Liu and Lin — Ultrastructural study and lipid formation of Isochrysis sp. 207Bot. Bull. Acad. Sin. (2001) 42: 207-214
*Corresponding author. Fax: 886-2-23626455; E-mail:
m046@ccms.ntu.edu.tw
Ultrastructural study and lipid formation of Isochrysis sp.
CCMP1324
Ching-Piao Liu and Liang-Ping Lin*
Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan, Republic of China
(Received May 22, 2000; Accepted December 12, 2000)
Abstract. This study investigates methods for extracting lipids from microalgae and analyzes the effects of culture
media as well as culture conditions on PUFA yields and total fatty acid contents. Experimental results of an optimal
culturing of Isochrysis spp. were based on a 3.2% salinity culture medium. These microalgae were cultured in a 1-
2 L Roux’s flat-flask and a 5 L jar fermentor. The optimum culture temperature and initial pH for DHA production
were 25°C and 8.0, respectively. Pigments included chlorophylls a and c. The DHA yield increased with cultivation
time until the eighth day. Optimum DHA amounts in the cells were reached under aeration with 10% CO2 and with
continuous illumination of 10 klux. The biomass dry weight reached 4 g per liter of culture, and the DHA produc-
tion reached 16 mg per liter of culture. Lipid bodies in Isochrysis spp. and related genera were observed during
culture by light and transmission electron microscopy; 0.5~3.0 µm sized lipid bodies were confirmed by staining
with Sudan Black B in cells from log stage to stationary stage cultures. These results demonstrated that DHA-
containing lipid bodies in cells can be produced and accumulated in marine Isochrysis spp.
Keywords: Docosahexaenoic acid; Isochrysis sp.; Lipid formation; Polyunsaturated fatty acids (PUFA); Ultrastructure.
Introduction
Marine microalgae such as Isochrysis have received in-
creasing interest because of their ability to produce the
polyunsaturated fatty acid docosahexaenoic acid (DHA),
one of the n-3 fatty acids believed to provide health ben-
efits associated with the consumption of certain marine
fish and their oils. DHA, a C22-polyunsaturated fatty acid,
and its derivatives help prevent and treat pathologies such
as coronary heart disease and atherosclerosis (Norday and
Hansen, 1994), inflammatory problems, and some cancers,
and are believed to play a role in infant nutrition (Conner
and Neuringer, 1987). DHA accumulates in the membranes
of nervous, visual, and reproductive tissues (Dratz and
Deese, 1986). Polyunsaturated fatty acids are especially
helpful in preventing heart and circulatory disease and fa-
cilitating brain development in infants (Yongmanitchai and
Ward, 1991). Fish oils may not be an ideal source of n-3
PUFAs due to their scarcity and odor, as well as geo-
graphical and seasonal variations in quality (Varela et al.,
1990).
Isochrysis has been widely used as a mariculture feed
due to its high content of long chain polyunsaturated fatty
acids (PUFAs) (Jeffrey et al., 1994). However, the lipid class
and fatty acid compositions of microalgal cells at differ-
ent growth phases can differ significantly (Emdadi and
Berland, 1989), and can change with variations in culture
conditions e.g. nutrient status, temperature, salinity, pH,
photoperiod, light intensity and light quality (reviewed by
Yongmanitchai and Ward, 1989; Roessler, 1990). The cell
structure of Isochrysis has attracted the attention of many
investigators. Earlier studies (Green and Pienaar, 1977;
Hori and Green, 1985; 1991) on Isochrysis galbana have
mainly focused on its flagellar root system.
In this study, EM technologies were employed to sur-
vey this alga since previous papers have lacked detailed
investigations of lipid formation in marine Isochrysis. This
work also examines marine microalgae Isochrysis spp. as
an alternative source of PUFAs and analyzes the culture
medium and culture conditions that affect yields of PUFAs
and their content in the total fatty acids. Lipid bodies in
Isochrysis spp. and related genera are observed by light
and transmission electron microscopy. Lipid granules are
confirmed by staining with Sudan Black B in cells from the
stationary cultures. The results suggest that DHA-con-
taining lipid bodies in cells can be produced by marine
Isochrysis. The possible commercial production of biom-
ass and DHA-rich oil for use as food and feed ingredi-
ents is also predicted.
Materials and Methods
Cell Growth
Microalgal strains CCMP 463, 1324, 1325 and Pavlova
salina were obtained from the Provasoli-Guillard Center for
Culture of Marine Phytoplankton (West Boothbay Harbor,
Maine USA). Isochrysis galbana TK1, TK2, were origi-
nally isolated by the Tungkang Marine Laboratory
(Pingstung, Taiwan). Nannochloropsis oculata and Chlo-
