F o h a Microbiol.

31, 4 4 - - 4 9 (1986)

Identification of Sterols and Alcohols Produced

by Green Algae of the Genera Chlorella
and Scenedesmus by Means of Gas
Chromatography Mass Spectrometry
T. I~]~ZA~KA, O. VY~XLEK and M. 1)ODOJIL
Institute of Microbiology, Czechoslovak Academy of Sciences,
142 20 Prague 4

Received A ovember Y, 19~4

ABSTIIACT. Sterols a n d alcohols isolated f r o m a collection of i n d u s t r i a l l y i m p o r t a n t f r e s h - w a t e r

g r e e n algae Chlorella kessleri, Scenedesmus aruminc~tus, S. acutns and' S. aeut~ls var. Ton~,,xdti
c u l t i v a t e d h e t e r o t r o p h i c a l l y or a u t o t r o p h i e a l l y u n d e r pilot p l a n t conditions, were identifio~l
and quantified by gas chromatography--mass spectrometry. Previsously demonstrated sterols
w i t h d o u b l e b o n d s in p o s i t i o n 5 a n d 7 were d e t e c t e d . I n a d d i t i o n , cholesta-5,7,22-trien-3,%ol
t h a t h a d n o t y e t been described in green algae was f o u n d in S. acut~s. Alcohols were f o u m l o~dy
in C. kessleri c u l t i v a t e d u n d e r h e t e r o t r o p h i c conditions. S o m e s a t u r a t e d a n d u n s a t u r a t e d ah-ollolu
were d e t e c t e d for t h e first t i m e in green algae.

The sterol content of algae was discussed in several review articles (Patterson 1971; Goodwin 1974). Major sterols produced b y the genera Chlorel7~
and Scenedesmus have 28 and 29 carbon atoms and contain methyl or ethyl
groups on carbon C(~4) (fungisterol, chondrillasterol, schotenol). Using gas
c h r o m a t o g r a p h y - - m a s s spectrometry (GC--MS) ergosterol, ergostadienol,
corbisterol, 7-ergosterol, 5-ergosterol, poriferosterol and clionasterol were
gradually detected as minor components (Bdlanger et al. 1973; Paoletti et ~t.
1976; Milkova et al. 1977).
Paoletti et al. (1976) described three aliphatie alcohols in S c e n e d e s ~ s
quadricauda and ~qpirulina platensis that have not been characterized in more
detail. Volkman et al. (1981) described alcohols with an even number carbon
atoms (C14--C20) in wax of marine algae. Alcohols with an even number of
carbon atoms (C14--C22) were detected in photosynthetic microorganisms
of the genera Euglena and Chroomonas (Hilenski et al. 1976; Nichols et al.

19s3).
In this work we studied the occurrence of sterols and aliph~tic alcohols
in two industrially i m p o r t a n t genera of green algae (Chlorella and Scenedesmus). In addition, in the genus Chlorella we compared the production of
these compounds under heterotrophic and autotrophic cultivation conditions.

1986

S T E R O L S A N D A L C O H O L S I N GREEN" A L G A E

4S

TABLE I. Conditions of g a s c h r o m a t o g r a p h y - - m a s s s p e c t r o m e t r y u s e d to d e t e r m i n e sterols a n d

alcohols
Apparatus and analyzed
compounds

Column type
S t a t i o n a r y pha~e
Length, m
ID, mm
T h i c k n e s s of s t a t i o n a r y
phase, ~m
Injector temperature, ~
hfitial column temperature, ~
T e m p e r a t u r e increase, K / l n i n
~inal cohnnn temperature, ~
H e l i u m flow, m L / r a i n
Split
Ionization energy, aJ
Mass range, mu
Sweep rate, m/z X l / s

MATERIAL

H P 5995 B
sterol.~

alcohols

WCOT
SE-30
12.5
0.25

~VCOT
SE-30
25
0.25

0.25
300
240
1
280
0.54
1 : 47
11
4 0 - - 600
680

0.25
250
100
5
280
0.54
1 : 47
11
4 0 - - 600
680

AND METHODS

Cultures. Chlorella kessleri (cultivated both autotrophically and heterotrophically), ~Scenedesmus acuminatus (cultivated autotrophically) and Scenedesmus acutus (cultivated autotrophically) were prepared in the Department
of Autotrophic Microorganisn~ts of the Institute of Microbiology, Czechoslovak
Academy of Sciences (T~ebofi, Czechoslovakia) in the form of dried algal
biomass (about 5 % moisture). Scenedesmus acutus var. Tomasseli (cultivated
autotrophically) was a gift of the Institute of Algology, Bulgarian Academy
of Sciences, Sofia.
Preparation of the sterol and aliphatic alcohol fraction. Algal biomass (10 g)
was saponified by 2-h boiling with 300 mL 2 ~I NaOH in 50 % ethanol. The
reaction mixture was diluted with 1 L water and extracted with three 500-mL
portions of diethyl ether. Combined organic extracts were washed with
three 300-mL volumes of water, dried with sodium sulfate and evaporated
to dryness. Preparative TLC (plates 200 200 ram, Silica. gel H, Merck,
system light petroleum--diethyl ether, 3 : 1 ) yielded the sterol fraction
(RF 0.35--0.50). I n C. kessleri cultivated under heterotrophic conditions the
alcohol fraction was also isolated (Rr 0.5--0.6).
Identification of sterols and alcohols. Analytical methods used for the determination of sterols and alcohols by GC--MS method are summarized
in Table I. Sterols were analyzed as trimethylsilyl derivatives (Brooks et al.
1973). Aliphatie alcohols were determined as the corresponding trimethylsilyl ethers (Odham and Stenhagen 1972). UV spectra were measured in the
Cary 118 (Varian) apparatus in ethanol within the range of 200--350 rim.
RESULTS AND DISCUSSION

Sterols were identified on the basis of fragmentation of their mass spectra

according to Brooks et al. (1973) and Knights (1967). The distribution of
sterols (Table II) is characterized here primarilly by the presence of 7-ergo-

46

T. I~EZANKA et al.

Vol. .~I.

"5

Y2~Z

1986

STEROLS AND ALCOHOLS IN GREEN ALGAE

47

sten01 (23--57 ~o) and 7-chondrillastenol (6--55 ~o). Small quantities of

other sterols are present in C. kessleri, among them three unusual sterols,
24-methyl- and 24-ethyl-5~-cholest-8(14)-en-3~-ol and 4-methylchondrillasterol. These sterols are usually found primarily in algae cultivated in the
presence of inhibitors of sterol biosynthesis (Patterson et al. 1973; our unpublished results).
TABLE I I I . A l c o h o l c o n t e n t in C. t'essleri c u l t i v a t e d u ~ d e r h e t e r o t r o p hie c o n d i t i o n s (mass %)
Alcohol
( t r i m e t h y l s i l y l ether)

RRT a

M+

Previously described (in photosynthetic microorganisms) :

1-Tetradecanolb
1 - H e x a d e e a n o lb
l - O k t a d e c a n o lb
Phytol c

0.634
0.821
0.958
1.000

286
314
342
368

3.02
9.94
1.60
73.07

Not yet described (in photosynthetic microorganisms):

Deeen- 1~
l-Decanol
2-Undecanol
2-Tridecanol
1-Dodeeanol
1-Tetradecanold
1-Pentadecanol
Hexadecen-l-ol
E i k o s e n - 1-ol d
E i k o s a d i e n - 1-ol
E i k o s e n - 1-ol d
E i k o s e n - 1-ol

_Vot identified
~'ot identified
a
b
c
d
e

0.265
0.273
0.297
0.388
0.452
0.577
0.717
0.774
0.866
1.007
1.043
1.072
1.179
1.201

228
230
244
272
258
286
300
312
368
366
368
368
456 e
143 e

1.90
1.56
0.22
0.24
1.94
0.43
0.80
0.62
0.38
2.46
0.17
1.08
0.37
0.20

R e l a t i v e r e t e n t i o n t i m e r e l a t e d to p h y t o l (28.90 rain).
V o l k m a n et (d. (]981); Nichols et al. {1983).
P a o l e t t i et ~d. {1976).
B r a n c h i n g of t h e c h a i n n o t d e t e r m i n e d .
H i g h e s t ion d e t e c t e d .

The identification of eholesta-5,7,22-trien-3~-ol t h a t has not y e t been

described in green algae is of primary importance. It has been found only in
the red alga P o r p h y r i d i u m c r u e n t u m (Beastall et al. 1 9 7 1 ) a n d also in
marine animals, such as molluscs ( C a c o s p o n g i a mollior - - Cafieri and De Napoli
1976; A x i n e l l a c a n n a b i n a - - Cafieri et al. 1975; T e r ~ o s zeteki - - Delseth et al.
] 979a; B i e m n a f o r t i s - - Delseth et al. 1979b).
Its identification was further confirmed b y its UV spectrum which exhibits
a pronounced absorption at 293, 282.5 and 272 nm, indicating the presence
of a conjugated homoannular diene, as measured in the model UV spectrum
of ergosterol. Cafieri et al. (1975) and Cafieri and De Napoli (1976) described
a very low intensity of the molecular ion of cholesta-5,7,22-trien-3~-ol. This
ion was also not detected in this work b u t its characteristic fragmentation

45

T. I ~ E Z A N K A et al.

Ve]..21

and presence of ions m/z 364 and 253 are in agreement with the proposed
structure.
Interpretation of the mass spectra of trimethylsilyl ethers ( 0 d h a m and
Stenhagen 1972) and retention characteristics of the alcohol fraction isolated
from C. lcessleri, and mass spectra and retention characteristics of standards
of alcohols (C12--C29 even saturated and C ~ and Cis monoene) a total of
fifteen alcohols were identified, among them phytol as the major one. I~ is
apparently formed b y degradation of chlorophyll (Paoletfi et al. 1976). Among
other aliphatic alcohols, five saturated alcohols with an even number carbon
atoms, three with an odd number carbon atoms, three monoene and one
diene alcohol were detected. The hydroxyl group is always in position 1 of
the chain, only two unsaturated alcohols have hydroxyl group in position 2.
Two 1-tetradecanols and three eikosene-l-ols differing in branching were
described b u t could not be accurately determined b y the GC--MS method.
Alcohols detected in C. ]cessleri are summarized in Table III. In green alg~(~
(Chlorophyceae) aliphatic alcohols were not described, only three incompletely
characterized alcohols were briefly mentioned b y Paoletti et al. (1976). In
nature aliphatic alcohols are present primarilly in the form of waxes and
are assumed to play a physiological role, serving as energy reserves of the
cell (Withers and Nevenzel 1977).
Determinable amounts of aliphatic alcohols (200 ppm, as related to dry
mass) were detected only in heterotrophically cultivated C. kessleri, whereas
in autotrophic cultures of C. kessleri, S. acutus and S. acumi~atus these
compounds were not detected (if they are present, then only in quantities
lower them 10 ppm as related to the dry mass). The results referred to here
clearly demonstrate the u t m o s t significance of the way of cultivation of al~::~e
(hetero- and autotrophy) for the production of alcohols and are in agreemen~
with the data of Antia et al. (1974), who detected an increased productiot~
of waxes b y photosynthetic microorganisms cultivated under heterotropMc
conditions.
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STEROLS AND ALCOHOLS IN G R E E N ALGAE

4?

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