Phycological Research 2001; 49: 73–77
Effects of sodium chloride on the fatty acids composition in
Boekelovia hooglandii (Ochromonadales, Chrysophyceae)
Shuhei Fujii,* Miwa Uenaka, Shin Nakayama, Ryoichi Yamamoto and Shiro Mantani
Laboratory of Biology and Chemistry, Tezukayama College, Nara 631-8585, Japan
SUMMARY
Composition of fatty acids in Boekelovia hooglandii
Nicolai et Baas Becking (Chrysophyceae) was investigated as a function of salinity. It was confirmed by
gas chromatography that the composition of fatty acids
in cells cultured in a 50 mmol L–1 NaCl medium consisted of C14:0, C15:0, C16:0, C16:1, C18:0, C18:1,
C18:2, C18:3, C18:4, C20:0, C20:4, C20:5, C22:5
and C22:6, in which C14:0, C16:0, C16:1, C18:4,
C20:0, C20:5, C22:5 and C22:6 were main constituents. When the cells were cultured in a medium
with different concentrations of NaCl ranging from 50
to 800 mmol L–1, the mole percentage of fatty acids
such as C14:0, C16:0 and C16:1 decreased with
increases in the salinity, while the mole percentage of
highly polyunsaturated fatty acids such as C18:4,
C20:5, C22:5 and C22:6 increased. When the cells
were transferred from a 200 mmol L–1 NaCl medium to
a 600 mmol L–1 NaCl medium, a decrease in mole percentage of C14:0, C16:0 and C16:1, and an increase
in C18:4, C20:5, C22:5 and C22:6 were observed
within 4 h. However, no change in the compositions of
fatty acids was observed within 4 h when the cells were
transferred from a 600 mmol L–1 NaCl medium to a
200 mmol L–1 NaCl one. The increase in the content of
highly polyunsaturated fatty acids was considered to
reflect the rapid response to upshock and to be the
characteristic of salt tolerance in B. hooglandii.
Key words: Boekelovia hooglandii, docosahexaenoic
acid, eicosapentaenoic acid, fatty acid, salinity
INTRODUCTION
Boekelovia hooglandii Nicolai et Baas Becking is a unicellular microalga belonging to Chromophyta (Toon
1987; Volkman et al. 1989) and occurs most abundantly in saline environments characterized by high
ratios of monovalent to divalent cations (Barklay et al.
1991).
There have been some investigations regarding the
effects of salinity on lipids and fatty acid properties in
plants and fungi (Curtain et al. 1983; Watanabe and
Takakuwa 1984, 1987; Peeler et al. 1989; Yamaoka
et al. 1992). In Dunaliella salina Teodoresco, which is
known to have an extremely halotolerant character, the
fatty acid compositions of polar lipids have been
reported to change significantly in response to changes
in NaCl concentration (Peeler et al. 1989). A halotolerant yeast strain, Zygosaccharomyces rouxiii Kikuchi
et Imai, has been reported to increase the content of
sterol ester, free fatty acids and oleic acid, and decrease
that of triacylglycerol and linoleic acid in response to
salinity (Watanabe and Takakuwa 1984, 1987).
Recently, Allakhverdiev et al. (1999) reported that the
unsaturation of fatty acids in membrane lipids of Synechocystis sp. is associated with the ability of the
photosynthetic machinery to tolerate salt stress.
According to Barclay et al. (1991), the total lipid
content in B. hooglandii increased with an increase in
the concentration of NaCl in the ambient medium. They
did not mention changes in fatty acid composition. In
addition to the increase in total lipid content, alterations in the composition of fatty acids in B. hooglandii
might occur in response to salinity.
In this study, we report effects of NaCl on the composition of fatty acids in B. hooglandii.
MATERIALS AND METHODS
Growth conditions
Boekelovia hooglandii was cultured in a glass bottle
containing 1 L of medium. The basal medium consisted
of 10 mmol L–1 Tris-HCl (pH 8.0), 13 mmol L–1 KCl,
9.9 mmol L–1 KHCO3, 7.4 mmol L–1 MgCl2, 5.0 mmol
L–1 KNO3, 0.19 mmol L–1 CaCl2, 0.1 mmol L–1 MgSO4,
0.3 mmol L–1 KH2PO4, vitamins (2 nmol L–1 biotin,
4 nmol L–1 vitamin B12 and 0.3 µmol L–1 thiamine-HCl)
and micronutrients (Fujii and Hellebust 1994). Cells
were cultured in medium with various concentrations of
NaCl in 3% CO2 enriched air at 25°C under continuous
illumination with fluorescent lamps (500 µmol photons
m–2 s–1 at the surface of the bottle).
*To whom correspondence should be addressed.
Email: [email protected]
Communicating editor: A. Murakami.
Received 17 March 2000; accepted 30 October 2000.
74
S. Fujii et al.
Extraction of lipids in Boekelovia
hooglandii
Lipids were extracted from cells using the procedure of
Bligh and Dyer (1959). Algal suspension (100 mL; ca.
4 × 106 cells mL–1) was centrifuged for 10 min at
2000 g. The collected cells were resuspended in
3.75 mL of a mixture of chloroform and methanol
(1:2 v/v). The suspension was vigorously shaken and
left at room temperature for 30 min to extract lipids.
Then, 1.25 mL of chloroform and 1.25 mL of 1% KCl
were added to the suspension. After mixing, the suspension was centrifuged at 1000 g for 10 min. After
the upper layer was discarded, 2 mL of methanol/H2O
(10:9 v/v) were added. The mixture was vigorously
shaken and centrifuged at 1000 g for 10 min. The
obtained lower layer was dried in vacuo to obtain lipids.
Analysis of fatty acids by gas
chromatography
The obtained lipids were dissolved in 1 mL of benzene.
Then 2 mL of 0.5 mol L–1 sodium metoxide methanol
solution were added and the solution was heated at
50°C for 10 min. Next, 0.1 mL of glacial acetic acid
and 5 mL of H2O were added to the solution. To extract
methyl esters of fatty acids, 5 mL of N-hexane were
added. A layer of hexane was obtained and dried in
vacuo. After the obtained methyl ester of fatty acids
was dissolved in 100 µL of hexane, 2 µL of the solution
was subjected to gas chromatograph (GC-17 A,
Shimadzu, Kyoto) with a flame ionization detector
operated with nitrogen (1 mL min–1) as carrier gas. A
capillary column (0.25 mm inner diameter × 30 m,
0.25 µm film thickness, Spelco 2–4136 OMEGAWAX,
Spelco Japan, Tokyo) was employed with an oven programmed for the temperature to increase from 150°C to
210°C at 2°C per min. Identification of fatty acids in
B. hooglandii was conducted in relation to the retention
time of authentic fatty acids. Heptadecanoic acid was
used as an internal standard for quantity. The percentages of individual fatty acids were calculated as a ratio
of area on a chromatogram.
RESULTS
Figure 1 shows a gas chromatograph profile of fatty
acids in B. hooglandii cells which were cultured in a
medium with 50 mmol L–1 NaCl at 25°C under continuous illumination. By comparing with the retention time
of authentic fatty acids, it was confirmed that B. hooglandii contained fatty acids such as C14:0, C15:0,
C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C18:4,
C20:4, C20:5, C22:5 and C22:6. Among the identified
fatty acids, C14:0, C16:0, C16:1, C18:4, C20:0,
C20:5, C22:5 and C22:6 were the main constituents.
Fig. 1. Gas chromatographic profile of fatty acids extracted from
Boekelovia hooglandii cells incubated in a 500 mmol L–1 NaCl.
Heptadecanoic acid (C17:0) was used as an internal standard.
The optimum concentrations of NaCl for growth of
B. hooglandii ranged from 50 to 400 mmol L–1 (Fujii
and Hellebust 1994). We examined effects of salinity
on the composition of fatty acids in B. hooglandii. Cells
were cultured in a medium with various NaCl concentrations ranging from 50 mmol L–1 to 800 mmol L–1 for
1 week. As seen in Fig. 2a, the mole percentage of
C14:0, C16:0 and C16:1 decreased as the concentrations of NaCl increased. However, the mole percentage
of highly unsaturated fatty acids such as C18:4, C20:5,
C22:5 and C22:6 increased with an increase in NaCl
concentrations. Figure 2b shows the effects of NaCl
concentration on the sum of mole percentage of fatty
acids, C14:0, C16:0 and C16:1 or C18:4, C20:5,
C22:5 and C22:6. Apparently, the sum of C14:0,
C16:0 and C16:1 decreased from approximately 46%
to approximately 36% with the increase in concentration of NaCl, while that of C18:4, C20:5, C22:5 and
C22:6 increased from 41% to 53%. These results
suggest that the level of highly polyunsaturated fatty
acids was enhanced in accordance with the increase in
NaCl concentration.
In Fig. 2, we analyzed the fatty acid composition
in cells, which had been cultured for 1 week. If the
enhancement of the level of highly unsaturated fatty
Fatty acids in B. hooglandii
Fig. 2. Effects of NaCl concentration on the composition of fatty
acids in Boekelovia hooglandii. Cells were incubated for about
1 week in a medium with different concentration of NaCl at 25°C
under continuous illumination. (a) Effects of NaCl concentration
on mole percentage of individual fatty acids. ( ) 50 mmol L–1;
( ) 200 mmol L–1 () 400 mmol L–1 () 800 mmol L–1.
(b) Effects of NaCl concentration on the sum of mole percentage
of C14:0, C16:0 and C16:1 (), and that of C18:4, C20:5,
C22:5 and C22:6 ().
acids is involved in the mechanism of osmotic adjustment, there is a possibility that such a change occurs
even within a short time after the application of osmotic
shock. Therefore, we examined the changes in the composition of fatty acids after B. hooglandii cells were
75
Fig. 3. Effects of upshock treatment on the composition of fatty
acids in Boekelovia hooglandii. The cell suspension that was
cultured in a 200 mmol L–1 NaCl medium was divided into three
parts. The obtained cells by centrifugation were resuspended into
a 600 mmol L–1 NaCl medium. Data represent means of results
from three experiments (± SD). (a) Changes in mole percentage
of individual fatty acids after the application of upshock. ( )
initial; ( ) 2 h incubation; () 4 h incubation. (b) Changes in
the sum of mole percentage of C14:0, C16:0 and C16:1 (), and
that of C18:4, C20:5, C22:5 and C22:6 () after the application
of upshock.
transferred from a 200 mmol L–1 NaCl medium to a
600 mmol L–1 NaCl medium (Fig. 3a). By the application of upshock, the percentage of C14:0, C16:0 and
76
S. Fujii et al.
Changes in the level of highly polyunsaturated fatty
acids in response to upshock seemed to occur within
2 h as seen in Fig. 3b.
It is also expected that the downshock decreases the
level of highly polyunsaturated fatty acids. Thus, we
examined the effect of downshock on the composition
of fatty acids by transferring cells from a 600 mmol L–1
NaCl medium to a 200 mmol L–1 NaCl one. As seen in
Fig. 4a, the percentage of individual fatty acids did not
change after the application of downshock. The sum of
C14:0, C16:0 and C16:1 and that of C18:4, C20:5,
C22:5 and C22:6 also remained constant even after 4 h
of the application of downshock. The treatment of
downshock did not affect the composition of fatty acids
within 4 h, which was different to that of upshock.
DISCUSSION
Fig. 4. Effects of downshock treatment on the composition of
fatty acids in Boekelovia hooglandii. The cell suspension that was
cultured in a 600 mmol L–1 NaCl medium was divided into three
parts. The cells obtained by centrifugation were resuspended into
a 200 mmol L–1 NaCl medium. Data represent means of results
from three experiments (± SD). (a) Changes of individual fatty
acids after the application of downshock. ( ) initial; () 2 h
incubation; () 4 h incubation. (b) Changes in the sum of mole
percentage of C14:0, C16:0 and C16:1 (), and that of C18:4,
C20:5, C22:5 and C22:6 () after the application of downshock.
C16:1 decreased, while that of C18:4, C20:5, C22:5
and C22:6 increased. As shown in Fig. 3b, the sum of
C14:0, C16:0 and C16:1 decreased from 44.5% to
41.5%, while the sum of C18:4, C20:5, C22:5 and
C22:6 increased from 47% to 50% within 4 h.
It is well known that a plant cell membrane undergoes
a number of alterations in its lipid and fatty acid composition in response to changes in environmental
factors such as temperature and salinity. For example,
in Sorghum plants, exposure to low temperature induces
an increase in the level of unsaturated fatty acids such
as C18:2 and C18:3 (Pham Thi et al. 1989). In general,
it is considered that such an increase in unsaturated
fatty acids serves to maintain the fluidity of the membrane even at low temperatures. Moreover, in transgenic
tobacco plants, unsaturation of fatty acids in thylakoid
membranes has been reported to stabilize the photosynthetic machinery against low-temperature photoinhibition (Moon et al. 1995).
Although NaCl is an important environmental factor,
effect of NaCl on the composition of lipids and fatty
acid composition have not been investigated well. As
shown in Fig. 1, highly polyunsaturated fatty acids such
as C18:4, C20:5, C22:5 and C22:6 were identified in
B. hooglandii, which is in good agreement with results
reported by Barclay et al. (1991). It has been reported
that highly polyunsaturated fatty acids, such as C20:5
and C22:6, are the major constituents in many marine
microalgae (Borowitzka 1988; Volkman et al. 1989). In
Pavlova lutheri (Droop) Green, most of C20:5 and
C22:6 has been identified as acyl moieties of betaine
lipids (Kato et al. 1995). In B. hooglandii, the total
lipid extract was used for the analysis of fatty acids.
Therefore, it is unclear which lipids contain these
highly polyunsaturated fatty acids.
The mole percentage of C14:0, C16:0 or C16:1 in
B. hooglandii decreased with the increase in NaCl
concentrations in an ambient medium. Conversely, the
percentage of highly polyunsaturated fatty acids such
as C18:4, C20:5, C22:5 and C22:6 increased with
increasing in NaCl concentrations (Fig. 2). Moreover,
the application of upshock by transferring cells from a
0.2 mol L–1 NaCl medium to a 0.6 mol L–1 NaCl one
affected the composition of fatty acids in B. hooglandii,
Fatty acids in B. hooglandii
resulting in an increase in the percentage of highly
polyunsaturated fatty acids (Fig. 3). These results
indicate that fatty acid unsaturation in B. hooglandii
can be induced in response to NaCl concentration,
similar to low temperature-induced changes in fatty
acid composition in other organisms (Russell 1974;
Wada et al. 1987; Pham Thi et al. 1989). However, the
increase of polyunsaturated fatty acid in response to
salinity is puzzling. In the halotolerant yeast, Z. rouxii,
the increase in the content of C18:1 and the decrease
in that of C18:2 have been observed after the increase
in the concentration of NaCl in the ambient medium
(Watanabe and Takakuwa 1984). In the plasma membrane of D. salina, the degree of fatty acid saturation
increased in a higher concentration of NaCl (Peeler
et al. 1989). The increase in the degree of fatty acid
saturation has been hypothesized to make membranes
less permeable for NaCl (Kuiper 1984). Since we
analyzed here the composition of fatty acids using
whole cells, the obtained results can not be directly
compared with those in D. salina. However, the increase
in the level of highly polyunsaturated fatty acids is
considered to reflect the adaptation of B. hooglandii to
salinity. However, changes in the compositions of fatty
acids were not observed within 4 h after the application
of downshock (Fig. 4). The adjustment to downshock
might take much more time than that of upshock.
The activation or de novo synthesis of related
enzymes might be involved in the increase in the level
of highly polyunsaturated fatty acids in B. hooglandii
caused by upshock, although the mechanism needs to
be explored in further studies.
ACKNOWLEDGEMENTS
We thank Prof. R. Dunham at Tezukayama College for
his comments and corrections on the manuscript. This
work was partially supported by a grant from Tezukayama
Academy.
REFERENCES
Allakhverdiev, S. I., Nishiyama, Y., Suzuki, I., Tasaka, Y. and
Murata, N. 1999. Genetic engineering of the unsaturation
of fatty acids in membrane lipids alters the tolerance of
Synechocystis to salts stress. Proc. Natl. Acad. Sci. USA
96: 5862–7.
Barklay, W. R., Johansen, J. W., Terry, K. L. and Toon, S. P.
1991. Influence of ionic parameters on the growth and
distribution of Boekelovia hooglandii (Chromophyta).
Phycologia 30: 355–64.
Bligh, E. G. and Dyer, W. J. 1959. A rapid method of total
lipid extraction and purification. Can. J. Biochem. Physiol.
37: 911–7.
Borowitzka, M. A. 1988. Microalgae as sources of essential
fatty acids. Aust. J. Biotech. 1: 58–62.
77
Curtain, C. C., Looney, F. D., Regan, D. L. and Ivancic, N. M.
1983. Changes in the ordering of lipids in the membrane
of Dunaliella in response to osmotic-pressure changes.
Biochem. J. 213: 131–6.
Fujii, S. and Hellebust, J. A. 1994. Growth and osmoregulation of Boekelovia hooglandii in relation to salinity. Can. J.
Bot. 72: 823–8.
Kato, M., Hajiro-Nakanishi, K., Sano, H. and Miyachi, S.
1995. Polyunsaturated fatty acids and betaine lipids from
Pavlova lutheri. Plant Cell Physiol. 36: 1607–11.
Kuiper, P. J. C. 1984. Lipid metabolism of higher plants as a
factor in environmental adaptation. In Siegenthaler, P. A.
and Eichenberger, W. (Eds) Structure, Function and Metabolism of Plant Lipids. Elsevier, New York, pp. 525–30.
Moon, B. Y., Higashi, S., Gombos, Z. and Murata, N. 1995.
Unsaturation of the membrane lipids of chloroplasts
stabilizes the photosynthetic machinery against lowtemperature photoinhibition in transgenic tobacco plants.
Proc. Natl. Acad. Sci. USA 92: 6219–23.
Peeler, T. C., Stephenson, M. B., Einsphar, K. J. and Thompson, G. A. Jr. 1989. Lipid characterization of an enriched
plasma membrane fraction of Dunaliella salina grown in
media of varying salinity. Plant Physiol. 89: 970–6.
Pham Thi, A. T., Sidibe-Andrieu, M. D., Zully-Fodll, Y. and
Vieira da Silva, J. 1989. Effect of low temperature on lipid
and fatty acid composition of Sorghum roots and shoots. In
Biacs, P. A., Grutz, K. and Kremmer, T. (Eds) Biological
Role of Plant Lipids. Akademiai Klado, Budapest and
Plenum Publishing Corporation, New York and London,
pp. 555–8.
Russell, N. J. 1974. The lipid composition of the psychrophilic bacterium Micrococcus cryophilus. J. Gem. Microbiol. 80: 217–25.
Toon, S. 1987. The ultrastructure and taxonomic position of
Boekelovia Hooglandii Nicolai & Baas-Becking. MSc
Thesis, Department of Biology, University of Arkansas,
Fayettevile, 50 pp.
Volkman, J. K., Jeffrey, S. W., Nichols, P. D., Rogers, G. I. and
Garland, C. D. 1989. Fatty acid and lipid composition of
10 species of microalgae used in mariculture. J. Exp. Mar.
Biol. Ecol. 128: 219–40.
Wada, M., Fukunaga, N. and Sasaki, S. 1987. Effect of
growth temperature on phospholipid and fatty acid
compositions in a psychrotrophic bacterium, Pseudomonas
sp. strain E-3. Plant Cell Physiol. 28: 1209–17.
Watanabe, Y. and Takakuwa, M. 1984. Effect of sodium
chloride on lipid composition of Saccharomyces rouxii.
Agric. Biol. Chem. 48: 2415–22.
Watanabe, Y. and Takakuwa, M. 1987. Changes of lipid composition of Zygosaccharomyces rouxii after transfer to high
sodium chloride culture medium. J. Ferment. Technol. 65:
365–9.
Yamaoka, Y., Takimura, O., Fuse, H. and Kamimura, K. 1992.
Effect of culture conditions on fatty acid composition of
Heterosigma akashiwo. Nippon Nogeikagaku Kaishi 66:
1089–91 (in Japanese with English abstract).