Biotechnol Lett (2010) 32:1911–1914

DOI 10.1007/s10529-010-0371-0

ORIGINAL RESEARCH PAPER

Ultraviolet-B radiation improves astaxanthin accumulation

in green microalga Haematococcus pluvialis
Zhengyun Wu • Guanqun Chen •
Siukuen Chong • Nai-ki Mak • Feng Chen •

Yue Jiang

Received: 15 May 2010 / Accepted: 28 July 2010 / Published online: 10 August 2010
Ó Springer Science+Business Media B.V. 2010

Abstract Ultraviolet-B (UV-B) radiation has sig-
nificantly negative effect on cell survival rate
(P \ 0.01) and positive effect on astaxanthin accu-
mulation (P \ 0.01) of Haematococcus pluvialis.
H. pluvialis accumulated 3.2 mg/g of astaxanthin
when being exposed to 5 W/m2 of UV-B for 60 min
prior to 72 h of high light treatment, which was 122%
higher than that of the control. This UV-B treatment
also significantly stimulated lipid peroxidation and
the value of malondialdehyde and glutathione
Zhengyun Wu and Guanqun Chen contributed equally to this
work.
The authors would like the paper to appear in the section
‘‘Microbial and Enzyme Technology’’.
Z. Wu
G. Chen
S. Chong
N. Mak
Y. Jiang (&)
Department of Biology and Kwong Living Trust Food
Safety & Analysis Laboratory, Hong Kong Baptist
University, Kowloon Tong, Hong Kong
e-mail: yjiang@hkbu.edu.hk
F. Chen
School of Biological Sciences, The University of Hong
Kong, Pokfulam Road, Hong Kong
Present Address:
Z. Wu
Department of Food Engineering, College of Light
Industry & Food Engineering, Sichuan University,
Chengdu 610065, China
G. Chen
Department of Agricultural, Food & Nutritional Science,
University of Alberta, Edmonton, AB T6G 2P5, Canada
peroxidase activities were 156 and 166% higher than
those of control, respectively (P \ 0.01).
Keywords Antioxidant enzyme
Astaxanthin

Haematococcus pluvialis
Ultraviolet-B radiation
Introduction
Ultraviolet (UV) radiation, especially UV-A
(320–400 nm) and UV-B (280–315 nm), can induce
reactive oxygen species (ROS) and has significant
effects on the growth and productivity of microalgae
and plants (Salguero et al. 2005; Selvakumar 2008;
Xu et al. 2008; Zhang and Björn 2008). Microalgae
and plant have developed two major classes of
antioxidants to mitigate the damage from UV-induced
ROS: enzymatic antioxidants such as superoxide
dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GPx) and non-enzymatic antioxidant such
as ascorbate, flavonoids, and carotenoids (Xu et al.
2008). The exposure of some microalgae to UV-A
radiation, such as Dunaliella bardawil, could induce
the accumulation of total carotenoids and enhanced
cell growth (Salguero et al. 2005).
Green microalga Haematococcus pluvialis (Chlo-
rophyceae) is the known richest source of astaxan-
thin, a high value ketocarotenoid with powerful
antioxidant capacity. To find specific environmental
stress conditions that lead to large accumulation of

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astaxanthin in H. pluvialis is currently an attractive
task in the field of microalgal biotechnology. In
addition, H. pluvialis has been used as a model
microorganism to elucidate the biological function of
astaxanthin as a major non-enzymatic antioxidant
(Steinbrenner and Sandmann 2006). Since astaxan-
thin has a superior protective role to human cells
against ROS mediated damage in vitro in a dose-
dependent manner (Pashkow et al. 2008), astaxanthin
may function as a protective agent against oxidative
stress damage in H. pluvialis cell in vivo.
Although UV-B can induce oxidative stress dam-
age in microalgae (Lee and Shiu 2009), limited
information is available on the effect of UV-B
radiation on astaxanthin formation and the response
of antioxidant defense system of H. pluvialis. The
focus of this study, therefore, was to investigate the
impact of UV-B radiation on cell growth, astaxanthin
accumulation and enzymatic antioxidant system of
H. pluvialis. The result could help to understand the
relationship between the roles of carotenoid accumu-
lation and the enzymatic defense system under UV-B
stress in this microalga.
Materials and methods
Microorganism and culture conditions
Haematococcus pluvialis Flotow NIES 144 was
kindly provided by the National Institute for Envi-
ronmental Studies in Tsukuba, Japan. Algal cells
were grown aseptically in 250 ml Erlenmeyer flasks
containing 100 ml of basal growth medium (Hu et al.
2008). Cultures were incubated at 22°C with light
intensity of 26 lmol photons/m2s under a 12 h:12 h
light/dark cycle (low light condition) for 4 days
before being treated with UV-B.
Determination of cell survival rate, cell dry
weight and astaxanthin content
Cell numbers were determined using a hemacytom-
eter under a light microscope. The cell survival rate
was calculated with the following formula: cell
survival rate (%) = [(cell number of UV-B treated
culture)/(cell number of the cultures under low light
condition)] 9 100%. A 3 ml aliquot of the cultiva-
tion broth was sampled aseptically and filtered
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Biotechnol Lett (2010) 32:1911–1914
through a preweighed filter paper (Whatman GF/C).
The filter paper with cell residue was dried at 80°C
oven to constant weight to determine the cell dry
weight. Astaxanthin of the cells was analyzed by
HPLC (Boussiba et al. 1999).
Quantitative assay of lipid peroxidation
and antioxidant enzyme activities
The lipid peroxidation in cells was estimated by
measuring the formation of malondialdehyde (MDA)
with the MDA Detection Kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China). Activities
of SOD, CAT and GPx were measured with assay kits
purchased from Nanjing Jiancheng Bioengineering
Institute. The protein content was determined using
Bio-Rad DC protein Assay Kit (Bio-Rad Laboratories).
Experiment design and statistical analysis
A 3 9 3 9 3 factorial design was used for UV-B
exposure experiment, such as three UV-B intensities
(0, 3, 5, or 9 W/m2), three exposure time (20, 60 or
100 min) and each treatment was repeated triplicate.
Four-day culture was transferred aseptically into
sterile glass petridishes (diameter of 150 mm) and
exposed to UV-B radiation. The intensity of UV-B
radiation was measured with a LP 471 UV-B probe
equipped with SICRAM module (Delta OHM S.r.l.,
Padua, Italy). The liquid temperature in the petridish
was kept at 28°C throughout the process. After UV-B
treatment, cells were aseptically transferred to sterile
conical flasks (250 ml) and cultured under continuous
high light (170 lmol photons/m2s, HLT) for 72 h.
Student Newman–Keuls test and factorial analysis
were performed using the Statistical Analysis System
(SAS Institute, Inc., Cary, NC, USA).
Results and discussion
Effect of UV-B radiation on cell growth
and astaxanthin production
UV-B radiation had no significant influence on the
increase of cell dry weight (P [ 0.05, Table 1). How-
ever, the increase of either UV-B exposure time or
UV-B intensity prior to HLT showed significantly
negative effect on cell survival rate (P \ 0.01, Table 1),
Biotechnol Lett (2010) 32:1911–1914 1913

which might reflect the UV-B induced oxidative
damage on cell growth. It was reported that the amount
of DNA damage was related to the dose of UV-B in
perennial herb Gunnera magellanica (Rousseaux et al.
1999). In alga Gigartina skottsbergii, the negative effect
of UV-B radiation on cell growth was also observed,
which might be due to the cytological damage caused by
UV-B (Garbary et al. 2004).
High dose of UV-B radiation (3 W/m2 for
100 min, or 5 W/m2 for no less than 20 min, or
9 W/m2 for no less than 20 min) significantly
induced astaxanthin production than that of control
(P \ 0.05, Table 1). The highest astaxanthin content
of 3.2 mg/g was obtained when H. pluvialis cells
were exposed to 5 W/m2 of UV-B for 60 min prior to
72 h HLT. If there was no HLT, no significant
influence of UV-B on cellular astaxanthin content
was observed. With 72 h HLT, however, both the
intensity and the exposure time of UV-B have
significant effect on astaxanthin production (P \
0.01), whereas their interactive effect is insignificant
(P [ 0.05) based on the factorial analysis. According
to multiple comparisons, the cellular astaxanthin
contents obtained under higher dose of UV-B expo-
sure were significantly higher than that of the control
without UV-B. The exposure to UV-B could accel-
erate the production and the quantity of astaxanthin in
H. pluvialis. As UV-B radiation can be easily applied
and removed as required in contrast to other nutrient
stresses (Salguero et al. 2005), our results may
provide a valuable method to enhance astaxanthin
productivity by H. pluvialis in commercial scale,
especially when using column bioreactor.
Effect of UV-B radiation on lipid peroxidation
and antioxidant enzyme activities
The increase of astaxanthin content and susceptibility
to high light intensity of H. pluvialis might indicate an
oxidative protection role of astaxanthin in the cells
cultured under stress condition, but the oxidative stress
response of algal cells could not solely come from the
presence of astaxanthin (Hu et al. 2008). In this study,
cells exposed to 3 and 5 W/m2 of UV-B for 60 min
were selected for the analyses of lipid peroxidation and
antioxidant enzyme activities. UV-B slightly stimu-
lated lipid peroxidation in H. pluvialis (Fig. 1a),
whereas had no obvious influence on SOD activity
(Fig. 1b). UV-B showed slightly negative effect on
CAT activity (Fig. 1c), but had significantly positive
influence on GPx activity (P \ 0.01, Fig. 1d) based on
factorial analysis. No significant interactive effect
between UV-B and HLT was identified for either the
MDA content or the activities of the antioxidant
enzymes.

Table 1 Effect of ultraviolet-B radiation on cell dry weight, survival rate and cellular astaxanthin content of H. pluvialis
UV-B Exposure Cell dry weight (g/l) Survival rate (%) Astaxanthin content (mg/g)
intensity time (min)
(W/m2) UV-B UV-B ? 72 h UV-B UV-B ? 24 h UV-B ? 72 h UV-B UV-B ? 72 h
HLT HLT HLT HLT

0 0 0.42 ± 0.00e 0.84 ± 0.01d 100 ± 0a 97 ± 2a 90 ± 1ab 0.76 ± 0.04d 1.44 ±0.11cd
e cd a ab abc
3 20 0.42 ± 0.01 0.87 ± 0.01 100 ± 2 91 ± 10 70 ± 10 0.72 ± 0.04d 2.00 ± 0.17bc
e bcd a ab abc
60 0.42 ± 0.01 0.88 ± 0.01 100 ± 2 82 ± 9 67 ± 8 0.82 ± 0.09d 2.00 ± 0.25bc
e bcd a abc abcd
100 0.41 ± 0.00 0.90 ± 0.06 97 ± 7 80 ± 4 60 ± 8 0.79 ± 0.02d 3.04 ± 0.38a
5 20 0.41 ± 0.00e 0.91 ± 0.01abcd 96 ± 13a 89 ± 7ab 75 ± 17abc 0.72 ± 0.01d 2.67 ± 0.04ab
e a a ab abcd
60 0.41 ± 0.00 0.97 ± 0.01 95 ± 1 82 ± 6 63 ± 7 0.67 ± 0.02d 3.20 ± 0.29a
e abc ab abc abcd
100 0.41 ± 0.01 0.94 ± 0.01 88 ± 1 67 ± 11 60 ± 3 0.75 ± 0.06d 3.05 ± 0.31a
9 20 0.39 ± 0.01e 0.89 ± 0.01bcd 85 ± 5ab 76 ± 8abc 63 ± 5abcd 0.74 ± 0.03d 2.56 ± 0.24ab
60 0.38 ± 0.01e 0.95 ± 0.00ab 75 ± 7abc 63 ± 0abcd 53 ± 9bcd 0.71 ± 0.01d 3.17 ± 0.31a
100 0.37 ± 0.00e 0.90 ± 0.01bcd 62 ± 4abcd 44 ± 4 cd
31 ± 1d 0.74 ± 0.02d 2.63 ± 0.15ab
There are significant differences among values with different superscripts (P \ 0.05). The values with the same letter are not
significantly different. All experiments were performed in triplicate
HLT high light treatment, UV-B cells were exposed to UV-B for a certain time and then harvested for analysis, UV-B ? HLT UV-B
exposure followed by 24 or 72 h of HLT

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MDA content (nmol/mg prot.)

Fig. 1 Malondialdehyde
(MDA) content and 3.0 a b

SOD activity (U/mg prot.)

activities of antioxidant 2.5 70
defense enzymes in
2.0
H. pluvialis with varied
ultraviolet-B (UV-B) 1.5 60
intensities (triangle, 1.0
5 W/m2; circle, 3 W/m2; 0.5 50
square, 0 W/m2) prior to
0.0
high light treatment (HLT).
0 36 72 0 36 72
Prot. protein, SOD
superoxide dismutase, HLT (h) HLT (h)
CAT catalase, GPx
c d

GPx activity (U/mg prot.)

CAT activity (U/mg prot.)
glutathione peroxidase. All 16 120
experiments were 100
performed in triplicate 12
80
8 60

4 40
20
0 0
0 36 72
0 36 72
HLT (h)
HLT (h)

Lipid peroxidation has been regarded as an index of
UV-B induced membrane injury in a dose-dependent
manner in higher plants Vigna unguiculata and
Crotalaria juncea (Selvakumar 2008). The result
from this study, however, showed that lipid peroxi-
dation in H. pluvials caused by UV-B was insignif-
icant compared to HLT. The responses of antioxidant
enzymes to stress condition in different organisms
were species specific. For example, UV-B radiation
caused a significant increase of SOD activity but the
decrease of the activities of other antioxidant enzymes
in algae Ptercladiella capillacea and Gelidium aman-
sii (Lee and Shiu 2009). Our result indicated that GPx
might be an important component among the antiox-
idant enzymes in H. pluvialis against UV-B damage.
Acknowledgment The authors acknowledge the support
from Faculty Research Grant of Hong Kong Baptist University.
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