Algal Research 10 (2015) 210217

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Algal Research

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Pavlova lutheri is a high-level producer of phytosterols

Faruq Ahmed a, Wenxu Zhou b,c, Peer M. Schenk a,
a
Algae Biotechnology Laboratory, School of Agriculture and Food Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia
b
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia
c
Centre of Excellence for Plant Metabolomics, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Plant sterols are well-known for their ability to reduce low density lipoprotein (LDL)-cholesterol and promote
Received 27 January 2015 cardiovascular health, as well as anti-inammatory, anti-atherogenicity, anti-cancer and anti-oxidative activities
Received in revised form 13 May 2015 and protection against nervous system disorders. As considerable amounts of phytosterols are present in
Accepted 19 May 2015
microalgae, we screened out of hundreds of Australian isolates, ten microalgal species for sterol proles and
Available online 23 May 2015
total sterol content. The top three sterol producers at 0.42.6% dry weight were Pavlova lutheri, Tetraselmis sp.
Keywords:
M8 and Nannochloropsis sp. BR2. To further increase sterol accumulation, the highest sterol producer, P. lutheri
Microalgae was subjected to nutrient and salinity changes. Although no signicant immediate effects were observed for al-
Pavlova lutheri tered total sterol contents, individual sterols varied and, signicantly higher total sterol amounts (up to a 2-fold
Plant sterols increase) were found after prolonged cultivation. Total sterol accumulation of 5.1% dry weight was achieved in
Sterol proles P. lutheri. Combined with the high areal productivity, commercial phytosterol production from microalgae
could be considered.
2015 Published by Elsevier B.V.

1. Introduction
Phytosterols or plant sterols belong to the chemical group known as
triterpenes and they are structurally and functionally similar to choles-
terol in the sense that they have four-ring steroid nucleus, the 3-
hydroxyl group and often a 5,6-double bond and also because they
play a major role in maintaining stability of the phospholipid bilayers
in cell membranes. The differences with cholesterol include the number
of carbon atoms in side-chains (cholesterol has eight carbon atoms
whereas phytosterols have 9 or 10) [1]. Moreover, phytosterols can
only be obtained by animals through their diets [2].
Sterols are important constituents of cell membranes, being
intertwined with phospholipid bilayers in all eukaryotes. Through
such existence, the sterols play signicant roles in the maintenance of
cellular structural stability, and acclimation to membrane temperature
[3]. Sterols help maintain uidity and permeability by controlling move-
ment of fatty acid chains within the membrane [4]. More than 200 dif-
ferent sterols have been reported in plants, whereas the most
dominant sterol reported in animals is cholesterol [5,6]. The distribution
of sterols in plants varies, with some species producing only one or two
types, whereas others harbour ten or more different sterols [7]. Sterols
have become useful biomarkers for the determination of the origin of
organic matter in the sediments, due to the stability of these compounds
over geologic time and thus have been used extensively for chemotax-
onomic and phylogenetic comparisons [8].
Corresponding author.
E-mail address: p.schenk@uq.edu.au (P.M. Schenk).
Phytosterols have become well-known for their ability to lower cho-
lesterol. It is well established that high total or low density lipoprotein
(LDL)-cholesterol is the major contributor to coronary heart disease
(CHD) as well as morbidity and mortality in developed countries and
1% reduction in total cholesterol may reduce the risk of CHD by 3%.
The cholesterol lowering ability of phytosterols was rst reported in
1951 which led to the development of the product Cytellin marketed
by Eli Lilly. Phytosterols reduce the intestinal absorption of dietary and
biliary cholesterol and studies have suggested that an intake of 2 g/day
can cause a 10% reduction in the levels of LDL-cholesterol in the blood
and for this reason phytosterols have been used as additives in various
food items such as margarine, yoghurt and milk during processing [9].
Besides lowering of cholesterol, phytosterols have been reported to be
involved in anti-inammatory and anti-atherogenicity, anti-cancer
and anti-oxidative activities. In addition, evidence has been provided
showing that phytosterols provide protection against nervous system
disorders such as autoimmune encephalomyelitis, amyotrophic lateral
sclerosis or Alzheimer's disease [10,11].
Phytosterols are also used as raw materials for therapeutic steroids
through processes involving bio and chemical transformations and the
world market for such steroids was estimated at 1000 tonnes per year.
In addition, phytosterols have already been introduced in the cosmetics
industries e.g. in creams and lipstick [1]. With expansion of this market,
research is focussing on identifying new natural sources of phytosterols
and microalgae have been demonstrated as a suitable alternative source
of these functional compounds [12]. Currently, the main industrial
sources of phytosterols are tallow and vegetable oils, but with the devel-
opment of suitable induction, recovery and purication methods,

http://dx.doi.org/10.1016/j.algal.2015.05.013
2211-9264/ 2015 Published by Elsevier B.V.
 F. Ahmed et al. / Algal Research 10 (2015) 210217
microalgae may become a suitable environmentally friendly substitute
for phytosterol production [8]. Microalgae have recently created a
wide interest due to several advantages: they possess high areal pro-
ductivities, are relatively easy to cultivate, do not need to compete
with food production for arable land or fresh water, and can adapt to en-
vironmentally changing conditions by producing a wide variety of sec-
ondary metabolites. Moreover, microalgae can be used to purify and
take up nutrients from wastewater [13]. The biosynthesis of phytos-
terols can be triggered by metabolic engineering, either by controlling
the cultivation conditions or by using genetic engineering. Currently,
phytosterols have a global market of nearly USD $1 billion and this is ex-
pected to grow by 79% per annum. Due to expanding market demand
for nutraceuticals from natural sources, phytosterols from microalgae
could potentially contribute signicantly if their capacity to produce
these secondary metabolites is fully explored [12].
Although many microalgal species have been studied for distribu-
tion of sterol compounds as reviewed by Volkman [14], screening stud-
ies comparing the quantity of sterols in different species have been
reported scarcely so far, with comparisons of sterol contents available
on only nine species [15]. However, it has been reported that changes
in growth conditions such as the renewal rate of semicontinuous cul-
tures [16], salt concentration in the growth medium [8,17,18], type of
photobioreactor [19], light and phosphorus content [20], temperature
and silicate content [3] and the stage of growth [21] can affect the pro-
duction of sterols in the different microalgae species studied so far.
In this study, we screened for the rst time ten microalgal species
(currently used in aquaculture and the nutraceuticals industry) for phy-
tosterols on a per gramme dry weight basis. The highest phytosterol-
producing species, Pavlova lutheri, was further analysed by applying
nutrient and salinity changes in the growth medium and prolonged cul-
tivation, leading to phytosterol accumulation over 5% DW, the highest
reported so far for any microalga.
2. Materials and methods
2.1. Screening for phytosterol content in microalgal species
Out of several hundred microalgal isolates that were collected in
coastal or brackish water and maintained at the Algae Biotechnology
Laboratory at the University of Queensland, Australia, ten species were
selected, mainly based on their rapid growth and ease of handling com-
pared to other cultures. Microalgal species Dunaliella salina, Isochrysis
galbana, Nannochloropsis sp. BR2, Pavlova lutheri, P. salina, Chaetoceros
muelleri, Tetraselmis chui, Tetraselmis suecica, Tetraselmis sp. M8 have
been described previously [22]. Phaeodactylum tricornutum was obtain-
ed from Commonwealth Scientic and Industrial Research Organisation
(CSIRO)'s collection of living microalgae (Strain ID: CS 29/8; GenBank
Accession: EF140622.1). Pure cultures were obtained as described pre-
viously [23]. Microalgal cultures (100 mL each) were inoculated from
master cultures of various species in 250 mL asks with F/2 medium
(AlgaBoost F/2 2000) and were grown at 25 C with constant aera-
tion under 16:8 h light/dark photoperiod of uorescent white light
(80 mol photons m2 s1). When cultures reached the late exponen-
tial growth phase (doubling times were twice as long as during the
highest exponential growth), the cultures were centrifuged, the super-
natant was decanted and the remaining biomass was washed in MilliQ
water before freeze-drying. The freeze-dried samples were kept at
20 C until extraction for phytosterol analysis.
2.2. Cultivation of P. lutheri at different nutrient levels and salinities
Pure P. lutheri cultures from the University of Queensland's Algae Bio-
technology Laboratory master culture collection were inoculated in three
2 L-asks with F/2 medium (AlgaBoost F/2 2000) at 35 salinity.
When these separately-grown cultures reached the late exponential
growth phase (as described previously), they were transferred to
211
200 mL F/2 medium in 250 mL asks. These cultures were either grown
at different nutrient levels or different salinities (three separately-
grown cultures per treatment). The late exponential phase was chosen
based on the assumption that the level of biomass in the culture would
be the highest at that phase. Therefore, it was hypothesized that the re-
sponse in terms of secondary metabolite accumulation would be trig-
gered to the highest level at this phase.
Flasks containing nitrate and phosphate levels, three and six times of
the normal F/2 medium content were designated as 3N, 6N, 3P and 6P,
respectively. Another set of P. lutheri cultures containing regular F/2 me-
dium was grown as controls. Biomass (25 mL culture) was collected
regularly from each ask until 10 days since the initiation of the nutrient
manipulation treatment. Medium was added every two days (addition
of 100 L 2000 F/2 stock solution) to avoid any effect of nutrient dep-
rivation during these 10 days. In a separate experiment, the P. lutheri
cultures were grown until the nitrate and phosphate was used up (con-
rmed by using an API nutrient testing kit according to the
manufacturer's instructions). Afterwards, these cultures were grown
in nitrate deplete (designated as N), phosphate deplete (P), or nitrate
and phosphate replete (F/2) medium. A set of cultures without nitrate
and phosphate in the medium was grown as controls (C). All treated
cultures were grown in triplicates. Biomass (25 mL culture) was collect-
ed for 2 days after the initiation of nutrient deprivation treatments. All
collected biomass was centrifuged (3000 g; 3 min), washed in Milli-
Q water, freeze-dried and kept at 20 C until extraction.
For the salinity experiment, biomass (25 mL samples) from cultures
grown in 15, 25, 35 or 45 was collected on days 2, 4 and 6 after trans-
ferring the cultures to the 250 mL asks, and processed as described
above. F/2 medium was added every two days (addition of 100 L
2000 F/2 stock solution) to prevent nutrient deprivation. On day 11,
the cultures were 50% diluted by addition of medium of the same salin-
ity. Further biomass samples were collected on days 12, 14, 16 and proc-
essed as described above.
2.3. Proling and quantication of microalgal sterols
The proling and quantication of microalgal sterols were conduct-
ed following the methods described previously [24] with minor modi-
cations. In brief, the frozen dried microalgal biomass was transferred to
2 mL microcentrifuge tubes. Cholestanol (purchased from Sigma, and
puried by HPLC) in toluene was added to each tube as internal stan-
dard (2.5 g/sample). The samples were rst saponied to reduce the
fatty acids interference for the subsequent analysis. To saponify the
sample, 0.5 mL of 10% methanolic KOH was added and incubated at
75 C for 2 h. After cooling to room temperature and addition to
250 L of 0.9% NaCl solution, the nonsaponiable fraction (NSF) was ex-
tracted from the mixture with n-hexane. The NSF was dried under vac-
uum and sterols in NSF were converted to their trimethylsilyl esters by
addition of 10 L BSTFA with TCSM (BSTFA:TCSM = 99:1) with 10 L of
anhydride pyridine as catalyst, and subjected for sterol analysis using
Agilent 6890 gas chromatograph (GC) coupled with a 5975 mass selec-
tive detector. A total of 1 L of sterols was injected for gas chromatogra-
phy/mass spectrometry (GC/MS). A DB-5 type column (Varian factor4
VF, 30 m 0.25 mm 0.25 m) was used for sterol analysis. The GC
was programmed as follows: initial temperature 100 C and hold for
1 min. The temperature was ramped to 280 C at 37 C/min then to
325 C at rate of 10 C/min followed by holding at the same temperature
for the next 2 min. Sterols were eluted from the column from 15 to
20 min, and the sterols were identied based on their retention times
and their fragmentation patterns of mass spectra. The amount of each
sterol was calculated based on its response (total ion current, TIC) rela-
tive to that of the internal standard. Most sterols were identied based
on the NIST 08 mass spectra library, with a cut-off score at 80%. Others
were identied based on the GC/MS proles of sterols of Pavlova report-
ed previously [25,26].
212 F. Ahmed et al. / Algal Research 10 (2015) 210217
2.4. Statistical analysis
The effects of salt concentration in the medium and sampling days
were compared by repeated measures two-way ANOVA followed by
Tukey's test in order to compare the differences between individual
treatments. The differences were considered signicant when P values
were below 0.05. Data were expressed in average SE. The statistical
software GraphPad Prism 6.0 was used for the analyses.
3. Results
3.1. Screening for phytosterols in microalgal species isolated from brackish
and seawater
Among the 10 microalgal species screened for sterols, the highest
content by far was found in P. lutheri (26.05 mg/g dry weight (DW)),
followed by Tetraselmis sp. M8 (4.32 mg/g DW), and Nannochloropsis
sp. BR2 (4.04 mg/g DW; Fig. 1). The lowest content was measured in
P. salina (0.16 mg/g DW). A total of 25 sterol compounds were detected
in the 10 microalgal species screened (Table 1; see Supplementary
Table S1 for chemical structures). There was variability in the number
and distribution of sterol compounds in the ten microalgal species
with the highest number found in P. lutheri (11), D. salina (7), P. salina
(6), and C. muelleri (5; Table 1). As expected, the most dominant sterol
compounds were identical within the microalgal species of the same
genus (24-methylcholest-5-enol for Tetraselmis and poriferasterol for
Pavlova; Table 1).
3.2. Induction of phytosterol production in P. lutheri
Previous studies have shown that availability of nutrients and salinity
of the cultivation medium may inuence phytosterol biosynthesis in
microalgae [3,8,17,18]. When P. lutheri was grown under high nitrate
or phosphate concentrations, the sterol contents varied from 5.0
0.8 mg/g DW (6P on day 2) to 7.6 0.6 mg/g DW (control on day 10)
during the 10 days of cultivation (Supplementary Fig. S1). However, no
signicant difference in sterol content between different nitrate or phos-
phate levels and the control at different sampling days could be found
(two way ANOVA; p N 0.05). In the nutrient-starved biomass, the sterol
content varied from 8.6 0.9 mg/g DW (control on day 2) to 9.5
0.8 mg/g DW (F/2 on day 2) during the 2 days of rearing (Supplementary
Fig. S2). But similar to the high nutrient experiment, no signicant differ-
ence was found in sterol content among the sampling days or the differ-
ent nutrient levels (two way ANOVA; p N 0.05). This suggests that
nutrient levels may not play a major role for phytosterol biosynthesis
in P. lutheri.
Fig. 1. Ranking of ten microalgal species by total sterol (mg/g dry weight; sum of identied
sterols).
Next, the effect of salinity on phytosterol production was tested in
P. lutheri, both as a short term effect during 26 days after cultures
were placed from 35 to higher or lower salt concentrations, and
over a longer period up to 16 days after cultures were diluted for further
growth on day 11. Total sterol contents during the initial responses to
salinity changes (26 days) varied from 20.29 0.96 mg/g DW (day
4; 25 salinity) to 29.44 3.8 mg/g DW (day 2; 15 salinity) but no
signicant differences were found between treatments (Fig. 2). Howev-
er, when cultures were diluted on day 11 and adapted to the varying salt
concentrations in the medium the total sterol contents slightly in-
creased and varied from 25.98 4.8 mg/g DW (day 12 at 45 salinity)
to 51.86 3.8 mg/g DW (day 14 at 35 salinity). Signicantly higher
sterol levels were found for cultures grown at 25 on the 16th day
(46.7 10.1 mg/g DW compared to day 4: 20.29 0.96 mg/g DW;
Tukey's test: p b 0.05) and at 35 on the 14th day when compared to
the non-adapted cultures (51.86 3.8 mg/g DW compared to day 4:
23.94 0.1 mg/g DW or day 6: 21.42 1.9 mg/g DW; Tukey's test:
p b 0.05; Fig. 2). Statistical analysis conrmed that signicant differ-
ences were observed between the sampling days but not for the differ-
ent salinities (Table 2). As this effect was independent of whether or not
cultures underwent a salinity change, it is likely that the increase in total
phytosterol was supported by the dilution of cells on day 11, leading to a
lower cell density and a higher light exposure per cell. Notably, the re-
sults show that microalgal phytosterol contents higher than 5% DW
are achievable in P. lutheri.
3.3. Proling of P. lutheri phytosterols at different salinities
A total of nineteen sterol compounds were detected in P. lutheri bio-
mass and their contents varied at different salinities of the medium
(Table 3). Among these compounds, the concentrations of epicam-
pestanol, ergost-7-enol, dihydrochondrillasterol, chondrillasterol, and
an unknown compound (named as unknown-2) were very low (gener-
ally 1020 g/g DW) and therefore these compounds were not consid-
ered for statistical analysis due to the assumption that they had very
little effect on the overall sterol content. Six other compounds, namely
clinosterol, 4-alpha-methylporiferast-22-enol, methylpavlovol, 22-
dehydroethylpavlovol, and two other unknown compounds (named
as unknown-1 and unknown-3) did not vary signicantly at different
salinities or sampling days (repeated measures ANOVA; p N 0.05).
Among the remaining eight compounds, poriferasterol, epicampesterol,
ethylpavlovol, epibrassicasterol, and cholesterol showed signicant dif-
ferences in the rst six days after the salinity change (repeated mea-
sures ANOVA; p b 0.05) but no differences were found between the
salinities or sampling days after cultures were adapted to the salinities
(repeated measures ANOVA; p N 0.05; Fig. 3). Poriferasterol in the
35 cultures decreased over time when compared to other salinities
and was the lowest on the 6th day (Tukey's test: p b 0.05); however,
the levels were higher on the 14th and 16th days in all salinities except
45 (Fig. 3). Similarly, ethylpavlovol had the highest levels on the 6th
day after the salinity change (15 and 25) and it continued to go
up in the adapted cultures during later time points (Table 3; Fig. 3).
On the contrary, epicampesterol and epibrassicasterol were at the
highest level on the 2nd day after the salinity change to 15 and the
levels declined in the next four days (Tukey's test: p b 0.05; Fig. 3). Sim-
ilarly, cholesterol was also at the highest level on the 2nd day for all sa-
linities tested (Tukey's test: p b 0.05) and it continued to decline as the
cultures got older (Fig. 3). On the contrary, 22-dehydromethylpavlovol
differed signicantly on days 1216 when the cultures were adapted
to the salinities (repeated measures ANOVA; p b 0.05) and was at the
highest level for 15 and 25 cultures on the 16th day (Tukey's test:
p b 0.05) but did not differ in the rst six days after osmotic shock (re-
peated measures ANOVA; p N 0.05; Fig. 3). The remaining two com-
pounds, 4-alpha-methylergost-22-enol and 4-alpha-methylergostanol
differed at both early and late timepoints (repeated measures ANOVA;
p b 0.05; Fig. 3).
 F. Ahmed et al. / Algal Research 10 (2015) 210217 213

Table 1
Composition (% of total sterols) of sterol in 10 microalgae species from subtropical coastal and brackish water.

Sterols Dunaliella Tetraselmis Isochrysis Tetraselmis Tetraselmis Pavlova Pavlova Chaetoceros Nannochloropsis Phaeodactylum
salina sp. M8 galbana chui suecica salina lutheri muelleri sp. BR2 tricornutum

Cholesterol + (5.2) + (0.34) + (1.76) + (0.15) + (0.23) n.d. n.d. +++ +++ (77.73) + (2.71)
(66.1)
Epibrassicasterol n.d. n.d. +++ n.d. n.d. n.d. + (0.53) + (1.82) n.d. +++ (95.34)
(98.24)
Ergosterol ++ n.d. n.d. n.d. n.d. + (1.43) n.d. n.d. n.d. n.d.
(28.32)
24-Methylenecholest-5-enol n.d. n.d. n.d. n.d. n.d. n.d. n.d. + (1.88) n.d. n.d.
Ergost-5-enol n.d. +++ n.d. +++ +++ +(9.85) +(8.36) n.d. n.d. + (1.94)
(99.66) (99.85) (99.77)
24-Methylcholesta-7,22-dienol + (1.14) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Epicampestanol n.d. n.d. n.d. n.d. n.d. n.d. + (0.39) n.d. n.d. n.d.
Poriferasterol n.d. n.d. n.d. n.d. n.d. ++ ++ + (1.07) n.d. n.d.
(35.2) (35.61)
24-Ethylcholest-22-enol n.d. n.d. n.d. n.d. n.d. n.d. + (4.31) n.d. n.d. n.d.
Fungisterol + (4.05) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
24-Ethylcholesta-5,7,22-trienol ++ n.d. n.d. n.d. n.d. + (3.6) n.d. n.d. n.d. n.d.
(58.5)
Fucosterol n.d. n.d. ++ (29.1) n.d. n.d. n.d. n.d. n.d. + (8.28) n.d.
Clinasterol n.d. n.d. n.d. n.d. n.d. ++ ++ n.d. n.d. n.d.
(24.62) (24.26)
Chondrillasterol + (0.64) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
24-Ethylcholestanol n.d. n.d. n.d. n.d. n.d. n.d. + (0.09) n.d. n.d. n.d.
Isofucosterol n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. ++ (13.98) n.d.
4-Alpha-methylergost-22-enol n.d. n.d. n.d. n.d. n.d. n.d. + (2.05) n.d. n.d. n.d.
4-Alpha-methylporiferast-22-enol n.d. n.d. n.d. n.d. n.d. ++ ++ (22) n.d. n.d. n.d.
(15.74)
Dihydrochondrillasterol + (2.1) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
22-Dehydroethylpavlovol n.d. n.d. n.d. n.d. n.d. n.d. + (0.42) n.d. n.d. n.d.
Methylpavlovol n.d. n.d. n.d. n.d. n.d. n.d. +(1.98) n.d. n.d. n.d.

n.d.: not detectable.

+++: high (N50% of total sterol content); ++: average (50% of total sterol content); +: low (b10% of total sterol content).
Total sterol concentrations of the 10 microalgae species are summarised in Fig. 1.

4. Discussion
To our knowledge this is the rst report on screening for sterols on a
per gramme DW basis of several microalgal species that are currently
used in the aquaculture and neutraceuticals industry. We also report
here for the rst time the distribution of individual sterol compounds
and total sterol content in T. chui, P. salina and C. muelleri. Among the
other seven species previously reported, only I. galbana and T. suecica
had similar total sterol contents (Table 4). Apart from T. suecica, the
number of sterol compounds detected in the previous studies has
been different from what has been found in the present study. The dif-
ferences can likely be attributed to the variation in the origin and type
of species, condition of growth, or the analytical procedures adopted
by the different researchers. Our screening data also conrms the previ-
ous ndings that P. lutheri is the highest phytosterol producer among
the studied microalgae species [15,27].
4.1. Effect of different nutrient and salinity of growth media on microalgal
sterol content
Nutrients especially phosphorus and silicate (for diatoms) have
been reported to cause changes in the levels of sterols in the freshwater
phytoplankton species studied by Piepho et al. [3]. However, no report
on the nutrient effects (e.g. manipulation of nitrogen and phosphorus
content or deprivation of nitrogen or phosphorus in the medium) of
sterol accumulation in other high producing species such as Dunaliella
tertiolecta, D. salina or P. lutheri is available. In our study, no signicant
change in sterol content could be found at high nitrate or phosphate
levels or even at nitrate- and/or phosphate-deprived conditions. The
results have some similarities with the ndings of Piepho et al. [3,20]
who reported no effect of phosphorus on sterol contents in Cryptomonas
ovata but interactive effects of light, temperature and nutrients (e.g.
phosphorus and/or silicate) on sterol content in Scenedesmus
quadricauda and Cyclotella meneghiniana. The lack of effect of phospho-
rus in the medium on sterol content in C. ovata was attributed to the
mixotrophic nature of the species with the hypothesis that C. ovata
switched to heterotrophy under unfavourable conditions, avoiding
maintenance of the functions of the photosynthetic apparatus by chang-
ing the biochemical composition of chloroplast membranes [3]. This

Table 2
Repeated measures two-way analysis of variance of salinity and sampling days for total
sterol from Pavlova lutheri.

Source of variation df SS MS F P

Salinity 3 313.6 104.5 0.5809 0.6582

Fig. 2. Total sterol (mg/g dry weight) in Pavlova lutheri at different salinities under adap-
Time 5 2123 424.7 6.438 0.001
tation and non-adaptation scenarios. Shown are mean values and SEs from three separate-
Salinity Time 15 828.9 55.26 0.8377 0.6317
ly-grown cultures. Different letters indicate statistically signicant difference (Tukey's
test; p b 0.05). Highly signicant p b 0.01.
214 F. Ahmed et al. / Algal Research 10 (2015) 210217

differs with the present study where P. lutheri was solely grown under

0.02
0.15
1.69
0.15

1.23
0.01
5.12
0.47
8.38

0.01
0.13
6.53
0.07

0.39
0.06
7.8

0.7
nd
45
autotrophic conditions.

1
Changes of salinity in the culture medium have been reported to af-
0.02
0.17
1.57
0.12

1.76
0.01

0.48
8.76

0.05
0.19
6.16
0.06
0.66
0.05
0.04
0.38
8.1

4.3

nd
35

fect sterol contents since the 1980's [8,17,18]. The studies were carried
out on Dunaliella spp. probably because of their ability to withstand var-
0.02
0.34
2.47
0.02

2.84
0.02

0.66

0.01
0.37

0.08
0.77
0.08
0.04
0.59
ious salinity levels, and due to the hypothesis that quick adjustment of
13.1

10.7
5.2

0.1

9.4
25

sterol in the cell membrane may offer an advantage for salinity adapta-
Day 16

tion. These authors reported a decrease in sterol contents with an in-

0.01
0.26
2.15
0.08
11.2
2.26
0.02
5.32
0.45
9.47

0.38
10.3

0.87
0.08
0.07
0.77
0.1

0.1
nd
15

crease in salt concentration, postulating that the hyperosmotic shock

caused a two-stage response in the cell membrane where at rst the ste-
0.03
0.08

0.08

0.92
0.01
5.15
0.45
8.34
0.01
0.01
0.12
5.55
0.06
0.87
0.05
0.08
0.47
1.5

6.6

rols act to increase the low membrane lipid order followed by a de-
45

crease in sterol contents as the new status of membrane lipid order

0.05
0.26

0.31

2.47
0.06
7.28

0.02
0.01
0.34

0.11
1.19
0.17

0.42
was achieved due to the stability of the new osmotic equilibrium [8].
12.3

12.6

10.7
2.9

0.8

0.4
35

In the present study, P. lutheri cultures were initially grown in 35

salt medium but then transferred to 15, 25, 35 and 45 salt medium
0.02

2.06
0.05

1.95
0.02
4.98

0.01
0.01
0.36
9.65
0.11
0.85
0.19
0.02
0.33
0.2

9.6

0.5

which did not show any difference in sterol contents over the next six
11
25

days. This agrees with the ndings of Peeler et al. [17] but differs with
Day 14

the ndings of Zelazny et al. [18] and Francavilla et al. [8], indicating
0.02

1.63
0.06

0.01
5.67
0.24
6.96

0.01
0.32
7.82
0.09

0.13
0.02
0.31
0.1

8.8

0.8
nd
15

that the postulation outlined by Francavilla et al. [8] for the decrease
in sterol contents due to hyperosmotic shock may not be applicable
0.04
0.09

0.01

0.97
0.01
5.07
0.26
5.41
0.01
0.01
0.13
5.44
0.03
0.69
0.05
0.03
0.22
1.6
45

for P. lutheri. The theory is not applicable for P. lutheri also because of
6

our ndings that the sterol contents were high at 15, 25 and 35
0.03
0.09
1.47
0.04

1.16
0.01
5.42

8.26

0.01
0.18
5.78
0.06
0.72
0.06
0.02
0.24
7.3

0.3
Composition of sterol (mg/g) in P. lutheri grown at four salt concentrations on different sampling days after adaptation or non-adaptation to the growth medium.

even 16 days after the osmotic shock indicating that sterols were still
nd
35

playing a major role in reaching the membrane lipid order a long time
0.02
0.09
1.74
0.01

1.41
0.01
5.69
0.29
9.61
0.01
0.01
0.34
7.65
0.08
0.73
0.07
0.01

after the salinity shock was provided. Possible reasons for the signi-
7.6

0.3
25

cantly higher contents of sterols in cultures grown for longer times

Day 12

(Fig. 2) may be caused by the accumulation of certain nutrient compo-

0.02
0.08
1.75
0.01

1.05
0.01
7.09
0.22

0.01
0.01
0.27
9.09

1.38

0.02
0.25
8.2

0.1

0.1
15

nents or exudates in the growth medium, or by the dilution of cultures

on day 11, leading to lower cell densities and increased light exposure
0.09
0.11
1.71
0.02

0.81
0.01
4.86
0.18
3.88
0.01
0.01
0.11
3.54
0.05
0.46
0.17
0.02
0.19
6.4

on a per cell basis. Indeed, high light (e.g. 500 mol photons m2 s2)
45

in combination with high phosphorus contents in the medium has

0.06
0.11
1.62
0.02

0.78
0.01
4.76
0.13
3.24
0.07
0.01
0.12
3.31
0.04
0.39
0.15
0.04
0.32
6.3

been reported to play an important role in increasing sterol content in

35

S. quadricauda [20]. Our ndings may be explained by the results report-

0.06
0.19

0.03

1.19
0.01
5.07
0.33

0.01
0.12

0.02

0.17
0.03
0.32

ed for Nannochloropsis oceanica [28]. These authors reported that light

2.2

3.5

4.2

0.4
nd
25

(e.g. 100 to 300 mol photons m2 s2) played a signicant role in ste-
Day 6

rol production through changes in several genes involved in the sterol

0.02
0.31
2.36
0.03

1.59
0.01
4.75
0.28
3.29

0.01
0.15
4.47
0.02
0.41
0.18
0.04
9.8

0.4
nd
15

biosynthesis pathway (e.g. SMT1, SMT2, DWF5, DWF7; Fig. 4). They
also reported higher production of sterols at mid to late log phase of
0.09
0.16
1.94
0.03

0.99
0.01

0.24
3.73

0.01

3.34
0.03
0.36
0.13
0.04
0.36
7.5

5.1

0.1
nd
45

growth of N. oceanica cultures due to higher upregulation of sterol bio-

synthesis genes during these phases. The 50% dilution of P. lutheri on day
0.13
0.16
1.89
0.02

1.09
0.01
5.13
0.21
3.57

0.01
0.08
3.13
0.03
0.29

0.03
0.31

11 in our study may have taken the cultures to the mid to late log phase
7.8

0.1
nd
35

coupled with the exposure of higher number of cells to a light level

0.08

1.75
0.01

1.02
0.01
4.35
0.19
2.63

0.01
0.08
2.32
0.02
0.21
0.11
0.03
0.31

below the saturation point. This may have led to efcient light utilization
0.2

nd
25

for photosynthetic system and upregulation of sterol biosynthesis genes

Day 4

resulting in higher phytosterol production on days 14 to 16 (Fig. 2).

0.07
0.25
2.26
0.01

1.23
0.01
5.38

0.01
0.01
0.09

0.02
0.22
0.08
0.03
0.35
8.4

0.2
2.6

2.9
15

However, further studies on the effect of light as well as in-depth studies

on the gene/enzyme expression proles of the P. lutheri biomass are war-
1.97
0.02

0.97
0.01
4.28
0.19
3.36

0.01

3.22
0.02
0.33
0.04
0.02
0.23
0.1
0.2

8.6

0.1
nd
45

ranted to understand the mechanism of high sterol accumulation in this

alga and ne-tune the biosynthetic pathway to make this alga commer-
0.17

0.02

1.24
0.02
5.65
0.24

0.01
0.01
0.09

0.03
0.27
0.07
0.03
0.33

cially suitable for large-scale production of phytosterols.

0.2
2.4

9.7

4.1
35

3
0.32

0.01

0.01
5.26
0.25
3.61

0.01
0.09
4.22
0.03
0.32
0.11
0.03
0.45

4.2. Effect of salinity and adaptation to salt concentration on individual ste-

0.1

2.6

8.5
1.6

nd
25

rol compounds
Day 2

0.48

0.01
11.2
1.47
0.01
5.11
0.25
2.63

0.13
4.23
0.02
0.31
0.08
0.02
0.33
0.1

3.1

nd
nd
15

The composition of sterols was similar in all salt concentrations test-

ed (Fig. 3), conrming the assertion that the prole of sterols can be use-
4-Alpha-methylporiferast-22-enol

ful for chemotaxonomic and phylogenetic comparisons, as the sterol

4-Alpha-methylergost-22-enol

proles can be the characteristic of microalgae taxa [29,30]. Among

22-Dehydromethylpavlovol
4-Alpha-methylergostanol

22-Dehydroethylpavlovol

the different sterol compounds identied at the adaptation and non-

Dihydrochondrillasterol

adaptation scenarios, signicant differences were observed for 4-

alpha-methylergost-22-enol and 4-alpha-methylergostanol, which
Epibrassicasterol

Chondrillasterol
Epicampestanol

Methylpavlovol
Epicampesterol

constitute a minor portion of the total sterol (~6%; Fig. 3). This indicates
Ethylpavlovol
Ergost-7-enol
Poriferasterol

Unknown-1
Unknown-2
Unknown-3
Clionasterol
Cholesterol

that these two compounds are actively involved during the response to
the osmotic shock process and may also have been the intermediaries in
Sterol
Table 3

the sterol biosynthetic pathway which explains the small percentage in

the overall sterol content of P. lutheri. In the present study, although
 F. Ahmed et al. / Algal Research 10 (2015) 210217 215

Fig. 3. Composition of sterol (g/g dry weight) in Pavlova lutheri under adaptation and non-adaptation scenarios at different salinities. Shown are mean values and SEs from three sepa-
rately-grown cultures. Different letters indicate statistically signicant difference (Tukey's test; p b 0.05).

total sterol contents did not differ, a higher number of compounds var-
ied signicantly at different salinity concentrations soon after the os-
motic shock, compared to when P. lutheri was allowed to adapt to the
different salt concentrations in the medium. This indicates that soon
after the salinity change most of the sterol compounds present in
P. lutheri actively participated in the cell recovery process, but with
the passage of time as the equilibrium is restored through adjustment
to the new osmotic condition in the cell membrane, fewer sterol com-
pounds varied. The subtle sterol prole changes after prolonged growth
in the adaptation scenario may have been due to their involvement in

Table 4
Comparison of total sterol content of microalgae species used in the current study with previous reports.

Microalgae Total sterols in previous studies (mg/g) Current study (mg/g) No of sterol compounds detected in previous studies Current study

Isochrysis galbana 2.25 [39]; 2.5 [40] 2.44 4 [39]; 2 [40] 3

Nannochloropsis oculata 2.3 [40] 4.04 6 [40] 3
Dunaliella salina 8.89 [8] 2.26 12 [8] 7
Pavlova lutheri 8.45 [39] 26.05 8 [39] 11
Phaeodactylum tricornutum 1.1 [16] 3.37 1 [16] 3
Tetraselmis suecica 2.1 [16] 2.51 2 [16] 2
216 F. Ahmed et al. / Algal Research 10 (2015) 210217

In addition to cholesterol-lowering activity in hypercholesterolemia-

Fig. 4. Generalized sterol biosynthesis pathway for microalgae. Details of the abbreviations
used: HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A; IPP: isopentenyl pyrophos-
phate; DMAPP: dimethylallyl pyrophosphate; FPP: farnesyl pyrophosphate; SQE: squa-
lene epoxidase; CAS: cycloartenol synthase 1; CPI1: cyclopropyl isomerase; SMO1: sterol
4-alpha-methyl oxidase 1; SMT1: sterol methyltransferase 1; SMT2: sterol methyltrans-
ferase 2; DWF1: (24)-sterol reductase (Dwarf1); DWF5: sterol delta7 reductase
(Dwarf5); DWF7: delta7 sterol C-5 desaturation (Dwarf7); CYP51: cytochrome P450 51.
Adapted from Sasso et al. [42] and Lu et al. [28].
the turning over of sterols, as pointed out by Zelazny et al. [18], rather
than adaptation to the salinity levels or the medium dilution on day
11, as the passage of the 14 or 16 days may be too long to be still
responding to the osmotic imbalance in the cell membrane.
In the present study, the major three sterol compounds in P. lutheri
were 4-alpha-methylporiferast-22-enol, poriferasterol, and clionasterol.
Table 5
Comparison of phytosterol levels in food and microalgae.
induced rabbits [31], these compounds have been conrmed to have
anti-inammatory activity [32], in vitro and in vivo anticancer activity,
especially against hepatocellular carcinoma, and these compounds
were able to restore the levels of liver enzymes such as alanine transam-
inase (ALT), aspartate aminotransferase (AST) and albumin [33]. More-
over, a combination of these sterol compounds with cocoa polyphenols
and extracts of a composition of plants containing these sterol com-
pounds were reported to treat, prevent or reduce the risks of heart ar-
rhythmias, diseases involving defective gap junctional communication
between cells such as cognitive dysfunction, stimulate immune system,
promote prostate health, and assist treatments of chronic and degenera-
tive diseases involving malignant tumours [34,35]. The other major ste-
rol compound, epicampesterol or 22-dihydrobrassicasterol (synonym
of 24-campesterol) has been reported to possess antimicrobial activity
against Staphylococcus aureus and Escherichia coli [36]. Another major
sterol compound, methylpavlovol has been found in several Pavlova spe-
cies [25,37] and is reported to contribute signicantly to metabolic activ-
ities in scallops (e.g. Pecten maximus) and oysters (e.g. Crassostrea gigas)
[19]. However, its impact on human cells is yet to be studied.
Currently, exploitation of microalgae for the purpose of producing
phytosterol is an unexplored area due to the suggestion that the average
levels are very low going as far as 0.57% for diatoms and 3% in dinoa-
gellates [14]. The ndings in our study of 5.1% sterol content in
P. lutheri is higher than such estimates and could make P. lutheri a prom-
ising candidate for further studies targeting the commercial production
of these benecial compounds. Phytosterols from P. lutheri microalgae
present the highest percentage reported among various vegetables,
fruits, and crude vegetable oils (Table 5). The high sterol content in
P. lutheri presents an additional benet from this species as a highly
sought after microalga because P. lutheri is already commercially used
in shellsh hatcheries and well-known as a good source of omega 3
fatty acids [38] and its high phytosterol content may make this species
amenable for a microalgal biorenery concept.
5. Conclusion
The haptophyte P. lutheri was identied as a high phytosterol-
producing microalga after screening ten fast-growing species of impor-
tance in the aquaculture and nutraceutical industries. Medium nutrient
alterations or exposure to osmotic shock by salinity changes did
not cause a signicant increase in phytosterol production. However,
allowing the cultures to grow for a longer period of time caused higher
production of sterols. To our knowledge, a phytosterol content over 5%
DW, as found in the present study for P. lutheri, is the highest reported
for any microalgae so far, suggesting that commercial production of
phytosterols from microalgae could be further developed.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.algal.2015.05.013.

Food Sterol content (mg/100 g) References Acknowledgements

Vegetable oil (crude): [1]
Corn 850 We wish to thank the Australian Research Council (LP0990558) for
Rapeseed 820 nancial support.
Sunower 430
Soya 350
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