|Electronic Journal of Biotechnology
9 No. 4, Issue of July 15, 2006
|© 2006 by Pontificia Universidad Católica
de Valparaíso -- Chile
19, 2005 / Accepted November 21, 2005
Modulation of unsaturated fatty
acids content in algae Spirulina platensis and Chlorella
minutissima in response to herbicide SAN 9785
Centre for Biochemical Technology
Mall Road, New Delhi, India
M. Singh Jolly
City University of New
Brooklyn NY 11210,
Tel: 1 718 951 5720
E mail: firstname.lastname@example.org
Coimbatore 641004 India
Tel: 91 422 4344777
Fax: 91 422 2573833
minutissima, fatty acid desaturation, polyunsaturated fatty acids,
Spirulina platensis, SAN 9785.
polyunsaturated fatty acids
The accumulation of
polyunsaturated fatty acids by algae Spirulina platensis and
Chlorella minutissima was studied. Response of these organisms
to the substituted pyridazinone, SAN 9785, an inhibitor of the long
chain fatty acid desaturase, indicated that fatty acid synthesis and
their desaturation were regulated differently in these organisms.
While the pool of palmitic acid, the precursor for the unsaturated
C18 fatty acids, was stringently maintained in the green
alga C. minutissima, in the cyanobacterium S. platensis
the level of palmitic acid was liberally maintained in spite of the
enhanced accumulation of unsaturated C18 fatty acids.
health benefits of polyunsaturated fatty acids (PUFA) has spurred
interest in their commercial production. Microalgae have been an attractive
source of PUFA (Benemann et al. 1987) due to their
inherently high PUFA content, de novo biosynthetic capabilities,
amenability to genetic manipulation, as well as to the employability
of biochemical selection strategies, and the possibility of cultivation
to high densities in limited space. Several algae, both prokaryotic
and eukaryotic, have inherently high amounts of PUFA, and further
respond to stress conditions by altering their PUFA concentration.
Cyanobacterium Spirulina is rich in γ-linolenic acid (GLA)
(and poor in the α-isomer) and thus is a good source for the
purification of this PUFA (Mahajan and Kamat, 1995).
Chlorella minutissima is an eukaryotic alga, with a fast growth
rate and high PUFA content (Seto et al. 1984) and
could be another important source of a PUFA-rich nutraceutical supplement.
Since these algae could be relatively easily cultivated at different
stress conditions, they offer the prospect of a good source of PUFA
for the nutraceutical market.
et al. had shown that the substituted pyridazinone SAN 9785 inhibits
the desaturation of long chain fatty acids (Murphy et
al.1985), and this finding has been used for obtaining strains
overproducing PUFA (Cohen et al. 1993). We have compared
the response of Spriulina platensis and C. minutissima,
to SAN 9785 to study the mechanisms behind overproduction of unsaturated
fatty acids in these organisms.
platensis was obtained from Ballarpur Industries Limited, New
Delhi, and Chlorella minutissima from
Sammlung von Algenkulturen, Göttingen.
SAN 9785, developed by BASF,
was obtained from the laboratory of Dr. J. St. John, US Department
of Agriculture, Beltsville, MD.
platensis was grown in Zarrouk medium (Starr and
Zeikus, 1993) at 28ºC
under cyclic fluorescent illumination (14 hrs light: 10 hrs dark;
3000 lux). C. minutissima was grown in artificial sea water
medium (Starr and Zeikus, 1993) in a glass house
maintained at 32ºC, illuminated by indirect sunlight.
To the culture medium SAN 9785 stock prepared in sterile DMSO was
added as per the experimental design and inoculated with exponentially
growing cultures. Tolerant cultures were obtained by repeated subculture
at 4 d interval in growth medium supplemented with SAN 9785 at 0.2 mM concentration. Stable tolerant
cultures of both the algae were obtained in 2 months.
concentration of S. platensis was estimated by measuring the
optical density (OD) at 560 nm of appropriately diluted cultures.
Cell density of C. minutissima was counted using a hemocytometer.
Both OD and cell number measurements were caliberated with dry weight
estimations of several culture samples. Lipids were extracted from
exponentially growing cultures in chloroform methanol mixture (2:1
v/v), at 28ºC
for 24 hrs in darkness and filtered. The extract was mixed thoroughly
with half volume of 0.9% NaCl solution and the organic phase containing
free fatty acids was separated (Jayaraman, 1981).
The solvent evaporated under nitrogen and the lipid content was gravimetrically
estimated. Fatty acid composition of extracted lipid was determined
after transmethylation with methanol-acetyl chloride (Cohen
and Cohen, 1991). Gas chromatographic analysis was carried out
using a DEGS column (30 m x 0.38 mm, Nucon Instruments, New
Delhi) maintained at 190ºC
and N2 as the carrier gas at the rate of 30 ml/min. The
injector and detector ports were maintained at 220ºC.
Peaks were identified using an FID detector by comparison with reference
standards (Sigma Chemical Co., St.
Louis, MO.) and area measured using an integrator. The data shown
are the mean of four independent replicate cultures.
of SAN 9785 on the growth of the algae
evaluate the response of the algae to SAN 9785, cultures of Spirulina
platensis and Chlorella minutissima were grown in media
containing different concentration of SAN 9785, and the concentration
of biomass as a function of time was monitored (Figure
1). S. platensis cells exposed to 0.8 mM SAN 9785 were completely lyzed
within a week. Cells exposed to 0.2 mM SAN 9785 exhibited a prolonged
lag phase before increase in cell mass could be observed, while cells
exposed to 0.4 mM
SAN 9785 were static for more than one month. The surviving cells
exhibited exponential growth, which however was significantly lower
than that in cultures not exposed to the herbicide. Although the cells
that were growing had acclimatized to the lower herbicide concentration,
they did not exhibit the growth rate observed under the herbicide-free
condition. This is in contrast to the observation of Cohen
et al. (1993), who report an enhanced growth rate in herbicide
resistant S. platensis isolates. The reduced growth rate suggested
that the herbicide interfered with metabolic activities required for
normal growth rate, and the herbicide itself was not inactivated under
the prevailing conditions of the culture, like high alkalinity, exposure
to light, high dissolved oxygen content, etc.
minutissima was generally more tolerant to SAN 9785 as compared
to S. platensis. At the highest concentration examined, C.
minutissima cells were substantially lyzed, and the remaining
cells did not grow any further in 10 weeks. However, at lower concentrations
of SAN 9785 (0.2 and 0.4 mM),
the growth rate was suppressed, and the level of suppression was proportional
to the concentration of SAN 9785
in the culture. However, C. minutissima
cells acclimatized to SAN 9785 more readily, since at 0.2
mM concentration the growth rate suppression
was only marginal compared to control culture, while in S. platensis
the difference in growth rate was substantial. This suggested that
the metabolic processes affected by SAN 9785 was less influential
on the overall growth of C. minutissima, as compared with the
case in S. platnensis.
lipid content of the cells tolerant to SAN 9785
in both the algae did not show significant difference.
In S. platensis cultures the lipid content was 7.5 ± 0.2% of
the dry weight in both the control as well as tolerant cultures. Similarly
in C. minutissima the lipid content was 7.1 ± 0.3% of the dry
weight in both the control and tolerant cultures. Otles
and Pire (2001) report similar value for S. platensis,
but much higher value for other Chlorella species (C. pyrenoidosa
and C. vulgaris). The variability in the lipid content
observed in both the algae in our study was more influenced by the
environmental and growth conditions rather than by their response
to the herbicide (data not shown).
of SAN 9785 on the free fatty acid composition of S. platensis
S. platensis growing in 0.2 mM SAN 9785
the amount of palmitic acid (16:0) decreased
gradually and stabilized at around 52% of the palmitic
acid content observed in control culture. This reduced amount was
maintained as long as the cultures were exposed to SAN 9785. Upon
removal of the herbicide however, palmitic acid content began to increase
to a concentration as high as observed in cultures not exposed to
the herbicide at all. In contrast, stearic acid (saturated fatty acid,
18:0) which was not detectable in control culture rose to detectable
level in cells exposed to and surviving in the presence of SAN 9785.
The amount of the C18 unsaturated fatty acids - oleic (18:1),
linoleic (18:2) and linolenic (18:3) acids - initially decreased when
the cultures were exposed to SAN 9785. Upon extended cultivation in
the presence of the herbicide the amount of these unsaturated fatty
acids increased and became a substantial fraction of the free fatty
acids. However, this enhanced amount of the unsaturated fatty acids
was detectable only as long as the cultures were grown in the presence
of the herbicide. Upon removal of the herbicide, these unsaturated
fatty acids and their presumed immediate saturated precursor, stearic
acid, decreased while the content of the C16 fatty acid
palmitic acid increased, approaching a profile seen in cultures not
exposed to the herbicide.
of SAN 9785 on the free fatty acid composition of C. minutissima
C. minutissima growing in 0.2 mM SAN 9785, in the first ten days the amount
of palmitic acid decreased by about 15%, but upon continuous exposure
to the herbicide it reached a level as high as that observed in control
culture. Upon removal of the herbicide the palmitic acid content increased
by about 15% in ten days, compared to its level in untreated culture.
In contrast to this, the stearic acid content did not show much variation
and was observed to be at a low concentration in the cultures irrespective
of their SAN 9785 exposure status. The amount of the unsaturated fatty
acids, oleic, linoleic and linolenic acids initially decreased by
62%, 91% and 75%, respectively, when the culture was exposed to SAN
9785. But upon extended growth in the presence of the herbicide, the
concentration of these acids showed substantial recovery. While the
amount of palmitic, stearic, oleic and linoleic acids was comparable
to control culture, the amount of linolenic acid, in contrast, after
an initial decrease upon exposure to 0.2 mM SAN 9785, increased to an amount
almost five times to that found in control culture. However, upon
removal of the herbicide, the amount of oleic and linoleic acids accumulated
to a concentration higher than that found in control culture while
the elevated amount of linolenic acid was reduced to a concentration
closer to that found in control culture. Thus, in the presence of
fatty acid desaturase inhibitor, the tolerant cells of both S.
platensis and C. minutissima overcompensate with enhanced
accumulation of linolenic acid.
results indicate the difference in the regulation of fatty acid synthesis
but not the desaturase activity between S. platensis and C.
minutissima. In both the algae, response to continuous exposure
to SAN 9785 was by elevating their linolenic acid content. It is likely
that prolonged subculture in 0.2 mM SAN 9785 had selected for cells
with enhanced fatty acid desaturation efficiency whereby the level
of desaturated free fatty acid was elevated. Although other investigators
have suggested that SAN 9785 effects could be pleiotropic in eucaryotic
algae and some higher plants like maize (Murphy et al.
1985) and eustigmatophyte (Khozin and Cohen, 1996),
the organisms studied in this report showed similar response as far
as linolenic acid level enhancement was concerned. However, the fate
of its precursors in the algae differed. While in S. platensis
there appeared to be no compensation of palmitic acid while its utilization
for the synthesis of the unsaturated fatty acids had increased upon
exposure of the culture to SAN 9785, in C. minutissima the
increased consumption of the saturated fatty acids, as a consequence
of increased conversion to unsaturated fatty acids was compensated.
This suggests that in C. minutissima there was an increased
de novo fatty acid synthesis. While in C. minutissima
the amount of saturated fatty acids, palmitic and stearic acid, were
stringently maintained at amounts observed in untreated cultures,
in S. platensis their amounts were greatly influenced by their
conversion to the desaturated fatty acids. Further, in S. platensis,
similar trend of accumulation of the different unsaturated fatty acids
suggests that the enzymes involved in the individual step of the desaturation
reactions had similar response to SAN 9785. The three genes involved
in desaturase activity in S. platensis have different specificity
(Apiradee et al. 2004), but may respond to SAN 9785
similarly. In the case of C. minutissima, it is not clear whether
different enzymes are used or the same enzyme carries out the succeeding
steps of desaturation. Thus, the non-uniform change in the amount
of different unsaturated fatty acids in C. minutissima suggests
that the influence of the herbicide on each step of reduction, or
the activity of the different desaturating enzymes for the succeeding
steps of the reaction was likely to be different. It is suggested
that the C. minutissima desaturases are more similar to the
multiple fatty acid desaturases identified in plants (Sprecher
et al. 1995; Heppard et al. 1996). Also, it is
likely that C. minutissima possesses both chloroplastic as
well as cytoplastic fatty acid metabolisms, responding to SAN 9785
differently, as suggested by Khozin and Cohen (1996).
suggest that S. platensis responds to SAN 9785 by driving the
fatty acid desaturation pathway without compensating for the enhanced
conversion of the precursor palmitic acid, while C. minutissima
responds by adequate compensation of the fatty acid precursor pool,
possibly through enhanced de novo synthesis. Perhaps due to
this robustness of C. minutissima, it was able to tolerate
higher doses of SAN 9785.
thank Dr. MP Kapoor, Director, Thapar Institute of Engineering and
Technology, Patiala for providing infrastructure facilities and Dr.
H.A. Norman and Dr. J. St. John of USDA, Beltsville, MD, for the generous
gift of SAN 9785 used in these studies. SJ acknowledges the Research
Fellowship provided by the Council of Scientific and Industrial Research,
Hongsthong; KALYANEE, Paithoonrangsarid; PONGSATHON, Prapugrangkul;
PATCHARAPORN, Deshnium; MATURA, Sirijuntarut; SANJUKTA, Subhudhi;
SUPAPON, Cheevadhanarak and MORAKOT, Tanticharoen. The expression
of three desaturase genes of Spirulina platensis in Escherichia
coli DH5a - heterologous expression of Spirulina-desaturase
genes. Molecular Biology Reports, September 2004, vol. 31,
no. 3, p. 177-189. [CrossRef]
John R.; TILLETT, David M. and WEISSMAN, Joseph C. Microalgae biotechnology.
Trends in Biotechnology, February 1987, vol. 5, no. 2, p.
Zvi and COHEN, Simon. Preparation of eicosapentaenoic acid (EPA)
concentrate from Porphyridium cruentum. Journal of the
American Oil Chemists Society, January 1991, vol. 68, no. 1,
Zvi; REUNGJITCHACHAWALI, Marasri; SIANGDUNG, Wipawan; TANTICHAROEN,
Morakot and HEIMER, Yair M. Herbicide-resistant lines of microalgae:
Growth and fatty acid composition. Phytochemistry, November
1993, vol. 34, no. 4, p. 973-978. [CrossRef]
E.P.; KENNEY, A.J.; STECCA, K.L. and MIAO, G.H. Developmental and
growth temperature regulation of two different microsomal w-6 desaturase
genes in soybeans. Plant Physiology, January 1996, vol. 110,
no. 1, p. 311-319.
J. Laboratory Manual in Biochemistry. New
Delhi, New Age Publishers, 1981, 180 p. ISBN
Inna and COHEN, Zvi. Differential response of microalgae to the
substituted pyridazinone, sandoz 9785, reveal different pathways
the biosynthesis of eicosapentaenoic acid. Phytochemistry,
July 1996, vol. 42, no. 4, p. 1025-1029. [CrossRef]
G. and KAMAT, M. γ-Linolenic production from Spirulina platensis.
Applied Microbiology and Biotechnology, July 1995, vol. 43,
no. 3, p. 466-469. [CrossRef]
Denis J.; HARWOOD, John N.; LEE, Kevin A.; ROBERTO, Francisco; STUMPF,
Paul K. and ST. JOHN, Judith B. Differential responses of a range
of photosynthetic tissues to a substituted pyridazinone, Sandoz
9785. Specific effects on fatty acid desaturation. Phytochemistry,
September 1985, vol. 24, no. 9, p. 1923-1929. [CrossRef]
Semih and PIRE, Ruhşen. Fatty acid composition of Chlorella
and Spirulina microalgae species. Journal of AOAC International,
November 2001, vol. 84, no. 6, p. 1708-1714.
Akira; WANG, H.L. and HESSELTINE, C.W. Culture conditions affect
eicosapentaenoic acid content of Chlorella minutissima. Journal
of the American Oil Chemists Society, May 1984, vol. 61,
no. 5, p. 892-894.
H.; LUTHRIA, D.L.; MOHAMMED, B.S. and BAYKOUSHEVA, S.P. Reevaluation
of the pathways for the biosynthesis of polyunsaturated fatty acids.
Journal of Lipid Research, September 1995, vol. 36, no. 12,
Richard C. and ZEIKUS, Jeffrey A. UTEX–The culture collection of
algae at the University of Texas
at Austin 1993 list
of cultures. Journal of Phycology, April 1993, vol. 29, no.
2 (supplement), p. 1-106. [CrossRef]
Note: Electronic Journal
of Biotechnology is not responsible if on-line references cited on
manuscripts are not available any more after the date of publication.