanthin
content were obtained in algal cells under complete medium
(BBM) and nitrogen starvation medium (BG-11), respec-
tively. The endogenous ROS levels, MAPK and astaxanthin
biosynthetic genes expressions were related to increased
13

Fig. 6  Effect of different induction media on the expressions levels of plastid terminal oxidase (PTOX), ptox2 (a); phytoene synthase, psy (b);
β-carotene ketolase, bkt (c) and β-carotene oxygenase, chy (d) of H. pluvialis LUGU under induction period. d days
astaxanthin accumulation. The crosstalk between ROS and
MAPK-signalling transduction and astaxanthin production
is uncharacterized and requires further experimental studies.
Acknowledgements  The National Natural Science Foundation of
China (no. 21766012 to X. Yu), the Key Science and Technology
Project of Yunnan Province, China (2018ZG003 to X. Yu), and the
National Natural Science Foundation of China (no. 21666012 to P.
Zhao) supported this study.
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44. Ding W, Peng J, Zhao Y, Zhao P, Xu JW, Li T, Yu X (2018) A Publisher’s Note Springer Nature remains neutral with regard to
strategy for boosting astaxanthin accumulation in green microalga jurisdictional claims in published maps and institutional affiliations.
Haematococcus pluvialis by using combined diethyl aminoethyl
hexanoate and high light. J Appl Phycol. https​://doi.org/10.1007/
s1081​1-018-1561-8

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