1. Studies on fermentative production of squalene
P. Bhattacharjee, V.B. Shukla, R.S. Singhal* and P.R. Kulkarni
Food and Fermentation Technology Division, University Department of Chemical Technology (U.D.C.T), Matunga,
Mumbai-400 019, India
*Author for correspondence: Fax: +91-22-4145614, E-mail: rekha@foodbio.udct.ernet.in
Received 3 May 2001; accepted 12 September 2001
Keywords: Chromatography, fermentative production, lipid extract, spectroscopy, squalene
Summary
Fermentative production of squalene under anaerobic conditions using commercially available compressed baker’s
yeast (Saccharomyces cerevisiae), and a strain of Torulaspora delbrueckii isolated from molasses was studied. Yield
of squalene from S. cerevisiae and T. delbrueckii were found to be 41.16 and 237.25 lg g)1
respectively, dry weight
of yeast cells. Isolation and purification of squalene from the lipid extracts obtained by cell lysis of either strain were
achieved chromatographically. The purified squalene was characterized spectroscopically against an authentic
standard.
Introduction
Squalene is a naturally occuring constituent found in
plant oils such as olive oil (Roncero & Janer 1962), palm
oil, wheat germ oil, amaranth oil (Singhal & Kulkarni
1990), rice bran oil, fish oils as well as in human sebum.
It is the principal hydrocarbon in human surface lipids,
amounting to about 10% of the total surface fat, and is
the biosynthetic precursor of cholesterol. The richest
source is the liver of Aizame (dogfish) shark (Squalas
spp) of the southern Pacific oceans of Australia. It is a
highly unsaturated aliphatic hydrocarbon (2, 6, 10, 15,
19, 23-hexamethyltetracosa-2, 6, 10, 14, 18, 22-hexaene;
molecular formula – C30H50 belonging to the triterpene
group of oils) isolated from fish oil by vacuum distilla-
tion. It is a clear, brilliant and almost colourless oil with
a faint odour and taste. It serves as a detoxification
factor, as a skin and eye antioxidant, in providing cells
with oxygen, and as a bactericidal and fungicidal agent.
It has wide applications as an antistatic agent and
emollient in cosmetics and pharmaceuticals, and also in
fine chemicals, magnetic tape and low-temperature
lubricants. Olive oil containing 0.8% squalene is widely
used for cooking and salad dressing. Oryzanol is
dissolved in squalene and added to foods as an
antioxidant (Ishitani 1980). Squalene is also used an
additive in animal feed.
The limited availability of squalene has compelled
manufacturers to switch over to its substitutes such as
squalane, squaliformes etc. A 500 mg capsule of squa-
lene is priced at 0.125 US$. The present work was aimed
at production of squalene using microbial sources.
There are only a few reports on microbial production
of squalene: Saccharomyces (Jollow et al. 1968; Kata-
yoka et al. 1992; Kawai 1992; Kamimura et al. 1994;
Socaciu et al. 1995; Ciesarova et al. 1996), Pseudomonas
(Uragami & Koga 1986), Candida (Tsujiwaki et al.
1995a, b), the algae Euglena (Kawaura et al. 1995;
Kawaura & Matsuda, 1996) and in general yeasts
(Mauricio et al. 1993) are the organisms used for this
purpose.
The biosynthetic pathway of squalene production in
Saccharomyces spp. starts with the synthesis of meval-
onate from acetate, then conversion of mevalonate to
two activated isoprenes, condensation of six activated
isoprene units to form squalene and finally conversion
of squalene to the 4-ring lanosterol (Lehninger et al.
1993a).
Commercially available compressed baker’s yeast
(Saccharomyces cerevisiae) and a strain of Torulaspora
delbrueckii isolated from molasses were used for fer-
mentative production of squalene. The yield of squalene
in mg g)1
of wet biomass, under anaerobic conditions,
was studied as a function of time and inoculum size of
the aerobic culture. Downstream processing of squalene
to purify it was attempted and ascertained spectroscop-
ically using an authentic standard.
Materials and Methods
Materials
Glucose, yeast extract powder, malt extract powder,
peptone were from Himedia Laboratories, Mumbai,
aluminium HPTLC plates coated with silica gel 60 (F254)
World Journal of Microbiology & Biotechnology 17: 811–816, 2001. 811
Ó 2001 Kluwer Academic Publishers. Printed in the Netherlands.
2. from E. Merck, Germany, silicic acid (100–200 mesh for
lipid chromatography) of Spectrochem Pvt. Ltd, Mum-
bai. All solvents used were of AR grade. Commercially
available compressed baker’s yeast (S. cerevisiae) and a
strain of T. delbrueckii were used for the isolation of
squalene. Standard squalene was procured from Sigma-
Aldrich Corporation, USA.
Methods
Aerobic culture of microorganisms
Compressed baker’s yeast (S. cerevisiae). Two millilitre
of 10% suspension of compressed yeast in sterile
distilled water, was inoculated into 100 ml growth
medium having composition – glucose 20 mg ml)1
,
yeast extract powder 5 mg ml)1
, peptone (bacteriolog-
ical) 5 mg ml)1
and malt extract powder 5 mg ml)1
(pH
4–5) in 250 ml shake flask. The flask was incubated at
30 ± 2 °C for 48 h on a rotary shaker at 44 · g.
T. delbrueckii
Molasses serially diluted with sterile distilled water was
inoculated into growth medium (Long & Ward 1989)
having the composition: glucose 20 mg ml)1
, yeast
extract powder 10 mg ml)1
and peptone 20 mg ml)1
(pH 5.5) and incubated at 30 ± 2 °C for 48 h on a
rotary shaker at 44 · g. Growth was plated on solid
medium containing 29 mg agar ml)1
. The organisms
from plates were selected and isolated on the basis of
colony and morphological characteristics. As many as 16
isolates were obtained. One of the isolates, which was
used for biotransformation of benzaldehyde to L-Pheny-
lacetylcarbinol (Shukla & Kulkarni 2000) and identified
as T. delbrueckii was deposited in Microbial Type
Culture Collection, Institute of Microbial Technology,
Chandigarh, India and was given culture number MTCC
3417. The culture had many characteristics similar to S.
cerevisiae which is known to be a squalene producer.
Hence, this isolate was chosen for a comparative study.
One loopful of active culture from the slant was
inoculated into 100 ml growth medium having compo-
sition the same as the above, in a 250 ml shake flask.
The flask was incubated at 30 ± 2 °C for 48 h on a
rotary shaker at 44 · g.
Anaerobic production of squalene
The procedure for both the cultures chosen in the study
was similar. After 48 h, 1, 2, 3 and 5% of active culture
from the aerobic medium were inoculated into fresh
100 ml medium having the composition: glucose
40 mg ml)1
, yeast extract powder 10 mg ml)1
and
peptone 20 mg ml)1
(pH 5.5) (Lodder 1970) contained
in 250 and 100 ml conical flasks for both species
separately. To maintain anaerobic conditions, a layer
of sterile light liquid paraffin about 1 cm thick was
spread on the surface of the medium and the flasks were
tightly sealed. The culture was left standing under
anaerobic conditions for 24–72 h.
Recovery and detection of squalene from the biomass
After 24, 48 and 72 h, the culture was centrifuged at
20,000 · g for 20 min at 28 ± 2 °C, to collect the cell
mass. As chloroform is reported to cause yeast cell lysis
and is the solvent commonly used for extraction of
neutral lipids, it was used to recover the intracellular
squalene from the biomass after lysis. But chloroform
did not allow quantitative recovery of the biomass from
the centrifuge tubes owing to the stickiness of the yeast
cell on the walls of the tubes. Petroleum ether, reported
to be one of the best solvents for the extraction of oils,
was also tested. But the yeast cells formed an agglo-
merate in the centrifuge tube which could not be scraped
off. Microscopic observation indicated a complete loss
of individuality of the yeast cells. Finally, the cell mass
were dispersed in chloroform/methanol (2:1, v/v) mix-
ture. This solvent has been reported for squalene
recovery from Euglena (Kawaura & Matsuda 1996).
With this mixture, quantitative recovery of the biomass
was ensured. The commonly reported method for lipid
extraction using chloroform/methanol/water (1:2:0.8, v/
v) (Lehninger et al. 1993b) was not used as the aim was
to selectively extract only the neutral lipid. Methanol–
water mixture is polar and could have extracted all
triglycerides and other sterol esters. About 150 ml of the
chloroform/methanol solvent mixture was used for
quantitative transfer of the biomass from each anaero-
bic culture flask. The dispersed cells were subjected to
shaking extraction in 250 ml shake flasks on a rotary
shaker at 34 · g at 30 ± 2 °C for 8–10 h to allow
complete cell lysis. The biomass was removed by
filtration using non-absorbent cotton–wool and the
filtrate was passed through activated molecular sieves
to remove the residual moisture. The biomass on the
cotton was washed 2–3 times with fresh chloroform–
methanol solvent system (20 ml each time). Solvent
from the extract was then removed by a rotary evapo-
rator operating at 500 mm Hg at 45 °C to yield a
viscous oily substance. The crude extract was observed
to be a bilayered product – the upper layer, a white
greasy substance and a pale yellow coloured oily
substance in the lower layer. The extract was character-
ized by gas chromatography (GC) using a capillary
column, FT-IR HPTLC and UV analysis, using stan-
dard squalene for comparison.
Gas chromatographic analysis of the extract
The white and yellow portions of the extract were
analysed by GC separately. The two fractions after
thorough drying were redissolved in n-hexane and
analysed by GC using Chemito 8510 model equipped
with a flame ionization detector connected to Oracle 1
computing integrator. A BP-5 capillary column (2.2 mm
o.d. · 50 m length) with the following specifications was
used. The carrier gas was Iolar grade hydrogen with a
flow rate of 40 ml min)1
at room temperature. Purge and
split were 5 and 60 ml min)1
respectively. About 0.4 ll of
812 P. Bhattacharjee et al.
3. the extract was used for analysis. The column was heated
from 120 to 280 °C at 10 °C min)1
and held at 280 °C for
30 min. Injector and detector temperatures were 250 and
260 °C respectively. Squalene standard and that in the
extract had a retention time of 27.76 ± 2 min. Since the
pure compound could not be flushed out completely in a
single run from the capillary column but was carried over
to the consecutive runs; GC could not quantify the
squalene in the extract. Various temperature program-
ming were tried with no success. Squalene could be
detected only in the yellow portion of the extract. Though
quantification of squalene using gas-liquid chromato-
graphy is reported as a rapid and accurate technique for
vegetable oils (Lanzon et al. 1995), such as olive oils
(Leonardis et al. 1998) and in amaranth oils (Singhal
1989). It could only be used for qualitative detection of
squalene with the microbial lipid extract.
UV scan of extracts for kmax
Pure squalene of 0.5% w/v and the yellow portion of the
crude extracts, dissolved in chloroform (Spectro grade)
were scanned in the UV region of 400–200 nm by the
Hitachi Spectrophotometer (Model U 2001). kmax of
272 nm obtained for both pure and the extracts con-
firmed presence of squalene in the extracts (Singhal
Kulkarni 1990).
Colorimetric estimation of squalene
The above extract was thoroughly dried by purging
commercial grade nitrogen and quantification of squa-
lene was carried out colorimetrically at 400 nm as
described by Rothblat et al. (1962) using Elico Spectro-
photometer (Model CL-27).
Densitometric estimation of squalene
For densitometric assay, several solvent systems were
tried. Cyclohexane was found to give the best resolution,
in which squalene recorded an Rf value of 0.60 ± 0.02.
The extracts were spotted on aluminium plates coated
with silica gel 60 (F254) by use of Camag Linomat IV.
The extracts, dissolved in n-hexane (AR grade), were
applied to the plates in the form of bands, each 6 mm
wide, spacing between consecutive bands being 8 mm.
Nitrogen gas was used at a low flow rate of 4 bar for
spotting. The plates were developed at 26 ± 2 °C in a
glass chamber containing cyclohexane. The spot corre-
sponding to squalene could be easily detected on
exposure to iodine vapours. Spectrum scanning of the
spots after development was carried out in Camag
HPTLC unit (TLC scanner II). Squalene recorded a
kmax value of 200 nm (95% transmission which de-
creased to 86% at 203–204 nm). Densitometric studies
were performed at 200 nm and the area under the curve
for squalene was recorded. A standard curve was plotted
for pure squalene at 200 nm (area under the curve as
recorded by Camag HPTLC vs. lg of pure squalene
spotted).
Isolation and purification of squalene from the lipid
extract by chromatographic technique
Since the colorimetric test (Rothblat et al. 1962) based
on H2SO4–formaldehyde chromogen also shows a
positive colour reaction with lanosterol or ergosterol
(if produced from squalene) attempts were made to
purify the cell extract by silicic acid chromatography.
Silicic acid (100–200 mesh for lipid chromatography)
was washed with methanol and water to remove fines
and impurities and activated at 110–120 °C overnight.
Approximately 35–38 g of silicic acid was used for the
column of length 180–200 mm and ID 25 mm. A slurry
of silicic acid in 6% (v/v) benzene in hexane was poured
into the column. About 1 g of the lipid extract obtained
by cell lysis was applied to the column. The crude
extract after thorough drying in a rotary evaporator was
adsorbed onto 1.5 times its weight of silicic acid and
loaded on to the column. Squalene was eluted with 6%
benzene in n-hexane (Horning et al. 1960) in the initial
fractions itself which was confirmed by HPTLC, but
pure squalene was not obtained. Since sterols and sterol
esters are eluted along with hydrocarbons in the initial
fractions (Weber 1969; Stoller Weber 1970), the
impurity peaks may have been due to ergosterol and/or
lanosterol. The Liebermann–Bucchard test for the
presence of the steroid nucleus was positive for the
eluates. So further purification of squalene was attempt-
ed by alumina chromatography. The silicic acid column
fractions in which squalene was detected by TLC were
combined, saponified and loaded on to an alumina
column as described (Firestone 1995). The hydrocarbon
fraction containing squalene was eluted in petroleum
ether, concentrated by rotary evaporator at 45 °C and
500 mm Hg vacuum and further purified by preparative
TLC using silica gel-coated glass plates and cyclohexane
as solvent. The band with Rf value 0.6 was scraped off
and characterized by FT-IR, 1
H-NMR, 13
C-NMR and
GC-MS. Squalene isolated from both the microbial
sources was purified and characterized. The spectrum of
squalene isolated from T. delbrueckii had been shown to
be similar to that obtained from S. cerevisiae.
FT-IR spectra of the purified compound
The FT-IR spectra for both authentic and purified
squalene from T. delbrueckii were recorded on Perkin-
Elmer-783 spectrophotometer using CHCl3 as solvent.
IR (neat, cm)1
): 2914 (CAH stretching), 2728, 1668
(alkene, non-conjugated), 1446 (alkane, CH2), 1382
(alkane, CH3), 1330, 1224, 1151, 1188, 964 (alkene,
disubstituted trans), 835 (two adjacent hydrogen atoms),
722.1 (due to CHCl3 solvent used for dissolution of
isolated squalene). The FT-IR spectra of the purified
squalene compared well with the authentic standard.
Squalene production by yeasts 813
4. NMR spectra of the purified compound
1
H-NMR for both authentic and purified squalene from
T. delbrueckii were recorded in CDCl3 using 300 MHz
Brucker ACP-300 model. In 1
H-NMR spectra, chemical
shifts are reported in ¶ units (ppm) relative to TMS as
internal standard. 1
H-NMR (CDCl3, 300 MHz): ¶ 5.1
(t, 6H), 1.93–2.09 (m, 20H), 1.68 (s, 6H), 1.60 (s, 18H).
13
C-NMR spectra were recorded in CDCl3. In 13
C-
NMR spectra, coupling constants (J) are reported in
hertz (Hz). 13
C-NMR (CDCl3, 125 MHz): 135.00 (d,
J = 25.7 Hz), 131.10 (s), 124.42 (d, J = 13.7 Hz),
77.24 (t, J = 31.9 Hz; due to CDCl3 solvent), 39.73
(s), 28.2 (s), 26.77 (d, J = 14.3 Hz), 25.63 (s), 17.61 (s),
15.98 (d, J = 3.36 Hz). Both 1
H-NMR and 13
C-NMR
spectra of the purified squalene showed good correlation
with those of the authentic sample.
GC-MS of the purified compound
The compound was analyzed in a GC-7A Shimadzu
model coupled to a QP-5000 MS equipped with a flame
ionization detector and electron ionization (EI) detector
respectively. EI mass spectra was obtained with an
ionization voltage of 70 eV. The column was a fused
silica capillary column (0.3 mm o.d. · 50 m length). It is
a non-polar column packed with DB-5 (polymethyl
siloxane with 5% phenyl modification). The carrier gas
was helium at 21 ml min)1
at 25 °C. The programming
was as described above. The retention time for squalene
was 24.47 min. EI mass spectra data of squalene
(purified from T. delbrueckii) are reported in the form
m/z (relative abundance). m/z 41 (M+
, 0.07), 149 (7.14),
136 (14.2), 95 (14.5), 81 (43.5), 69 (100), 41 (14.8).
Results and discussion
Table 1 shows a comparative data on squalene produc-
tion as estimated colorimetrically by S. cerevisiae and T.
delbrueckii in 250 ml shake flasks. After 48 h of anaero-
bic fermentation, the best yield of squalene was obtained
with 5% inoculum. A decrease in squalene content was
observed after 72 h. This could be due to utilization of
squalene as a carbon source by the yeast cells for
sustenance or due to conversion into some other
unknown product. There is a report on utilization of
squalene as a carbon source by C. famata US-238
(Tsujiwaki et al. 1995a, b). Yeast at a glucose concen-
tration above 0.4% is known to exhibit the Crabtree
effect, in which it will forego respiration and carry out
fermentation. Yeast will first utilize oxygen to synthesize
sterols for cell wall building. So the ergosterol and/or
lanosterol generated in 2% glucose medium in aerobic
phase gets extracted by solvents later when cell wall is
ruptured for lipid extraction. Hence, the colorimetric
estimation assays both sterols and squalene. Up to 5%
inoculum level, the media nutrients are sufficient to
promote good growth of yeast cells and consequently
squalene production in anaerobic phase. But 10 and
20% inoculum levels are too high to be sustained. The
growth of yeast cells may have been arrested in the
aerobic phase and the media nutrients in the anaerobic
phase have been utilized for maintenance rather than
channelized for squalene production. Five percent of
inoculum gave the best yield of squalene after 24 h of
anaerobic fermentation for T. delbrueckii. The trend in
squalene production is just the reverse with that
observed with S. cerevisiae. This may be probably due
to greater utilization of squalene by T. delbrueckii as
compared to compressed yeast.
Since oxygen is the primary limiting factor in yeast
growth, it was thought that squalene production would
be better in 100 ml flasks. No definite trend in squalene
production was observed and hence the study was
discontinued.
For both the organisms, the set that gave the best
yield in shake flask studies was quantified by the
densitometric method as shown in Tables 2 and 3. This
assay established T. delbrueckii to be a better squalene
producer than S. cerevisiae. The values for squalene
estimated colorimetrically were higher compared to that
estimated densitometrically as ergosterol and/or lano-
sterol contributed to the values in the colour test.
Isolation and purification of squalene from the lipid
extracts of both S. cerevesiae and T. delbrueckii by
column chromatography has confirmed the authenticity
of squalene. This study could be valuable in the
production of squalene, which due to limited availability
has compelled manufacturers to switch over to substi-
tutes.
Table 1. Comparative production of squalene by S. cerevisiaea
and T.
delbrueckiib
in mg g)1
of wet biomassc,d
.
Percentage (%) of
inoculum added to
anaerobic medium
from aerobic medium
Time of fermentation (h)
24 48 72
1a
0.22 ± 0.05 0.27 ± 0.05 0.34 ± 0.07
1b
1.07 ± 0.08 0.49 ± 0.09 0.34 ± 0.08
2a
0.27 ± 0.09 0.37 ± 0.08 0.38 ± 0.08
2b
1.09 ± 0.06 0.52 ± 0.08 0.41 ± 0.07
3a
0.40 ± 0.09 0.40 ± 0.10 0.41 ± 0.08
3b
1.30 ± 0.07 0.64 ± 0.08 0.52 ± 0.07
5a
0.42 ± 0.06 1.38 ± 0.06e
0.35 ± 0.06
5b
1.89 ± 0.06f
0.51 ± 0.08 0.34 ± 0.08
10a
1.25 ± 0.08 0.40 ± 0.04 0.36 ± 0.07
10b
1.53 ± 0.08 0.43 ± 0.04 0.30 ± 0.05
20a
0.99 ± 0.09 0.23 ± 0.04 0.22 ± 0.06
20b
1.04 ± 0.08 0.34 ± 0.08 0.34 ± 0.08
a
Data for S. cerevisiae.
b
Data for T. Delbrueckii.
c
The results are expressed as mean ± S.D of four individual
production runs.
d
Yield of squalene in terms of dry weight of biomass could not be
done as complete removal of the adhered media broth to the
biomass was not possible.
e
The optimum yield of squalene from S. cerevisiae.
f
The optimum yield of squalene from T. delbruekii.
814 P. Bhattacharjee et al.
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Table 3. Densitometric assay of squalene from T. delbrueckii.
Lipid extract applied
on the plate (lg)
Y1
a
Y2
a
X1
b
= Y1/
m · 10)3
X2
b
= Y2/
m · 10)3
Mean
· 10)3
SD (n)1,
n = 2) · 10)6
% RSD =
(SD/mean)
· 100
Squalene/g
of lipid
extract (lg)
Squalene/g of
dry
biomass (lg)
1.53 113.4 114.5 0.31 0.35 0.35 2.48 0.70 496.73 237.25
3.06 294.8 292.7 0.91 0.90 0.91 4.60 0.51 640.29
4.59 493.0 487.8 1.52 1.51 1.51 11.60 0.77 712.47
6.12 528.9 521.4 1.63 1.61 1.62 16.26 1.00 572.39
7.65 543.5 540.2 1.68 1.67 1.67 7.07 0.42 472.23
The wet weight of the biomass obtained by centrifugation was 7.3875 g with a moisture content of 80.49%.
Concentration of lipid extract taken for HPTLC analysis: 15.3 lg ll)1
.
a
Y1 and Y2 are areas under the curves for squalene when the lipid extract is spotted in replica in one TLC plate.
b
X1 and X2 are concentration of squalene in the extract evaluated from standard curve of pure squalene with slope, m = 323830,
R2
= 0.9927.
816 P. Bhattacharjee et al.
