Journal of Biotechnology 236 (2016) 7177

Contents lists available at ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Enhancement of astaxanthin production from Haematococcus pluvialis

mutants by three-stage mutagenesis breeding
Ni Wang, Bin Guan , Qing Kong, Han Sun, Zhaoyan Geng, Liangfei Duan
School of Food Science and Engineering, Ocean University of China, Qingdao, Shandong 266003, China

a r t i c l e i n f o a b s t r a c t

Article history: Haematococcus pluvialis was modied for higher astaxanthin production compatible with the superi-
Received 31 March 2016 orities of high biomass and high activity by three-stage mutagenesis breeding. UV irradiation mutants
Received in revised form 10 August 2016 named UV11-4 made an increase on cell dry weight, but showed a longer growth circle than the wild
Accepted 11 August 2016
type. On the basis of UV mutants, ethyl methane sulphonate (EMS) mutants E2-5 cut down the latent
Available online 12 August 2016
phase, brought forward and extended the logarithmic phase. The inhibitor diphenylamine (DPA) was
employed to screen high-yield astaxanthin producer by the color change of colonies from green to red on
Keywords:
solid medium. Via the contravariant cultivation, proliferation and transformation, the mutant DPA12-2
Haematococcus pluvialis
Astaxanthin
possessed an 1.7-fold astaxanthin production compared to the wild type, reaching 47.21 3.30 mg/g dry
Three-stage mutagenesis cells.
Diphenylamine 2016 Published by Elsevier B.V.

1. Introduction in liquid medium to screen mutants with high astaxanthin pro-

Astaxanthin (3,3 -dihydroxy-, -carotene-4,4 -dione) exhibits
high antioxidant activity superior to most of the hydrophobic
antioxidants (Guerin et al., 2003), showing pharmaceutical and
nutraceutical value to be developed in commercial production
(Higueraciapara et al., 2006). Additionally, astaxanthin has been
used as pigmentation source in aquaculture (Augusti et al., 2008).
Haematococcus pluvialis, the unicellular green alga, synthesizes
a larger amount of astaxanthin than some other microorganisms
such as Phaffa rhodozyma (Liu et al., 2008), Paracoccus sp. (Ide et al.,
2012) and Agrobacterium aurantiacum (Lorenz and Cyseqski, 2000).
H. pluvialis was cultivated to multiply cells and accumulate astax-
anthin in a continuous process known as one-step cultivation (Del
Rio et al., 2005). Two-stage cultivation has been involved, mark-
ing off two sequential stages separately to breed green agellate in
temperate situation and red cyst under stress condition (Suh et al.,
2006; Fabregas et al., 2001), which is compatible with the cell cycle
and growth particularity of H. pluvialis, contributing to the increase
of biomass and astaxanthin production (Hong et al., 2012).
Chen et al. (2003) isolated astaxanthin-hyper producing
mutants of H. pluvialis by UV and ethyl methane sulphonate (EMS)
compound mutagenesis and accumulated 2.5% (mg/100 mg dry
cells) astaxanthin. Tripathi et al. (2001) used multiple herbicides
Corresponding author.
E-mail address: guanbin@ouc.edu.cn (B. Guan).
duction and the highest outcome was 0.76 mg/100 mg dry cells.
Chumpolkulwong et al. (1997) selected Phaffa rhodozyma mutants
in solid medium by detecting the salmon-colored colonies with the
inhibition of diphenylamine (DPA) and accumulated 2.0-fold higher
astaxanthin than the wild type did.
The quality of H. pluvialis seed is a signicant factor in commer-
cial production. However, traditional mutagenesis of H. pluvialis
is of heavy workload and large randomness. Previous researches
focused on the astaxanthin production as the result of mutagen-
esis directly, namely one-step mutagenesis, but the biomass and
cell activity before high-yield astaxanthin screening were ignored.
The seed breeding of H. pluvialis is expected to be connected with
the two-stage cultivation. In this study, the three-stage mutagene-
sis breeding method was proposed to modify the H. pluvialis strain.
The partition of the mutagenesis stage was based on the cell cycle
of H. pluvialis. The strains were aimed at high biomass in green ag-
ellate period, while high-yield of astaxanthin in red cyst period, so
the activity of the algal cells had been ltrated before cells turned
red. Moreover, the preliminary screening of superior strains was
selected among the colonies on solid agar plates instead of liq-
uid medium, which provided more alternatives and guaranteed
the purebred strain multiplied from a single cell. Thus, the three
stages consist of high biomass breeding, high activity breeding
and high-yield astaxanthin breeding, among which the high-yield
astaxanthin strain was identied by the change in color of colonies
on agar plated containing DPA.

http://dx.doi.org/10.1016/j.jbiotec.2016.08.009
0168-1656/ 2016 Published by Elsevier B.V.
72 N. Wang et al. / Journal of Biotechnology 236 (2016) 7177
2. Materials and methods
2.1. Strains and culture conditions
H. pluvialis strain 712 was provided by Bioengineering Labora-
tory, College of Food Science and Engineering, Ocean University
of China, and cultivated in conical asks or on agar plates. The
medium consisted of 0.6 g/l KNO3 , 0.02 g/l KH2 PO4 , 0.2 g/l MgSO4
7H2 O, 0.02 g/l CaCl2 , 100 l/l micronutrients and 100 l/l Fe-
EDTA solution. Among them, the micronutrients (in g/l) included
12.37 g/l H3 PO4 , 84.51 g/l MnSO4 H2 O, 71.89 g/l ZnSO4 , 62.42 g/l
CuSO4 5H2 O, 7.26 g/l Na2 MoO4 2H2 O, 4.76 g/l CoCl2 2H2 O. The Fe-
EDTA solution consisted of 3.72 g/l EDTA2Na, 4.17 g/l FeSO4 7H2 O.
The algal strain grew at 22 C, with 12 h light intensity of
2500 lx and 12 h dark alternating, after which, they were exposed
to continuous light intensity of 7500 lx at 30 C for accumulating
astaxanthin.
2.2. Screening of UV mutants
H. pluvialis strain (1 105 cells/ml) in logarithmic phase were
illuminated for 711 min at 20 cm below a UV lamp, of which the
light wavelength was 254 nm. After irradiation, the cells were kept
in dark for 12 h, then spread on agar plates and incubated for
15 days. Large and green colonies were chosen and inoculated in
12-well cell culture clusters for 5 days, then transferred to 30 ml
medium in 100-ml conical asks for 15 days.
2.3. Screening of EMS mutants
10 ml H. pluvialis culture solution (1 105 cells/ml) was cen-
trifuged (3000 rpm) at 4 C for 6 min. The precipitate was suspended
in EMS-phosphate buffer (pH 7.2) for 1 h, in which the content of
EMS was 1.5% to 2.0% (V/V). After mutagenic treatment, the algal
cells were washed with phosphate buffer twice and kept in dark
for 12 h, then cultured as described above in 2.2.
2.4. Screening of diethyl sulphate (DES) mutants
10 ml H. pluvialis culture solution (1 105 cells/ml) was cen-
trifuged and treated by DES for 40 min in the volume range of 0.1%
to 1.5% (V/V). Then the cells were washed, kept in dark for 12 h and
incubated as described above in 2.2.
2.5. Isolation of high-yield astaxanthin strains
H. pluvialis mutants were spread on agar plates containing
12 mg/l DPA, which was an inhibitor from synthesizing astaxan-
thin. The cells were cultivated at 22 C, 2500 lx day light to form
green colonies. Then the plates were moved to a stress condition at
30 C, 7500 lx continuous light. The cells which were still compe-
tent to synthesize astaxanthin in DPA medium turned red. The red
colonies were picked out and transferred to moderate environment
for propagation.
2.6. Measurement of cell dry weight and growth rate
Cell dry weight (g/l) was measured after drying at 60 C in hot air
oven till constant weight had been attained. Taking the derivative
of cell dry weight on culture date, the growth rate of H. pluvialis
strain was estimated.
2.7. Measurement of chlorophyll content
The chlorophyll was extracted with 80% acetone at 55 C for
30 min and measured with spectrophotometer (Wellburn, 1994).
The chlorophyll concentration was calculated with the equation,
c (mg/l) = 8.02 A663 + 20.21 A645 (Chakraborty et al., 2011). To
divide the chlorophyll concentration (mg/l) by cell dry weight (g/L)
at the same growing point, the chlorophyll content (mg/g dry cells)
revealed the proportion of chlorophyll in the cells.
2.8. Measurement of astaxanthin production
The astaxanthin concentration was measured by spectropho-
tometry at 490 nm (Sun et al., 2015). The red cells were centrifuged
at 4800 rpm for 10 min, heated in water bath of 5% KOH and
30% methanol (v/v = 1:1) at 65 C to remove chlorophyll (Boussiba
et al., 1999). The culture solution was added to dimethyl sulfoxide
(DMSO) (Sedmak et al., 1990) and smashed by high speed disper-
sion homogenizer at 23000 rpm. The extraction operation had been
repeated until the cells were faded. The red supernatant was col-
lected for measuring absorbance. The astaxanthin concentration
was calculated with the equation, c(mg/L) = 4.5 A490 (Va /Vb ) f,
in which c is the astaxanthin concentration, Va (ml) is the volume
of DMSO, Vb (ml) is the volume of algal sample, f is the dilution
ratio for absorbance measuring. To divide the astaxanthin concen-
tration (mg/l) by cell dry weight (g/l) at the same growing point,
the astaxanthin content (mg/g dry cells) was calculated.
3. Results
3.1. Mutation breeding for high-biomass strains
The wild strain was treated with UV irradiation, followed by
chemical mutagens EMS or DES. After UV irradiation and incu-
bation, 30 green colonies were chosen and transferred to liquid
medium. 20 days later, the top three strains of the highest biomass
were preserved and subcultured for another 10 days in 250-ml con-
ical asks (Fig. 1a). 7-min irradiated mutants degenerated as the cell
passage increased, while 11-min irradiated mutants appeared to
be productive stably. Then, the mutant named UV11-4 was treated
with EMS (Fig. 1b) and DES (Fig. 1c) respectively, and screened by
the same method with UV mutants. Treated by 2% EMS or 0.1%
DES, the H. pluvialis strain was still high-yield after being cultured
for more than 5 generations, while the degenerated mutants by
DNA restoration for the cells themselves were weeded out. With
acclimatization to the culturing condition, the selected mutants
trended to grow better for more generations (Fig. 1d).
3.2. Screening for high-activity strains
The growth curves of wild type, UV11-4, E2-5 and D0.1-1 were
detected (Fig. 2a). During a 15-day growth stage, the cell dry weight
of wild type reached 1.29 g/l. The rst three days after inoculation,
the wild type was in the latent phase with a continuous low growth
rate. During the 4th to 8th days, it was in the logarithmic phase with
high cell activity. Since the 9th day after it was inoculated, the wild
type had been in the stagnate phase (Fig. 2b).
UV11-4 produced a cell dry weight of 1.79 g/l, making an
increase of 32.2% than the wild type, however, it showed a longer
growth circle (Fig. 2c), for it was still growing at a high rate
after 15 days. Although a misty boundary remained between latent
phase and logarithmic phase, the latent phase was shortened than
the wild type.
The production of E2-5 was 1.80 g/l, almost equivalent with that
of UV11-4. The growth rate curve of E2-5 presented to be vaulted
(Fig. 2d), which showed an inconspicuous latent phase and a longer
logarithmic phase than the wild type, and a shorter growth circle
than UV11-4 obviously.
DES-treated cells spent much time growing into colonies, indi-
cating low cell activity after mutagenesis. The cell dry weight of
 N. Wang et al. / Journal of Biotechnology 236 (2016) 7177 73

wild type
UV7-7 wild type
(a) 0.90 (b) E1.5-6
UV11-4 1.5
UV11-8 E2-5
0.85 1.4
E2-6
0.80 1.3
0.75 1.2
0.70 1.1
cell dry weight (g/L)

cell dry weight (g/L)

0.65 1.0
0.60 0.9
0.55 0.8
0.50 0.7
0.45 0.6
0.40 0.5
0.4
0.35
0.3
0.30
0.2
0.25
0.1
0.20
1 2 3 4 5 1 2 3 4 5
generation generation

UV11-4
E2-5
(d) 2.2 D0.1-1
(c)
wild type wild type
D0.1-1 2.0
1.2 D1-6
D1.5-3 1.8
1.1
cell dry weight (g/L)
1.0 1.6
cell dry weight (g/L)

0.9
1.4
0.8

0.7 1.2
0.6
1.0
0.5

0.4 0.8
0.3
0.6
1 2 3 4 5 6 7 8 9 10 11 12
generation generation

Fig. 1. The tests of genetics stability in cell dry weights of H. pluvialis mutants. (a) UV mutants; (b) Ethyl methane sulphonate mutants; (c) Diethyl sulphate mutants; (d)
Selected mutants for more generations. The values were shown in mean SD for at least three independent experiments.

D0.1-1 was 1.55 g/L, less than UV-treated strains. D0.1-1 showed
a uctuant growth rate (Fig. 2e), and a longer growth circle than
E2-5.
The chlorophyll contents (W/W) of H. pluvialis strains were mea-
sured (Fig. 3). Its indicated that the chlorophyll content was related
to the growth stage of the strain. At the rst two days, almost all
the four strains had high chlorophyll contents. During the 3rd day
to the 7th day, the strains presented lower chlorophyll levels. Since
the 8th day, the chlorophyll contents trended to be stable. In par-
ticular, the change of chlorophyll level was one day earlier than
the one of growth rate, and both of them could reveal the growth
situation and activity of the H. pluvialis strain.
The consumption of nitrate nitrogen (mg/g dry cells) was also
assessed by spectrophotometry (Fig. 4). It is observed that the
mutants utilized nitrate nitrogen more effectively. Particularly,
UV11-4 saved 34.7% of nitrogen compared to the wild type.
3.3. Screening for high-yield astaxanthin strains
colonies on agar plates containing DPA, and no colonies formed
on the plates in which the concentration of DPA exceed 20 mg/l. In
the DPA low-dose group, the colonies became red completely under
stress condition, while in the medium-dose group, a part of colonies
remained green, but appeared to be lackluster and darker (Table 1).
The red colonies in 12-mg DPA group and control group were picked
out and reversely cultivated from red cyst to green agellate in
DPA-free medium for 15 days at appropriate environment. After
being measured for the cell dry weights respectively, the strains
were moved to accumulate astaxanthin for another 10 days. The
mutant DPA12-1 selected by DPA acquired 47.21 3.30 mg/g dry
cells, about 1.7-fold astaxanthin (W/W) than the wild type (Fig. 5a).
The total production of astaxanthin was related to cell density and
producing capacity of each cell. The screening for high biomass
strains enhanced cell numbers, while DPA mutagenesis provided
strains with higher producing capacity. Thus, both the two stages
were helpful for accumulating astaxanthin. The total astaxanthin
production (mg/L) was demonstrated in Fig. 5b, showing high cell
density of green cells contributed to the total astaxanthin yield.

Considering the screening result of biomass and activity com-

prehensively, E2-5 was chosen as the parent strain to seek 4. Discussion
high-yield astaxanthin producer. DPA was employed to distin-
guished the mutants that were strongly capable of compounding H. pluvialis cells regulate the metabolism in different culture
astaxanthin. The cells spent longer time growing into green condition. The cells are green at suitable environment, red at inap-
74 N. Wang et al. / Journal of Biotechnology 236 (2016) 7177

(a) 2.0
wild type
1.8 UV11-4
1.6 E2-5
D0.1-1
1.4

cell dry weight (g/L)

1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
culture time (day)

(b) (c)
0.18 0.20

0.16 0.18

0.14 0.16

0.12 0.14
derivative

derivative

0.10 0.12

0.10
0.08

0.08
0.06
0.06
0.04
0.04
0.02
0.02
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

culture time (day) culture time (day)

(d) (e)
0.16
0.20

0.14 0.18

0.12 0.16

0.14
0.10
derivative

0.12
derivative

0.08
0.10
0.06
0.08

0.04 0.06

0.02 0.04

0.02
0.00
0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

culture time (day) culture time (day)

Fig. 2. The growth curves and growth rates of the wild type and mutants in H. pluvialis. (a) The growth curves of the wild type and mutants; (b) The growth rate of the
wild type; (c) The growth rate of UV mutant; (d) The growth rate of EMS mutant; (e) The growth rate of DES mutant. The values were shown in mean SD for at least three
independent experiments.

propriate environment. Red cells produce astaxanthin to protect tion with three specic targets, including fostering high-biomass
themselves from high temperature, strong light or nutrition de- strains, high-activity strains and high-yield astaxanthin strains.
ciency (Fan et al., 1994; De la Fuente et al., 2010). Because of the Enhancing biomass of H. pluvialis strain was the foundation of
growth particularities, the mutation breeding of H. pluvialis was improving astaxanthin production. Physical-chemical composite
innovatively divided into three stages in matched culture condi- mutation method was applied. UV irradiation modied the algal
 N. Wang et al. / Journal of Biotechnology 236 (2016) 7177 75

wild type
UV11-4
70 E2-5
D0.1-1
60

50
chlorophyll content (mg/g)

40

30

20

10

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
culture time (day)
Fig. 3. The chlorophyll contents of the wild type and mutants in H. pluvialis. The values were shown in mean SD for at least three independent experiments.

Table 1
Effect of DPA on conversion in cell cycle of H. pluvialis mutants.

DPA concentration (mg/L) Proportion of red colonies (%) Density of colonies Size of colonies

0 99.4 0.2 Dense Common

2 93.6 0.8 Dense Common
4 78.4 2.6 Medium Common
8 64.2 2.9 Medium Common
12 21.1 3.2 Medium Common
16 8.9 1.1 Sparse Small
20 Not any colony

50 industrial production from H. pluvialis. Comparing DES mutagene-

sis with EMS mutagenesis, DES-treated strains retrogressed while
EMS-treated strains remained stable in biomass, thus UV-EMS com-
40 posite mutation method was effective and practical in breeding
high-biomass and high-activity strains.
nitrogen consumption (mg/g)

The chlorophyll content was correlated with the activity of H.

30
20
10
0 gen among the nutrients was the main variation in the medium. All
wild type UV11-4
Fig. 4. The nitrogen consumption of the wild type and mutants in H. pluvialis. The
values were shown in mean SD for at least three independent experiments.
seeds and acquired considerably higher biomass strains than the
wild type, but prolonged the growth circle. On the basis of UV muta-
genesis, EMS and DES shortened the incubation time, especially
brought forward and extended the logarithmic phase, cut down
the latent phase, which was greatly meaningful to the astaxanthin
pluvialis cells (Wang et al., 2011). In this research, it is found that
the change of chlorophyll level was one day earlier than the one
of growth rate, which implied the change of chlorophyll was the
premise of cell numbers shooting up. It is also remarkable that
the chlorophyll content uctuated regularly during the logarithmic
phase. It is inferred that chlorophyll synthesis was behind the erup-
tion of new cell numbers, making the average values of chlorophyll
content waved periodically.
The utilization efciency of nutrients was also investigated to
index the quality of the strains. The concentration of nitrate nitro-
E2-5 D0.1-1 the selected mutants decreased the consumption of nitrate nitro-
gen, which was benecial of saving raw material in large scale
production. On the other hand, reducing the initial concentra-
tion of nitrate nitrogen could release the inhibiting effect at the
beginning of growth, shorten the culture time before transform-
ing into red cells, and reduce material cost in practical production.
Apart from nitrogen, phosphorus is also important for H. pluvialis in
autotrophic condition, and the consumption of nitrogen and phos-
phorus by the wild type was shown in Fig. S1. Nitrogen demand
76 N. Wang et al. / Journal of Biotechnology 236 (2016) 7177

cell dry weight

4.0 astaxanthin content
(a)
50
3.5

3.0 40

astaxanthin content (mg/g)

cell dry weight (g/L) 2.5
30
2.0

1.5 20

1.0
10
0.5

0.0 0
wild type DPA12-1 DPA12-5 DPA12-8

(b) 100
90
total astaxanthin production ( mg/L)

80

70

60

50

40

30

20

10

0
wild type DPA12-1 DPA12-5 DPA12-8

Fig. 5. Astaxanthin production and cell dry weights of the wild type and mutants for H. pluvialis. (a) Cell dry weights and astaxanthin contents; (b) Total astaxanthin
production. The values were shown in mean SD for at least three independent experiments.

of mutants was lower than the wild type, thus the N/P ratio could
be reduced. According to Tocquin et al. (2012), the low N/P condi-
tions were benecial to growth of H. pluvialis and activity for cell
dividing. Increasing N supply and P supply together also reduced
the positive effect of high P in green growth stage.
DPA was employed to screen high-yield astaxanthin strains.
DPA is considered to be a common inhibitor for some carotenoid-
producing microorganisms, such as Phaffa rhodozyma (Bon et al.,
1997), a frequently-used astaxanthin producer. However, DPA has
been hardly used to select H. pluvialis mutants, because it is far
more difcult for H. pluvialis to grow on agar plates with inhibitors
than yeast. Phaffa rhodozyma mutants grew into salmon colonies,
while wild type grew into white ones (An et al., 1989). Differ-
ently, H. pluvialis strains were cultivated into green colonies at
the rst stage, then the mutants turned red and the wild type
remained green. Hence, DPA was effective to regulate the carotene
synthesis metabolism of H. pluvialis. -carotene was utilized in
astaxanthin biosynthesis of H. pluvialis, and there are two common
pathways to form astaxanthin for the wild type (Kobayashi et al.,
1997). In one pathway, -carotene is oxidized into echinenone,
then canthaxabthin, adonirubin and astaxanthin are produced in
sequence. In another pathway, -carotene is hydroxylated into -
cryptoxanthin, then zeaxanthin, adonixanthin and astaxanthin are
formed successively. Chumpolkulwong et al. (1997), indicated that
the target enzyme of DPA was likely to be phytoene desaturase
in Phaffa rhodozyma. Kamath et al. (2008) reported that herbicide-
glufosinate promoted the lycopene cyclase activity in H. pluvialis.
The mechanism why DPA-resistant mutants produced more astax-
anthin in detail is expected to be investigated.
Particularly, the contravariant cultivation from red cyst to green
agellate for H. pluvialis was a difculty and key point in DPA
screening, only by which, could the proliferation and conserva-
tion of mutants be implemented. It is inferred that zoospores were
available in red cells, and were diffused when casting off the stress
condition, reecting the protective function of astaxanthin for H.
pluvialis cells under stress condition.
High-quality seeds are regarded as the precondition and
key point of fermentation industry. The specic and targeted
mutagenesis of H. pluvialis is signicantly practical for commer-
cial production of astaxanthin. DPA was introduced to identify
high-yield astaxanthin strains, proving to be a direct and effec-
tive screening indicator, with which the astaxanthin production
was improved observably. The three-stage mutagenesis method
has adjusted to the staged growing particularity of H. pluvialis,
 N. Wang et al. / Journal of Biotechnology 236 (2016) 7177
bred superior mutants equipped with specialties including high
biomass, high activity and high-yield astaxanthin, ameliorated
the blindness of traditional mutagenesis conspicuously. Thus, the
three-stage mutation method would be a promising practice for
astaxanthin commercial production from H. pluvialis.
Acknowledgements
The nancial supports by the 12th Five-year-plan in National Sci-
ence and Technology for the Rural Development Project of China
(2013BAD10B02-06), the Development of Science and Technol-
ogy Project of Shandong Province (2014GSF121029) and National
Natural Science Foundation of China (31471657) are gratefully
acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jbiotec.2016.08.
009.
References
An, G.H., Schuman, D.B., Johnson, E.A., 1989. Isolation of Phafa rhodozyma mutants
with increased astaxanthin content. Appl. Environ. Microbiol. 55, 116124.
Augusti, P.R., Conterato, G.M.M., Somacal, S., Sobieski, R., Spohr, P.R., Torres, J.V.,
Charao, M.F., Moro, A.M., Rocha, M.P., Garcia, S.C., Emanuelli, T., 2008. Effect of
astaxanthin on kidney function impairment and oxidative stress induced by
mercuric chloride in rats. Food Chem. Toxicol. 46, 212219.
Bon, J.A., Leathers, T.D., Jayaswal, R.K., 1997. Isolation of
astaxanthin-overproducing mutants of Phafa rhodozyma. Biotechnol. Lett. 19,
109112.
Boussiba, S., Wang, B., Yuan, J.P., Zarka, A., Feng, C., 1999. Changes in pigments
prole in the green alga Haeamtococcus pluvialis exposed to environmental
stresses. Biotechnol. Lett. 21, 601604.
Chakraborty, D., Kumar, S.A., Sen, M., Apte, S.K., Das, S., Acharya, R., Das, T., Reddy,
A.V.R., Roychaudhury, S., Rajaram, H., Seal, A., 2011. Manganese and iron both
inuence the shoot transcriptome of Typha angustifolia despite distinct
preference towards manganese accumulation. Plant Soil 342, 301317.
Chen, Y., Li, D., Lu, W., Xing, J., Hui, B., Han, Y., 2003. Screening and characterization
of astaxanthin-hyperproducing mutants of Haematococcus pluvialis.
Biotechnol. Lett. 25, 527529.
Chumpolkulwong, N., Kakizono, T., Nagai, S., Nishio, N., 1997. Increased
astaxanthin production by phafa rhodozyma mutants isolated as resistant to
diphenylamine. J. Ferment. Bioeng. 83, 429434.
De la Fuente, J.L., Rodriguez-Saiz, M., Schleissner, C., Diez, B., Peiro, E., Barredo, J.,
2010. High-titer production of astaxanthin by the semi-industrial
fermentation of Xanthophyllomyces dendrorhous. J. Biotechnol. 148, 144146.
77
Del Rio, E., Acien, G., Garcia-Malea, M.C., Rivas, J., Molina-Grima, E., Guerrero, M.G.,
2005. Efcient one-step production of astaxanthin by the microalga
Haematococcus pluvialis in continuous culture. Biotechnol. Bioeng. 91,
808815.
Fabregas, J., Otero, A., Maseda, A., Dominguez, A., 2001. Two-stage cultures for the
production of Astaxanthin from Haematococcus pluvialis. J. Biotechnol. 89,
6571.
Fan, L., Vonshak, A., Boussiba, S., 1994. Effect of temperature and irradiance on
growth of Haematococcus pluvialis. J. Phycol. 30, 829833.
Guerin, M., Huntley, M.E., Olaizola, M., 2003. Haematococcus</it> astaxanthin:
applications for human health and nutrition. Trends Biotechnol. 21, 210216.
Higueraciapara, I., Flixvalenzuela, L., Goycoolea, F.M., 2006. Astaxanthin: a review
of its chemistry and applications. Crit. Rev. Food Sci. 46, 185196.
Hong, M.E., Choi, S.P., Park, Y.I., Kim, Y.K., Chang, W.S., Kim, B.W., Sim, S.J., 2012.
Astaxanthin production by a highly photosensitive Haematococcus mutant.
Process Biochem. 47, 19721979.
Ide, T., Hoya, M., Tanaka, T., Harayama, H., 2012. Enhanced production of
astaxanthin in Paracoccus sp: strain N-81106 by using random mutagenesis
and genetic engineering. Biochem. Eng. J. 65, 3743.
Kamath, B., Vidhyavathi, R., Sarada, R., Ravishankar, G., 2008. Enhancement of
carotenoids by mutation and stress induced carotenogenic genes in
Haematococcus pluvialis mutants. Bioresour. Technol. 99, 86678673.
Kobayashi, M., Kurimura, Y., Kakizono, T., Nishio, N., Tsuji, Y., 1997. Morphological
changes in the life cycle of the green alga Haematococcus pluvialis. J. Ferment.
Bioeng. 84, 9497.
Liu, Z.Q., Zhang, J.F., Zheng, Y.G., Shen, Y.C., 2008. Improvement of astaxanthin
production by a newly isolated Phafa rhodozyma with low-energy ion beam
implantation. J. Appl. Microbiol. 104, 861872.
Lorenz, R.T., Cyseqski, G.R., 2000. Commercial potential for Haematococcus
microalgae as a natural source of astaxanthin. Trends Biotechnol. 18, 160167.
Sedmak, J.J., Weerasinghe, D.K., Jolly, S.O., 1990. Extraction and quantitation of
astaxanthin from Phafa rhodozyma. Biotechnol. Tech. 4, 107112.
Suh, I.S., Joo, H.N., Lee, C.G., 2006. A novel double-layered photobioreactor for
simultaneous Haematococcus pluvialis cell growth and astaxanthin
accumulation. J. Biotechnol. 125, 540546.
Sun, H., Kong, Q., Geng, Z.Y., Duan, L.F., Yang, M., Guan, B., 2015. Enhancement of
cell biomass and cell activity of astaxanthin-rich Haematococcus pluvialis.
Bioresour. Technol. 186, 6773.
Tocquin, P., Fratamico, A., Franck, F., 2012. Screening for a low-cost Haematococcus
pluvialis medium reveals an unexpected impact of a low N/P ratio on
vegetative growth. J. Appl. Phycol. 24, 365373.
Tripathi, U., Venkateshwaran, G., Sarada, R., Ravishankar, G.A., 2001. Studies on
Haematococcus pluvialis for improved production of astaxanthin by
mutagenesis. World J. Microbiol. Biotechnol. 17, 143148.
Wang, H.C., Cho, M.G., Riznichenko, G., Rubin, A.B., Lee, J.H., 2011. Investigation of
the maximum quantum yield of PS II in Haematococcus pluvialis cell cultures
during growth: effects of chemical or high-intensity light treatment. J.
Photochem. Photobiol. B 104, 394398.
Wellburn, A.R., 1994. The spectral determination of chlorophylls a and b, as well as
total carotenoids, using various solvents with spectrophotometers of different
resolution. J. Plant Physiol. 144, 307313.