2906 J. Agric. Food Chem.

2007, 55, 2906−2910

Lipid Characterization of Mangrove Thraustochytrid −

Schizochytrium mangrovei
KING-WAI FAN,† YUE JIANG,*,‡ YUN-WING FAAN,† AND FENG CHEN*,†
Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, and Department
of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong

Lipid class composition and distribution of fatty acids within the lipid pool of microalga, Schizochytrium
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mangrovei FB3 harvested at the late exponential phase, was studied, with special emphasis on the
distribution of docosahexaenoic acid (C22:6 n-3, DHA). Neutral lipids were the major lipid constituents
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(95.90% of total lipids) in which triacylglyerol (TAG) was the predominant component and accounted
for 97.20% of the neutral lipids. Phosphatidylcholine (PC) was the major polar lipid. Phosphatic acid
and phosphatidylserine were the two classes in phospholipids reported for the first time in
thraustochytrids. Both TAG and PC were primarily saturated and consisted of C16:0 at approximately
50% of total fatty acids. DHA was found to be distributed in all lipid classes and to be the major
polyunsaturated fatty acid. TAG contained the highest amount of DHA, although the percentage of
DHA in total fatty acids in TAG (29.74%) was lower than that in PC (39.61%). The result from this
study would be useful for further optimization of DHA production by S. mangrovei.

KEYWORDS: Lipid; docosahexaenoic acid (DHA); Schizochytrium mangrovei

INTRODUCTION productivity. It has long been recognized that environmental

factors are essential for determining the quantity and quality of
Thraustochytrids are heterotrophic eukaryotic organisms and lipids produced by microalgae. The alteration of lipid composi-
more closely related to the heterokont algae based on 18S rRNA tion in microalgal species in response to environmental stimuli
analysis (1, 2). They have been isolated from a wide range of is critical for their survival and the production of lipid-based
habitats throughout the world, including hyper-saline lakes and compounds, such as fatty acids (12, 13). A full understanding
deep sea. Recently, thraustochytrids have also been found in of lipid composition and the distribution of DHA in the lipid
subtropical mangroves and suggested as one of the major pool is therefore the key point for the further study on
colonizers on mangrove detritus (3, 4). It is supposed that environmental influences on lipid metabolism and the increase
mangrove thraustchytrids are associated with decaying mangrove of DHA productivity in thraustochytrids.
leaves in carbon cycle and represent valuable sources of The production and storage of lipids by microalgae in
nutrients, such as polyunsaturated fatty acids in the microbial response to environmental factors are species-specific. Up to
loop in marine sediment (5). Our previous report indicated that now, few studies focused on the lipid composition as well as
a newly isolated mangrove thraustochytrid in Hong Kong, the distribution of fatty acids in individual lipid class in
Schizochytrium mangoVei FB3, is a potential producer of thraustochytrids. Schizochytrium mangroVei FB3 used in our
docosahexaenoic acid (DHA, C22:6), an essential ω-3 polyun- previous study contained a rich source of PUFAs with DHA
saturated fatty acid (PUFA) (6). content of 39.14% (of total fatty acids) under unoptimized
It is well known that DHA plays an important role in the condition (6). The main objective of this research was to
normal cognitive, neurological, and visual developments of elucidate the lipid class composition of S. mangroVei FB3 with
humans, especially during the infancy stage (7). The intake of special emphasis on its DHA distribution in the lipid pool. The
sufficient quantity of DHA is required for both adults and infants results from this study would be helpful for further optimizing
(8). Thraustochytrids are currently being explored for their DHA the culture conditions for lipid and DHA production using this
production potential due to their ability to produce substantial alga.
biomass with rich DHA content heterotrophically (4, 9-11).
The focuses of those studies, however, were mainly on the MATERIALS AND METHODS
immediate influences of environmental conditions on DHA
Heterotrophic Growth. Schizochytrium mangroVei FB3 was isolated
from decaying Kandelia candel leaves in the intertidal zone from local
* Authors to whom correspondence should be addressed [(Y.J.) telephone mangrove in Sai Keng, Hong Kong, according to the isolation method
852-3411-7062; fax 852-3411-5995; e-mail [email protected]; (F.C.) described by Fan et al. (14) and Raghukumar (15). Cultures were
telephone 852-2299-0309; fax 852-2299-0311; e-mail [email protected]].
† The University of Hong Kong. maintained in yeast extract-peptone agar slants with 1 mL of 15%
‡ Hong Kong Baptist University. (v/v) sterile artificial seawater (ASW) and subcultured on a monthly

10.1021/jf070058y CCC: $37.00 © 2007 American Chemical Society

Published on Web 03/24/2007
Lipid Characterization of Mangrove Thraustochytrid J. Agric. Food Chem., Vol. 55, No. 8, 2007 2907

basis. An inoculum was prepared in 250 mL Erlenmeyer flasks, each

containing 50 mL of glucose medium consisting of (per liter) 10 g of
glucose, 1 g of yeast extract, and 15 g of ASW at 25 °C for 2 days
with orbital shaking at 200 rpm in darkness (6). A glucose-yeast extract
medium consisting of (per liter) 30 g of glucose, 3 g of KH2PO4, 5 g
of yeast extract, and 15 g of ASW was used for the growth experiment.
The inoculum size was 5%, and the growth culture was incubated at
25 °C in an orbital shaker at 200 rpm in the dark.
Determination of Cell Dry Weight. The cell dry weight was
determined according to Jiang et al. (6).
Determination of Glucose Concentration. Residual glucose con-
centration in culture medium was determined by 3,5-dinitrosalicylic
acid method (16).
Fatty Acids and Lipid Analysis. The lyophylized cells of S.
mangroVei FB3 were stored at -20 °C prior to the analyses of fatty
acids and lipid classes. Total lipids were obtained from triplicate
measurements. Lyophylized samples (200 mg) were extracted with a
Figure 1. Cell growth and glucose consumption of Schizochytrium
solvent mixture of chloroform-methanol-water (1:2:0.8, v/v) accord-
ing to Lewis et al. (17). The extracted lipids were resuspended in mangrovei FB3 cultured at 25 °C. b, Biomass concentration; 1, glucose
chloroform containing 80 mg/L BHT and were stored at -20 °C under concentration. Values are expressed as mean ± standard deviation of
nitrogen to prevent oxidation. triplicates.
The lipids were separated into neutral lipid (NL), glycolipid (GL),
and phospholipid (PL) by solid-phase extraction using silica gel Table 1. Kinetic Growth Parameters of Schizochytrium mangrovei
cartridge (sep-pak) according to Christie (18). The eluted NL, GL, and FB3a
PL were concentrated under nitrogen and resuspended in 100 µL of
chloroform. The lipid fractions were subjected to one-dimensional thin- parametersb Schizochytrium mangrovei FB3
layer chromatography (TLC) for lipid class separation and identification µ (h-1) 000.069
using TLC plates (20 × 20 cm) coated with silica gel 60 (Merck). Yx/glu (g/g) 000.410
NLs were eluted with a solvent mixture of hexane-diethyl ether- Xmax (g/L) 012.200
acetic acid (70:30:1, v/v), whereas GLs and PLs were eluted with DHA (mg/g) 201.48
TFA (mg/g) 680.420
chloroform-acetone-methanol-acetic acid-water (50:20:10:10:5, v/v)
(18). Two-dimensional TLC was performed to further confirm the lipid
a Data are expressed as mean of triplicates. b µ, specific growth rate; X
classes, using chloroform-methanol-28% aqueous ammonia (65:35: max,

5, v/v) as the first solvent and chloroform-acetone-methanol-acetic maximum biomass concentration; Yx/glu, growth yield coefficient based on glucose;
acid-water (50:20:10:10:5, v/v) as the second solvent on the same type DHA, DHA content in cells; TFA, total fatty acid content in cells.
of TLC plates (18).
Bands of lipid classes were identified by co-chromatography with other well-known fast growth microbial species, for example,
authentic standards (Sigma) and staining with specific stains for Saccharomyces cereVisae, etc. (21).
confirmation. 2,7-Dichlorofluorescein (Sigma) was used to visualize Lipid Class Composition. As shown in Figure 2A and B,
different lipid classes on TLC plate under UV light. The following the lipids of S. mangroVei FB3 in late exponential phase were
reagents were used as specific stains for lipid class identification: mostly NLs (95.89% of total lipids and 650 mg/g of cell dry
Dragendorff reagent (Sigma) for lipids containing choline and betaine,
weight) of which triacylglycerol (TAG) was the major compo-
H2O-acetic acid for sterol, molybdenum blue spray reagent (Sigma)
for lipids containing phosphorus, ninhydrin spray for phospholipids nent (97.19% of neutral lipids). Monoacylglycerol (2.31 mg/g
containing a free amino group, and orcinol-sulfuric acid for glycolipids of cell dry weight), diacylglycerol (10.52 mg/g of cell dry
(18). weight), free fatty acids, and sterol were also present in NLs
After visualization and identification, lipid bands were immediately but in smaller proportions (Figure 2B). The presence of
and carefully scraped out for direct trans-methylation with methanol- monoacylglycerol and diacylglycerol in S. mangroVei FB3 is
acetyl chloride for fatty acid analysis using an HP 6890 capillary gas the same as the other algal species as they are the key
chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a metabolites in TAG metabolism (22, 23). In microalgae, TAG
flame-ionization detector and a J&W Scientific Innowax capillary is abundant during the resting phase associated with nitrogen
column (30 m × 0.25 mm). The nonadecanoic acid (C19:0) was used deficiency or cessation of cellular division (24, 25). The same
as internal standard, and the quantities of individual fatty acid methyl
phenomenon was also observed in other microorganisms. Under
esters were estimated according to the peak areas on the chromatogram
(6).
low nitrogen condition, the fungi showed reduced production
of nitrogenous substances (i.e., serine, ethanolamine, choline)
resulting in a reduced phospholipid synthesis and a correspond-
RESULTS AND DISCUSSION
ing increase in triacylglycerol (26). Although lipid accumulation
Heterotrophic Growth Characteristics. Thraustochytrids in heterotrophically grown thraustochytrid during growth phase
possess the ability to use organic carbon sources for their was not closely related to the exhaustion of nitrogen supply
survival and growth (19). Glucose is the most conventional (27), TAG was still the most abundant lipid fraction and
carbon source in the fermentation industry (20). In this study, accounted for 93.19% of total lipids in S. mangroVei FB3 in
S. mangroVei FB3 grew well heterotrophically in the glucose- the late exponential phase, which was in agreement with the
yeast extract medium containing 30 g/L of glucose. As shown previous analysis (28). On the other hand, it was reported that
in Figure 1, the cells reached the late exponential phase after changes in growth rate might strongly influence the distribution
3 days of growth when the glucose was completely consumed. of lipid classes in microlagae. In chemostat culture of Isochrysis
Table 1 presents the kinetic growth parameters of S. mangroVei galbana, a photosynthetic DHA-producing microalga, NLs were
FB3. From the table, it could be seen that the specific growth abundant at slow growth rate, while a progressive reduction of
rate (0.069 h-1) and the growth yield coefficient based on neutral lipids with a concomitant increase of polar lipids was
glucose (0.41 g/g) of S. mangroVei FB3 were comparable to observed with increasing growth rate (29). According to the
2908 J. Agric. Food Chem., Vol. 55, No. 8, 2007 Fan et al.

Figure 2. Fractionation of lipid extracted from Schizochytrium mangrovei FB3. (A) Distribution of NL, GL, and PL in lipids; (B) distribution of lipid classes
in NL; and (C) distribution of lipid classes in PL. NL, neutral lipids; GL, glycolipids; PL, phospholipids. Values are expressed as mean ± standard
deviation of triplicates. DAG, diacylglycerols; FFA, free fatty acids; MAG, monoacylglycerols; SE, steroid ester; TAG, triacylglyceride; DPG,
diphosphatidylacylglycerols; LPC, lysophosphatidylcholine; PA, phosphatic acid; PC, phosphatidylcholine; PE, phosphatidylalethanolamine; PG,
phosphatidylglycerols; PI, phosphatidylinositol; PS, phosphatidylserine; PX, unidentified phospholipid class.
Monod model (µ ) µmaxS/Ks + S, where Ks is the half-saturation important constituent of mitochondrial lipids, and its biosynthetic
constant, S is the concentration of substrate, and µmax is the precursor phosphatidylglycerol (PG) were also detected in S.
maximum specific growth rate), the specific growth rate, µ, mangroVei FB3, but their amounts to total lipids were relatively
decreased with the consumption of the major nutrients (i.e., low as compared to other heterotrophic DHA-producing mi-
glucose as shown in Figure 1) at the late exponential phase. croalgae (31). Unlike NLs and PLs, glycolipids were a minor
This might also result in a higher TAG content in S. mangroVei constituent, which accounted for less than 1% of total lipids in
FB3. S. mangroVei FB3 (Figure 2A), much lower than that of
PLs, the essential components of cell membranes, constituted Thraustochytrium sp. KK17-3 (9). Further characterization of
the major polar lipids in S. mangroVei FB3. They were detected glycolipids was not attempted in this study.
to be 23.40 mg/g of cell dry weight and accounted for 83.75% Fatty Acid Composition of Individual Lipid Class. The
of polar lipids and 3.45% of total lipids, respectively (Figure total fatty acids of S. mangroVei FB3 at the late exponential
2A). Phosphatidylcholine (PC) was the major PL (47.78% of phase were 680.42 mg/g cell dry weight, among which 93.20%
phospholipids, Figure 2C), but the amount was lower than that of the fatty acids were distributed in TAG (Tables 1 and 2).
of Thraustochytrium sp. ATCC 26185, in which PC accounted As shown in Table 2, the lipid of S. mangroVei FB3 was
for 76% of phospholipids (11). Phosphatidylethanolamine (PE) composed primarily of C16:0 and DHA (50.36% and 29.71%
was the second most abundant phospholipid, which made up of total lipids, respectively). By fractionating the lipid, it is
9.41% of the phospholipids. PC and PE are the common obvious that most of the fatty acids were accumulated in TAG,
phospholipids in eukaryotic microorganisms, but the proportions whereas only 6.79% of fatty acids were distributed in other lipid
vary widely among different species (30). In other DHA- classes. As for DHA, it was primarily concentrated in TAG,
producing heterotrophic microalgae, the percentage of PC and which accounted for 93.60% of total DHA. The profile of fatty
PE in PLs was also found to be high (31, 32), similar to that in acids in TAG that consists mainly of 16:0 (50.57%), 22:5
S. mangroVei FB3. Besides PC and PE, lysophosphatidylcholine (8.24%), and DHA (29.74%) was similar to that in total lipid
(LPC), phosphatidic acid (PA), and phosphatidylinositol (PI) of S. mangroVei FB3 (Table 2). This result was comparable to
were equally abundant, ranging from 8.59% to 10.60% of PL the fatty acid profiles of other Schizochytrium, such as
(Figure 2C). Although PA and phosphatidylserine (PS) have Schizochytrium sp. SR2 (32) and S. limacinum SR21 (34), in
been detected in other DHA-producing microalga (31, 33), this which these three fatty acids make up nearly 88.2-90.4% of
study was the first report of the presence of PA and PS in total fatty acids in TAG. The abundance of C16:0 in these DHA-
thraustochytrid. The presence of PA in S. mangroVei might be producing Schizochytrium might be due to the interlock of DHA
a metabolic necessity for lipid production catalyzed by acetyl- with C16:0 in their lipid bodies (28).
CoA carboxylase because PA could serve as the common In contrast to TAG, a higher percentage of PUFAs (as
precursor leading to the synthesis of both PLs and TAGs (26, indicated by high 3/mol value) was obtained in PL, especially
27). The diphosphatidylacylglycerol (DPG, or cardiolipin), an in PC and PG (Table 2), although the total amount of DHA
Table 2. Fatty Acid Composition of Individual Lipid Class of Schizochytrium mangrovei FB3 Grown at 25 °Ca
lipid classesb
total lipid DAG FFA MAG SE TAG GL DPG LPC PA PC PE PG PI PS PX
Saturated Fatty Acids
12:00 0.10 ± 0.00 0.11 ± 0.01 0.17 ± 0.02 0.19 ± 0.02 0.79 ± 0.21 0.07 ± 0.00 1.42 ± 0.37 5.89 ± 0.47 0.30 ± 0.21 0.75 ± 0.07 0.07 ± 0.01 0.47 ± 0.17 0.37 ± 0.004 0.18 ± 0.11 0.77 ± 0.16 1.59 ± 0.16
13:00 0.09 ± 0.01 0.07 ± 0.01 0.52 ± 0.04 1.83 ± 0.14 3.03 ± 0.57 0.02 ± 0.001 4.53 ± 0.35 2.21 ± 0.33 0.78 ± 0.04 0.81 ± 0.08 0.14 ± 0.04 0.69 ± 0.12 1.20 ± 0.17 0.40 ± 0.02 1.44 ± 0.11 3.58 ± 0.21
14:00 3.31 ± 0.09 3.30 ± 0.02 3.33 ± 0.02 3.35 ± 0.07 2.76 ± 0.25 3.38 ± 0.02 1.93 ± 0.17 4.31 ± 0.71 2.92 ± 0.68 2.56 ± 0.11 1.17 ± 0.27 1.51 ± 0.21 0.60 ± 0.03 1.64 ± 0.10 2.03 ± 0.17 3.20 ± 0.47
15:00 5.04 ± 0.12 3.15 ± 0.05 4.74 ± 0.26 3.58 ± 0.03 4.33 ± 0.26 5.20 ± 0.05 1.73 ± 0.11 3.21 ± 0.21 4.72 ± 0.95 3.99 ± 0.28 1.66 ± 0.21 2.47 ± 0.02 0.98 ± 0.07 2.23 ± 0.03 0.00 ± 0.00 3.97 ± 0.29
16:00 50.36 ± 2.53 52.62 ± 0.88 52.77 ± 0.96 50.47 ± 3.99 43.69 ± 3.38 50.57 ± 0.80 36.46 ± 2.76 32.95 ± 2.89 47.00 ± 2.25 45.77 ± 2.15 47.32 ± 1.82 45.81 ± 0.24 40.84 ± 0.47 48.69 ± 0.99 49.36 ± 1.99 45.20 ± 2.12
17:00 1.06 ± 0.02 0.90 ± 0.01 3.20 ± 0.25 1.65 ± 0.25 1.68 ± 0.17 1.01 ± 0.05 5.61 ± 0.35 2.06 ± 0.42 1.76 ± 0.41 2.63 ± 0.95 0.49 ± 0.08 1.16 ± 0.04 1.45 ± 0.29 1.18 ± 0.09 2.80 ± 0.09 3.41 ± 0.15
18:00 1.05 ± 0.02 3.25 ± 0.50 5.99 ± 0.56 10.80 ± 0.68 2.14 ± 0.66 0.82 ± 0.01 15.32 ± 1.26 11.41 ± 1.47 2.65 ± 0.19 3.44 ± 0.41 0.39 ± 0.02 2.67 ± 0.47 1.42 ± 0.19 2.30 ± 0.15 1.54 ± 0.10 5.17 ± 0.67
22:00 0.27 ± 0.01 0.35 ± 0.03 1.24 ± 0.07 0.39 ± 0.007 0.35 ± 0.005 0.26 ± 0.002 0.97 ± 0.18 0.64 ± 0.01 0.25 ± 0.07 0.20 ± 0.02 0.27 ± 0.01 0.30 ± 0.02 0.41 ± 0.004 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Monoenoic Fatty Acids
18:1 n-9 0.14 ± 0.00 0.10 ± 0.10 4.66 ± 0.86 2.78 ± 0.38 3.70 ± 0.56 0.04 ± 0.01 3.68 ± 0.69 1.89 ± 0.38 0.29 ± 0.004 0.64 ± 0.16 0.65 ± 0.01 0.14 ± 0.14 0.82 ± 0.15 0.00 ± 0.00 16.10 ± 0.24 0.62 ± 0.04
Polyunsaturated Fatty Acids
18:3 n-3 0.14 ± 0.00 0.03 ± 0.004 0.09 ± 0.002 1.16 ± 0.17 1.07 ± 0.19 0.08 ± 0.01 7.37 ± 0.73 2.19 ± 0.24 0.05 ± 0.004 0.06 ± 0.003 0.04 ± 0.003 0.40 ± 0.004 1.84 ± 0.41 0.53 ± 0.03 0.48 ± 0.02 0.00 ± 0.00
20:3 n-6 0.12 ± 0.00 0.22 ± 0.07 0.24 ± 0.02 0.65 ± 0.03 2.43 ± 0.38 0.08 ± 0.01 3.61 ± 0.24 0.73 ± 0.02 0.16 ± 0.01 0.34 ± 0.004 0.16 ± 0.01 0.13 ± 0.04 0.00 ± 0.00 0.31 ± 0.02 0.03 ± 0.002 1.49 ± 0.41
20:4 n-6 0.39 ± 0.01 0.25 ± 0.03 1.11 ± 0.25 0.38 ± 0.005 5.52 ± 0.49 0.37 ± 0.01 0.00 ± 0.00 0.19 ± 0.01 0.44 ± 0.02 0.90 ± 0.19 0.31 ± 0.03 1.07 ± 0.05 0.46 ± 0.06 0.60 ± 0.03 0.00 ± 0.00 0.00 ± 0.00
20:5 n-3 0.16 ± 0.01 0.45 ± 0.08 0.68 ± 0.30 1.38 ± 0.04 0.00 ± 0.00 0.12 ± 0.01 1.17 ± 0.11 0.88 ± 0.08 0.57 ± 0.02 2.27 ± 0.21 0.49 ± 0.01 0.72 ± 0.02 1.14 ± 0.09 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
22:5 n-6 8.06 ± 0.36 5.63 ± 0.19 3.91 ± 0.55 4.24 ± 0.28 4.91 ± 0.35 8.24 ± 0.69 1.24 ± 0.24 4.68 ± 0.61 6.00 ± 0.44 3.50 ± 0.41 7.24 ± 0.18 10.12 ± 0.34 8.23 ± 1.04 8.79 ± 0.52 2.05 ± 0.07 4.97 ± 0.32
22:6 n-3 29.71 ± 1.01 29.58 ± 0.36 17.35 ± 2.48 17.14 ± 0.92 23.59 ± 2.11 29.74 ± 1.70 14.98 ± 2.08 26.76 ± 2.11 32.12 ± 3.76 32.14 ± 0.63 39.61 ± 2.35 32.34 ± 2.65 40.25 ± 3.70 33.16 ± 1.74 23.39 ± 0.89 26.80 ± 2.01
Lipid Characterization of Mangrove Thraustochytrid

b
TFA mg/g 680.42 ± 25.32 10.52 ± 1.62 3.67 ± 0.33 2.31 ± 0.33 1.85 ± 0.11 634.14 ± 18.47 4.54 ± 0.60 0.72 ± 0.07 2.48 ± 0.27 2.01 ± 0.41 11.18 ± 0.31 2.63 ± 0.16 0.82 ± 0.06 2.42 ± 0.33 0.82 ± 0.14 0.32 ± 0.02
3/molc 2.16 ± 0.08 2.10 ± 0.03 1.37 ± 0.12 1.41 ± 0.10 2.02 ± 0.38 2.22 ± 0.40 1.39 ± 0.10 2.00 ± 0.19 2.28 ± 0.26 2.27 ± 0.02 2.80 ± 0.15 2.54 ± 0.17 2.97 ± 0.17 2.48 ± 0.11 1.68 ± 0.13 1.91 ± 0.12
DHA mg/gd 201.48 ± 10.18 3.12 ± 0.25 0.64 ± 0.15 0.40 ± 0.08 0.41 ± 0.02 188.59 ± 4.03 0.07 ± 0.03 0.19 ± 0.04 0.74 ± 0.08 0.65 ± 0.05 4.44 ± 0.10 0.83 ± 0.05 0.32 ± 0.01 0.80 ± 0.07 0.19 ± 0.04 0.09 ± 0.02

a Data are expressed as mean ± standard deviation of triplicates and statistically analyzed at a level of p < 0.05. DAG, diacylglycerols; FFA, free fatty acids; MAG, monoacylglycerols; SE, steroid ester; TAG, triacylglyceride; GL, glycolipid; DPG,
diphosphatidylacylglycerols; LPC, lysophosphatidylcholine; PA, phosphatic acid; PC, phosphatidylcholine; PE, phosphatidyalethanolamine; PG, phosphatidylglycerols; PI, phosphatidylinositol; PS, phosphatidylserine; PX, unidentified phospholipid class. b TFA
) mg of total fatty acids/g of cell dry weight. c 3/mol: the degree of fatty acid unsaturation. This value was calculated according to the formula 3/mol ) [1.0(% monoene) + 3.0(% triene) + 4.0(% tetraene) + 5.0(% pentaene) + 6.0(% hexaene)]/100.d DHA
) mg of DHA/g of cell weight.

comments.
mangroVei.

637-647.
components (9).

LITERATURE CITED
ACKNOWLEDGMENT

1989; pp 388-398.

Elsevier Science: New York, 2006; Chapter 11.

J. Agric. Food Chem., Vol. 55, No. 8, 2007

carbon into lipids instead of converting them into biomass.

Philos. Trans. R. Soc. London, Ser. B 1994, 346, 378-397.

(3) Margulis, L.; Corliss, J. O.; Melkonian, M.; Chapman, D. J.

be a structural necessity for their normal functions as membrane

A.; Schaumann, K.; Higashihara, T. Molecular phylogeny of

added products from renewable resources; Yang, S. T., Ed.;

(4) Fan, K. W.; Chen, F. Production of high-value products by the
nitrogen source, the culture at resting stage, the cell at low
cellular level through manipulation on the net carbon flux
found in TAG, although PLs contained a higher percentage of

18S ribosomal RNA gene. J. Eukaryotic Microbiol. 1999, 46,

labyrinthulids and thraustochytrids based on the sequencing of
lead to TAG accumulation as well (38). It is also expected that

We sincerely thank the reviewers for their constructive

to further increase the DHA content in S. mangroVei FB3 at
37). The limitation of other nutrients, such as phosphorus, may
induce a series of reactions leading to the continuous supply of
fore, TAG accumulation should be enhanced by the manipula-
that DHA was present in both neutral and polar lipids in
specialized membrane components that regulate membrane

are chromists not fungi: 18s rRNA signatures of Heterokonta.

growth rate, etc., on the accumulation of lipid and DHA in S.
environmental factors, for example, the medium with limiting
DHA production process on an industrial scale might be
with TAG comprised of 97.20% in this lipid fraction. In all
Neutral lipid was found to be the predominant lipid component
using thin-layer chromatography and gas chromatography.
the fast assimilation of carbon and might channel the surplus

established through systematic investigation on the effects of

leading to the accumulation of TAG. As DHA and TAG
DHA in total fatty acids. The results suggested the possibility
acetyl-CoA, an essential building block of the polyketide
because nitrogen limitation in the oleaginous cell culture can
tion of culture age and growth conditions to further increase
major lipid component for DHA production (Table 2). There-
organisms, did produce large amounts of TAG, and TAG is a
fluidity in response to salinity and temperature variations. The

with an excess of carbon and limiting nitrogen may be required

Thraustochytrium aureum ATCC 34304 (9, 35). Moreover, the
2909

accumulated in PL was extremely low. In fact, PLs are highly

(as % of total fatty acids) in the lipid fractions except

syntheses are highly environmentally dependent, a cost-effective

Conclusion. In this study, the lipid of S. mangroVei FB3 and
synthase system for DHA formation in thraustochytrids (27, 36,
results proved that S. mangroVei FB3, like the other oleaginous
By lipid fractionation of S. mangroVei FB3, it was confirmed

(1) Cavalier-Smith, T.; Allsopp, M. T.; Chao, E. E. Thraustochytrids

marine microalgae thraustochytrids. In Bioprocessing for Value-

Handbook of Protoctista; Jones and Bartlett Publishers: Boston,
lipid classes, DHA was the major PUFA with the highest amount
the cells of S. mangroVei FB3 with slow growth rate might allow

the distribution of DHA within the lipid pool were characterized

DHA content in S. mangroVei FB3. To meet this end, a medium

(2) Honda, D.; Yokochi, T.; Nakahara, T.; Raghukumar, S.; Nakagiri,
high degree of unsaturation of the fatty acyl groups in PLs may

thraustochytrids and PLs contained a higher proportion of DHA

2910 J. Agric. Food Chem., Vol. 55, No. 8, 2007 Fan et al.

(5) Bongiorni, L.; Pignataro, L.; Santangelo, G. Thraustochytrids green algae during one growth phase. Phytochemistry 1984, 23,
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