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Ultrastructural Study on Lipid Formation and DHA Production in Isochrysis sp. Algae - Prof, Papers of Cell Biology

A research study on the ultrastructural investigation of lipid formation and docosahexaenoic acid (dha) production in isochrysis sp. Marine microalgae. The study explores the effects of culture media and conditions on pufa yields and total fatty acid contents. The presence of lipid bodies in isochrysis sp. Is confirmed through staining techniques and microscopic observations.

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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-
pf3
pf4
pf5
pf8

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Liu and Lin — Ultrastructural study and lipid formation ofBot. Bull. Acad. Sin. (2001) 42: 207-214 Isochrysis sp. 207

*Corresponding author. Fax: 886-2-23626455; E-mail: m046@ccms.ntu.edu.tw

Ultrastructural study and lipid formation of Isochrysis sp.

CCMP

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% CO 2 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 C 22 -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-

PUFAs due to their scarcity and odor, as well as geo-

graphical and seasonal variations in quality (Varela et al.,

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-

208 Botanical Bulletin of Academia Sinica, Vol. 42, 2001

rella minutussima UTEX 2341 were obtained from our

laboratory’s previously collected strains. The cultures

were grown under constant illumination in f/2 medium

(Guillard and Ryther, 1962) at 25°C, pH 8, and an air-spe-

cific supply rate of 250 mL min -1^. Artificial seawater was

sterilized in an autoclave at 120°C for twenty minutes.

Microalgae were cultured in 1-2 L Roux’s flat-flasks and 5

L jar fermentors. The different salinities (NaCl

concentrations) examined were 0.8, 1.6, 2.4 and 3.2%. So-

dium acetate 10~50 mM was applied to the mixotrophic cul-

ture of microalgae. All cultures were harvested by

centrifugation during the stationary growth phase for sub-

sequent analysis. The algal biomass was lyophilized and

stored at -30°C, and the lyophilized cells were utilized for

analysis within two weeks. A lipid analysis was performed

after the saponification and methylation. The extracted

pigment concentrations of chl a and chl c were estimated

by spectrophotometry (Jeffrey and Humphrey, 1975).

Light and Electron Microscopies

The lipid bodies in cells from the stationary cultures

were stained with Sudan Black B (Weete et al., 1997) and

observed under a Normarski differential interference con-

trast light microscope (LM). The algal cells were collected

by centrifugation at a higher concentration for viewing by

a Normarski DIC on a Nikon E-600 microscope.

The algal cells for electron microscopy (EM) were col-

lected by centrifugation at 3,000 g , and fixed with 2.5% (v/

v) glutaraldehyde in 0.2 M sodium cacodylate buffer at pH

7.2. The cells were postfixed for two hours with 1% os-

mium tetroxide in a cacodylate buffer. The fixed material

was washed once in a cacodylate buffer prior to

dehydration. The samples were dehydrated in a series of

30, 50, 70, 85, 90, 95 and 100% (v/v) acetone solutions for

ten minutes each. The dehydrated cells were suspended

in a 50:50 mixture of Spurr’s resin and acetone for one hour

and then embedded in 100% Spurr’s resin. The embed-

ded samples were polymerized at 65°C for twenty-four

hours and sectioned using an LKB ultramicrotome. Thin

sections were picked up on 300-mesh copper grids and

post-stained with uranyl acetate for thirty minutes. After

rinsing with distilled water, the ultra-sections were post-

stained with lead citrate for four minutes and finally rinsed

with distilled water. The sections were examined under a

transmission electron microscope (JEOL JEM 1200 EXII)

at an accelerating voltage of 80 kV.

Total Lipid and Fatty Acid Analysis

The total lipid was extracted from dry cells (ca. 20 mg)

using the Bligh and Dyer (1959) procedure. The dry cells

were re-extracted two or three times with small portions of

CHCl 3 -MeOH (2:1, v/v) and purified by removing nonlipid

contaminants (Folch et al., 1957). The total amount of lipid

was calculated from the gas chromatographic data. The

methyl ester derivatives of the fatty acid methyl esters

were directly prepared from the cells, after adding

pentadecanoic acid FAME (fatty acid methyl ester) as an

internal standard. All FAME were analyzed by FID-GC,

using a capillary column (RTX 225, 30 m length, 0.32 mm

in diameter) in a Hewlett Packard 5890 gas chromatograph.

The initial oven temperature was set at 150°C, followed

by a temperature program of 4°C min -1^ to a final oven tem-

perature of 210°C. The injector and detector temperature

were set at 230°C, and the flow rates for hydrogen and air

were 30 and 400 mL min -1, respectively. Fatty acid con-

tents were determined by comparing their peak areas with

that of the internal standard.

Results

The growth and biomass production of Isochrysis spp.

(in Roux’s flat flask cultures) were examined after eight

days of growth, the cells were harvested and the solvent

extractable lipid content of cells was determined. The DHA

Table 1. The fatty acid composition of several marine microalgae (% total fatty acids).

Fatty Pavlova Isochrysis sp. Isochrysis sp. Isochrysis Isochrysis Pavlova lutheri Nannochloropsis Chlorella

acid salina CCMP 463 CCMP 1324 galbana

galbana CCMP 1325 oculata

minutussima TK1 TK2 UTEX

14:0 10.1±0.2 10.4±0.9 10.2±0.5 17.5±1.0 16.3±0.9 10.3±0.5 5.1±0.4 4.5±0. 16:0 23.4±0.4 14.9±0.7 17.6±0.6 14.3±0.4 12.9±0.7 20.8±1.2 32.1±1.4 33.9±1. 16:1n-7 6.2±0.1 4.5±0.2 3.9±0.1 6.3±0.5 4.0±0.1 18.4±0.4 24.9±1.7 23.2±1. 18:0 0.9±0.1 N.D. N.D. N.D. N.D. 0.4±0.1 2.7±0.2 2.9±0. 18:1n-9 16.9±0.3 29.8±1.2 32.0±1.4 15.1±0.7 28.1±1.0 3.3±0.5 16.5±0.9 20.4±1. 18:2n-6 7.8±0.4 5.7±0.4 4.1±0.3 8.8±0.4 3.0±0.1 1.9±0.1 1.9±0.3 3.4±0. 18:3n-3 3.1±0.1 6.4±0.5 6.4±0.3 8.2±0.3 5.5±0.2 1.5±0.2 N.D. N.D. 18:4n-3 5.9±0.1 17.5±0.9 15.1±0.6 24.9±1.4 18.9±0.3 6.8±0.5 N.D. N.D. 20:4n-6 1.6±0.1 N.D. N.D. N.D. N.D. N.D. 2.8±0.2 1.9±0. 20:5n-3 11.8±0.3 N.D. N.D. N.D. N.D. 21.0±0.5 9.4±0.7 8.7±0. 22:6n-3 4.4±0.4 10.7±0.5 10.9±0.3 8.2±0.6 11.1±0.4 6.2±0.3 N.D. N.D. ΣUn-3 25.2 34.6 32.3 41.4 31.8 35.5 12.2 8. ΣUn-6 9.4 5.7 4.1 8.8 3.0 1.9 4.7 5. n-3/n-6 2.7 6.1 7.9 4.7 10.6 18.7 2.6 1.

N.D. = None detected.

210 Botanical Bulletin of Academia Sinica, Vol. 42, 2001

Table 2. Influence of different salinities on the fatty acid composition of Isochrysis sp. CCMP 1324 (% total fatty acids).

Fatty acids

NaCl conc

0.8% 1.6% 2.4% 3.2%

14:0 18.4±0.9 18.6±0.9 16.9±1.2 16.3±0. 16:0 15.5±0.7 13.4±0.7 13.4±0.7 12.9±0. 16:1n-7 5.6±0.3 5.5±0.4 4.3±0.2 4.0±0. 18:0 N.D. N.D. N.D. N.D. 18:1n-9 27.7±1.1 27.9±1.7 28.0±1.4 28.1±1. 18:2n-6 4.3±0.2 6.2±0.7 5.0±0.4 3.0±0. 18:3n-3 5.1±0.2 4.8±0.2 4.9±0.3 5.5±0. 18:4n-3 14.9±0.9 14.6±0.7 17.1±0.8 18.9±1. 20:5n-3 N.D. N.D. N.D. N.D. 22:6n-3 9.4±0.9 8.9±0.7 10.5±0.9 11.2±0. Total n-3 29.4 28.4 32.4 35. PUFA 33.6 34.9 37.4 38.

N.D. = None detected.

Figure 2. Transmission electron micrographs of Isochrysis sp. CCMP 1324, showing the lipid body (arrow) formation in chloro- plast (A~C), their size from small to large and finally rounded in spherical form (D) at early log phase. Scale bar = 200 nm.

Liu and Lin — Ultrastructural study and lipid formation of Isochrysis sp. 211

Figure 3. Transmission electron micrographs of Isochrysis sp. CCMP 1324 vegetative cell. After the fourth day of growth, no oil drop was observed (A), but oil droplets (arrows) could be observed in the stationary phase (6~11th day) (B~D). Scale bar = 500 nm.

Table 3. Variation in fatty acid composition of Isochrysis sp. CCMP 1324 in different sodium acetate concentration (% total fatty acids).

Fatty acids

CH 3 COONa

10 mM 20 mM 30 mM 40 mM 50 mM 14:0 17.8±0.9 16.5±0. 8 16.4±0.4 15.9±0.7 16.9±0. 16:0 12.8±0.6 11.9±0.5 12.0±0.4 11.7±0.4 12.7±0. 16:1n-7 4.7±0.2 4.5±0.2 3.7±0.1 4.6±0.1 4.8±0. 18:0 N.D. N.D. N.D. N.D. N.D. 18:1n-9 30.7±1.2 31.8±1.4 32.9±1.2 32.4±1.5 33.5±1. 18:2n-6 5.7±0.2 4.8±0.1 4.7±0.2 4.9±0.2 3.8±0. 18:3n-3 5.1±0.3 4.4±0.2 4.7±0.1 4.7±0.4 3.9±0. 18:4n-3 12.8±0.5 13.1±0.4 13.6±0.7 13.0±0.5 12.7±0. 20:5n-3 N.D. N.D. N.D. N.D. N.D. 22:6n-3 10.3±0.6 13.1±0.5 11.9±0.5 12.7±0.8 12.1±0. Total n-3 28.3 30.8 30.2 30.4 28. PUFA 34.0 35.4 34.9 35.3 32. N.D. = None detected.

Liu and Lin — Ultrastructural study and lipid formation of Isochrysis sp. 213

culatory diseases, and also has general health benefits.

However, Isochrysis sp. (clone T-ISO) contained the un-

usual very-long-chain unsaturated methyl and ethyl

alkenones, hydrocarbons, and methyl and ethyl esters of

a 36:2 fatty acid (Volkman et al., 1980; Marlowe et al., 1984).

The role of these compounds in Isochrysis sp. is not

known, but it seems likely that they are associated with

membrane structure (Dunstan et al., 1993). These

alkenones are significant components in stationary phase

cultures of Isochrysis (25.6% of total lipid), a fact which

must be taken into account when calculating fatty acid

content from total lipid (Lopez Alonso et al., 1992). It may

also affect the lipid formation in this microalga, especially

for the distribution of lipid body’s density.

The EM photographs illustrate that several oil drops

accumulated during the late phase of growth. The lipid

body formed in the inner thylakoid spaces of the chloro-

plast structure. Their sizes ranged from small to large in

different growth phase stages, and they finally formed a

rounded shape. The accumulated dense lipid granules

were partially dissolved and diffused into cytosol and

formed less dense large lipid globules. The oil droplet ac-

cumulation was surveyed at each growth stage and is il-

lustrated in the EM photographs (Figure 3). These show

prominent lipid bodies similar to those in a previous re-

port (Weete et al., 1997). Lipid bodies of 0.5 to 3.0 μm

existed in cells containing 3~7 granules. These structures

seem to be closely associated with lipid synthesis. The

concentrations of polyunsaturated fatty acids increased

significantly as the culture reached stationary stage, which

correlates with data in a report by Moreton (1987). LM

and EM technologies were employed to survey this alga

since no investigations of the marine Isochrysis sp. CCMP

1324 lipid formation have previously been done. The DHA

in algal oil exhibited a greater degree of oxidative stability

than that in fish oil. It also lacks the fishy odor or taste

present in fish oils (Varela et al., 1990). The oil from

Isochrysis sp. CCMP 1324, which contains various poly-

unsaturated fatty acids, has an advantage in the DHA pu-

rification process. The haptophyte Isochrysis sp. is a

common marine unicellular algae for aquaculture (Sukenik

and Wahnon, 1991). The developed strain can be culti-

vated in the tropics as a planktonic feed for mariculture

since it can grow at relatively high temperatures and con-

tains a high quantity of n-3 PUFAs (Zhu and Lee, 1997).

Haptophytes are also rich in B, C, D, and K vitamins. The

cells are easily assimilated by larval animals because of

their small size and absence of a tough cell wall. Other at-

tributes include fast growth rates, easy mass-culture, wide

temperature and salinity tolerance, and absence of toxins

(Jeffrey et al., 1994). Therefore, Isochrysis spp. are a po-

tential source of DHA where marine microalgae are used.

Isochrysis sp. CCMP 1324 appears be an another promis-

ing source for microbial DHA production since it has a

simple polyunsaturated fatty acid profile and is quite

productive. We believe that DHA-containing lipid bod-

ies in cells can be produced and accumulated in sufficient

quantity for mass culturing and DHA production.

Acknowledgements. We are especially grateful to Ms. Ji, S. J. for technical assistance. The material in this paper is part of the dissertation submitted to the National Taiwan University, Taipei, Taiwan, in partial fulfillment of the requirements for the degree of Doctor in Agricultural Chemistry.

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DHA

10% CO 2 10 klux

16

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